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Sample records for alternative human cd133

  1. Promoter hypomethylation regulates CD133 expression in human gliomas

    Institute of Scientific and Technical Information of China (English)

    Kouichi Tabu; Ken Sasai; Taichi Kimura; Lei Wang; Eiko Aoyanagi; Shinji Kohsaka; Mishie Tanino; Hiroshi Nishihara; Shinya Tanaka

    2008-01-01

    Brain tumor-initiating cells (BTICs) have been enriched using antibodies against the cell surface protein CD133;however,the biological relevance and the regulatory mechanism of CD133 expression in human gliomas are not yet understood.In this study,we initially demonstrated that CD133 was overexpressed in high-grade human glioblastomas where CD133-positive cells were focally observed as a micro-cluster.In addition,CD133 transcripts with exon 1A,1B,or 1C were predominantly expressed in glioblastomas.To elucidate the mechanism regulating this aberrant expression of CD133,three proximal promoters (P1,P2,and P3) containing a CpG island were isolated.In U251MG and T98Gglioblastoma cells,the P1 region flanking exon 1A exhibited the highest activity among the three promoters,and this activity was significantly inactivated by in vitro methylation.After treatment with the demethylating agent 5-azacytidine and/or the histone deacetylase inhibitor valproic acid,the expression level of CD133 mRNA was significantly restored in glioma cells.Importantly,hypomethylation of CpG sites within the P1,P2,and P3 regions was observed by bisulfite sequencing in human glioblastoma tissues with abundant CD133 mRNA.Taken together,our results indicate that DNA hypomethylation is an important determinant of CD133 expression in glioblastomas,and this epigenetic event may be associated with the development of BTICs expressing CD133.

  2. Selective lentiviral gene delivery to CD133-expressing human glioblastoma stem cells.

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    N Sumru Bayin

    Full Text Available Glioblastoma multiforme (GBM is a deadly primary brain malignancy. Glioblastoma stem cells (GSC, which have the ability to self-renew and differentiate into tumor lineages, are believed to cause tumor recurrence due to their resistance to current therapies. A subset of GSCs is marked by cell surface expression of CD133, a glycosylated pentaspan transmembrane protein. The study of CD133-expressing GSCs has been limited by the relative paucity of genetic tools that specifically target them. Here, we present CD133-LV, a lentiviral vector presenting a single chain antibody against CD133 on its envelope, as a vehicle for the selective transduction of CD133-expressing GSCs. We show that CD133-LV selectively transduces CD133+ human GSCs in dose-dependent manner and that transduced cells maintain their stem-like properties. The transduction efficiency of CD133-LV is reduced by an antibody that recognizes the same epitope on CD133 as the viral envelope and by shRNA-mediated knockdown of CD133. Conversely, the rate of transduction by CD133-LV is augmented by overexpression of CD133 in primary human GBM cultures. CD133-LV selectively transduces CD133-expressing cells in intracranial human GBM xenografts in NOD.SCID mice, but spares normal mouse brain tissue, neurons derived from human embryonic stem cells and primary human astrocytes. Our findings indicate that CD133-LV represents a novel tool for the selective genetic manipulation of CD133-expressing GSCs, and can be used to answer important questions about how these cells contribute to tumor biology and therapy resistance.

  3. Isolation and tumorigenicity of CD133 + subpopulation in human hepatocellular carcinoma%人肝癌CD133+细胞亚群的分离及致瘤性

    Institute of Scientific and Technical Information of China (English)

    李时兵; 郑进方; 张震生; 陈劲松; 孙启刚

    2015-01-01

    Objective To separate the CD133 + subpopulation in human hepatocellular carcinoma (HCC) and investigate the tumorigenicity.Methods The human liver cancer tissues were subcutaneously transplanted into nude mice to generate xenograft tumors which were then isolated to prepare single cell suspension.The expression of CD133 + subpopulation was further detected using flow cytometry.The CD133 + subpopulations were separated and depurated with magnetic-activated cell sorting system.Immunofluorescence was performed to identify the histological phenotype of CD133 + subpopulation.The in vitro and in vivo clone formation assay and in vivo xenograft formation assay were performed, respectively.Results Flow cytometry analysis revealed that a percentage of (4.1 ± 0.6) % CD133 + cells were detected in xenografts.Immunofluorescence studies showed that (86.8 ± 7.5) % of the isolated cells were CD133 +.Compared with CD133-population, CD133 + cells showed a higher capability to generate clone sphere in vitro and a higher tumorigenicity in nude mice (P < 0.05).Conclusion The CD133 + subpopulation in human hepatocellular carcinoma had a potent tumorigenicity and was enriched in cancer stem cells.%目的 从人肝癌组织中分离CD133+细胞亚群并探讨其致瘤能力.方法 将人肝癌组织种植于裸鼠皮下形成移植瘤,将移植瘤标本消化制成单细胞悬液.流式细胞仪检测CD133+的表达.免疫磁珠分选法进一步分离纯化CD133+细胞.免疫荧光检测CD133+细胞亚群的组织表型.对不同亚群细胞进行体外克隆形成实验和裸鼠体内移植瘤形成实验.结果 流式细胞仪检测显示,肝癌组织中(4.1±0.6)%的细胞为CD133+细胞.免疫荧光显示分离纯化的细胞中,CD133+细胞占(86.8±7.5)%.体外培养显示CD133+亚群比CD133-亚群具有更强的克隆球形成能力,在裸鼠体内具有更强的肿瘤形成能力(P<0.05).结论 从人肝癌组织中分离的CD133+细胞亚群具有高致瘤性,可能为肝癌干细胞.

  4. 3-Bromopyruvate inhibits cell proliferation and induces apoptosis in CD133+ population in human glioma.

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    Xu, Dong-Qiang; Tan, Xiao-Yu; Zhang, Bao-Wei; Wu, Tao; Liu, Ping; Sun, Shao-Jun; Cao, Yin-Guang

    2016-03-01

    The study was aimed to investigate the role of 3-bromopyruvate in inhibition of CD133+ U87 human glioma cell population growth. The results demonstrated that 3-bromopyruvate inhibited the viability of both CD133+ and parental cells derived from U87 human glioma cell line. However, the 3-bromopyruvate-induced inhibition in viability was more prominent in CD133+ cells at 10 μM concentration after 48 h. Treatment of CD133+ cells with 3-bromopyruvate caused reduction in cell population and cell size, membrane bubbling, and degradation of cell membranes. Hoechst 33258 staining showed condensation of chromatin material and fragmentation of DNA in treated CD133+ cells after 48 h. 3-Bromopyruvate inhibited the migration rate of CD133+ cells significantly compared to the parental cells. Flow cytometry revealed that exposure of CD133+ cells to 3-bromopyruvate increased the cell population in S phase from 24.5 to 37.9 % with increase in time from 12 to 48 h. In addition, 3-bromopyruvate significantly enhanced the expression of Bax and cleaved caspase 3 in CD133+ cells compared to the parental cells. Therefore, 3-bromopyruvate is a potent chemotherapeutic agent for the treatment of glioma by targeting stem cells selectively.

  5. CD133+ adult human retinal cells remain undifferentiated in Leukaemia Inhibitory Factor (LIF

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    Mayer Eric J

    2009-02-01

    Full Text Available Abstract Background CD133 is a cell surface marker of haematopoietic stem and progenitor cells. Leukaemia inhibitory factor (LIF, sustains proliferation and not differentiation of embryonic stem cells. We used CD133 to purify adult human retinal cells and aimed to determine what effect LIF had on these cultures and whether they still had the ability to generate neurospheres. Methods Retinal cell suspensions were derived from adult human post-mortem tissue with ethical approval. With magnetic automated cell sorting (MACS CD133+ retinal cells were enriched from post mortem adult human retina. CD133+ retinal cell phenotype was analysed by flow cytometry and cultured cells were observed for proliferative capacity, neuropshere generation and differentiation with or without LIF supplementation. Results We demonstrated purification (to 95% of CD133+ cells from adult human postmortem retina. Proliferating cells were identified through BrdU incorporation and expression of the proliferation markers Ki67 and Cyclin D1. CD133+ retinal cells differentiated whilst forming neurospheres containing appropriate lineage markers including glia, neurons and photoreceptors. LIF maintained CD133+ retinal cells in a proliferative and relatively undifferentiated state (Ki67, Cyclin D1 expression without significant neurosphere generation. Differentiation whilst forming neurospheres was re-established on LIF withdrawal. Conclusion These data support the evidence that CD133 expression characterises a population of cells within the resident adult human retina which have progenitor cell properties and that their turnover and differentiation is influenced by LIF. This may explain differences in retinal responses observed following disease or injury.

  6. 喉癌Hep-2细胞CD44+CD133+生物学特性研究%Research on the biological characteristics of CD44+CD133+of human laryngeal carcinoma Hep-2 cells

    Institute of Scientific and Technical Information of China (English)

    孔令帅; 温树信; 高伟; 王珏; 付荣; 杨丽娟; 李飞; 杨雨燕

    2016-01-01

    Objective To isolate, culture and identify laryngeal cancer stem cells from laryngeal carcinoma Hep-2 cell line and observe its biological characteristics in vitro. Methods Isolated, cultured and indentified human laryngeal carcinoma Hep-2 cells by MACS to obtain CD44+CD133+,CD44+CD133-,CD44-CD133+,CD44-CD133-four subsets of stem cells and plot-ted, the growth curves of four kinds of cells. The positive rate was detected by flow cytometry. The transwell chamber invasion assay, cell adhesion experiment and cloneformation assay were performed to evaluate the invasive capability, adhesion ability and the cloneforming ability respectively. The drug resistance was detected by CCK8 method. Results The biological charac-teristics of the five kinds of cells in Hep-2, CD44+CD133+, CD44+CD133-, CD44-CD133+and CD44-CD133-were analyzed. The ability of proliferation, invasion, adhesion, clone formation and drug resistance of CD44+CD133+cell subsets were higher than those of other four. CD44+CD133+>CD44-CD133+>Hep2>CD44+CD133->CD44-CD133-. Conclusion MACS is an effective method for sorting CD44+CD133+ cells. CD44+CD133+ cell subsets have obvious characteristics of cancer stem cells , and may make some new exploration for the high expression markers of laryngeal cancer cells.%目的:分选培养和鉴定人喉癌Hep-2细胞珠,并研究其体外生物学特性。方法通过磁珠细胞分选(mag-netic bead cell sorting,MACS)方法,分选培养和鉴定CD44+CD133+喉癌干细胞,应用免疫磁珠分选技术分选CD44+CD133+、CD44+CD133-、CD44-CD133+、CD44-CD133-4种亚群,对4种细胞绘制生长曲线,应用流式细胞仪检测其阳性率,应用侵袭实验、黏附实验和克隆形成实验评价其侵袭能力、黏附能力和克隆形成能力,用CCK8法检测其耐药性。结果对Hep-2、CD44+CD133+亚群、CD44+CD133-亚群、CD44-CD133+亚群、CD44-CD133-亚群5种细胞进行生物学特性研究,CD44+CD133+细胞亚群的增殖、侵

  7. Impact of short-term liquid storage on human CD133(+) stem cells.

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    Lux, Cornelia A; Mark, Peter; Klopsch, Christian; Laupheimer, Michael; Tu-Rapp, Hoang; Li, Wenzhong; Ma, Nan; Steinhoff, Gustav; David, Robert

    2015-01-01

    Stem cell transplantation is a viable strategy for regenerative medicine. However, it is inevitable to have cells undergo storage for several hours or days due to processing and transportation. Therefore, it is crucial to have rigidly controlled conditions ensuring the therapeutic benefit of isolated stem cells. In the present study, we investigated the impact of short-term storage on human CD133(+) cells. CD133(+) cells were isolated from human bone marrow and kept at standardized nonfreezing storage conditions for up to 72 h. Cell viability (apoptosis/necrosis) and expression of CD133 and CXCR4 were analyzed by flow cytometry. Metabolic activity was determined using an MTT assay; colony-forming ability, as well as endothelial-like differentiation, was further evaluated. A qRT-PCR array was employed to investigate the expression of stemness genes. CD133 and CXCR4 expressions were preserved at all time points. After 30 h, cell number and metabolic activity decreased, although no significant changes were detected in cell viability and proliferation as well as endothelial-like differentiation. Cell viability and proliferation decreased significantly only after 72 h of storage. Our results indicate that storage of isolated human CD133(+) bone marrow stem cells in liquid allows for high viability and functionality. However, storage time should be limited in order to avoid cell loss.

  8. In vivo myogenic potential of human CD133+ muscle-derived stem cells: a quantitative study.

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    Negroni, Elisa; Riederer, Ingo; Chaouch, Soraya; Belicchi, Marzia; Razini, Paola; Di Santo, James; Torrente, Yvan; Butler-Browne, Gillian S; Mouly, Vincent

    2009-10-01

    In recent years, numerous reports have identified in mouse different sources of myogenic cells distinct from satellite cells that exhibited a variable myogenic potential in vivo. Myogenic stem cells have also been described in humans, although their regenerative potential has rarely been quantified. In this study, we have investigated the myogenic potential of human muscle-derived cells based on the expression of the stem cell marker CD133 as compared to bona fide satellite cells already used in clinical trials. The efficiency of these cells to participate in muscle regeneration and contribute to the renewal of the satellite cell pool, when injected intramuscularly, has been evaluated in the Rag2(-/-) gammaC(-/-) C5(-/-) mouse in which muscle degeneration is induced by cryoinjury. We demonstrate that human muscle-derived CD133+ cells showed a much greater regenerative capacity when compared to human myoblasts. The number of fibers expressing human proteins and the number of human cells in a satellite cell position are all dramatically increased when compared to those observed after injection of human myoblasts. In addition, CD133+/CD34+ cells exhibited a better dispersion in the host muscle when compared to human myoblasts. We propose that muscle-derived CD133+ cells could be an attractive candidate for cellular therapy.

  9. In Vivo Myogenic Potential of Human CD133+ Muscle-derived Stem Cells: A Quantitative Study

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    Negroni, Elisa; Riederer, Ingo; Chaouch, Soraya; Belicchi, Marzia; Razini, Paola; Di Santo, James; Torrente, Yvan; Butler-Browne, Gillian S; Mouly, Vincent

    2009-01-01

    In recent years, numerous reports have identified in mouse different sources of myogenic cells distinct from satellite cells that exhibited a variable myogenic potential in vivo. Myogenic stem cells have also been described in humans, although their regenerative potential has rarely been quantified. In this study, we have investigated the myogenic potential of human muscle–derived cells based on the expression of the stem cell marker CD133 as compared to bona fide satellite cells already used in clinical trials. The efficiency of these cells to participate in muscle regeneration and contribute to the renewal of the satellite cell pool, when injected intramuscularly, has been evaluated in the Rag2−/− γC−/− C5−/− mouse in which muscle degeneration is induced by cryoinjury. We demonstrate that human muscle–derived CD133+ cells showed a much greater regenerative capacity when compared to human myoblasts. The number of fibers expressing human proteins and the number of human cells in a satellite cell position are all dramatically increased when compared to those observed after injection of human myoblasts. In addition, CD133+/CD34+ cells exhibited a better dispersion in the host muscle when compared to human myoblasts. We propose that muscle-derived CD133+ cells could be an attractive candidate for cellular therapy. PMID:19623164

  10. Isolation and identification of CD133 + cells from human hepatocellular carcinoma cell lines Huh-7%人肝癌细胞系Huh-7中CD133+细胞的分离及鉴定

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    孙岚; 宋东颖; 刘岩磊; 刘岩; 张英鸽

    2013-01-01

    目的 通过表面标志分选法富集人肝癌Huh-7细胞中CD133+细胞,并初步鉴定其特性.方法 采用流式细胞分选技术从人肝癌细胞系Huh-7中分选出CD133+细胞,并进行干细胞比例分析;通过对CD133+细胞体外成球能力及增殖能力检测,考察CD133+细胞的自我更新能力;观察CD133+细胞在非肥胖性糖尿病/重度联合免疫缺陷小鼠(NOD/SCID)体内的成瘤情况.结果 分选获得的CD133+细胞经无血清培养后阳性比例达90%以上;CD133+细胞体外无血清培养3d即可成球且生长速度较CD133-细胞快;CD133+细胞在NOD/SCID小鼠体内21 d左右即可形成异种移植瘤.结论 CD133+表面标志物分选方法可以高纯度富集CD133+细胞,利用CD133抗体分选获得的CD133+细胞具有肿瘤干细胞样特性.%Objective To enrich CD133+ cells from human hepatocellular carcinoma cell lines Huh-7 cells through fluorescence activated cell sorting and identify their biological characteristics. Methods CD133 + cells were sorted by flow cytometry and the percentage of them in cultured Huh-7 cells was analyzed. The self-renewing and sphere-forming ability of CD133 + cell were observed by light microscope in vitro in comparison with CD133+ cells. Tumor-forming ability of CD133+ cells was observed by xenografts of them in NOD/SCID mice. Results Flow cytometry analysis indicated that the purity of CD133 + subset cells exceeded 90% , CD133 + subset cells were verified multipotent with the ability of forming tumor spheres within 3 culture days. And CD133 + subset cells were higher proliferative in vitro and had higher tumorigenitic ability in vivo than those of CD133+ subset cells in mice for 21 d. Conclusion CD133 + cells super marker sorting method can enrich CD133 + cells in high purity, and CD133 + cells sorted with CD133 antibody possess the characteristics of tumor stem cells.

  11. CD133(+) human umbilical cord blood stem cells enhance angiogenesis in experimental chronic hepatic fibrosis.

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    Elkhafif, Nagwa; El Baz, Hanan; Hammam, Olfat; Hassan, Salwa; Salah, Faten; Mansour, Wafaa; Mansy, Soheir; Yehia, Hoda; Zaki, Ahmed; Magdy, Ranya

    2011-01-01

    The in vivo angiogenic potential of transplanted human umbilical cord blood (UCB) CD133(+) stem cells in experimental chronic hepatic fibrosis induced by murine schistosomiasis was studied. Enriched cord blood-derived CD133(+) cells were cultured in primary medium for 3 weeks. Twenty-two weeks post-Schistosomiasis infection in mice, after reaching the chronic hepatic fibrotic stage, transplantation of stem cells was performed and mice were sacrificed 3 weeks later. Histopathology and electron microscopy showed an increase in newly formed blood vessels and a decrease in the fibrosis known for this stage of the disease. By immunohistochemical analysis the newly formed blood vessels showed positive expression of the human-specific angiogenic markers CD31, CD34 and von Willebrand factor. Few hepatocyte-like polygonal cells showed positive expression of human vascular endothelial growth factor and inducible nitric oxide synthase. The transplanted CD133(+) human stem cells primarily enhanced hepatic angiogenesis and neovascularization and contributed to repair in a paracrine manner by creating a permissive environment that enabled proliferation and survival of damaged cells rather than by direct differentiation to hepatocytes. A dual advantage of CD133(+) cell therapy in hepatic disease is suggested based on its capability of hematopoietic and endothelial differentiation.

  12. Enhanced cell growth and tumorigenicity of rat glioma cells by stable expression of human CD133 through multiple molecular actions.

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    Fang, Kuan-Min; Lin, Tzu-Chien; Chan, Ti-Chun; Ma, Shi-Zhang; Tzou, Bo-Cheng; Chang, Wen-Ruei; Liu, Jun-Jen; Chiou, Shih-Hwa; Yang, Chung-Shi; Tzeng, Shun-Fen

    2013-09-01

    CD133 (Prominin-1/AC133) is generally treated as a cell surface marker found on multipotent stem cells and tumor stem-like cells, and its biological function remains debated. Genetically modified rat glioma cell lines were generated by lentiviral gene delivery of human CD133 into rat C6 glioma cells (hCD133(+) -C6) or by infection of C6 cells with control lentivirus (mock-C6). Stable hCD133 expression promoted the self-renewal ability of C6-formed spheres with an increase in the expression of the stemness markers, Bmi-1 and SOX2. Akt phosphorylation, Notch-1 activation, and Notch-1 target gene expression (Hes-1, Hey1 and Hey2) were increased in hCD133(+) -C6 when compared to mock-C6. The inhibition of Akt phosphorylation, Notch-1 activation, and Hes-1 in hCD133(+) -C6 cells effectively suppressed their clonogenic ability, indicating that these factors are involved in expanding the growth of hCD133(+) -C6. An elevated expression of GTPase-activating protein 27 (Arhgap27) was detected in hCD133(+) -C6. A decline in the invasion of hCD133(+) -C6 by knockdown of Arhgap27 expression indicated the critical role of Arhgap27 in promoting cell migration of hCD133(+) -C6. In vivo study further showed that hCD133(+) -C6 formed aggressive tumors in vivo compared to mock-C6. Exposure of hCD133(+) -C6 to arsenic trioxide not only reduced Akt phosphorylation, Notch-1 activation and Hes-1 expression in vitro, but also inhibited their tumorigenicity in vivo. The results show that C6 glioma cells with stable hCD133 expression enhanced their stemness properties with increased Notch-1/Hes-1 signaling, Akt activation, and Arhgap27 action, which contribute to increased cell proliferation and migration of hCD133(+) -C6 in vitro, as well as progressive tumor formation in vivo.

  13. Human α(2)β(1)(HI) CD133(+VE) epithelial prostate stem cells express low levels of active androgen receptor.

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    Williamson, Stuart C; Hepburn, Anastasia C; Wilson, Laura; Coffey, Kelly; Ryan-Munden, Claudia A; Pal, Deepali; Leung, Hing Y; Robson, Craig N; Heer, Rakesh

    2012-01-01

    Stem cells are thought to be the cell of origin in malignant transformation in many tissues, but their role in human prostate carcinogenesis continues to be debated. One of the conflicts with this model is that cancer stem cells have been described to lack androgen receptor (AR) expression, which is of established importance in prostate cancer initiation and progression. We re-examined the expression patterns of AR within adult prostate epithelial differentiation using an optimised sensitive and specific approach examining transcript, protein and AR regulated gene expression. Highly enriched populations were isolated consisting of stem (α(2)β(1)(HI) CD133(+VE)), transiently amplifying (α(2)β(1)(HI) CD133(-VE)) and terminally differentiated (α(2)β(1)(LOW) CD133(-VE)) cells. AR transcript and protein expression was confirmed in α(2)β(1)(HI) CD133(+VE) and CD133(-VE) progenitor cells. Flow cytometry confirmed that median (±SD) fraction of cells expressing AR were 77% (±6%) in α(2)β(1)(HI) CD133(+VE) stem cells and 68% (±12%) in α(2)β(1)(HI) CD133(-VE) transiently amplifying cells. However, 3-fold lower levels of total AR protein expression (peak and median immunofluorescence) were present in α(2)β(1)(HI) CD133(+VE) stem cells compared with differentiated cells. This finding was confirmed with dual immunostaining of prostate sections for AR and CD133, which again demonstrated low levels of AR within basal CD133(+VE) cells. Activity of the AR was confirmed in prostate progenitor cells by the expression of low levels of the AR regulated genes PSA, KLK2 and TMPRSS2. The confirmation of AR expression in prostate progenitor cells allows integration of the cancer stem cell theory with the established models of prostate cancer initiation based on a functional AR. Further study of specific AR functions in prostate stem and differentiated cells may highlight novel mechanisms of prostate homeostasis and insights into tumourigenesis.

  14. Common molecular pathways involved in human CD133+/CD34+ progenitor cell expansion and cancer

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    Vêncio Ricardo Z

    2007-06-01

    Full Text Available Abstract Background Uncovering the molecular mechanism underlying expansion of hematopoietic stem and progenitor cells is critical to extend current therapeutic applications and to understand how its deregulation relates to leukemia. The characterization of genes commonly relevant to stem/progenitor cell expansion and tumor development should facilitate the identification of novel therapeutic targets in cancer. Methods CD34+/CD133+ progenitor cells were purified from human umbilical cord blood and expanded in vitro. Correlated molecular changes were analyzed by gene expression profiling using microarrays covering up to 55,000 transcripts. Genes regulated during progenitor cell expansion were identified and functionally classified. Aberrant expression of such genes in cancer was indicated by in silico SAGE. Differential expression of selected genes was assessed by real-time PCR in hematopoietic cells from chronic myeloid leukemia patients and healthy individuals. Results Several genes and signaling pathways not previously associated with ex vivo expansion of CD133+/CD34+ cells were identified, most of which associated with cancer. Regulation of MEK/ERK and Hedgehog signaling genes in addition to numerous proto-oncogenes was detected during conditions of enhanced progenitor cell expansion. Quantitative real-time PCR analysis confirmed down-regulation of several newly described cancer-associated genes in CD133+/CD34+ cells, including DOCK4 and SPARCL1 tumor suppressors, and parallel results were verified when comparing their expression in cells from chronic myeloid leukemia patients Conclusion Our findings reveal potential molecular targets for oncogenic transformation in CD133+/CD34+ cells and strengthen the link between deregulation of stem/progenitor cell expansion and the malignant process.

  15. The importance of the stem cell marker prominin-1/CD133 in the uptake of transferrin and in iron metabolism in human colon cancer Caco-2 cells.

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    Erika Bourseau-Guilmain

    Full Text Available As the pentaspan stem cell marker CD133 was shown to bind cholesterol and to localize in plasma membrane protrusions, we investigated a possible function for CD133 in endocytosis. Using the CD133 siRNA knockdown strategy and non-differentiated human colon cancer Caco-2 cells that constitutively over-expressed CD133, we provide for the first time direct evidence for a role of CD133 in the intracellular accumulation of fluorescently labeled extracellular compounds. Assessed using AC133 monoclonal antibody, CD133 knockdown was shown to improve Alexa488-transferrin (Tf uptake in Caco-2 cells but had no impact on FITC-dextran or FITC-cholera-toxin. Absence of effect of the CD133 knockdown on Tf recycling established a role for CD133 in inhibiting Tf endocytosis rather than in stimulating Tf exocytosis. Use of previously identified inhibitors of known endocytic pathways and the positive impact of CD133 knockdown on cellular uptake of clathrin-endocytosed synthetic lipid nanocapsules supported that CD133 impact on endocytosis was primarily ascribed to the clathrin pathway. Also, cholesterol extraction with methyl-β-cyclodextrine up regulated Tf uptake at greater intensity in the CD133(high situation than in the CD133(low situation, thus suggesting a role for cholesterol in the inhibitory effect of CD133 on endocytosis. Interestingly, cell treatment with the AC133 antibody down regulated Tf uptake, thus demonstrating that direct extracellular binding to CD133 could affect endocytosis. Moreover, flow cytometry and confocal microscopy established that down regulation of CD133 improved the accessibility to the TfR from the extracellular space, providing a mechanism by which CD133 inhibited Tf uptake. As Tf is involved in supplying iron to the cell, effects of iron supplementation and deprivation on CD133/AC133 expression were investigated. Both demonstrated a dose-dependent down regulation here discussed to the light of transcriptional and post

  16. Cystogenic potential of CD133+ progenitor cells of human polycystic kidneys.

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    Carvalhosa, Raquel; Deambrosis, Ilaria; Carrera, Paola; Pasquino, Chiara; Rigo, Francesca; Ferrari, Maurizio; Lasaponara, Fedele; Ranghino, Andrea; Biancone, Luigi; Segoloni, Giuseppe; Bussolati, Benedetta; Camussi, Giovanni

    2011-09-01

    In autosomal dominant polycystic kidney disease, cysts arise focally and disrupt normal renal tissue leading to renal failure. In the present study, we show that cyst-lining cells express the stem cell marker CD133. CD133+ progenitor cells isolated from polycystic kidney, carrying mutations of PKD genes, showed a dedifferentiated phenotype similar to CD133+ progenitor cells from normal kidney. However, these cells were more proliferative and presented a defective epithelial differentiation phenotype with respect to normal renal CD133+ cells as they were not able to express all tubular epithelial cell markers when cultured in epithelial differentiation medium. Polycystic CD133+ cells, in contrast to normal renal CD133+ cells, formed cysts in vitro in a three-dimensional culture system and in vivo when injected subcutaneously within Matrigel in SCID mice. Rapamycin treatment reduced in vitro proliferation of polycystic CD133+ cells and decreased cystogenesis both in vitro and in vivo. The in vitro epithelial differentiation was only partially improved by rapamycin. These results indicate that polycystic CD133+ cells retain a dedifferentiated phenotype and the ability to generate cysts.

  17. CD133+人脐血造血祖细胞的干性维持培养及鉴定%Long-term culture and identification of CD133 + hematopoietic progenitor cells from human umbilical cord blood

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    吴小金; 陈芳; 陆滟霞; 杨慧; 彭璠莉; 袁理; 刘国炳; 李学农

    2012-01-01

    Objective To isolate CD133+ hematopoietic progenitor cells from human umbilical cord blood and optimize the culture condition for maintaining their stem cell characteristics. Methods CD133+ hematopoietic progenitor cells were isolated from human umbilical cord blood using magnetic cell sorting system, and the cells were detected by flow cytometry. Four methods were used for culturing cells. After 8 weeks' culture, cytomorphology, flow cytometry, immunocytochemistry and immunofluorescence assay were used to identify the characteristics of the stem cells. Results Over 80% of CD133 * hematopoietic progenitor cells were isolated from human umbilical cord blood using magnetic cell sorting system. The cells were effectively expanded using optimized serum-free medium after 8 weeks of cell culture, whereas the cells in other media differentiated into adherent cells in a poor state. Conclusion The optimized serum-free medium allows effective expansion of CD133+ hematopoietic progenitor cells that maintain stem cell characteristics after a long-term culture.%目的 从人脐带血中分选出CD133+造血祖细胞,并进行长时间干性维持培养.方法 通过免疫磁珠法分选出人脐带血中的CD133+造血祖细胞,经流式细胞仪检测免疫磁珠分选后的CD133+造血祖细胞.采用五种方法扩增培养该细胞,8周后,再通过细胞形态学、流式细胞术、免疫细胞化学和免疫荧光对细胞进行干性鉴定,探索最佳干性维持培养方法.结果 通过免疫磁珠法可以从人脐带血中分选出80%以上的CD133+造血祖细胞.采用优化的无血清培养基培养8周之后,CD133+造血祖细胞可得到有效扩增.而其他的培养基会使CD133+造血祖细胞由半悬浮细胞分化为梭形贴壁细胞,并且细胞状态欠佳.结论 利用免疫磁珠法分选出的CD133+造血祖细胞,采用优化的无血清培养基能够有效扩增该细胞,并可长期有效的维持其干性.

  18. Characterization of a new human melanoma cell line with CD133 expression.

    Science.gov (United States)

    Gil-Benso, Rosario; Monteagudo, Carlos; Cerdá-Nicolás, Miguel; Callaghan, Robert C; Pinto, Sandra; Martínez-Romero, Alicia; Pellín-Carcelén, Ana; San-Miguel, Teresa; Cigudosa, Juan C; López-Ginés, Concha

    2012-06-01

    A novel human malignant melanoma cell line, designated MEL-RC08, was established from a pericranial metastasis of a malignant melanoma of the skin. The cell line has been subcultured for more than 150 passages and is tumorigenic in nude mice. Growth kinetics, cytogenetics, flow cytometry, and molecular techniques for analysis of the genes implicated in cell cycle control; mutations in BRAF, NRAS, C-KiT, RB, and TP53 genes; and amplification of MDM2, CDK4, and cyclin D1 have been studied. Cytogenetically, the tumor and the cell line showed a hypertriploid karyotype with many clonal numeric and structural abnormalities. DNA flow cytometry showed an aneuploid peak with a DNA index value of 1.5. Mutations in TP53 and BRAF genes were demonstrated in both tumor and cell line. Furthermore, stem cell marker CD133 expression was detected in most cells, together with other stem cell markers, suggesting the presence of cells with tumor-initiating potential in this cell line.

  19. The High Yield Expansion and Megakaryocytic Differentiation of Human Umbilical Cord Blood CD133+ Cells

    Directory of Open Access Journals (Sweden)

    Mahin Nikougoftar Zarif

    2011-01-01

    Full Text Available Objective: Despite of many benefits, umbilical cord blood (UCB hematopoietic stem cell(HSC transplantation is associated with low number of stem cells and slow engraftment;in particular of platelets. So, expanded HSCs and co-transfusion of megakaryocyte (MKprogenitor cells can shorten this period. In this study, we evaluated the cytokine conditionsfor maximum expansion and MK differentiation of CD133+ HSCs.Materials and Methods: In this experimental study, The CD133+ cells were separatedfrom three cord blood samples by magnetic activated cell sorting (MACS method, expandedin different cytokine combinations for a week and differentiated in thrombopoietin(TPO for the second week. Differentiation was followed by the flow cytometry detectionof CD41 and CD61 surface markers. Colony forming unit (CFU assay and DNA analysiswere done for colonogenic capacity and ploidy assay.Results: CD133+ cells showed maximum expansion in the stem span medium with stemcell factor (SCF + FMS-like tyrosine kinase 3-ligand (Flt3-L + TPO but the maximum differentiationwas seen when CD133+ cells were expanded in stem span medium with SCF+ Interleukin 3 (IL-3 + TPO for the first and in TPO for the second week. Colony FormingUnit-MK (CFU-MK was formed in three sizes of colonies in the mega-cult medium. In theDNA analysis; 25.2 ± 6.7% of the cells had more than 2n DNA mass.Conclusion: Distinct differences in the MK progenitor cell count were observed when thecells were cultured in stem span medium with TPO, SCF, IL-3 and then the TPO in thesecond week. Such strategy could be applied for optimization of CD133+ cells expansionfollowed by MK differentiation.

  20. CD133阳性/阴性肺癌细胞的分选、鉴定及差异基因的筛选%Isolation and Identification of CD133 Positive and Negative Cells from Human Lung Cancer and Screening of the Differential Genes between the Positive and Negative Cells

    Institute of Scientific and Technical Information of China (English)

    郑少秋; 李书华; 王红艳; 谢晓斌; 张雅洁

    2015-01-01

    Background and objective It has been proven that cancer stem cell existed in variety of cancer, which an significant dierence of biological characteristics was observed between the cancer stem cells and non-cancer stem cells. And CD133 is considered to be cancer stem cell marker. So there may be significant dierences in CD133- positive cells and CD133-negative cells. e aim of this study is to isolate CD133+ cells and CD133- cells from lung cancer cell line A549, ex-plore their biological characteristics and screen the metastasis-related genes. Methods MACS was applied to isolate CD133+cells and CD133- cells from human lung cancer cell line A549. To observe the formation of sphere, CD133+ cells and CD133-cells were cultured in serum-free DMEM-F12 medium (containing EGF, bFGF) in vitro. e colony formaing eciency of CD133+ cells, CD133- cells and cells without sorting was tested by colony-forming assay. e dierentiation of sphere was in-duced by culturing in DMEM-F12 medium (containing serum). e metastasis-related genes (84 genes) of CD133+ cells and CD133- cells were detected by using DNA microarray. Immunohistochemistry was used to detect the expression of CD133 protein in Human lung cancer tissue. Results CD133+ cells formed sphere in serum-free DMEM-F12 medium,while the CD133- cells failed to form sphere. e rates of CD133+ cell colony formation (57.1%) was significantly higher than that of CD133- cells (3.3%). Sphere (CD133+/CK7-) was induced to dierentiate, and CK7 expression was found in dierentiated cells. e expression levels of 19 metastasis-related genes from CD133+ cells and CD133- cells were significant dierent. Lile CD133 positive cells which distributing around the cancer nests were found in lung cancer tissue. e expression of CD133 was not related to tumor types, cell dierentiation or TNM stage. Conclusion CD133+ cells exhibit the characteristics of can-cer stem cells. e dierence of metastasis

  1. Wnt interaction and extracellular release of prominin-1/CD133 in human malignant melanoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Rappa, Germana [Cancer Research Program, Roseman University of Health Sciences, 10530 Discovery Drive. Las Vegas, NV 89135 (United States); College of Pharmacy, Roseman University of Health Sciences, Henderson, NV 89104 (United States); Mercapide, Javier; Anzanello, Fabio [Cancer Research Program, Roseman University of Health Sciences, 10530 Discovery Drive. Las Vegas, NV 89135 (United States); Le, Thuc T. [Nevada Cancer Institute, Las Vegas, NV 89135 (United States); Johlfs, Mary G. [Cancer Research Program, Roseman University of Health Sciences, 10530 Discovery Drive. Las Vegas, NV 89135 (United States); Center for Diabetes and Obesity Prevention, Treatment, Research and Education, Roseman University of Health Sciences, Henderson, NV 89104 (United States); Fiscus, Ronald R. [Cancer Research Program, Roseman University of Health Sciences, 10530 Discovery Drive. Las Vegas, NV 89135 (United States); College of Pharmacy, Roseman University of Health Sciences, Henderson, NV 89104 (United States); Center for Diabetes and Obesity Prevention, Treatment, Research and Education, Roseman University of Health Sciences, Henderson, NV 89104 (United States); Wilsch-Bräuninger, Michaela [Max-Planck-Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, 01307 Dresden (Germany); Corbeil, Denis [Tissue Engineering Laboratories (BIOTEC) and DFG Research Center and Cluster of Excellence for Regenerative Therapies Dresden (CRTD), Technische Universität Dresden, Tatzberg 47–49, 01307 Dresden, Germany Technische Universitat Dresden, Dresden (Germany); Lorico, Aurelio, E-mail: alorico@roseman.edu [Cancer Research Program, Roseman University of Health Sciences, 10530 Discovery Drive. Las Vegas, NV 89135 (United States); College of Pharmacy, Roseman University of Health Sciences, Henderson, NV 89104 (United States)

    2013-04-01

    Prominin-1 (CD133) is the first identified gene of a novel class of pentaspan membrane glycoproteins. It is expressed by various epithelial and non-epithelial cells, and notably by stem and cancer stem cells. In non-cancerous cells such as neuro-epithelial and hematopoietic stem cells, prominin-1 is selectively concentrated in plasma membrane protrusions, and released into the extracellular milieu in association with small vesicles. Previously, we demonstrated that prominin-1 contributes to melanoma cells pro-metastatic properties and suggested that it may constitute a molecular target to prevent prominin-1-expressing melanomas from colonizing and growing in lymph nodes and distant organs. Here, we report that three distinct pools of prominin-1 co-exist in cultures of human FEMX-I metastatic melanoma. Morphologically, in addition to the plasma membrane localization, prominin-1 is found within the intracellular compartments, (e.g., Golgi apparatus) and in association with extracellular membrane vesicles. The latter prominin-1–positive structures appeared in three sizes (small, ≤40 nm; intermediates ∼40–80 nm, and large, >80 nm). Functionally, the down-regulation of prominin-1 in FEMX-I cells resulted in a significant reduction of number of lipid droplets as observed by coherent anti-Stokes Raman scattering image analysis and Oil red O staining, and surprisingly in a decrease in the nuclear localization of beta-catenin, a surrogate marker of Wnt activation. Moreover, the T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter activity was 2 to 4 times higher in parental than in prominin-1-knockdown cells. Collectively, our results point to Wnt signaling and/or release of prominin-1–containing membrane vesicles as mediators of the pro-metastatic activity of prominin-1 in FEMX-I melanoma. - Highlights: ► First report of release of prominin-1–containing microvesicles from cancer cells. ► Pro-metastatic role of prominin-1–containing microvesicles in

  2. Expression and function of Galectin-1 in CD133+ human pulmonary adenocarcinoma cells%半乳糖凝集素1在CD133+肺腺癌细胞中的表达和功能

    Institute of Scientific and Technical Information of China (English)

    周雪峰; 但攀; 张力; 朱少平; 赵金平

    2011-01-01

    Objective To investigate the expression and function of Galectin-1 in CD133+ pulmonary adenocarcinoma cells. Methods CD133 + cells were separated by magnetic activated cell sorting (MACS) from excised pulmonary adenocarcinoma speciments of 9 patiens. The proportion of CD133 + cells was measured by flow cytometry (FCM). The expression of Galectin-1 in CD133 + or CD133 - cells was quantitated by fluorescent quantitation real-time polymerase chain reaction (fqRT-PCR), Western blotting and enzyme linked immunosorbent assay (ELISA) respectively. CD133 + cells were transfected with small interfering RNA (siRNA) of Galectin-1 to study the effect of Galectin-1 inhibition on cancer cells growth and clonality. Tumorigenesis in nude mice was also performed in vivo. Results 92.6% cells separated by MACS were positive for CD133, which was proved by FCM. The expression of Galectin-1 in CD133 + cells was 1. 748 folds and 1. 135 folds higher than that in CD133 - celts detected by fqRT-PCR and Western blot respectively. Downregulation of Galectin-1 ex vivo also resulted in (36. 75 ±: 1.35 ) % decrease of proliferation rate of cancer cells. Conclusion Galectin-1 which can be efficiently inhibited by Galectin-1 siRNA was significantly highly expressed in CD133 + cells and associated with the proliferation and clonality of CD133 + cells.%目的 观察肺腺癌CD133+细胞中半乳糖凝集素1(Galectin-1)的表达和功能.方法 磁珠分选出9例患者肺腺癌中CD133+细胞并以流式细胞术检测分选效率,荧光实时定量聚合酶链反应(fqRT-PCR)、Western blot和酶联免疫吸附试验(ELISA)检测CD133+细胞中或上清液中Galectin-1的表达,Galectin-1小干扰RNA(siRNA)转染CD133+细胞后检测其对肿瘤细胞生长以及克隆形成能力的影响.予裸鼠皮下注射Galectin-1 siRNA转染后的CD133+细胞并观察肿瘤的生长.结果 流式细胞术结果表明磁珠分选出的细胞中CD133+细胞率为92.6%.fqRT-PCR和Western blot

  3. Characterization of CD133~+ parenchymal cells in the liver: Histology and culture

    Institute of Scientific and Technical Information of China (English)

    Seiichi Yoshikawa; Yoh Zen; Takahiko Fujii; Yasunori Sato; Tetsuo Ohta; Yutaka Aoyagi; Yasuni Nakanuma

    2009-01-01

    AIM: To reveal the characteristics of CD133~+ cells in the liver.METHODS: This study examined the histological characteristics of CD133~+ cells in non-neoplastic and neoplastic liver tissues by immunostaining, and also analyzed the biological characteristics of CD133~+ cells derived from human hepatocellular carcinoma (HCC) or cholangiocarcinoma cell lines.RESULTS: Immunostaining revealed constant expression of CD133 in non-neoplastic and neoplastic biliary epithelium, and these cells had the immunophenotype CD133~+/CK19+/HepPar-1~-. A small number of CD133~+/CK19~-/HepPar-1~+ cells were also identified in HCC and combined hepatocellular and cholangiocarcinoma. In addition, small ductal structures, resembling the canal of Hering, partly surrounded by hepatocytes were positive for CD133. CD133 expression was observed in three HCC (HuH7, PLC5 and HepG2) and two cholangiocarcinoma cell and HepG2) and two cholangiocarcinoma cell lines (HuCCT1 and CCKS1). Fluorescence-activated cell sorting (FACS) revealed that CD133~+ and CD133~-cells derived from HuH7 and HuCCT1 cells similarly produced CD133~+ and CD133~-cells during subculture. To examine the relationship between CD133~+ cells and the side population (SP) phenotype, FACS was performed using Hoechst 33342 and a monoclonal antibody against CD133. The ratios of CD133~+/CD133~-cells were almost identical in the SP and non-SP in HuH7. In addition, four different cellular populations (SP/CD133~+, SP/CD133~-, non-SP/CD133~+, and non-SP/CD133~-) could similarly produce CD133~+ and CD133~-cells during subculture. CONCLUSION: This study revealed that CD133 could be a biliary and progenitor cell marker in vivo. However, CD133 alone is not sufficient to detect tumor-initiating cells in cell lines.

  4. An alternatively spliced variant of CXCR3 mediates the metastasis of CD133+ liver cancer cells induced by CXCL9

    Science.gov (United States)

    Ding, Qiang; Xia, Yujia; Ding, Shuping; Lu, Panpan; Sun, Liang; Liu, Mei

    2016-01-01

    Metastasis of liver cancer is closely linked to tumor microenvironment, in which chemokines and their receptors act in an important role. The CXCR3, the receptor of chemokine CXCL9, belongs to a superfamily of rhodopsin-like seven transmembrane GPCRs and CXCR subfamily. In HCC tissues, CXCR3 was frequently upregulated and correlated with tumor size, tumor differentiation, portal invasion and metastasis. In the study, CXCR3-A isoform that was bound by CXCL9 was found to cause significant change of ERK1/2 phosphorylation level in the MAPK signaling pathway, consequently upregulating the MMP2 and MMP9 expression and promoting invasion and metastasis of CD133+ liver cancer cells. Also, CXCR3-A suppressed the adhesion ability of CD133+ liver cancer cells that stimulated by CXCL9 for 24h. These findings suggest that CXCR3 and its ligand CXCL9 could promote the metastasis of liver cancer cells and might be a potential target for the intervention of liver cancer metastasis. PMID:26883105

  5. MicroRNAs as markers for neurally committed CD133+/CD34+ stem cells derived from human umbilical cord blood.

    Science.gov (United States)

    Hafizi, Maryam; Atashi, Amir; Bakhshandeh, Behnaz; Kabiri, Mahboubeh; Nadri, Samad; Hosseini, Reza Haji; Soleimani, Masoud

    2013-04-01

    Neural differentiation of the CD133+/CD34+ subpopulation of human umbilical cord blood stem cells was investigated, and neuro-miR (mir-9 and mir-124) expression was examined. An efficient induction protocol for neural differentiation of hematopoietic stem cells together with the exclusion of retinoic acid in this process was also studied. Transcription of some neural markers such as microtubule-associated protein-2, beta-tubulin III, and neuron-specific enolase was evaluated by real-time PCR, immunocytochemistry, and western blotting. Increased expression of neural indicators in the treated cells confirmed the appropriate neural differentiation, which supported the high efficiency of our defined neuronal induction protocol. Verified high expression of neuro-miRNAs along with neuronal specific proteins not only strengthens the regulatory role of miRNAs in determining stem cell fate but also introduces these miRNAs as novel indicators of neural differentiation. These data highlight the prominent therapeutic potential of hematopoietic stem cells for use in cell therapy of neurodegenerative diseases.

  6. In vitro characterization of a human neural progenitor cell coexpressing SSEA4 and CD133

    DEFF Research Database (Denmark)

    Barraud, Perrine; Stott, Simon; Møllgård, Kjeld;

    2007-01-01

    The stage-specific embryonic antigen 4 (SSEA4) is commonly used as a cell surface marker to identify the pluripotent human embryonic stem (ES) cells. Immunohistochemistry on human embryonic central nervous system revealed that SSEA4 is detectable in the early neuroepithelium, and its expression d...

  7. Human CD34+ CD133+ hematopoietic stem cells cultured with growth factors including Angptl5 efficiently engraft adult NOD-SCID Il2rγ-/- (NSG mice.

    Directory of Open Access Journals (Sweden)

    Adam C Drake

    Full Text Available Increasing demand for human hematopoietic stem cells (HSCs in clinical and research applications necessitates expansion of HSCs in vitro. Before these cells can be used they must be carefully evaluated to assess their stem cell activity. Here, we expanded cord blood CD34(+ CD133(+ cells in a defined medium containing angiopoietin like 5 and insulin-like growth factor binding protein 2 and evaluated the cells for stem cell activity in NOD-SCID Il2rg(-/- (NSG mice by multi-lineage engraftment, long term reconstitution, limiting dilution and serial reconstitution. The phenotype of expanded cells was characterized by flow cytometry during the course of expansion and following engraftment in mice. We show that the SCID repopulating activity resides in the CD34(+ CD133(+ fraction of expanded cells and that CD34(+ CD133(+ cell number correlates with SCID repopulating activity before and after culture. The expanded cells mediate long-term hematopoiesis and serial reconstitution in NSG mice. Furthermore, they efficiently reconstitute not only neonate but also adult NSG recipients, generating human blood cell populations similar to those reported in mice reconstituted with uncultured human HSCs. These findings suggest an expansion of long term HSCs in our culture and show that expression of CD34 and CD133 serves as a marker for HSC activity in human cord blood cell cultures. The ability to expand human HSCs in vitro should facilitate clinical use of HSCs and large-scale construction of humanized mice from the same donor for research applications.

  8. Modulation of Chemokine Gene Expression in CD133 Cord Blood-Derived Human Mast Cells by Cyclosporin A and Dexamethasone

    DEFF Research Database (Denmark)

    Holm, Mette; Kvistgaard, Helene; Dahl, Christine;

    2006-01-01

    We have recently developed a protocol for generating huge numbers of mature and functional mast cells from in vitro differentiated umbilical cord blood cells. Using CD133 as a positive selection marker to isolate haematopoietic progenitors we routinely expand the number of recovered cells at least...... 150-fold, which vastly exceeds the yields of conventional protocols using CD34(+) cells as a source of progenitors. Taking advantage of the large quantities of in vitro differentiated mast cells, here we assess at the levels of transcription and translation the kinetics of chemokine gene induction...... following receptor mediated mast cell activation or following pharmacological activation of specific signal transduction cascades that become activated upon classical FcepsilonRI receptor crosslinking. We demonstrate that chemokine genes encoding IL-8, MCP-1, MIP-1alpha, and MIP-1beta are induced...

  9. Molecular and phenotypic characterization of CD133 and SSEA4 enriched very small embryonic-like stem cells in human cord blood.

    Science.gov (United States)

    Shaikh, A; Nagvenkar, P; Pethe, P; Hinduja, I; Bhartiya, D

    2015-09-01

    Very small embryonic-like stem cells (VSELs) are immature primitive cells residing in adult and fetal tissues. This study describes enrichment strategy and molecular and phenotypic characterization of human cord blood VSELs. Flow cytometry analysis revealed that a majority of VSELs (LIN(-)/CD45(-)/CD34(+)) were present in the red blood cell (RBC) pellet after Ficoll-Hypaque centrifugation in contrast to the hematopoietic stem cells (LIN(-)/CD45(+)/CD34(+)) in the interphase layer. Thus, after lyses of RBCs, VSELs were enriched using CD133 and SSEA4 antibodies. These enriched cells were small in size (4-6 μm), spherical, exhibited telomerase activity and expressed pluripotent stem cell (OCT4A, OCT4, SSEA4, NANOG, SOX2, REX1), primordial germ cell (STELLA, FRAGILIS) as well as primitive hematopoietic (CD133, CD34) markers at protein and transcript levels. Heterogeneity was noted among VSELs based on subtle differences in expression of various markers studied. DNA analysis and cell cycle studies revealed that a majority of enriched VSELs were diploid, non-apoptotic and in G0/G1 phase, reflecting their quiescent state. VSELs also survived 5-fluorouracil treatment in vitro and treated cells entered into cell cycle. This study provides further support for the existence of pluripotent, diploid and relatively quiescent VSELs in cord blood and suggests further exploration of the subpopulations among them.

  10. Paracrine proangiopoietic effects of human umbilical cord blood-derived purified CD133+ cells--implications for stem cell therapies in regenerative medicine.

    Science.gov (United States)

    Ratajczak, Janina; Kucia, Magda; Mierzejewska, Kasia; Marlicz, Wojciech; Pietrzkowski, Zbigniew; Wojakowski, Wojciech; Greco, Nicholas J; Tendera, Michal; Ratajczak, Mariusz Z

    2013-02-01

    CD133+ cells purified from hematopoietic tissues are enriched mostly for hematopoietic stem/progenitor cells, but also contain some endothelial progenitor cells and very small embryonic-like stem cells. CD133+ cells, which are akin to CD34+ cells, are a potential source of stem cells in regenerative medicine. However, the lack of convincing donor-derived chimerism in the damaged organs of patients treated with these cells suggests that the improvement in function involves mechanisms other than a direct contribution to the damaged tissues. We hypothesized that CD133+ cells secrete several paracrine factors that play a major role in the positive effects observed after treatment and tested supernatants derived from these cells for the presence of such factors. We observed that CD133+ cells and CD133+ cell-derived microvesicles (MVs) express mRNAs for several antiapoptotic and proangiopoietic factors, including kit ligand, insulin growth factor-1, vascular endothelial growth factor, basic fibroblast growth factor, and interleukin-8. These factors were also detected in a CD133+ cell-derived conditioned medium (CM). More important, the CD133+ cell-derived CM and MVs chemoattracted endothelial cells and display proangiopoietic activity both in vitro and in vivo assays. This observation should be taken into consideration when evaluating clinical outcomes from purified CD133+ cell therapies in regenerative medicine.

  11. Resistance to hypoxia-induced, BNIP3-mediated cell death contributes to an increase in a CD133-positive cell population in human glioblastomas in vitro.

    Science.gov (United States)

    Kahlert, Ulf Dietrich; Maciaczyk, Donata; Dai, Fangping; Claus, Rainer; Firat, Elke; Doostkam, Soroush; Bogiel, Tomasz; Carro, Maria Stella; Döbrössy, Mate; Herold-Mende, Christel; Niedermann, Gabriele; Prinz, Marco; Nikkhah, Guido; Maciaczyk, Jaroslaw

    2012-12-01

    In addition to intrinsic regulatory mechanisms, brain tumor stemlike cells (BTSCs), a small subpopulation of malignant glial tumor-derived cells, are influenced by environmental factors. Previous reports showed that lowering oxygen tension induced an increase of BTSCs expressing CD133 and other stem cell-related genes and more pronounced clonogenic capacity in vitro. We investigated the mechanisms responsible for hypoxia-dependent induction of CD133-positive BTSCs in glioblastomas. We confirmed that cultures exposed to lowered oxygen levels showed a severalfold increase of CD133-positive BTSCs. Both the increase of CD133-positive cells and deceleration of the growth kinetics were reversible after transfer to normoxic conditions. Exposure to hypoxia induced BNIP3 (BCL2/adenovirus E1B 19-kDa protein-interacting protein 3)-dependent apoptosis preferentially in CD133-negative cells. In contrast, CD133-positive cells proved to be more resistant to hypoxia-induced programmed cell death. Application of the demethylating agent 5'-azacitidine resulted in an increase of BNIP3 expression levels in CD133-positive cells. Thus, epigenetic modifications led to their better survival in lowered oxygen tension. Moreover, the, hypoxia-induced increase of CD133-positive cells was inhibited after 5'-azacitidine treatment. These results suggest the possible efficacy of a novel therapy for glioblastoma focused on eradication of BTSCs by modifications of epigenetic regulation of gene expression.

  12. Full GMP-compliant validation of bone marrow-derived human CD133(+) cells as advanced therapy medicinal product for refractory ischemic cardiomyopathy.

    Science.gov (United States)

    Belotti, Daniela; Gaipa, Giuseppe; Bassetti, Beatrice; Cabiati, Benedetta; Spaltro, Gabriella; Biagi, Ettore; Parma, Matteo; Biondi, Andrea; Cavallotti, Laura; Gambini, Elisa; Pompilio, Giulio

    2015-01-01

    According to the European Medicine Agency (EMA) regulatory frameworks, Advanced Therapy Medicinal Products (ATMP) represent a new category of drugs in which the active ingredient consists of cells, genes, or tissues. ATMP-CD133 has been widely investigated in controlled clinical trials for cardiovascular diseases, making CD133(+) cells one of the most well characterized cell-derived drugs in this field. To ensure high quality and safety standards for clinical use, the manufacturing process must be accomplished in certified facilities following standard operative procedures (SOPs). In the present work, we report the fully compliant GMP-grade production of ATMP-CD133 which aims to address the treatment of chronic refractory ischemic heart failure. Starting from bone marrow (BM), ATMP-CD133 manufacturing output yielded a median of 6.66 × 10(6) of CD133(+) cells (range 2.85 × 10(6)-30.84 × 10(6)), with a viability ranged between 96,03% and 99,97% (median 99,87%) and a median purity of CD133(+) cells of 90,60% (range 81,40%-96,20%). Based on these results we defined our final release criteria for ATMP-CD133: purity ≥ 70%, viability ≥ 80%, cellularity between 1 and 12 × 10(6) cells, sterile, and endotoxin-free. The abovementioned criteria are currently applied in our Phase I clinical trial (RECARDIO Trial).

  13. Full GMP-Compliant Validation of Bone Marrow-Derived Human CD133+ Cells as Advanced Therapy Medicinal Product for Refractory Ischemic Cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Daniela Belotti

    2015-01-01

    Full Text Available According to the European Medicine Agency (EMA regulatory frameworks, Advanced Therapy Medicinal Products (ATMP represent a new category of drugs in which the active ingredient consists of cells, genes, or tissues. ATMP-CD133 has been widely investigated in controlled clinical trials for cardiovascular diseases, making CD133+ cells one of the most well characterized cell-derived drugs in this field. To ensure high quality and safety standards for clinical use, the manufacturing process must be accomplished in certified facilities following standard operative procedures (SOPs. In the present work, we report the fully compliant GMP-grade production of ATMP-CD133 which aims to address the treatment of chronic refractory ischemic heart failure. Starting from bone marrow (BM, ATMP-CD133 manufacturing output yielded a median of 6.66 × 106 of CD133+ cells (range 2.85 × 106–30.84 × 106, with a viability ranged between 96,03% and 99,97% (median 99,87% and a median purity of CD133+ cells of 90,60% (range 81,40%–96,20%. Based on these results we defined our final release criteria for ATMP-CD133: purity ≥ 70%, viability ≥ 80%, cellularity between 1 and 12 × 106 cells, sterile, and endotoxin-free. The abovementioned criteria are currently applied in our Phase I clinical trial (RECARDIO Trial.

  14. The biological difference between CD13+CD133+ and CD13¬CD133¬liver cancer cells and its clinical significance

    Directory of Open Access Journals (Sweden)

    Shi-long JIN

    2013-09-01

    Full Text Available Objective To compare the biological difference between CD13+CD133+ and CD13-CD133- hepatocellular carcinoma (HCC cells in HuH7 cell line and its clinical significance. Methods The status of proliferation, phase of the cell cycle, tumor formation in vivo, differentiation, and their chemoresistance to 5-FU and pirarubicin of CD13+CD133+ and CD13-CD133-HCC cells were studied to analyze the clinical implication of CD13+CD133+HCC cell subset. Results The proliferation rate of CD13+CD133+HCC cells was significantly higher than that of CD13-CD133-HCC cells. The cell-cycle phase study showed that 78.45% of the CD13+CD133+HCC cells were in the G0/G1 phase, 2.19% in G2/M phase, and 19.36% in S phase, while 62.18% CD13-CD133-HCC cells were in the G0/G1 phase, 11.88% in G2/M phase, and 25.95% in S phase. Limiting dilution analysis of HuH7 cells revealed that 1×103 CD13+CD133+ cells could form the tumor, while 1×105 CD13-CD133- cells did. CD13+CD133+ cells showed chemoresistance to 5-FU and pirarubicin, while other three subsets succumbed to the drugs. Conclusion CD13+CD133+ cancer cells in HuH7 showed the characteristics of cancer stem cells (CSCs, which might contribute to the relapse and metastasis of liver cancer, and they may be the main target for chemotherapy in human liver cancer.

  15. STAT3 signaling pathway is necessary for cell survival and tumorsphere forming capacity in ALDH{sup +}/CD133{sup +} stem cell-like human colon cancer cells

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    Lin, Li, E-mail: lin.796@osu.edu [Center for Childhood Cancer, The Research Institute at Nationwide Children' s Hospital, Department of Pediatrics, Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH 43205 (United States); Division of Cardiology, Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030 (China); Fuchs, James; Li, Chenglong [Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210 (United States); Olson, Veronica [Center for Childhood Cancer, The Research Institute at Nationwide Children' s Hospital, Department of Pediatrics, Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH 43205 (United States); Bekaii-Saab, Tanios [Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH 43210 (United States); Lin, Jiayuh, E-mail: lin.674@osu.edu [Center for Childhood Cancer, The Research Institute at Nationwide Children' s Hospital, Department of Pediatrics, Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH 43205 (United States)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer The phosphorylated or activated form of STAT3 was expressed in colon cancer stem-like cells. Black-Right-Pointing-Pointer STAT3 inhibitor, FLLL32 inhibits P-STAT3 and STAT3 target genes in colon cancer stem-like cells. Black-Right-Pointing-Pointer Inhibition of STAT3 resulted in decreased cell viability and reduced numbers of tumorspheres. Black-Right-Pointing-Pointer STAT3 is required for survival and tumorsphere forming capacity in colon cancer stem-like cells. Black-Right-Pointing-Pointer Targeting STAT3 in cancer stem-like cells may offer a novel treatment approach for colon cancer. -- Abstract: Persistent activation of Signal Transducers and Activators of Transcription 3 (STAT3) is frequently detected in colon cancer. Increasing evidence suggests the existence of a small population of colon cancer stem or cancer-initiating cells may be responsible for tumor initiation, metastasis, and resistance to chemotherapy and radiation. Whether STAT3 plays a role in colon cancer-initiating cells and the effect of STAT3 inhibition is still unknown. Flow cytometry was used to isolate colon cancer stem-like cells from three independent human colon cancer cell lines characterized by both aldehyde dehydrogenase (ALDH)-positive and CD133-positive subpopulation (ALDH{sup +}/CD133{sup +}). The effects of STAT3 inhibition in colon cancer stem-like cells were examined. The phosphorylated or activated form of STAT3 was expressed in colon cancer stem-like cells and was reduced by a STAT3-selective small molecular inhibitor, FLLL32. FLLL32 also inhibited the expression of potential STAT3 downstream target genes in colon cancer stem-like cells including survivin, Bcl-XL, as well as Notch-1, -3, and -4, which may be involved in stem cell function. Furthermore, FLLL32 inhibited cell viability and tumorsphere formation as well as induced cleaved caspase-3 in colon cancer stem-like cells. FLLL32 is more potent than curcumin as evidenced with lower

  16. 肿瘤干细胞标记CD44和CD133在鼻咽癌细胞株中的表达检测%Expression of tumor stem cell marker CD44 and CD133 in human nasopharyngeal carcinoma cell line

    Institute of Scientific and Technical Information of China (English)

    苏进; 黄乔; 许新华; 鲁明骞; 易芳

    2011-01-01

    Objective To evaluate the expression of the tumor stem cell( TSC ) marker CD44 and CD133 in human nasopharyngeal carcinoma ( NPC ) SUNE - 1 5 - 8F cell line. Methods Immunocytochemistry and flow cytometry were used to assess the expression of CD44 and CD133 in SUNE - 1 5 - 8F cells. Fluorescence - activated cell sorting ( FACS ) was applied for purification of CD44 + cells. Results Immunofluorescence analysis revealed that CD44 was expressed on some of SUNE - 1 5 - 8F cells. CD44 positive cells accounted for 42. 4% ~ 52. 5% of the total cells. The purities of CD44+ and CD44 - cells after sorting were 98. 3% and 97.9% , respectively. Conclusion SUNE - 1 5 - 8F cell line with high expression of TSC marker is suitahle for study of NPC TSC. FACS is effective in cell sorting.%目的 探索肿瘤干细胞标记物CD44和CD133在鼻咽癌(NPC)中的表达量及其有效分选方式.方法 常规培养SUNE-1 5-8F细胞,采用免疫荧光技术及流式细胞学技术检测SUNE-1 5-8F细胞中CD44、CD133的表达,并用流式细胞仪分选CD44+、CD44+CD133+细胞.结果 激光共聚焦镜下鼻咽癌SUNE-1 5-8F细胞株细胞膜上可以观察到CD44分布;流式细胞学技术检测SUNE-1 5-8F中CD44+其表达率为42.4%~52.5%,经流式细胞仪分选得到的CD44+和CD44-细胞,其纯度分别达98.3%和97.9%.流式细胞仪检测到CD44+CD133+细胞(P2)在SUNE-1 5-8F细胞株中的表达约为3.1%.结论 SUNE-1 5-8F细胞株中肿瘤干细胞标记含量较高,适合鼻咽癌干细胞的研究,且流式细胞学技术不失为一种有效的分选细胞手段.

  17. CD133-enriched Xeno-Free human embryonic-derived neural stem cells expand rapidly in culture and do not form teratomas in immunodeficient mice

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    Daniel L. Haus

    2014-09-01

    Full Text Available Common methods for the generation of human embryonic-derived neural stem cells (hNSCs result in cells with potentially compromised safety profiles due to maintenance of cells in conditions containing non-human proteins (e.g. in bovine serum or on mouse fibroblast feeders. Additionally, sufficient expansion of resulting hNSCs for scaling out or up in a clinically relevant time frame has proven to be difficult. Here, we report a strategy that produces hNSCs in completely “Xeno-Free” culture conditions. Furthermore, we have enriched the hNSCs for the cell surface marker CD133 via magnetic sorting, which has led to an increase in the expansion rate and neuronal fate specification of the hNSCs in vitro. Critically, we have also confirmed neural lineage specificity upon sorted hNSC transplantation into the immunodeficient NOD-scid mouse brain. The future use or adaptation of these protocols has the potential to better facilitate the advancement of pre-clinical strategies from the bench to the bedside.

  18. CD133+ anaplastic thyroid cancer cells initiate tumors in immunodeficient mice and are regulated by thyrotropin.

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    Susan Friedman

    Full Text Available BACKGROUND: Anaplastic thyroid cancer (ATC is one of the most lethal human malignancies. Its rapid onset and resistance to conventional therapeutics contribute to a mean survival of six months after diagnosis and make the identification of thyroid-cancer-initiating cells increasingly important. METHODOLOGY/PRINCIPAL FINDINGS: In prior studies of ATC cell lines, CD133(+ cells exhibited stem-cell-like features such as high proliferation, self-renewal and colony-forming ability in vitro. Here we show that transplantation of CD133(+ cells, but not CD133(- cells, into immunodeficient NOD/SCID mice is sufficient to induce growth of tumors in vivo. We also describe how the proportion of ATC cells that are CD133(+ increases dramatically over three months of culture, from 7% to more than 80% of the total. This CD133(+ cell pool can be further separated by flow cytometry into two distinct populations: CD133(+/high and CD133(+/low. Although both subsets are capable of long-term tumorigenesis, the rapidly proliferating CD133(+/high cells are by far the most efficient. They also express high levels of the stem cell antigen Oct4 and the receptor for thyroid stimulating hormone, TSHR. Treating ATC cells with TSH causes a three-fold increase in the numbers of CD133(+ cells and elicits a dose-dependent up-regulation of the expression of TSHR and Oct4 in these cells. More importantly, immunohistochemical analysis of tissue specimens from ATC patients indicates that CD133 is highly expressed on tumor cells but not on neighboring normal thyroid cells. CONCLUSIONS/SIGNIFICANCE: To our knowledge, this is the first report indicating that CD133(+ ATC cells are solely responsible for tumor growth in immunodeficient mice. Our data also give a unique insight into the regulation of CD133 by TSH. These highly tumorigenic CD133(+ cells and the activated TSH signaling pathway may be useful targets for future ATC therapies.

  19. Expression of CD133 in acute leukemia.

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    Tolba, Fetnat M; Foda, Mona E; Kamal, Howyda M; Elshabrawy, Deena A

    2013-06-01

    There have been conflicting results regarding a correlation between CD133 expression and disease outcome. To assess CD133 expression in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) and to evaluate its correlation with the different clinical and laboratory data as well as its relation to disease outcome, the present study included 60 newly diagnosed acute leukemic patients; 30 ALL patients with a male to female ratio of 1.5:1 and their ages ranged from 9 months to 48 years, and 30 AML patients with a male to female ratio of 1:1 and their ages ranged from 17 to 66 years. Flow cytometric assessment of CD133 expression was performed on blast cells. In ALL, no correlations were elicited between CD133 expression and some monoclonal antibodies, but in AML group, there was a significant positive correlation between CD133 and HLA-DR, CD3, CD7 and TDT, CD13 and CD34. In ALL group, patients with negative CD133 expression achieved complete remission more than patients with positive CD133 expression. In AML group, there was no statistically significant association found between positive CD133 expression and treatment outcome. The Kaplan-Meier curve illustrated a high significant negative correlation between CD133 expression and the overall survival of the AML patients. CD133 expression is an independent prognostic factor in acute leukemia, especially ALL patients and its expression could characterize a group of acute leukemic patients with higher resistance to standard chemotherapy and relapse. CD133 expression was highly associated with poor prognosis in acute leukemic patients.

  20. Human prominin-1 (CD133) is detected in both neoplastic and non-neoplastic salivary gland diseases and released into saliva in a ubiquitinated form.

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    Karbanová, Jana; Laco, Jan; Marzesco, Anne-Marie; Janich, Peggy; Voborníková, Magda; Mokrý, Jaroslav; Fargeas, Christine A; Huttner, Wieland B; Corbeil, Denis

    2014-01-01

    Prominin-1 (CD133) is physiologically expressed at the apical membranes of secretory (serous and mucous) and duct cells of major salivary glands. We investigated its expression in various human salivary gland lesions using two distinct anti-prominin-1 monoclonal antibodies (80B258 and AC133) applied on paraffin-embedded sections and characterized its occurrence in saliva. The 80B258 epitope was extensively expressed in adenoid cystic carcinoma, in lesser extent in acinic cell carcinoma and pleomorphic adenoma, and rarely in mucoepidermoid carcinoma. The 80B258 immunoreactivity was predominately detected at the apical membrane of tumor cells showing acinar or intercalated duct cell differentiation, which lined duct- or cyst-like structures, and in luminal secretions. It was observed on the whole cell membrane in non-luminal structures present in the vicinity of thin-walled blood vessels and hemorrhagic areas in adenoid cystic carcinoma. Of note, AC133 labeled only a subset of 80B258-positive structures. In peritumoral salivary gland tissues as well as in obstructive sialadenitis, an up-regulation of prominin-1 (both 80B258 and AC133 immunoreactivities) was observed in intercalated duct cells. In most tissues, prominin-1 was partially co-expressed with two cancer markers: carcinoembryonic antigen (CEA) and mucin-1 (MUC1). Differential centrifugation of saliva followed by immunoblotting indicated that all three markers were released in association with small membrane vesicles. Immuno-isolated prominin-1-positive vesicles contained CEA and MUC1, but also exosome-related proteins CD63, flotillin-1, flotillin-2 and the adaptor protein syntenin-1. The latter protein was shown to interact with prominin-1 as demonstrated by its co-immunoisolation. A fraction of saliva-associated prominin-1 appeared to be ubiquitinated. Collectively, our findings bring new insights into the biochemistry and trafficking of prominin-1 as well as its immunohistochemical profile in certain types

  1. Human prominin-1 (CD133 is detected in both neoplastic and non-neoplastic salivary gland diseases and released into saliva in a ubiquitinated form.

    Directory of Open Access Journals (Sweden)

    Jana Karbanová

    Full Text Available Prominin-1 (CD133 is physiologically expressed at the apical membranes of secretory (serous and mucous and duct cells of major salivary glands. We investigated its expression in various human salivary gland lesions using two distinct anti-prominin-1 monoclonal antibodies (80B258 and AC133 applied on paraffin-embedded sections and characterized its occurrence in saliva. The 80B258 epitope was extensively expressed in adenoid cystic carcinoma, in lesser extent in acinic cell carcinoma and pleomorphic adenoma, and rarely in mucoepidermoid carcinoma. The 80B258 immunoreactivity was predominately detected at the apical membrane of tumor cells showing acinar or intercalated duct cell differentiation, which lined duct- or cyst-like structures, and in luminal secretions. It was observed on the whole cell membrane in non-luminal structures present in the vicinity of thin-walled blood vessels and hemorrhagic areas in adenoid cystic carcinoma. Of note, AC133 labeled only a subset of 80B258-positive structures. In peritumoral salivary gland tissues as well as in obstructive sialadenitis, an up-regulation of prominin-1 (both 80B258 and AC133 immunoreactivities was observed in intercalated duct cells. In most tissues, prominin-1 was partially co-expressed with two cancer markers: carcinoembryonic antigen (CEA and mucin-1 (MUC1. Differential centrifugation of saliva followed by immunoblotting indicated that all three markers were released in association with small membrane vesicles. Immuno-isolated prominin-1-positive vesicles contained CEA and MUC1, but also exosome-related proteins CD63, flotillin-1, flotillin-2 and the adaptor protein syntenin-1. The latter protein was shown to interact with prominin-1 as demonstrated by its co-immunoisolation. A fraction of saliva-associated prominin-1 appeared to be ubiquitinated. Collectively, our findings bring new insights into the biochemistry and trafficking of prominin-1 as well as its immunohistochemical profile in

  2. Quercetin induces cell cycle arrest and apoptosis in CD133+ cancer stem cells of human colorectal HT29 cancer cell line and enhances anticancer effects of doxorubicin

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    Atashpour, Shekoufeh; Fouladdel, Shamileh; Movahhed, Tahereh Komeili; Barzegar, Elmira; Ghahremani, Mohammad Hossein; Ostad, Seyed Nasser; Azizi, Ebrahim

    2015-01-01

    Objective(s): The colorectal cancer stem cells (CSCs) with the CD133+ phenotype are a rare fraction of cancer cells with the ability of self-renewal, unlimited proliferation and resistance to treatment. Quercetin has anticancer effects with the advantage of exhibiting low side effects. Therefore, we evaluated the anticancer effects of quercetin and doxorubicin (Dox) in HT29 cancer cells and its isolated CD133+ CSCs. Materials and Methods: The CSCs from HT29 cells were isolated using CD133 antibody conjugated to magnetic beads by MACS. Anticancer effects of quercetin and Dox alone and in combination on HT29 cells and CSCs were evaluated using MTT cytotoxicity assay and flow cytometry analysis of cell cycle distribution and apoptosis induction. Results: The CD133+ CSCs comprised about 10% of HT29 cells. Quercetin and Dox alone and in combination inhibited cell proliferation and induced apoptosis in HT29 cells and to a lesser extent in CSCs. Quercetin enhanced cytotoxicity and apoptosis induction of Dox at low concentration in both cell populations. Quercetin and Dox and their combination induced G2/M arrest in the HT29 cells and to a lesser extent in CSCs. Conclusion: The CSCs were a minor population with a significantly high level of drug resistance within the HT29 cancer cells. Quercetin alone exhibited significant cytotoxic effects on HT29 cells and also increased cytoxicity of Dox in combination therapy. Altogether, our data showed that adding quercetin to Dox chemotherapy is an effective strategy for treatment of both CSCs and bulk tumor cells. PMID:26351552

  3. CD133 Expression in Normal Skin and in Epithelial Cutaneous Tumors

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    S. H. Nam-Cha

    2013-01-01

    Full Text Available Background. Expression of human CD133 (human prominin-1 in cancer cells has been postulated to be a marker of stemness and is considered as a putative marker of cancer stem cells (CSCs. We designed a study to describe the expression pattern of CD133 in normal skin and in epithelial cutaneous neoplasms. Methods. The CD133 immunohistochemical expression of forty-three eccrine and apocrine tumors was compared to that observed in other epithelial tumors of the skin. In addition, flow cytometry was used to detect the CD133 expression of four epithelial skin neoplasms, including one porocarcinoma. Results. CD133 immunoreactivity at the apical or at the apicolateral surface of cells forming glandular structures was observed. Cells from solid areas of benign or malignant tumors were not stained. The porocarcinoma derived culture cells showed a 22% of CD133 positive cells using flow cytometry, while squamous cell carcinoma cultures contained less than 0.1%. Conclusions. These observations indicate that CD133 is a specific marker of glandular differentiation that could be included in the diagnostic panel of cutaneous tumors with possible eccrine or apocrine differentiation. However, the use of CD133 expression as a marker of CSCs should be interpreted with caution in experiments of skin.

  4. Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma

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    Lu Lizhi

    2006-12-01

    Full Text Available Abstract Background Recently, a small population of cancer stem cells in adult and pediatric brain tumors has been identified. Some evidence has suggested that CD133 is a marker for a subset of leukemia and glioblastoma cancer stem cells. Especially, CD133 positive cells isolated from human glioblastoma may initiate tumors and represent novel targets for therapeutics. The gene expression and the drug resistance property of CD133 positive cancer stem cells, however, are still unknown. Results In this study, by FACS analysis we determined the percentage of CD133 positive cells in three primary cultured cell lines established from glioblastoma patients 10.2%, 69.7% and 27.5%, respectively. We also determined the average mRNA levels of markers associated with neural precursors. For example, CD90, CD44, CXCR4, Nestin, Msi1 and MELK mRNA on CD133 positive cells increased to 15.6, 5.7, 337.8, 21.4, 84 and 1351 times, respectively, compared to autologous CD133 negative cells derived from cell line No. 66. Additionally, CD133 positive cells express higher levels of BCRP1 and MGMT mRNA, as well as higher mRNA levels of genes that inhibit apoptosis. Furthermore, CD133 positive cells were significantly resistant to chemotherapeutic agents including temozolomide, carboplatin, paclitaxel (Taxol and etoposide (VP16 compared to autologous CD133 negative cells. Finally, CD133 expression was significantly higher in recurrent GBM tissue obtained from five patients as compared to their respective newly diagnosed tumors. Conclusion Our study for the first time provided evidence that CD133 positive cancer stem cells display strong capability on tumor's resistance to chemotherapy. This resistance is probably contributed by the CD133 positive cell with higher expression of on BCRP1 and MGMT, as well as the anti-apoptosis protein and inhibitors of apoptosis protein families. Future treatment should target this small population of CD133 positive cancer stem cells in

  5. Overactivation of Ras signaling pathway in CD133+ MPNST cells.

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    Borrego-Diaz, Emma; Terai, Kaoru; Lialyte, Kristina; Wise, Amanda L; Esfandyari, Tuba; Behbod, Fariba; Mautner, Victor F; Spyra, Melanie; Taylor, Sarah; Parada, Luis F; Upadhyaya, Meena; Farassati, Faris

    2012-07-01

    Cancer stem cells (CSCs) are believed to be the regenerative pool of cells responsible for repopulating tumors. Gaining knowledge about the signaling characteristics of CSCs is important for understanding the biology of tumors and developing novel anti-cancer therapies. We have identified a subpopulation of cells positive for CD133 (a CSC marker) from human primary malignant peripheral nerve sheath tumor (MPNST) cells which were absent in non-malignant Schwann cells. CD133 was also found to be expressed in human tissue samples and mouse MPNST cells. CD133+ cells were capable of forming spheres in non-adherent/serum-free conditions. The activation levels of Ras and its downstream effectors such as ERK, JNK, PI3K, p38K, and RalA were significantly increased in this population. Moreover, the CD133+ cells showed enhanced invasiveness which was linked to the increased expression of β-Catenin and Snail, two important proteins involved in the epithelial to mesenchymal transition, and Paxilin, a focal adhesion protein. Among other important characteristics of the CD133+ population, endoplasmic reticulum stress marker IRE1α was decreased, implying the potential sensitivity of CD133+ to the accumulation of unfolded proteins. Apoptotic indicators seemed to be unchanged in CD133+ cells when compared to the wild (unsorted) cells. Finally, in order to test the possibility of targeting CD133+ MPNST cells with Ras pathway pharmacological inhibitors, we exposed these cells to an ERK inhibitor. The wild population was more sensitive to inhibition of proliferation by this inhibitor as compared with the CD133+ cells supporting previous studies observing enhanced chemoresistance of these cells.

  6. CD133/Src axis mediates tumor initiating property and epithelial-mesenchymal transition of head and neck cancer.

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    Yu-Syuan Chen

    Full Text Available BACKGROUND: Head and Neck squamous cell carcinoma (HNSCC is a human lethal cancer with clinical, pathological, phenotypical and biological heterogeneity. Caner initiating cells (CICs, which are responsible for tumor growth and coupled with gain of epithelial-mesenchymal transition (EMT, have been identified. Previously, we enriched a subpopulation of head and neck cancer initiating cells (HN-CICs with up-regulation of CD133 and enhancement of EMT. Others demonstrate that Src kinase interacts with and phosphorylates the cytoplasmic domain of CD133. However, the physiological function of CD133/Src signaling in HNSCCs has not been uncovered. METHODOLOGY/PRINCIPAL FINDING: Herein, we determined the critical role of CD133/Src axis modulating stemness, EMT and tumorigenicity of HNSCC and HN-CICs. Initially, down-regulation of CD133 significantly reduced the self-renewal ability and expression of stemness genes, and promoted the differentiation and apoptotic capability of HN-CICs. Additionally, knockdown of CD133 in HN-CICs also lessened both in vitro malignant properties including cell migration/cell invasiveness/anchorage independent growth, and in vivo tumor growth by nude mice xenotransplantation assay. In opposite, overexpression of CD133 enhanced the stemness properties and tumorigenic ability of HNSCCs. Lastly, up-regulation of CD133 increased phosphorylation of Src coupled with EMT transformation in HNSCCs, on the contrary, silence of CD133 or treatment of Src inhibitor inversely abrogated above phenotypic effects, which were induced by CD133 up-regulation in HNSCCs or HN-CICs. CONCLUSION/SIGNIFICANCE: Our results suggested that CD133/Src signaling is a regulatory switch to gain of EMT and of stemness properties in HNSCC. Finally, CD133/Src axis might be a potential therapeutic target for HNSCC by eliminating HN-CICs.

  7. Blocking the NOTCH pathway can inhibit the growth of CD133-positive A549 cells and sensitize to chemotherapy

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    Liu, Juntao; Mao, Zhangfan; Huang, Jie; Xie, Songping; Liu, Tianshu; Mao, Zhifu, E-mail: 48151660@qq.com

    2014-02-21

    Highlights: • Notch signaling pathway members are expressed lower levels in CD133+ cells. • CD133+ cells are not as sensitive as CD133− cells to chemotherapy. • GSI could inhibit the growth of both CD133+ and CD133− cells. • Blockade of Notch signaling pathway enhanced the effect of chemotherapy with CDDP. • DAPT/CDDP co-therapy caused G2/M arrest and elimination in CD133+ cells. - Abstract: Cancer stem cells (CSCs) are believed to play an important role in tumor growth and recurrence. These cells exhibit self-renewal and proliferation properties. CSCs also exhibit significant drug resistance compared with normal tumor cells. Finding new treatments that target CSCs could significantly enhance the effect of chemotherapy and improve patient survival. Notch signaling is known to regulate the development of the lungs by controlling the cell-fate determination of normal stem cells. In this study, we isolated CSCs from the human lung adenocarcinoma cell line A549. CD133 was used as a stem cell marker for fluorescence-activated cell sorting (FACS). We compared the expression of Notch signaling in both CD133+ and CD133− cells and blocked Notch signaling using the γ-secretase inhibitor DAPT (GSI-IX). The effect of combining GSI and cisplatin (CDDP) was also examined in these two types of cells. We observed that both CD133+ and CD133− cells proliferated at similar rates, but the cells exhibited distinctive differences in cell cycle progression. Few CD133+ cells were observed in the G{sub 2}/M phase, and there were half as many cells in S phase compared with the CD133− cells. Furthermore, CD133+ cells exhibited significant resistance to chemotherapy when treated with CDDP. The expression of Notch signaling pathway members, such as Notch1, Notch2 and Hes1, was lower in CD133+ cells. GSI slightly inhibited the proliferation of both cell types and exhibited little effect on the cell cycle. The inhibitory effects of DPP on these two types of cells were

  8. Expression of the "stem cell marker" CD133 in pancreas and pancreatic ductal adenocarcinomas

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    Sakariassen Per

    2008-02-01

    Full Text Available Abstract Background It has been suggested that a small population of cells with unique self-renewal properties and malignant potential exists in solid tumors. Such "cancer stem cells" have been isolated by flow cytometry, followed by xenograft studies of their tumor-initiating properties. A frequently used sorting marker in these experiments is the cell surface protein CD133 (prominin-1. The aim of this work was to examine the distribution of CD133 in pancreatic exocrine cancer. Methods Fifty-one cases of pancreatic ductal adenocarcinomas were clinically and histopathologically evaluated, and immunohistochemically investigated for expression of CD133, cytokeratin 19 and chromogranin A. The results were interpreted on the background of CD133 expression in normal pancreas and other normal and malignant human tissues. Results CD133 positivity could not be related to a specific embryonic layer of organ origin and was seen mainly at the apical/endoluminal surface of non-squamous, glandular epithelia and of malignant cells in ductal arrangement. Cytoplasmic CD133 staining was observed in some non-epithelial malignancies. In the pancreas, we found CD133 expressed on the apical membrane of ductal cells. In a small subset of ductal cells and in cells in centroacinar position, we also observed expression in the cytoplasm. Pancreatic ductal adenocarcinomas showed a varying degree of apical cell surface CD133 expression, and cytoplasmic staining in a few tumor cells was noted. There was no correlation between the level of CD133 expression and patient survival. Conclusion Neither in the pancreas nor in the other investigated organs can CD133 membrane expression alone be a criterion for "stemness". However, there was an interesting difference in subcellular localization with a minor cell population in normal and malignant pancreatic tissue showing cytoplasmic expression. Moreover, since CD133 was expressed in shed ductal cells of pancreatic tumors and was

  9. CD133-targeted gene transfer into long-term repopulating hematopoietic stem cells.

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    Brendel, Christian; Goebel, Benjamin; Daniela, Abriss; Brugman, Martijn; Kneissl, Sabrina; Schwäble, Joachim; Kaufmann, Kerstin B; Müller-Kuller, Uta; Kunkel, Hana; Chen-Wichmann, Linping; Abel, Tobias; Serve, Hubert; Bystrykh, Leonid; Buchholz, Christian J; Grez, Manuel

    2015-01-01

    Gene therapy for hematological disorders relies on the genetic modification of CD34(+) cells, a heterogeneous cell population containing about 0.01% long-term repopulating cells. Here, we show that the lentiviral vector CD133-LV, which uses a surface marker on human primitive hematopoietic stem cells (HSCs) as entry receptor, transfers genes preferentially into cells with high engraftment capability. Transduction of unstimulated CD34(+) cells with CD133-LV resulted in gene marking of cells with competitive proliferative advantage in vitro and in immunodeficient mice. The CD133-LV-transduced population contained significantly more cells with repopulating capacity than cells transduced with vesicular stomatitis virus (VSV)-LV, a lentiviral vector pseudotyped with the vesicular stomatitis virus G protein. Upon transfer of a barcode library, CD133-LV-transduced cells sustained gene marking in vivo for a prolonged period of time with a 6.7-fold higher recovery of barcodes compared to transduced control cells. Moreover, CD133-LV-transduced cells were capable of repopulating secondary recipients. Lastly, we show that this targeting strategy can be used for transfer of a therapeutic gene into CD34(+) cells obtained from patients suffering of X-linked chronic granulomatous disease. In conclusion, direct gene transfer into CD133(+) cells allows for sustained long-term engraftment of gene corrected cells.

  10. Chemoresistance of CD133+ cancer stem cells in laryngeal carcinoma

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    YANG Jing-pu; LIU Yan; ZHONG Wei; YU Dan; WEN Lian-ji; JIN Chun-shun

    2011-01-01

    Background Mounting evidence suggests that tumors are histologically heterogeneous and are maintained by a small population of tumor cells termed cancer stem cells. CD133 has been identified as a candidate marker of cancer stem cells in laryngeal carcinoma. This study aimed to analyze the chemoresistance of CD133+ cancer stem cells.Methods The response of Hep-2 cells to different chemotherapeutic agents was investigated and the expression of CD133 was studied. Fluorescence-activated cell sorting analysis was used to identify CD133,and the CD133+ subset of cells was separated and analyzed in colony formation assays,cell invasion assays,chemotherapy resistance studies,and analyzed for the expression of the drug resistance gene ABCG2.Results About 1%-2% of Hep-2 cells were CD133+ cells,and the CD133+ proportion was enriched by chemotherapy.CD133+ cancer stem cells exhibited higher potential for clonogenicity and invasion,and were more resistant to chemotherapy. This resistance was correlated with higher expression of ABCG2.Conclusions This study suggested that CD133+ cancer stem cells are more resistant to chemotherapy. The expression of ABCG2 could be partially responsible for this. Targeting this small population of CD133+ cancer stem cells could be a strategy to develop more effective treatments for laryngeal carcinoma.

  11. Blocking the NOTCH pathway can inhibit the growth of CD133-positive A549 cells and sensitize to chemotherapy.

    Science.gov (United States)

    Liu, Juntao; Mao, Zhangfan; Huang, Jie; Xie, Songping; Liu, Tianshu; Mao, Zhifu

    2014-02-21

    Cancer stem cells (CSCs) are believed to play an important role in tumor growth and recurrence. These cells exhibit self-renewal and proliferation properties. CSCs also exhibit significant drug resistance compared with normal tumor cells. Finding new treatments that target CSCs could significantly enhance the effect of chemotherapy and improve patient survival. Notch signaling is known to regulate the development of the lungs by controlling the cell-fate determination of normal stem cells. In this study, we isolated CSCs from the human lung adenocarcinoma cell line A549. CD133 was used as a stem cell marker for fluorescence-activated cell sorting (FACS). We compared the expression of Notch signaling in both CD133+ and CD133- cells and blocked Notch signaling using the γ-secretase inhibitor DAPT (GSI-IX). The effect of combining GSI and cisplatin (CDDP) was also examined in these two types of cells. We observed that both CD133+ and CD133- cells proliferated at similar rates, but the cells exhibited distinctive differences in cell cycle progression. Few CD133+ cells were observed in the G2/M phase, and there were half as many cells in S phase compared with the CD133- cells. Furthermore, CD133+ cells exhibited significant resistance to chemotherapy when treated with CDDP. The expression of Notch signaling pathway members, such as Notch1, Notch2 and Hes1, was lower in CD133+ cells. GSI slightly inhibited the proliferation of both cell types and exhibited little effect on the cell cycle. The inhibitory effects of DPP on these two types of cells were enhanced when combined with GSI. Interestingly, this effect was especially significant in CD133+ cells, suggesting that Notch pathway blockade may be a useful CSC-targeted therapy in lung cancer.

  12. Type 1 collagen as a potential niche component for CD133-positive glioblastoma cells.

    Science.gov (United States)

    Motegi, Hiroaki; Kamoshima, Yuuta; Terasaka, Shunsuke; Kobayashi, Hiroyuki; Houkin, Kiyohiro

    2014-08-01

    Cancer stem cells are thought to be closely related to tumor progression and recurrence, making them attractive therapeutic targets. Stem cells of various tissues exist within niches maintaining their stemness. Glioblastoma stem cells (GSCs) are located at tumor capillaries and the perivascular niche, which are considered to have an important role in maintaining GSCs. There were some extracellular matrices (ECM) on the perivascular connective tissue, including type 1 collagen. We here evaluated whether type 1 collagen has a potential niche for GSCs. Imunohistochemical staining of type 1 collagen and CD133, one of the GSCs markers, on glioblastoma (GBM) tissues showed CD133-positive cells were located in immediate proximity to type 1 collagen around tumor vessels. We cultured human GBM cell lines, U87MG and GBM cells obtained from fresh surgical tissues, T472 and T555, with serum-containing medium (SCM) or serum-free medium with some growth factors (SFM) and in non-coated (Non-coat) or type 1 collagen-coated plates (Col). The RNA expression levels of CD133 and Nestin as stem cell markers in each condition were examined. The Col condition not only with SFM but SCM made GBM cells more enhanced in RNA expression of CD133, compared to Non-coat/SCM. Semi-quantitative measurement of CD133-positive cells by immunocytochemistry showed a statistically significant increase of CD133-positive cells in Col/SFM. In addition, T472 cell line cultured in the Col/SFM had capabilities of sphere formation and tumorigenesis. Type 1 collagen was found in the perivascular area and showed a possibility to maintain GSCs. These findings suggest that type 1 collagen could be one important niche component for CD133-positive GSCs and maintain GSCs in adherent culture.

  13. CD133, CD15/SSEA-1, CD34 or side populations do not resume tumor-initiating properties of long-term cultured cancer stem cells from human malignant glio-neuronal tumors

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    Mihalescu-Maingot Maria

    2010-02-01

    Full Text Available Abstract Background Tumor initiating cells (TICs provide a new paradigm for developing original therapeutic strategies. Methods We screened for TICs in 47 human adult brain malignant tumors. Cells forming floating spheres in culture, and endowed with all of the features expected from tumor cells with stem-like properties were obtained from glioblastomas, medulloblastoma but not oligodendrogliomas. Results A long-term self-renewal capacity was particularly observed for cells of malignant glio-neuronal tumors (MGNTs. Cell sorting, karyotyping and proteomic analysis demonstrated cell stability throughout prolonged passages. Xenografts of fewer than 500 cells in Nude mouse brains induced a progressively growing tumor. CD133, CD15/LeX/Ssea-1, CD34 expressions, or exclusion of Hoechst dye occurred in subsets of cells forming spheres, but was not predictive of their capacity to form secondary spheres or tumors, or to resist high doses of temozolomide. Conclusions Our results further highlight the specificity of a subset of high-grade gliomas, MGNT. TICs derived from these tumors represent a new tool to screen for innovative therapies.

  14. hypoxia-inducible factors activate CD133 promoter through ETS family transcription factors.

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    Shunsuke Ohnishi

    Full Text Available CD133 is a cellular surface protein that has been reported to be a cancer stem cell marker, and thus it is considered to be a potential target for cancer treatment. However, the mechanism regulating CD133 expression is not yet understood. In this study, we analyzed the activity of five putative promoters (P1-P5 of CD133 in human embryonic kidney (HEK 293 cells and colon cancer cell line WiDr, and found that the activity of promoters, particularly of P5, is elevated by overexpression of hypoxia-inducible factors (HIF-1α and HIF-2α. Deletion and mutation analysis identified one of the two E-twenty six (ETS binding sites (EBSs in the P5 region as being essential for its promoter activity induced by HIF-1α and HIF-2α. In addition, a chromatin imunoprecipitation assay demonstrated that HIF-1α and HIF-2α bind to the proximal P5 promoter at the EBSs. The immunoprecipitation assay showed that HIF-1α physically interacts with Elk1; however, HIF-2α did not bind to Elk1 or ETS1. Furthermore, knockdown of both HIF-1α and HIF-2α resulted in a reduction of CD133 expression in WiDr. Taken together, our results revealed that HIF-1α and HIF-2α activate CD133 promoter through ETS proteins.

  15. Inhibitive effect of IL-24 gene on CD133+laryngeal cancer cells

    Institute of Scientific and Technical Information of China (English)

    Jin-Zhang Cheng; Dan Yu; Hui Zhang; Chun-Shun Jin; Yan Liu; Xue Zhao; Xin-Meng Qi; Xueshi-Bojie Liu

    2014-01-01

    Objective:To explore the inhibitive and apoptosis inductive effect of IL-24 genes on CD133+laryngeal cancer cells in Hep-2 line. Methods: Human peripheral blood monocytes were isolated. The total RNA was extracted by using Trizol method and reverse transcripted into cDNA using RT-PCR method. Primers P1 and P2 was designed for the amplification of human IL-24 genes. After confirmation of agarose gel electrophoresis tests, TA was cloned into pMD19-T simple vector. NheⅠand XhoⅠdouble digesting human IL-24 and pIRES2-ZsGreen1 and eukaryotic expression vector were used to establish the pIRES2-ZsGreen1-hIL-24 vector, and detected by enzyme digestion and gene sequencing methods. Flow cytometry (FCM) was used to isolate CD133+cells from Hep-2 cells. CD133+cells were transfected with pIRES2-ZsGreen1-hIL-24 through liposome 2000. After detection, MTT and FCM were used to observe the effect of IL-24 gene on CD133+laryngeal cancer Hep-2 cells. Results: Lipotin mediated transfection of recombinant pIRES2-ZsGreen1-hIL-24 plasmid into CD133+Hep-2 could expressed IL-24 gene in cells stably. MTT results showed that IL-24 transfected group was significantly suppressed compared to empty vector group and control group (P<0.05); FCM results showed that the apoptosis rate of experimental group increased significantly compared to empty vector group and control group (P<0.05). Conclusions:IL-24 gene expressions can inhibit proliferation of CD133+laryngeal cells in Hep-2 line and promote their apoptosis.

  16. CD105/CD133筛选鉴定SMMC-7721株干细胞表面标志物的实验研究%CD105/CD133 as cell surface markers for screening and identification of stem cell in SMMC-7721

    Institute of Scientific and Technical Information of China (English)

    吴立胜; 鲁旭; 耿小平; 卢寅; 张俊松

    2015-01-01

    目的:观察人肝癌细胞株 SMMC-7721中膜抗原CD133、CD105的表达情况并对不同亚群的生物学性状进行体内外实验研究。方法以含10%胎牛血清的 DMEM 对SMMC-7721株细胞培养;采用流式细胞仪方法分选检测CD133、CD105在SMMC-7721中表达情况并分选出CD133+/CD105+、CD133+/CD105-、CD133-/CD105+、CD133-/CD105-4个亚群;CCK-8和 Transwell侵袭实验分别检测4个亚群和未分选细胞组的增殖及侵袭能力,软琼脂克隆实验检测5组细胞成球能力;裸鼠成瘤实验了解 CD133+/CD105+、CD133-/CD105-亚群和未分选组的成瘤能力。结果流式细胞仪分选的 CD133+/CD105+、 CD133+/CD105-、CD133-/CD105+、CD133-/CD105-4种细胞亚群的比例分别为1.61%、0.01%、97.88%和0.50%。 CD133+亚群的增殖和成球能力较 CD133-亚群及未分选细胞组强,而CD105+亚群侵袭能力较 CD105-亚群及未分选细胞组强。CD133+/CD105+组与CD133-/CD105-组及未分选细胞组相比成瘤所需的时间短、所需细胞数少、成瘤的体积大。结论 CD133在人肝癌细胞株 SMMC-7721中的表达与其增殖成球能力有关,CD105与其侵袭能力有关,CD133+/CD105+具有体内高度的成瘤能力。 CD133+/CD105+亚群在人原发性肝癌细胞株SMMC-7721中具有肿瘤干细胞特性。%Objective The objective of this research is to compare the expression of CD105 and CD133 in mem-brane of human HCC cell line SMMC-7721 , different biological characters among the subpopulations in vitro and vi-vo. Methods SMMC-7721 cell line was cultured in DMEM containing 10%FBS. Flow cytometry was used to de-tect CD105, CD133 expression in SMMC-7721 cell in vitro. Four cell sub-populationsCD133 +/CD105 +,CD133 +/CD105 -,CD133 -/CD105 +, CD133 -/CD105 - were sorted by flow cytometry. Cell proliferation of these four sub-populations was detected by CCK-8 assay. Sphere formation ability of these four sub-populations was examined by soft agar test. Invasion ability

  17. CD133-targeted Gene Transfer Into Long-term Repopulating Hematopoietic Stem Cells

    NARCIS (Netherlands)

    Brendel, Christian; Goebel, Benjamin; Daniela, Abriss; Brugman, Martijn; Kneissl, Sabrina; Schwaeble, Joachim; Kaufmann, Kerstin B.; Mueller-Kuller, Uta; Kunkel, Hana; Chen-Wichmann, Linping; Abel, Tobias; Serve, Hubert; Bystrykh, Leonid; Buchholz, Christian J.; Grez, Manuel

    2015-01-01

    Gene therapy for hematological disorders relies on the genetic modification of CD34(+) cells, a heterogeneous cell population containing about 0.01% long-term repopulating cells. Here, we show that the lentiviral vector CD133-LV, which uses a surface marker on human primitive hematopoietic stem cell

  18. Investigating the link between molecular subtypes of glioblastoma, epithelial-mesenchymal transition, and CD133 cell surface protein.

    Directory of Open Access Journals (Sweden)

    Hadi Zarkoob

    Full Text Available In this manuscript, we use genetic data to provide a three-faceted analysis on the links between molecular subclasses of glioblastoma, epithelial-to-mesenchymal transition (EMT and CD133 cell surface protein. The contribution of this paper is three-fold: First, we use a newly identified signature for epithelial-to-mesenchymal transition in human mammary epithelial cells, and demonstrate that genes in this signature have significant overlap with genes differentially expressed in all known GBM subtypes. However, the overlap between genes up regulated in the mesenchymal subtype of GBM and in the EMT signature was more significant than other GBM subtypes. Second, we provide evidence that there is a negative correlation between the genetic signature of EMT and that of CD133 cell surface protein, a putative marker for neural stem cells. Third, we study the correlation between GBM molecular subtypes and the genetic signature of CD133 cell surface protein. We demonstrate that the mesenchymal and neural subtypes of GBM have the strongest correlations with the CD133 genetic signature. While the mesenchymal subtype of GBM displays similarity with the signatures of both EMT and CD133, it also exhibits some differences with each of these signatures that are partly due to the fact that the signatures of EMT and CD133 are inversely related to each other. Taken together these data shed light on the role of the mesenchymal transition and neural stem cells, and their mutual interaction, in molecular subtypes of glioblastoma multiforme.

  19. RNAi targeting CD133 enhances radiosensitivity in CD133 positive liver cancer stem cells%沉默CD133基因对CD133+肝癌干细胞放射敏感性的影响

    Institute of Scientific and Technical Information of China (English)

    王勇; 唐川; 兰曦; 冉雪琪; 邹冬玲; 曹姝; 李芳; 李少林

    2012-01-01

    Objective To determine the effects of CD133-downregulation on the radiosensitivity of CD133+ HepG2 cancer stem cells (CSCs) in hepatocellular carcinoma. Methods Magnetic-activated cell sorting (MACS) was used to isolate CD133+ and CD133- cells from HepG2 cells. Flow cytometry was used to detect the expression of CD133 before and after cells isolation. The NOD/SCID mice transplantation tumor experiments were performed to validate the cancer stem-like properties of sorted CD133 + cells. Targeted silencing towards CD133 gene was performed, and the cells were divided into blank control group, negative-transfec-tion group and positive-transfection group. RT-PCR and Western blotting were used to detect the mRNA and protein expression of CD133. Colony formation assay was applied to detect colony formation efficiency and survival rate after irradiation at different doses. Survival curve was drawn and radiobiology parameters D0, Dq, N and SER (sensitive enhancement ratio) were counted. Flow cytometry was used to test cell cycle and apoptosis. Results Flow cytometry indicated that the expression rate of CD133 was (1. 36 ± 0. 20) % and ( 87. 62± 1. 92)% respectively before and after MACS isolation. When the obtained CD133+ cells at a dose of 1×103/ml, subcutaneous tumor was found in the NOD/SCID mice after implantation. The expression levels of CD133 at mRNA and protein level were significantly decreased in positive-transfection group. After CD133-downregulation , the cells at G1 and S phase were decreased whereas those at G2 phase and apoptotic rate were increased significantly. Colony formation results showed that D0, Dq, N and SF2 were decreased while and SER was increased significantly in positive-transfection group (P <0. 05). Conclusion CD133 , as a marker of CSCs in liver cancer, could be a target for radiosensitization in liver CSCs treatment.%目的 研究沉默CD133基因对人肝癌CD133+-HepG2干细胞放射敏感性的影响.方法 免疫磁珠分选HepG2细

  20. CD133, Selectively Targeting the Root of Cancer

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    Jörg U. Schmohl

    2016-05-01

    Full Text Available Cancer stem cells (CSC are capable of promoting tumor initiation and self-renewal, two important hallmarks of carcinoma formation. This population comprises a small percentage of the tumor mass and is highly resistant to chemotherapy, causing the most difficult problem in the field of cancer research, drug refractory relapse. Many CSC markers have been reported. One of the most promising and perhaps least ubiquitous is CD133, a membrane-bound pentaspan glycoprotein that is frequently expressed on CSC. There is evidence that directly targeting CD133 with biological drugs might be the most effective way to eliminate CSC. We have investigated two entirely unrelated, but highly effective approaches for selectively targeting CD133. The first involves using a special anti-CD133 single chain variable fragment (scFv to deliver a catalytic toxin. The second utilizes this same scFv to deliver components of the immune system. In this review, we discuss the development and current status of these CD133 associated biological agents. Together, they show exceptional promise by specific and efficient CSC elimination.

  1. Clinicopathological characteristics and prognostic value of cancer stem cell marker CD133 in breast cancer: a meta-analysis

    Science.gov (United States)

    Li, Zhan; Yin, Songcheng; Zhang, Lei; Liu, Weiguang; Chen, Bo; Xing, Hua

    2017-01-01

    Background The association of CD133 overexpression with clinicopathological significance and prognosis in patients with breast cancer remains controversial. We thus performed a meta-analysis to evaluate the role of CD133 expression in the development and prognosis of breast cancer. Methods The databases PubMed, Embase, and Cochrane Library (updated to August 1, 2016) were searched. Pooled odds ratios (ORs) or hazard ratios (HRs) with 95% confidence intervals (95% CI) were used to evaluate the impact of CD133 expression on clinicopathological features, overall survival, and disease-free survival. Results A total of 1,734 patients from 13 studies were subject to final analysis. The results showed a significant association between overexpression of CD133 and estrogen receptor status (OR 0.35, 95% CI 0.18–0.70), progesterone receptor status (OR 0.56, 95% CI 0.43–0.74), human epidermal growth factor-2 status (OR 1.81, 95% CI 1.33–2.45), lymph node metastasis (OR 1.98, 95% CI 1.34–2.92), and tumor histological grade (OR 1.79, 95% CI 1.26–2.54) in breast cancer. However, no significant correlation was found between upregulation of CD133 expression and onset age (OR 1.03, 95% CI 0.70–1.53) or tumor size (OR 1.29, 95% CI 0.80–2.09). Moreover, CD133-positive breast cancer patients had a higher risk of mortality (HR 1.91, 95% CI 1.21–3.03) and disease progression (HR 2.70, 95% CI 1.05–6.95). Conclusion This meta-analysis suggested that CD133 might be a predictor of clinical outcomes as well as prognosis and could be a potentially new gene therapy target for breast cancer patients.

  2. Autophagy enhances chemotheragy resistance of CD133 + colon cancer stem cells to 5-FU%自噬增强结肠癌CD133+细胞对5-FU的化疗抵抗

    Institute of Scientific and Technical Information of China (English)

    司晓丽; 刘伟; 杨爱军; 王晨昱; 李敏

    2011-01-01

    Objective To observe the autophagy of CD133 + CSCs induced by 5-FU,and explore the effect of autophagy on the chemotherapy. Methods The cell proliferative inhibition rates and IC50 of 5-FU on human colon cancer cell lines SW480 were measured by MT1 assay. CD133 + CSCs were purified with MACS( magnetic activated cell-sorting system) CD133 cell isolation kit. Then the morphplpgical features of cells were observed using transmission election microscopy( TEM), and the autophagic vacuoles ( AV ) was observed using fluorescent microscope by monodansylcadaverin(MDC) staining. The effect of autophagy on the chemotherapey was evaluated by cell viability assay and colony-forming test. Results The vibility of human colon cancer cell line SW480 was obviously inhibited with different contrations of 5-FU, 50% inhibition concentration ( IC50 ) value was ( 1.09 ± 0. 18 ) μg/mL.Autophagsomes and autophagy vesica were observed in CD133 + CSCs. The combination treatment of 5-FU and 3-MA significantly decreased the viability of CD133 +CSCs from (81.8 ±4. 6)% to (56. 3 ±5.5)% (P <0. 05),and the colony-forming efficiency from (81.1 ±3.4)% to (64.4 ±4. 8)% (P <0.05), compared with 5-FU alone. Conclusion 5-FU may induce autophagy in CD133 + CSCs. Inhibition of autophagy may be employed to increase the cytotoxic effect of CD133 + CSCs to 5-FU.%目的 对5-FU诱导结肠癌CD133+细胞发生自噬的过程进行观察,初步探讨自噬对化疗的影响.方法 用MTT法检测细胞增殖抑制率及IC50;用免疫磁珠分选CD133+细胞;用透射电子显微镜(TEM)观察细胞的超微结构,用MDC染色,荧光显微镜观察自噬囊泡;联合应用细胞活性实验、集落形成实验研究自噬对化疗的影响.结果 不同浓度5-FU处理后,人结肠癌SW480细胞活性明显受到抑制,其半数抑制浓度IC50为(1.09±0.18)μg/mL.CD133+胞质出现大量的自噬体及自噬囊泡;相比较单独给予5-FU,联合给予5-FU及3-MA,CD133+细胞活性由(81

  3. Evaluation of the expansion of umbilical cord blood derived from CD133+ cells on biocompatible microwells

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    Mina Soufizomorrod

    2016-05-01

    Full Text Available Background: Hematopoietic stem cell transplantation (HSCT is a therapeutic approach for treatment of hematological malignancies and incompatibility of Bone marrow. Umbilical cord blood (UCB has known as an alternative for hematopoietic stem/progenitor cells (HPSC in allogeneic transplantation. The low volume of collected samples is the main hindrance in application of HPSC derived from umbilical cord blood. So, ex vivo expansion of HPSCs is the useful approach to overcome this restriction. The goal of using this system is to produce appropriate amount of hematopoietic stem cells, which have the ability of transplantation and long term haematopoiesis. Material & Methods: In current study CD133+ cells were isolated from cord blood (CB. Isolated cells were seeded on microwells. Then expanded cells proliferation rate and ability in colony formation were assessed and finally were compared with 2 Dimensional (2D culture systems. Results: Our findings demonstrated that CD133+ cells derived from UCB which were cultivated on microwells had significantly higher rate of proliferation in compared with routine cell culture systems. Conclusion: In Current study, it was shown that CD133+ cells’ proliferations which were seeded on PDMS microwells coated with collagen significantly increased. We hope that 3 dimensional (3D microenvironment which mimics the 3D structure of bone marrow can solve the problem of using UCB as an alternative source of bone marrow.

  4. Neuronal hypoxia in vitro: Investigation of therapeutic principles of HUCB-MNC and CD133+ stem cells

    OpenAIRE

    Emmrich Frank; Naumann Wilfried; Scholz Markus; Stahl Tobias; Hau Susann; Reich Doreen M; Boltze Johannes; Kamprad Manja

    2008-01-01

    Abstract Background The therapeutic capacity of human umbilical cord blood mononuclear cells (HUCB-MNC) and stem cells derived thereof is documented in animal models of focal cerebral ischemia, while mechanisms behind the reduction of lesion size and the observed improvement of behavioral skills still remain poorly understood. Methods A human in vitro model of neuronal hypoxia was used to address the impact of total HUCB-MNC (tMNC), a stem cell enriched fraction (CD133+, 97.38% CD133-positive...

  5. Strong expression of CD133 is associated with increased cholangiocarcinoma progression

    Institute of Scientific and Technical Information of China (English)

    Kawin Leelawat; Taweesak Thongtawee; Siriluck Narong; Somboon Subwongcharoen; Sa-ad Treepongkaruna

    2011-01-01

    AIM: To determine the role of CD133 in cholangiocar-cinoma progression.METHODS: CD133 protein expression was evaluated by immunohistochemistry in 34 cholangiocarcinoma specimens. In addition, proliferation, chemoresistance and invasive properties of CD133-enriched (CD133+) and CD133-depleted (CD133-) RMCCA1 cholangiocarci-noma cells were studied and compared.RESULTS: Strong CD133 expression was observed in 67.6% (23/34) of the cholangiocarcinoma specimens. Strong expression of CD133 was significantly associat-ed with nodal metastasis (P = 0.009) and positive sur-gical margin status (P = 0.011). In the in vitro study, both the CD133+ and CD133- cells had similar prolifera-tion abilities and resistance to chemotherapeutic drugs. However, the CD133+ cells had a higher invasive ability compared with CD133- cells.CONCLUSION: CD133+ cells play an important role in the invasiveness of cholangiocarcinoma. Targeting of the CD133+ cells may be a useful approach to improve treatment against cholangiocarcinoma.

  6. Differential number of CD34+, CD133+ and CD34+/CD133+ cells in peripheral blood of patients with congestive heart failure

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    Fritzenwanger M

    2009-03-01

    Full Text Available Abstract Background Endothelial progenitor cells (EPC which are characterised by the simulateous expression of CD34, CD133 and vascular endothelial growth receptor 2 (VEGF 2 are involved in the pathophysiology of congestive heart failure (CHF and their number and function is reduced in CHF. But so far our knowledge about the number of circulating hematopoietic stem/progenitor cells (CPC expressing the early hematopoietic marker CD133 and CD34 in CHF is spares and therefore we determined their number and correlated them with New York Heart Association (NYHA functional class. Methods CD34 and CD133 surface expression was quantified by flow cytometry in the peripheral venous blood of 41 healthy adults and 101 patients with various degrees of CHF. Results CD34+, CD133+ and CD34+/CD133+ cells correlated inversely with age. Both the number of CD34+ and of CD34+/CD133+ cells inversely correlated with NYHA functional class. The number of CD133+ cells was not affected by NYHA class. Furthermore the number of CD133+ cells did not differ between control and CHF patients. Conclusion In CHF the release of CD34+, CD133+ and CD34+/CD133+ cells from the bone marrow seems to be regulated differently. Modulating the releasing process in CHF may be a tool in CHF treatment.

  7. Malignant behaviorial characteristics of CD133(+/-) glioblastoma cells from a Northern Chinese population.

    Science.gov (United States)

    Liu, Xiaozhi; Chen, Lei; Jiang, Zhongmin; Wang, Junfei; Su, Zhiguo; Li, Gang; Yu, Shizhu; Liu, Zhenlin

    2013-01-01

    Following emergence of the tumor stem cell theory, the increasing number of related studies demonstrates the theory's growing importance in cancer research and its potential for clinical applications. Few studies have addressed the in vitro or in vivo properties of glioma stem cells from a Han Chinese population. In the present study, surgically obtained glioblastoma tissue was classified into two subtypes, CD133(+) and CD133(-). The hierarchy, invasiveness, growth tolerance under low nutrient conditions and colony forming abilities of the tissue samples were analyzed. Additionally, the characteristics of tumor cells transplanted subcutaneously or re-transplanted into nude mice were observed. The results demonstrated that CD133(+) glioblastoma cells derived from Han Chinese glioma specimens were more prone to primitive cell differentiation and more invasive than CD133(-) glioblastoma cells, leading to increased tumor malignancy compared with CD133(-) cells. The tumor formation rates of CD133(+) and CD133(-) cells in mice were 26/30 and 2/30, respectively. A comparison of tumor subtypes demonstrated that CD133(+) glioblastoma cells had a lower incidence of cell apoptosis in the tumor tissue and higher protein expression levels of Oct4, Sox2, PCNA, EGFR, Ang2, MMP2 and MMP9 compared with CD133(-) cells. Flow cytometry revealed that in the CD133(+) and CD133(-) glioblastoma cell-induced tumors, the percentage of CD133(+) cells was 2.47±0.67 and 0.44±0.14%, respectively. The tumor formation rates following the re-transplantation of CD133(+) or CD133(-) tumors into nude mice were 10/10 and 4/10, respectively. These findings suggest that the CD133(+) glioblastoma cell subpopulation has a stronger malignant cell phenotype than the CD133(-) subpopulation and that its recurrence rate is increased compared with the primitive tumorigenic rate following in vivo transplantation.

  8. Comparison of Biological Characteristics of CD133+ and CD133- Subpopulation of Hepatocellular Carcinoma Cell Line SMMC7721%肝癌细胞株SMMC7721中CD133+和CD133-亚群生物学特性的比较

    Institute of Scientific and Technical Information of China (English)

    朱殷; 郑青; 汪铮; 王园园; 萧树东

    2011-01-01

    背景:肝癌是常见的恶性肿瘤之一,易复发、转移,术后5年生存率较低.目前肿瘤干细胞学说已成为肿瘤研究的热点,CD133是一种肿瘤干细胞的标记物.目的:比较肝癌细胞株SMMC7721中CD133+和CD133-亚群的生物学特性差异,并初步探讨CD133+亚群的干细胞特性.方法:采用免疫磁珠法(MACS)分选SMMC7721细胞中CD133+和CD133-亚群,以流式细胞术检测CD133表达量,平板克隆形成实验检测CD133体外增殖能力,裸鼠成瘤实验检测体内致瘤性,CCK-8法检测对5-氟尿嘧啶(5-Fu)的敏感性.结果:MACS分选并培养1周后,CD133+亚群中CD133表达量明显下降.与CD133-亚群相比,CD133+亚群的体外克隆形成率明显增高,裸鼠肿瘤的体积明显升高,对5-Fu的敏感性降低,差异均有统计学意义(P<0.05).结论:肝癌SMMC7721细胞中CD133+亚群较CD133-亚群更具有肿瘤干细胞的特性.%Background: Hepatocellular carcinoma is a common malignant tumor with high recurrence rate and metastasis, and the 5-year survival rate is low. Cancer stem cell theory has become a hot spot of study on tumor and CD133 is a marker of cancer stem cells. Aims: To compare the biological characteristics of CD133+ with CD133- subpopulation of hepatocellular carcinoma cell line SMMC7721, and to explore tentatively the stem cell-like characteristics of CD133+ subpopulation.Methods: CD 133+ and CD133- subpopulation were isolated from hepatocellular carcinoma SMMC7721 cells by magnetic activated cell sorting (MACS). CD133 expression was determined by flow cytometry, and the potentials of CD133+ cells for colony formation and tumorigenicity were evaluated by cloning formation on plate and tumor formation in nude mice,respectively. 5-fluorouracil (5-Fu) sensitivity of CD133+ and CD133- cells was determined by CCK-8 assay. Results: After MACS and being cultured for 1 week, CD133 expression in CD133+ subpopulation was obviously decreased. The colonyforming capability was

  9. Rac1+ cells distributed in accordance with CD 133+ cells in glioblastomas and the elevated invasiveness of CD 133+ glioma cells with higher Rac1 activity

    Institute of Scientific and Technical Information of China (English)

    ZHANG Bin; SUN Jian; YU Sheng-ping; CHEN Cong; LIU Bin; LIU Zhi-feng; REN Bing-cheng; MING Hao-lang; YANG Xue-jun

    2012-01-01

    Background Recent studies have suggested that cancer stem cells are one of the major causes for tumor recurrence due to their resistance to radiotherapy and chemotherapy.Although the highly invasive nature of glioblastoma (GBM)cells is also implicated in the failure of current therapies,it is not clear how glioma stem cells (GSCs) are involved in invasiveness.Rac1 activity is necessary for inducing reorganization of actin cytoskeleton and cell movement.In this study,we aimed to investigate the distribution characteristics of CD133+ cells and Rac1+ cells in GBM as well as Rac1 activity in CD133+ GBM cells,and analyze the migration and invasion potential of these cells.Methods A series of 21 patients with GBM were admitted consecutively and received tumor resection in Tianjin Medical University General Hospital during the first half of the year 2011.Tissue specimens were collected both from the peripheral and the central parts for each tumor under magnetic resonance imaging (MRI) navigation guidance.Immunohistochemical staining was used to detect the CD133+ cells and Rac1+ cells distribution in GBM specimens.Double-labeling immunofluorescence was further used to analyze CD133 and Rac1 co-expression and the relationship between CD133+ cells distribution and Rac1 expression.Serum-free medium culture and magnetic sorting were used to isolate CD133+ cells from U87 cell line.Rac1 activation assay was conducted to assess the activation of Rac1 in CD133+ and CD133-U87 cells.The migration and invasive ability of CD133+ and CD133-U87 cells were determined by cell migration and invasion assays in vitro.Student's t-test and one-way analysis of variance (ANOVA) test were used to determine statistical significance in this study.Results In the central parts of GBMs,CD133+ cells were found to cluster around necrosis and occasionally cluster around the vessels under the microscope by immunohistological staining.In the peripheral parts of the tumors,CD133+ cells were lined up along

  10. Chemoresistance of CD133(+) colon cancer may be related with increased survivin expression.

    Science.gov (United States)

    Lee, Mi-Ra; Ji, Sun-Young; Mia-Jan, Khalilullah; Cho, Mee-Yon

    2015-07-31

    CD133, putative cancer stem cell marker, deemed to aid chemoresistance. However, this claim has been challenged recently and we previously reported that patients with CD133(+) colon cancer have benefit from 5-fluorouracil (5-FU) chemotherapy incontrast to no benefit in patients with CD133(-) cancer. To elucidate the role of CD133 expression in chemoresistance, we silenced the CD133 expression in a colon cancer cell line and determined its effect on the biological characteristics downstream. We comparatively analyzed the sequential changes of MDR1, ABCG2, AKT1 and survivin expression and the result of proliferation assay (WST-1 assay) with 5-FU treatment in CD133(+) and siRNA-induced CD133(-) cells, derived from Caco-2 colon cancer cell line. 5-FU treatment induced significantly increase of the mRNA expression of MDR1, ABCG2 and AKT1genes, but not protein level. CD133 had little to no effect on the mRNA and protein expression of these genes. However, survivin expression at mRNA and protein level were significantly increased in CD133(+) cells compared with siRNA-induced CD133-cells and Mock (not sorted CD133(+) cells) at 96 h after siRNA transfection. The cytotoxicity assay demonstrated notable increase of chemoresistance to 5-FU treatment (10 μM) in CD133(+) cells at 96 h after siRNA transfection. From this study, we conclude that CD133(+) cells may have chemoresistance to 5-FU through the mechanism which is related with survivin expression, instead of MDR1, ABCG2 and AKT1 expression. Therefore a survivin inhibitor can be a new target for effective treatment of CD133(+) colon cancer.

  11. Chemoresistance of CD133{sup +} colon cancer may be related with increased survivin expression

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Mi-Ra; Ji, Sun-Young; Mia-Jan, Khalilullah [Department of Pathology, Yonsei University, Wonju College of Medicine, Wonju (Korea, Republic of); Cho, Mee-Yon, E-mail: meeyon@yonsei.ac.kr [Department of Pathology, Yonsei University, Wonju College of Medicine, Wonju (Korea, Republic of); Institute of Genomic Cohort, Yonsei University, Wonju College of Medicine, Wonju (Korea, Republic of)

    2015-07-31

    CD133, putative cancer stem cell marker, deemed to aid chemoresistance. However, this claim has been challenged recently and we previously reported that patients with CD133{sup +} colon cancer have benefit from 5-fluorouracil (5-FU) chemotherapy incontrast to no benefit in patients with CD133{sup −} cancer. To elucidate the role of CD133 expression in chemoresistance, we silenced the CD133 expression in a colon cancer cell line and determined its effect on the biological characteristics downstream. We comparatively analyzed the sequential changes of MDR1, ABCG2, AKT1 and survivin expression and the result of proliferation assay (WST-1 assay) with 5-FU treatment in CD133{sup +} and siRNA-induced CD133{sup −} cells, derived from Caco-2 colon cancer cell line. 5-FU treatment induced significantly increase of the mRNA expression of MDR1, ABCG2 and AKT1genes, but not protein level. CD133 had little to no effect on the mRNA and protein expression of these genes. However, survivin expression at mRNA and protein level were significantly increased in CD133{sup +} cells compared with siRNA-induced CD133-cells and Mock (not sorted CD133{sup +} cells) at 96 h after siRNA transfection. The cytotoxicity assay demonstrated notable increase of chemoresistance to 5-FU treatment (10 μM) in CD133{sup +} cells at 96 h after siRNA transfection. From this study, we conclude that CD133{sup +} cells may have chemoresistance to 5-FU through the mechanism which is related with survivin expression, instead of MDR1, ABCG2 and AKT1 expression. Therefore a survivin inhibitor can be a new target for effective treatment of CD133{sup +} colon cancer. - Highlights: • We evaluate the role of CD133 in chemoresistance of colon cancer. • We compared the chemoresistance of CD133{sup +} cells and siRNA-induced CD133{sup −} cells. • CD133 had little to no effect on MDR1, ABCG2 and AKT1 expression. • Survivin expression and chemoresistance were increased in CD133{sup +} colon cancer cells.

  12. Increased expression of CD133 and reduced dystroglycan expression are strong predictors of poor outcome in colon cancer patients

    Directory of Open Access Journals (Sweden)

    Coco Claudio

    2012-09-01

    Full Text Available Abstract Background Expression levels of CD133, a cancer stem cell marker, and of the α-subunit of the dystroglycan (α-DG complex, have been previously reported to be altered in colorectal cancers. Methods Expression levels of CD133 and α-DG were assessed by immunohistochemistry in a series of colon cancers and their prognostic significance was evaluated. Results Scattered cells positive for CD133 were rarely detected at the bases of the crypts in normal colonic mucosa while in cancer cells the median percentage of positive cells was 5% (range 0–80. A significant correlation was observed with pT parameter and tumor stage but not with tumor grade and N status. Recurrence and death from disease were significantly more frequent in CD133-high expressing tumors and Kaplan-Meier curves showed a significant separation between high vs low expressor groups for both disease-free (p = 0.002 and overall (p = 0.008 survival. Expression of α-DG was reduced in a significant fraction of tumors but low α-DG staining did not correlate with any of the classical clinical-pathological parameters. Recurrence and death from the disease were significantly more frequent in α-DG-low expressing tumors and Kaplan-Meier curves showed a significant separation between high vs low expressor tumors for both disease-free (p = 0.02 and overall (p = 0.02 survival. Increased expression of CD133, but not loss of α-DG, confirmed to be an independent prognostic parameters at a multivariate analysis associated with an increased risk of recurrence (RR = 2.4; p = 0.002 and death (RR = 2.3; p = 0.003. Conclusions Loss of α-DG and increased CD133 expression are frequent events in human colon cancer and evaluation of CD133 expression could help to identify high-risk colon cancer patients.

  13. Prognostic value of cancer stem cell markers CD133, ALDH1 and nuclearβ-catenin in colon cancer

    Institute of Scientific and Technical Information of China (English)

    Eman H.Abdelbary; Hayam E.Rashed; Eman I.Ismail; Mohamed Abdelgawad

    2014-01-01

    Colon cancer is one of the most common human malignancies. Cancer stem cells (CSCs), despite being only a smal subset of cancer cells, have the capability to self renew and sustain the tumor. They also have the ability to proliferate. Multiple CSCs associated markers have been identified in colon cancer including CD133, ALDH1 andβ catenin. The aim of the work was to study the prognostic value of CSCs markers (CD133, ALDH1 andβ catenin), as wel as their rela tionship to clinicopathological features of colon cancer. Methods:CD133, ALDH1 andβ catenin proteins expression was as sessed immunohistochemical y in a series of colon cancers and their prognostic significance was evaluated. Results:CD133 expression showed significant relationship to tumor stage and lymph node metastasis (P value 0.004&<0.001 respectively), and near significant relationship to liver metastasis (P value 0.092). ALDH1 was significantly associated with tumor grade, stage and nodal metastasis (P value 0.021, 0.001 and 0.026 respectively), but its relationship to liver metastasis was near sig nificant (P value 0.068). Nuclearβ catenin was significantly related to tumor grade, stage, nodal and liver metastasis (P value 0.001,<0.001,<0.001 and 0.008 respectively). Overal survival (OS) was associated inversely with CD133, ALDH1 positivity, and directly with nuclearβ catenin positivity (P value<0.001, 0.0001 and<0.001 respectively). Also recurrence free survival (RFS) was associated inversely with CD133, ALDH1 and directly with nuclearβ catenin positivity (P value 0.0001, 0.001 and<0.001 respectively). Conclusion:CD133, ALDH1 andβ catenin expressions of tumor cells have significant impact upon malignant progression of colon cancer and thus patient survival and tumor recurrence. Hence they can be used to predict outcome of colon cancer patients.

  14. CD133 and membrane microdomains: Old facets for future hypotheses

    Institute of Scientific and Technical Information of China (English)

    Christine A Fargeas; Jana Karbanová; József Jászai; Denis Corbeil

    2011-01-01

    Understanding all facets of membrane microdomains in normal and cancerous cells within the digestive tract is highly important, not only from a clinical point of view, but also in terms of our basic knowledge of cellular transformation. By studying the normal and cancer stem cell-associated molecule CD133 (prominin-1), novel aspects of the organization and dynamics of polarized epithelial cells have been revealed during the last decade. Its association with particular membrane microdomains is highly relevant in these contexts and might also offer new avenues in diagnosis and/or targeting of cancer stem cells.

  15. Immunohistochemical detection of CD133 is associated with tumor regression grade after chemoradiotherapy in rectal cancer.

    Science.gov (United States)

    Hongo, Kumiko; Kazama, Shinsuke; Sunami, Eiji; Tsuno, Nelson H; Takahashi, Koki; Nagawa, Hirokazu; Kitayama, Joji

    2012-12-01

    CD133 has been identified as a putative cancer stem cell (CSC) marker in various cancers including colorectal cancer. The relation between CD133 expression and biological characteristics of colorectal cancer remains to be clarified. Protein expression of CD133 was immunohistochemically evaluated in surgical specimens of 225 patients with colorectal cancer who were treated by surgery, as well as those of 78 patients with rectal cancer who received preoperative chemoradiotherapy (CRT) followed by curative resection. The correlation between CD133 expression and clinicopathological features, tumor recurrence and overall survival was analyzed in both populations. Among 225 colorectal cancers without CRT, 93 (41.3%) were positive for CD133 expression, which was enhanced in cases with advanced T stage and venous invasion. Moreover, CD133 was positive in 47 (60.3%) of 78 cases with CRT, which was significantly higher than the CD133-positive rate in non-CRT specimens (P=0.05). Expression of CD133 was independently correlated with the histological tumor regression grade (P<0.01). These results suggest that CD133 is not a distinctive colorectal CSC marker; expression of CD133 is suggested to be one of the key factors associated with resistance to CRT in colorectal cancer.

  16. CXCL3 contributes to CD133+ CSCs maintenance and forms a positive feedback regulation loop with CD133 in HCC via Erk1/2 phosphorylation

    Science.gov (United States)

    Zhang, Lin; Zhang, Lixing; Li, Hong; Ge, Chao; Zhao, Fangyu; Tian, Hua; Chen, Taoyang; Jiang, Guoping; Xie, Haiyang; Cui, Ying; Yao, Ming; Li, Jinjun

    2016-01-01

    Although the chemotactic cytokine CXCL3 is thought to play an important role in tumor initiation and invasion, little is known about its function in hepatocellular carcinoma (HCC). In our previous study, we found that Ikaros inhibited CD133 expression via the MAPK pathway in HCC. Here, we showed that Ikaros may indirectly down-regulate CXCL3 expression in HCC cells, which leads to better outcomes in patients with CD133+ cancer stem cell (CSC) populations. CD133 overexpression induced CXCL3 expression, and silencing of CD133 down-regulated CXCL3 in HCC cells. Knockdown of CXCL3 inhibited CD133+ HCC CSCs’ self-renewal and tumorigenesis. The serum CXCL3 level was higher in HCC patients’ samples than that in healthy individual. HCC patients with higher CXCL3 expression displayed a poor prognosis, and a high level of CXCL3 was significantly associated with vascular invasion and tumor capsule formation. Exogenous CXCL3 induced Erk1/2 and ETS1 phosphorylation and promoted CD133 expression, indicating a positive feedback loop between CXCL3 and CD133 gene expression in HCC cells via Erk1/2 activation. Together, our findings indicated that CXCL3 might be a potent therapeutic target for HCC. PMID:27255419

  17. Increased CD133+ cell infiltration in the rat brain following fluid percussion injury

    Institute of Scientific and Technical Information of China (English)

    Ming Wei; Ziwei Zhou; Shenghui Li; Chengwei Jing; Dashi Zhi; Jianning Zhang

    2012-01-01

    The prominin-1/CD133 epitope is expressed in undifferentiated cells. Studies have reported that craniocerebral trauma in animal models of fluid percussion injury induces production of a specific stem cell subgroup. It has been hypothesized that fluid percussion injury induces CD133+ cell infiltration in the brain tissue. The present study established a traumatic brain injury model through fluid percussion injury. Immunohistochemical staining showed significantly increased CD133 antigen expression in the rat brain following injury. CD133+ cells were mainly distributed in hippocampal CA1-3 regions, as well as the dentate gyrus and hilus, of the lesioned hemisphere. Occasional cells were also detected in the cortex. In addition, reverse transcription-PCR revealed that no change in CD133 mRNA expression in injured brain tissue. These results suggested that fluid percussion injury induced CD133 antigen expression in the brain tissues as a result of conformational epitope changes, but not transcriptional expression.

  18. STAT3 is a key transcriptional regulator of cancer stem cell marker CD133 in HCC

    Science.gov (United States)

    Ghoshal, Sarani; Fuchs, Bryan C.

    2016-01-01

    Cancer stem cell (CSC) marker CD133 was found to be upregulated in many cancers including hepatocellular carcinoma (HCC). However, the molecular mechanism of CD133 regulation in the liver tumor microenvironment has remained elusive. In this study Won and colleagues report that interleukin-6 (IL-6) mediated signal transducer and activator of transcription factor 3 (STAT3) signaling and hypoxia enhance the expression of CD133 and promote the progression of HCC. PMID:27275460

  19. CD133 Immunohistochemisty in Glioblastoma – Identification of Tumor Stem Cells or a Matter of Coincidence?

    DEFF Research Database (Denmark)

    Hermansen, Simon Kjær; Christensen, Karina Garnier; Jensen, Stine Skov;

    The putative stem cell marker CD133 is the marker of choice for identifying brain tumor stem cells in gliomas, but the use of different antibody clones recognizing different epitopes with different glycosylation status, confuses the field. In this study, we sat out to highlight if current...... suggest that CD133 immunohistochemical studies take this in to consideration by using different CD133 antibody clones together with other stem cell markers and e.g. PCR techniques before too firm conclusions are drawn....

  20. Poly(lactic-co-glycolic acid nanoparticles conjugated with CD133 aptamers for targeted salinomycin delivery to CD133+ osteosarcoma cancer stem cells

    Directory of Open Access Journals (Sweden)

    Ni MZ

    2015-03-01

    Full Text Available Miaozhong Ni,1,* Min Xiong,1,* Xinchao Zhang,1,* Guoping Cai,1 Huaiwen Chen,2 Qingmin Zeng,1 Zuochong Yu1 1Department of Orthopedics, Jinshan Hospital, Fudan University, 2International Joint Cancer Institute, the Second Military Medical University, Shanghai, People’s Republic of China *These authors contributed equally to this work Background: Cancer stem cells (CSCs possess the characteristics associated with normal stem cells and are responsible for cancer initiation, recurrence, and metastasis. CD133 is regarded as a CSCs marker of osteosarcoma, which is the most common primary bone malignancy in childhood and adolescence. Salinomycin, a polyether ionophore antibiotic, has been shown to kill various CSCs, including osteosarcoma CSCs. However, salinomycin displayed poor aqueous solubility that hinders its clinical application. The objective of this study was to develop salinomycin-loaded nanoparticles to eliminate CD133+ osteosarcoma CSCs.Methods: The salinomycin-loaded PEGylated poly(lactic-co-glycolic acid nanoparticles (SAL-NP conjugated with CD133 aptamers (Ap-SAL-NP were developed by an emulsion/solvent evaporation method, and the targeting and cytotoxicity of Ap-SAL-NP to CD133+ osteosarcoma CSCs were evaluated.Results: The nanoparticles are of desired particle size (~150 nm, drug encapsulation efficiency (~50%, and drug release profile. After 48 hours treatment of the Saos-2 CD133+ osteosarcoma cells with drugs formulated in Ap-SAL-NP, SAL-NP, and salinomycin, the concentrations needed to kill 50% of the incubated cells were found to be 2.18, 10.72, and 5.07 µg/mL, respectively, suggesting that Ap-SAL-NP could be 4.92 or 2.33 fold more effective than SAL-NP or salinomycin, respectively. In contrast, Ap-SAL-NP was as effective as SAL-NP, and less effective than salinomycin in Saos-2 CD133- cells, suggesting that Ap-SAL-NP possess specific cytotoxicity toward Saos-2 CD133+ cells. Ap-SAL-NP showed the best therapeutic effect in Saos-2

  1. Lentivirus-mediated RNA interference of CD133 inhibits the proliferation of CD133+ liver cancer stem cells and increases their cisplatin chemosensitivity%RNA干扰抑制CD133表达对CD133+肝癌干细胞增殖和化疗敏感性的影响

    Institute of Scientific and Technical Information of China (English)

    兰曦; 王勇; 曹姝; 邹冬玲; 李芳; 李少林

    2012-01-01

    Objective To study the effects of CD133 suppression by lentivirus-mediated RNA interference (RNAi) on the proliferation and chemosensitivity of CD133+ cancer stem cells (CSCs) sorted from HepG2 cell line. Methods CD133+ and CD133-cells were sorted from HepG2 cell line by flow cytometry, and the expression of CD133 before and after cell sorting were detected. The stem cell property of sorted CD133+ cells were validated by sphere-forming assay in vitro and xenograft experiments in vivo. Lentivirus-mediated short haipin RNA (shRNA) targeting CD133 were transfected into CD133+ cells, and CD133 mRNA and protein expressions of the transfected cells were detected by RT-PCR and Western blotting, respectively. Before and after the transfection, the proliferative ability of CD133+ cells was evaluated by colony formation assay, and the cell growth inhibition rate and apoptosis following ciplatin exposure were detected using CCK-8 assay and flow cytometry. Results The sorted CD133+ cells showed a high purity of (88.74±3.19)%, as compared with the purity of (3.36±1.80)% before cell sorting. CD133+ cells showed a high tumor sphere formation ability and tumorigenesis capacity compared with CD133- cells. CD133 shRNA transfection significantly inhibited CD133 mRNA and protein expressions in CD133+ cells (P<0.01), resulting also in a significantly lowered cell proliferative ability (P<0.01) and an increased growth inhibition rate (P<0.01) and obviously increased cell apoptosis (P<0.05) after cisplatin exposure. Conclusions Lentivirus-mediated RNAi for CD133 suppression inhibits the proliferation of CD133+ liver cancer stem cells and increases their chemosensitivity to cisplatin.%目的 利用慢病毒介导的RNA干扰技术下调CD133+ HepG2肝癌干细胞中CD133的表达,探讨其对肝癌干细胞增殖及化疗敏感性的影响.方法 利用流式分选技术筛选CD133+ HepG2细胞并检测分选前后CD133表达量,体外成球及体内成瘤实验鉴定其“干性”;随后以CD

  2. High CD133 Expression Is Associated with Worse Prognosis in Patients with Glioblastoma.

    Science.gov (United States)

    Zhang, Wei; Chen, Huanran; Lv, Shengqing; Yang, Hui

    2016-05-01

    The CD133 antigen has been identified as a putative stem cell marker in gliomas. However, the prognostic significance of CD133 expression in glioblastoma patients remained controversial. A meta-analysis of published data was performed to comprehensively assess the prognostic role of CD133 expression in glioblastoma patients. Publications assessing the prognostic significance of CD133 expression in glioblastoma patients were identified in PubMed, Embase, and Web of Science up to November 2014. The pooled hazard ratio (HR) with 95% confidence interval (95% CI) was calculated using meta-analysis to evaluate the prognostic significance of CD133 expression in glioblastoma. Ten studies with a total of 715 glioblastoma patients were included into the meta-analysis. Overall, high CD133 expression was associated with poorer overall survival in patients with glioblastoma (HR = 1.96, 95% CI 1.46-2.64, P glioblastoma (HR = 2.03, 95% CI 1.43-2.88, P glioblastoma. Meta-analysis of studies with adjusted estimates further showed that high CD133 expression was an independent prognostic factor of glioblastoma. High CD133 expression is associated with worse prognosis in patients with glioblastoma. More prospective studies with well-design are needed to confirm this finding.

  3. CD133(+) niches and single cells in glioblastoma have different phenotypes

    DEFF Research Database (Denmark)

    Christensen, Karina; Schrøder, Henrik Daa; Kristensen, Bjarne Winther

    2011-01-01

    Putative CD133(+) brain tumor stem cells have been shown to be located in niches and as single cells. This is the first study providing insight into the different phenotypes of CD133(+) cells in glioblastoma according to localization. Paraffin sections were stained by double immunofluorescence...

  4. IL-6 promotes growth and epithelial-mesenchymal transition of CD133+ cells of non-small cell lung cancer.

    Science.gov (United States)

    Lee, Soo Ok; Yang, Xiaodong; Duan, Shanzhou; Tsai, Ying; Strojny, Laura R; Keng, Peter; Chen, Yuhchyau

    2016-02-09

    We examined IL-6 effects on growth, epithelial-mesenchymal transition (EMT) process, and metastatic ability of CD133+ and CD133- cell subpopulations isolated from three non-small cell lung cancer (NSCLC) cell lines: A549, H157, and H1299. We developed IL-6 knocked-down and scramble (sc) control cells of A549 and H157 cell lines by lentiviral infection system, isolated CD133+ and CD133- sub-populations, and investigated the IL-6 role in self-renewal/growth of these cells. IL-6 showed either an inhibitory or lack of effect in modulating growth of CD133- cells depending on intracellular IL-6 levels, but there was higher self-renewal ability of IL-6 expressing CD133+ cells than IL-6 knocked down cells, confirming the promoter role of IL-6 in CD133+ cells growth. We then examined tumor growth of xenografts developed from CD133+ cells of A549IL-6si vs. A549sc cell lines. Consistently, there was retarded growth of tumors developed from A549IL-6si, CD133+ cells compared to tumors originating from A549sc, CD133+ cells. The effect of IL-6 in promoting CD133+ self-renewal was due to hedgehog (Hhg) and Erk signaling pathway activation and higher Bcl-2/Bcl-xL expression. We also investigated whether IL-6 regulates the EMT process of CD133- and CD133+ cells differently. Expression of the EMT/metastasis-associated molecules in IL-6 expressing cells was higher than in IL-6 knocked down cells. Together, we demonstrated dual roles of IL-6 in regulating growth of CD133- and CD133+ subpopulations of lung cancer cells and significant regulation of IL-6 on EMT/metastasis increase in CD133+ cells, not in CD133- cells.

  5. CD133+细胞的干性鉴定及131I-CD133抗体对其体内外抑制作用%Identification of the stem property of CD133+ liver cancer cells and inhibition effect of 131I-labeled anti-CD133 mAb on them in vitro and vivo

    Institute of Scientific and Technical Information of China (English)

    段丽群; 侯妍利; 陈兴月; 唐敏; 康强强; 舒锦; 李少林

    2014-01-01

    目的:研究CD133+细胞的干性鉴定和131I-CD133抗体在体内外对人肝癌CD133+-HepG2干细胞的抑制作用.方法:氯胺T法制备并鉴定131I-CD133抗体;免疫磁珠(magnetic-activated cell sorting,MACS)分选CD133+-HepG2细胞;流式细胞仪(flow cytometry,FCM)检测分选前后CD133表达率;体外克隆形成实验、成球实验和体内成瘤实验验证其干细胞特性;将分选出的CD133+细胞分组为CD133抗体、131I、131I-CD133抗体和131I+CD133抗体4个组,MTT法检测不同处理后不同组中CD133+细胞生长抑制率;成功构建人肝癌CD133+-HepG2移植瘤模型;随机分4组,1次/2 d给予尾静脉用药,共14次.4周后,处死小鼠,比较肿瘤的体积、质量、计算抑瘤率;HE染色观察肿瘤组织病理学改变.结果:131I-CD133抗体标记率为89.34%,放化纯度为98.21%.流式显示分选前后CD133表达率分别为(1.78±0.54)%和(98.46±0.97)%.成球实验、克隆形成实验和裸鼠成瘤实验显现CD133+细胞相对于CD133-细胞更具有干细胞特性.131I-CD133抗体治疗组体外对细胞抑制率及体内抑瘤率明显高于其余各实验组,差异具有统计学意义(P<0.05).结论:131I-CD133抗体在体内外均能有效抑制人肝癌CD 133+-HepG2细胞的生长.

  6. The CD133+ cell as advanced medicinal product for myocardial and limb ischemia.

    Science.gov (United States)

    Bongiovanni, Dario; Bassetti, Beatrice; Gambini, Elisa; Gaipa, Giuseppe; Frati, Giacomo; Achilli, Felice; Scacciatella, Paolo; Carbucicchio, Corrado; Pompilio, Giulio

    2014-10-15

    Ischemic diseases are the major cause of death and morbidity in Western countries. In the last decade, cell therapy has been suggested to be a promising treatment both in acute/chronic myocardial and peripheral ischemia. Different cell lineages have been tested, including endothelial progenitor cells. A subpopulation of bone marrow-derived immature ECPs, expressing the highly conserved stem cell glycoprotein antigen prominin-1 or CD133 marker, was shown to possess pro-angiogenic and antiapoptotic effects on ischemic tissues. The mechanisms implicated in CD133+ cells ability to contribute to neovascularization processes have been attributed to their ability to directly differentiate into newly forming vessels and to indirectly activate pro-angiogenic signaling by paracrine mechanisms. A large body of in vivo experimental evidences has demonstrated the potential of CD133+ cells to reverse ischemia. Moreover, several clinical trials have reported promising beneficial effects after infusion of autologous CD133+ into ischemic heart and limbs exploiting various delivery strategies. These trials have contributed to characterize the CD133+ manufacturing process as an advanced cell product (AMP). The aim of this review is to summarize available experimental and clinical data on CD133+ cells in the context of myocardial and peripheral ischemia, and to focus on the development of the CD133+ cell as an anti-ischemic AMP.

  7. The Brain Microenvironment Preferentially Enhances the Radioresistance of CD133+ Glioblastoma Stem-like Cells

    Directory of Open Access Journals (Sweden)

    Muhammad Jamal

    2012-02-01

    Full Text Available Brain tumor xenografts initiated from glioblastoma (GBM CD133+ tumor stem-like cells (TSCs are composed of TSC and non-TSC subpopulations, simulating the phenotypic heterogeneity of GBMs in situ. Given that the discrepancies between the radiosensitivity of GBM cells in vitro and the treatment response of patients suggest a role for the microenvironment in GBM radioresistance, we compared the response of TSCs and non-TSCs irradiated under in vitro and orthotopic conditions. As a measure of radioresponse determined at the individual cell level, γH2AX and 53BP1 foci were quantified in CD133+ cells and their differentiated (CD133- progeny. Under in vitro conditions, no difference was detected between CD133+ and CD133- cells in foci induction or dispersal after irradiation. However, irradiation of orthotopic xenografts initiated from TSCs resulted in the induction of fewer γH2AX and 53BP1 foci in CD133+ cells compared to their CD133- counterparts within the same tumor. Xenograft irradiation resulted in a tumor growth delay of approximately 7 days with a corresponding increase in the percentage of CD133+ cells at 7 days after radiation, which persisted to the onset of neurologic symptoms. These results suggest that, although the radioresponse of TSCs and non-TSCs does not differ under in vitro growth conditions, CD133+ cells are relatively radioresistant under intracerebral growth conditions. Whereas these findings are consistent with the suspected role for TSCs as a determinant of GBM radioresistance, these data also illustrate the dependence of the cellular radioresistance on the brain microenvironment.

  8. Promoter hypermethylation and loss of CD133 gene expression in colorectal cancers

    Institute of Scientific and Technical Information of China (English)

    You-Kyung; Jeon; Sung-Hee; Kim; Seung-Ho; Choi; Kyung-Hee; Kim; Byong-Chul; Yoo; Ja-Lok; Ku; Jae-Gahb; Park

    2010-01-01

    AIM: To understand CD133 promoter hypermethyl-ation and expression in 32 colorectal cancer cell lines. METHODS: Nucleic acid was isolated from 32 colorectal cancer cell lines and CD133 expression levels were measured by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. Promoter methylation status of the CD133 gene was analyzed with a methylation-specific PCR after sodium-bisulfi te modification and by clonal sequencing analysis. The correlation between expression and promoter methy...

  9. Convenient and efficient enrichment of the CD133+ liver cells from rat fetal liver cells as a source of liver stem/progenitor cells.

    Science.gov (United States)

    Liu, Wei-hui; Li, Ren; Dou, Ke-feng

    2011-03-01

    Although the stem cells are commonly isolated by FACS or MACS, they are very expensive and these is no specific marker for liver stem/progentior cells (LSPCs). This paper applied a convenient and efficient method to enrich LSPCs. The fetal liver cells (FLCs) were firstly enriched by Percoll discontinuous gradient centrifugation (PDGC) from the rat fetal liver. Then the FLCs in culture were purified to be homogeneous in size by differential trypsinization and differential adherence (DTDA). Flow cytometric analysis revealed more than half of the purified FLCs expressed alternative markers of LSPCs (CD117, c-Met, Sca-1, CD90, CD49f and CD133). In other words, the purified FLCs were heterogeneous. Therefore, they were sequentially layered into six fractions by Percoll continuous gradient centrifugation (PCGC). Both CD133 and CD49f expressed decreasingly from fraction 1 to 6. In fraction 1 and 2, about 85% FLCs expressed CD133, which were revealed to be LSPCs by high expressions of AFP and CK-19, low expressions of G-6-P and ALB. To conclude, the purity of CD133(+) LSPCs enriched by combination of PDGC, DTDA and PCGC is close to that obtained by MACS. This study will greatly contribute to two important biological aspects: liver stem cells isolation and liver cell therapy.

  10. Prognostic significance of stem cell marker CD133 determined by promoter methylation but not by immunohistochemical expression in malignant gliomas.

    Science.gov (United States)

    Wu, Xing; Wu, Fenlang; Xu, Dongwen; Zhang, Tao

    2016-04-01

    CD133 has played a pivotal role in the identification and isolation of brain tumor stem cells. The correlation between CD133 expression in tumor tissues with patients survival is still controversial. CD133 expression is determinated by methylation status of the promoter region 1-3. Aberrant methylation of CD133 was observed in glioblastoma. To date, a direct link between CD133 methylation and patient outcome has not been established.To address this question, we studied CD133 expression and promoter methylation in a series of 170 gliomas of various grade and histology, and investigated the correlation of CD133 expression and promoter methylation with patient outcome.We detected five CD133 promoter methylation patterns in 170 glioma samples: methylation only (M+, U-), unmethylation only (M-, U+), both methylation and unmethylation equally (M+, U+), high methylation and low unmethylation (M+, Ul), and low methylation and high unmethylation (Ml, U+). By multivariate survival analysis, we found CD133 promoter methylation status was significant (P promoter methylation status was observed (Kw = -0.165).CD133 promoter methylation status in glioma is closely correlated with patient survival, which suggest CD133 promoter methylaiton pattern is a promising tool for diagnostic purposes.

  11. Rapid selection and proliferation of CD133+ cells from cancer cell lines: chemotherapeutic implications.

    Directory of Open Access Journals (Sweden)

    Sarah E Kelly

    Full Text Available Cancer stem cells (CSCs are considered a subset of the bulk tumor responsible for initiating and maintaining the disease. Several surface cellular markers have been recently used to identify CSCs. Among those is CD133, which is expressed by hematopoietic progenitor cells as well as embryonic stem cells and various cancers. We have recently isolated and cultured CD133 positive [CD133+] cells from various cancer cell lines using a NASA developed Hydrodynamic Focusing Bioreactor (HFB (Celdyne, Houston, TX. For comparison, another bioreactor, the rotary cell culture system (RCCS manufactured by Synthecon (Houston, TX was used. Both the HFB and the RCCS bioreactors simulate aspects of hypogravity. In our study, the HFB increased CD133+ cell growth from various cell lines compared to the RCCS vessel and to normal gravity control. We observed a +15-fold proliferation of the CD133+ cellular fraction with cancer cells that were cultured for 7-days at optimized conditions. The RCCS vessel instead yielded a (-4.8-fold decrease in the CD133+cellular fraction respect to the HFB after 7-days of culture. Interestingly, we also found that the hypogravity environment of the HFB greatly sensitized the CD133+ cancer cells, which are normally resistant to chemo treatment, to become susceptible to various chemotherapeutic agents, paving the way to less toxic and more effective chemotherapeutic treatment in patients. To be able to test the efficacy of cytotoxic agents in vitro prior to their use in clinical setting on cancer cells as well as on cancer stem cells may pave the way to more effective chemotherapeutic strategies in patients. This could be an important advancement in the therapeutic options of oncologic patients, allowing for more targeted and personalized chemotherapy regimens as well as for higher response rates.

  12. Side population rather than CD133+ cells distinguishes enriched tumorigenicity in hTERT-immortalized primary prostate cancer cells

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    Wolcott Karen

    2011-09-01

    Full Text Available Abstract Background Subpopulations of cancer cells with the capacity of generating solid tumors have been characterized. In various cancer types, including prostate cancer cells, a side population (SP and CD133-expressing cells have been proposed as containing a population cancer cells with stem-like ability. Therefore the aim of this work was to determine, in prostate cancer cell lines, the frequency and tumorigenic potential of SP and CD133+ cells. Results In vitro 2D colony-forming assay and sphere-forming assay, Flow cytometry analysis and magnetic cell sorting were utilized to sort CD133+, CD133- and Side population (SP cells. Our findings indicate that CD44 and integrin α-6 are uniformly expressed in the hTERT cell lines; however, CD133 is expressed only in a small population (in vitro and in vivo. Additionally, for the hTERT cells, SP rather than CD133 expression showed an 8-fold enhanced tumorigenic potential. The data suggest that SP cells, rather than those with CD133 marker, contain the rare population of CSC capable of producing prostate tumors. Conclusion Collectively, our data suggest that although CD133 is expressed only in a small population of hTERT-immortalized prostate cancer cells, it is not likely to be associated with stem cells, as CD133- and CD133+ cells exhibited similar tumorigenicity. However, SP isolated cells, appear to be enriched with tumorigenic stem-like cells capable of generating palpable tumors.

  13. DNA Damage in CD133-Positive Cells in Barrett’s Esophagus and Esophageal Adenocarcinoma

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    Raynoo Thanan

    2016-01-01

    Full Text Available Barrett’s esophagus (BE caused by gastroesophageal reflux is a major risk factor of Barrett’s esophageal adenocarcinoma (BEA, an inflammation-related cancer. Chronic inflammation and following tissue damage may activate progenitor cells under reactive oxygen/nitrogen species-rich environment. We previously reported the formation of oxidative/nitrative stress-mediated mutagenic DNA lesions, 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG and 8-nitroguanine, in columnar epithelial cells of BE tissues and cancer cells of BEA tissues. We investigated the mechanisms of BEA development in relation to oxidative/nitrative DNA damage and stem cell hypothesis. We examined 8-nitroguanine and 8-oxodG formation and the expression of stem cell marker (CD133 in biopsy specimens of patients with BE and BEA by immunohistochemical analysis in comparison with those of normal subjects. CD133 was detected at apical surface of columnar epithelial cells of BE and BEA tissues, and the cytoplasm and cell membrane of cancer cells in BEA tissues. DNA lesions and CD133 were colocalized in columnar epithelial cells and cancer cells. Their relative staining intensities in these tissues were significantly higher than those in normal subjects. Our results suggest that BE columnar epithelial cells with CD133 expression in apical surface undergo inflammation-mediated DNA damage, and mutated cells acquire the property of cancer stem cells with cytoplasmic CD133 expression.

  14. Importance of Quantitative Evaluation of Colorectal Cancer CD133+ Cells%结直肠癌CD133+细胞定量评估的意义

    Institute of Scientific and Technical Information of China (English)

    缪刚; 李尧; 赵艳阳; 黄美雄; 韦军民

    2012-01-01

    目的 研究CD133+结直肠癌干细胞与肿瘤增殖、转移的相关性.方法 取得术后新鲜的结直肠癌组织后,立刻进行清洗、消化、培养等过程,得到了具有活性的原代结直肠癌单细胞.特异性抗体CD133标记后运用流式细胞技术检测癌干细胞分布比例.癌组织同时进行免疫组化和分子生物学研究,确定增殖蛋白Ki - 67、抑制肿瘤转移相关蛋白e- cadherin和细胞凋亡相关蛋白caspase -3的表达.结果 结直肠癌原代细胞分离、纯化和检测技术简明可行,所得数据客观准确.研究病例按照CD133+癌干细胞比例≥3%和<3%分成两组.结果显示CD133+癌干细胞≥3%组在肿瘤大小和淋巴转移有增大和增多趋势;肿瘤增殖特异性蛋白Ki - 67增高具有显著性差异;抑制肿瘤转移相关蛋白e- cadherin的表达降低;凋亡相关蛋白caspase -3的表达降低.结论 本研究成功建立了一套可行性高的结直肠癌CD133+干细胞准确定量评估系统.结直肠癌干细胞的准确定量检测可以作为评估患者预后和化疗敏感度的一项重要指标.本技术也为进一步提纯结直肠癌干细胞,并且最终研究对此靶点的攻击提供了平台.%Objective To precisely quantify the percentage of CD133+ cancer stem cells by isolation and purification of primary colorectal cancer cells, and to clarify its relationship with cancer proliferation and metastasis. Methods Primary colorectal cancer cells were obtained after washing, digesting and culturing fresh colorectal cancer tissues. Cancer stem cells were labeled with specific CD133 antibody and analyzed by flow cytometry. Cancer tissues were also tested using immunostaining and Western blotting to detect expression of proliferative marker Ki - 67, invasion suppressor related protein e - cadherin, and apoptosis related protein caspase - 3. Results Isolation, purification and precisely quantification of primary colorectal cancer stem cells were

  15. Clinical value of CD133 and nestin in patients with glioma

    DEFF Research Database (Denmark)

    Dahlrot, Rikke H; Hansen, Steinbjørn; Jensen, Stine S

    2014-01-01

    133 and nestin separately and in combination using a novel quantitative approach in a well-characterized population-based cohort of glioma patients. The expression of CD133 and nestin was measured by systematic random sampling in stained paraffin sections from 239 glioma patients diagnosed between......-free survival (PFS) in multivariate analysis. High levels of co-localization were associated with poor PFS in patients with WHO grade II tumors, but not with OS. We conclude that CD133 was not an independent prognostic factor, but a high level of nestin was associated with poor PFS in patients with WHO grade II...... 2005 and 2009. We found that the expression of CD133 did not correlate with WHO grade, and there was no association with overall survival (OS). The level of nestin correlated positively with WHO grade. In patients with WHO grade II tumors, a high level of nestin was associated with short progression...

  16. Blocking NOTCH Pathway can Enhance the Effect of EGFR Inhibitor through Targeting CD133+ Endometrial Cancer Cells.

    Science.gov (United States)

    Shang, Chao; Lang, Bin; Meng, Li-Rong

    2016-10-28

    ABSTACT Although the molecular therapeutics targeting key biomarkers such as epithelial growth factor receptor (EGFR), PI3K/AKT/mTOR, and vascular endothelial growth factor (VEGF) shows some success in clinical trials, some internally existing challenges in endothelial cancer biology hinder the drug effects. One of the major challenges stems from cancer stem cell-derived drug resistance. CD133 positive cells are well believed as cancer stem cells (CSC) in endometrial cancers and NOTCH pathway plays a critical role in retaining CD133+ cells by promoting CSC self-renewal and chemoresistance. Here, we initiated a therapeutic strategy to improve effects of EGFR inhibition by targeting NOTCH pathway of CD133+ cells in endometrial cancers. We first detected and purified the CD133+ cell fraction in endometrial cancer cell line Ishikawa (IK), and validated activation of NOTCH pathway in the CD133+ cells that have higher proliferation rate and lower apoptosis rate, comparing to CD133- cells. Results of nude mouse xenograft experiments further demonstrated CD133+ cells retain higher tumorigenesis capacity than CD133- cells, indicating their tumor-initiating property. Last, we applied both NOTCH inhibitor DAPT and EGFR inhibitor AG1478 treatment on endometrial cancer lines IK and HEC-1A and the results suggested improvement effects of the combination therapy compared to the treatments of DAPT or AG1478 alone. These findings indicated targeting NOTCH pathway in CD133+ cells, combining with EGFR inhibition, which provides a novel therapeutic strategy for endometrial cancer diseases.

  17. Detection of the Hematopoietic Stem and Progenitor Cell Marker CD133 during Angiogenesis in Three-Dimensional Collagen Gel Culture

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    Masumi Akita

    2013-01-01

    Full Text Available We detected the hematopoietic stem and progenitor cell marker CD133 using immunogold labeling during angiogenesis in a three-dimensional collagen gel culture. CD133-positive cells were present in capillary tubes newly formed from aortic explants in vitro. The CD133-positive cell population had the capacity to form capillary tubes. Lovastatin strongly inhibited cell migration from aortic explants and caused the degradation of the capillary tubes. The present study provides insight into the function of CD133 during angiogenesis as well as an explanation for the antiangiogenic effect of statins.

  18. Increased plasma levels of microparticles expressing CD39 and CD133 in acute liver injury

    DEFF Research Database (Denmark)

    Schmelzle, Moritz; Splith, Katrin; Wiuff Andersen, Lars;

    2013-01-01

    BACKGROUND: We have previously demonstrated that CD133 and CD39 are expressed by hematopoietic stem cells (HSC), which are mobilized after liver injury and target sites of injury, limit vascular inflammation, and boost hepatic regeneration. Plasma microparticles (MP) expressing CD39 can block...... endothelial activation. Here, we tested whether CD133 MP might be shed in a CD39-dependent manner in a model of liver injury and could potentially serve as biomarkers of liver failure in the clinic. METHODS: Wild-type and Cd39-null mice were subjected to acetaminophen-induced liver injury. Mice were...

  19. Isolation of Cultured Endothelial Progenitor Cells in vitro from PBMCs and CD133~+ Enriched Cells

    Institute of Scientific and Technical Information of China (English)

    郑伟红; 万亚峰; 马小鹏; 李兴睿; 杨志芳; 殷茜; 易继林

    2010-01-01

    Two isolation methods for sorting of endothelial progenitor cells(EPCs):from peripheral blood mononuclear cells(PBMCs)and CD133+ enriched cells were compared,by defining the cell morphology,phenotype,reproductive activities and function in vitro,to provide a reference for clinical application of EPCs.PBMCs from healthy subjects were used either directly for cell culture or for CD133+ sorting.The two groups of cells were cultured in complete medium 199(M199)for 7 to 14 days and the phenotypes of EPCs were an...

  20. Autologous CD34~+ and CD133~+ stem cells transplantation in patients with end stage liver disease

    Institute of Scientific and Technical Information of China (English)

    Hosny; Salama; Abdel-Rahman; N; Zekri; Abeer; A; Bahnassy; Eman; Medhat; Hanan; A; Halim; Ola; S; Ahmed; Ghada; Mohamed; Sheren; A; Al; Alim; Ghada; M; Sherif

    2010-01-01

    AIM:To assess the utility of an autologous CD34 + and CD133 + stem cells infusion as a possible therapeutic modality in patients with end-stage liver diseases.METHODS:One hundred and forty patients with endstage liver diseases were randomized into two groups.Group 1,comprising 90 patients,received granulocyte colony stimulating factor for five days followed by autologous CD34 + and CD133 + stem cell infusion in the portal vein.Group 2,comprising 50 patients,received regular liver treatment only and served a...

  1. Colorectal cancers with aneuploids show high CD133 expression and poor prognosis

    Institute of Scientific and Technical Information of China (English)

    Dongdong Yu; Yonghong Zhang; You Zou; Ming Tian; Deding Tao; Junbo Hu; Jianping Gong

    2010-01-01

    Objective:The aim of the study was to investigate the relationship of DNA ploidy status in colorectal cancers with patients' prognosis and also the relationship of DNA ploidy status with expression of the colorectal cancer stem cell marker CD133.Methods:The DNA ploidy status and CD133 expression in colorectal cancers were detected by flow cytometry.The clinicopathological characteristics and progression-free survival analysis of patients was evaluated based on the clinical data.Results:DNA ploidy pattern did not correlated with gender,age,lesion diameter,histological type,depth of tumor invasion,lymphatic invasion and Dukes stage.Only primary lesion cite showed significant correlation with DNA ploidy pattern,more aneuploids were observed in colonic cancer than rectal cancer,P < 0.05.The 2-year progression-free survival rate and total progression-free time in patients with aneuploids were lower than that with diploids,P < 0.05.Tumors contained aneuploids showed higher expression of CD133 than tumors of only diploids,P < 0.05.Conclusion:Tumor DNA ploidy status is a significant prognostic factor in patients with colorectal cancer and also associated with the existence of CD133 positive colorectal cancer stem cells.

  2. Expression of CD133, PAX2, ESA, and GPR30 in invasive ductal breast carcinomas

    Institute of Scientific and Technical Information of China (English)

    LIU Qun; LI Ji-guang; ZHENG Xin-yu; JIN Feng; DONG Hui-ting

    2009-01-01

    Background Biomarkers in breast neoplasms provide invaluable information regarding prognosis and help determining the optimal treatment. We have examined the possible correlation between cancer stem cell (CSC)-Iike markers (CD133,paired box gene 2 protein (PAX2), epithelial specific antigen (ESA)), and a new membrane estrogen receptor (G-protein coupled receptor 30 (GPR30)) in invasive ductal breast carcinomas with known clinicopathological parameters, tumor recurrence, and expression of some known biomarkers.Methods In 74 invasive ductal breast carcinomas, we investigated the protein expression of these molecular markers by immunohistochemistry, and their associations with known clinicopathological parameters, tumor recurrence, and expression of some known biomarkers. We studied the interrelationship between the expressions of these proteins.Results CD133, a putative CSC marker, was positively related to tumor size, tumor stage, and lymph node metastasis.PAX2 was negatively correlated with tumor recurrence. ESA, one of the breast CSC markers, was an indicator of tumor recurrence. GPR30 was associated with hormone receptors. Despite the correlation between GPR30 and the nuclear estrogen receptor, the expression was dependent. Positive staining of GPR30 in tumors displayed a significant association with high C-erbB2 expression and a tendency for tumor recurrence. A positive relationship between GPR30 and CD133 existed.Conclusion Detecting the expression of CD133, PAX2, ESA, and GPR30 in invasive ductal breast carcinomas may be of help in more accurately predicting the aggressive properties of breast cancer and determining the optimal treatment.

  3. CD133 identifies perivascular niches in grade II-IV astrocytomas

    DEFF Research Database (Denmark)

    Christensen, Karina; Schrøder, Henrik; Kristensen, Bjarne

    2008-01-01

    The aim of the present study was to investigate the localization and distribution of the putative brain tumour stem cell marker CD133 in formalin fixed paraffin embedded astrocytomas. A retrospective analysis of 114 grade II, III and IV astrocytomas was undertaken. The immunohistochemical...

  4. Regulation of CD133 by HDAC6 Promotes β-Catenin Signaling to Suppress Cancer Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Anthony B. Mak

    2012-10-01

    Full Text Available The pentaspan membrane glycoprotein CD133 marks lineage-specific cancer progenitor cells and is associated with poor prognosis in a number of tumor types. Despite its utility as a cancer progenitor cell marker, CD133 protein regulation and molecular function remain poorly understood. We find that the deacetylase HDAC6 physically associates with CD133 to negatively regulate CD133 trafficking down the endosomal-lysosomal pathway for degradation. We further demonstrate that CD133, HDAC6, and the central molecule of the canonical Wnt signaling pathway, β-catenin, can physically associate as a ternary complex. This association stabilizes β-catenin via HDAC6 deacetylase activity, which leads to activation of β-catenin signaling targets. Downregulation of either CD133 or HDAC6 results in increased β-catenin acetylation and degradation, which correlates with decreased proliferation in vitro and tumor xenograft growth in vivo. Given that CD133 marks progenitor cells in a wide range of cancers, targeting CD133 may be a means to treat multiple cancer types.

  5. CD133 positive embryonal rhabdomyosarcoma stem-like cell population is enriched in rhabdospheres.

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    Dagmar Walter

    Full Text Available Cancer stem cells (CSCs have been identified in a number of solid tumors, but not yet in rhabdomyosarcoma (RMS, the most frequently occurring soft tissue tumor in childhood. Hence, the aim of this study was to identify and characterize a CSC population in RMS using a functional approach. We found that embryonal rhabdomyosarcoma (eRMS cell lines can form rhabdomyosarcoma spheres (short rhabdospheres in stem cell medium containing defined growth factors over several passages. Using an orthotopic xenograft model, we demonstrate that a 100 fold less sphere cells result in faster tumor growth compared to the adherent population suggesting that CSCs were enriched in the sphere population. Furthermore, stem cell genes such as oct4, nanog, c-myc, pax3 and sox2 are significantly upregulated in rhabdospheres which can be differentiated into multiple lineages such as adipocytes, myocytes and neuronal cells. Surprisingly, gene expression profiles indicate that rhabdospheres show more similarities with neuronal than with hematopoietic or mesenchymal stem cells. Analysis of these profiles identified the known CSC marker CD133 as one of the genes upregulated in rhabdospheres, both on RNA and protein levels. CD133(+ sorted cells were subsequently shown to be more tumorigenic and more resistant to commonly used chemotherapeutics. Using a tissue microarray (TMA of eRMS patients, we found that high expression of CD133 correlates with poor overall survival. Hence, CD133 could be a prognostic marker for eRMS. These experiments indicate that a CD133(+ CSC population can be enriched from eRMS which might help to develop novel targeted therapies against this pediatric tumor.

  6. In vitro identification and characterization of CD133(pos cancer stem-like cells in anaplastic thyroid carcinoma cell lines.

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    Giovanni Zito

    Full Text Available BACKGROUND: Recent publications suggest that neoplastic initiation and growth are dependent on a small subset of cells, termed cancer stem cells (CSCs. Anaplastic Thyroid Carcinoma (ATC is a very aggressive solid tumor with poor prognosis, characterized by high dedifferentiation. The existence of CSCs might account for the heterogeneity of ATC lesions. CD133 has been identified as a stem cell marker for normal and cancerous tissues, although its biological function remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: ATC cell lines ARO, KAT-4, KAT-18 and FRO were analyzed for CD133 expression. Flow cytometry showed CD133(pos cells only in ARO and KAT-4 (64+/-9% and 57+/-12%, respectively. These data were confirmed by qRT-PCR and immunocytochemistry. ARO and KAT-4 were also positive for fetal marker oncofetal fibronectin and negative for thyrocyte-specific differentiating markers thyroglobulin, thyroperoxidase and sodium/iodide symporter. Sorted ARO/CD133(pos cells exhibited higher proliferation, self-renewal, colony-forming ability in comparison with ARO/CD133(neg. Furthermore, ARO/CD133(pos showed levels of thyroid transcription factor TTF-1 similar to the fetal thyroid cell line TAD-2, while the expression in ARO/CD133(neg was negligible. The expression of the stem cell marker OCT-4 detected by RT-PCR and flow cytometry was markedly higher in ARO/CD133(pos in comparison to ARO/CD133(neg cells. The stem cell markers c-KIT and THY-1 were negative. Sensitivity to chemotherapy agents was investigated, showing remarkable resistance to chemotherapy-induced apoptosis in ARO/CD133(pos when compared with ARO/CD133(neg cells. CONCLUSIONS/SIGNIFICANCE: We describe CD133(pos cells in ATC cell lines. ARO/CD133(pos cells exhibit stem cell-like features--such as high proliferation, self-renewal ability, expression of OCT-4--and are characterized by higher resistance to chemotherapy. The simultaneous positivity for thyroid specific factor TTF-1 and onfFN suggest

  7. Role of ADAM17 in invasion and migration of CD133-expressing liver cancer stem cells after irradiation

    Science.gov (United States)

    Hong, Sung Woo; Hur, Wonhee; Choi, Jung Eun; Kim, Jung-Hee; Hwang, Daehee; Yoon, Seung Kew

    2016-01-01

    We investigated the biological role of CD133-expressing liver cancer stem cells (CSCs) enriched after irradiation of Huh7 cells in cell invasion and migration. We also explored whether a disintegrin and metalloproteinase-17 (ADAM17) influences the metastatic potential of CSC-enriched hepatocellular carcinoma (HCC) cells after irradiation. A CD133-expressing Huh7 cell subpopulation showed greater resistance to sublethal irradiation and specifically enhanced cell invasion and migration capabilities. We also demonstrated that the radiation-induced MMP-2 and MMP-9 enzyme activities as well as the secretion of vascular endothelial growth factor were increased more predominantly in Huh7CD133+ cell subpopulations than Huh7CD133− cell subpopulations. Furthermore, we showed that silencing ADAM17 significantly inhibited the migration and invasiveness of enriched Huh7CD133+ cells after irradiation; moreover, Notch signaling was significantly reduced in irradiated CD133-expressing liver CSCs following stable knockdown of the ADAM17 gene. In conclusion, our findings indicate that CD133-expressing liver CSCs have considerable metastatic capabilities after irradiation of HCC cells, and their metastatic capabilities might be maintained by ADAM17. Therefore, suppression of ADAM17 shows promise for improving the efficiency of current radiotherapies and reducing the metastatic potential of liver CSCs during HCC treatment. PMID:26993601

  8. DDX4 (DEAD box polypeptide 4) colocalizes with cancer stem cell marker CD133 in ovarian cancers

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ki Hyung [Department of Obstetrics and Gynecology, Pusan National University School of Medicine, Busan (Korea, Republic of); Biomedical Research Institute and Pusan Cancer Center, Pusan National University Hospital, Busan (Korea, Republic of); Kang, Yun-Jeong; Jo, Jin-Ok; Ock, Mee Sun [Department of Parasitology and Genetics, Kosin University College of Medicine, Busan (Korea, Republic of); Moon, Soo Hyun; Suh, Dong Soo; Yoon, Man Soo [Department of Obstetrics and Gynecology, Pusan National University School of Medicine, Busan (Korea, Republic of); Biomedical Research Institute and Pusan Cancer Center, Pusan National University Hospital, Busan (Korea, Republic of); Park, Eun-Sil [Vincent Center for Reproductive Biology, Massachusetts General Hospital, Harvard Medical School, MA (United States); Jeong, Namkung [Department of Obstetrics and Gynecology, The Catholic University, Seoul (Korea, Republic of); Eo, Wan-Kyu [Department of Internal Medicine, Kyung Hee University, Seoul (Korea, Republic of); Kim, Heung Yeol, E-mail: hykyale@yahoo.com [Department of Obstetrics and Gynecology, Kosin University College of Medicine, Busan (Korea, Republic of); Cha, Hee-Jae, E-mail: hcha@kosin.ac.kr [Department of Parasitology and Genetics, Kosin University College of Medicine, Busan (Korea, Republic of); Institute for Medical Science, Kosin University College of Medicine, Busan (Korea, Republic of)

    2014-05-02

    Highlights: • Germ cell marker DDX4 was significantly increased in ovarian cancer. • Ovarian cancer stem cell marker CD133 was significantly increased in ovarian cancer. • DDX4 and CD133 were mostly colocalized in various types of ovarian cancer tissues. • CD133 positive ovarian cancer cells also express DDX4 whereas CD133-negative cells did not possess DDX4. • Germ cell marker DDX4 has the potential of ovarian cancer stem cell marker. - Abstract: DDX4 (DEAD box polypeptide 4), characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD), is an RNA helicase which is implicated in various cellular processes involving the alteration of RNA secondary structure, such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. DDX4 is known to be a germ cell-specific protein and is used as a sorting marker of germline stem cells for the production of oocytes. A recent report about DDX4 in ovarian cancer showed that DDX4 is overexpressed in epithelial ovarian cancer and disrupts a DNA damage-induced G2 checkpoint. We investigated the relationship between DDX4 and ovarian cancer stem cells by analyzing the expression patterns of DDX4 and the cancer stem cell marker CD133 in ovarian cancers via tissue microarray. Both DDX4 and CD133 were significantly increased in ovarian cancer compared to benign tumors, and showed similar patterns of expression. In addition, DDX4 and CD133 were mostly colocalized in various types of ovarian cancer tissues. Furthermore, almost all CD133 positive ovarian cancer cells also express DDX4 whereas CD133-negative cells did not possess DDX4, suggesting a strong possibility that DDX4 plays an important role in cancer stem cells, and/or can be used as an ovarian cancer stem cell marker.

  9. Enhanced invasion in vitro and the distribution patterns in vivo of CD133+ glioma stem cells

    Institute of Scientific and Technical Information of China (English)

    YU Sheng-ping; YANG Xue-jun; ZHANG Bin; MING Hao-lang; CHEN Cong; REN Bing-cheng; LIU Zhi-feng; LIU Bin

    2011-01-01

    Background Recent studies have suggested that cancer stem cells cause tumor recurrence based on their resistance to radiotherapy and chemotherapy.Although the highly invasive nature of glioblastoma cells is also implicated in the failure of current therapies,it is not clear whether cancer stem cells are involved in invasiveness.This study aimed to assess invasive ability of glioma stem cells (GSCs) derived from C6 glioma cell line and the distribution patterns of GSCs in Sprague-Dawley (SD) rat brain tumor.Methods Serum-free medium culture and magnetic isolation were used to gain purely CD133+ GSCs.The invasive stem cell markers and luxol fast blue staining for white matter tracts were performed to show the distribution patterns of GSCs in brain tumor of rats and the relationship among GSCs,vessels,and white matter tracts.The results of matrigel invasion assay were estimated using the Student's t test and the analysis of Western blotting was performed using the one-way analysis of variance (ANOVA) test.Results CD133+GSCs(number:85.3±4.1)were significantly more invasive in vitro than matched CD133- cells(number:25.9±3.1) (t=14.5,P <0.005).GSCs invaded into the brain diffusely and located in perivascular niche of tumor-brain interface or resided within perivascular niche next to white fiber tracts.The polarity of glioma cells containing GSCs was parallel to the white matter tracts.Conclusions Our data suggest that CD133+ GSCs exhibit more aggressive invasion in vitro and GSCs in vivo probably disseminate along the long axis of blood vessels and transit through the white matter tracts.The therapies targeting GSCs invasion combined with traditional glioblastoma multiforme therapeutic paradigms might be a new approach for avoiding malignant glioma recurrence.

  10. Higher percentage of CD133+ cells is associated with poor prognosis in colon carcinoma patients with stage IIIB

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    Zhang Xin

    2009-07-01

    Full Text Available Abstract Background Cancer stem cell model suggested that tumor progression is driven by the overpopulation of cancer stem cells and eradicating or inhibiting the symmetric division of cancer stem cells would become the most important therapeutic strategy. However, clinical evidence for this hypothesis is still scarce. To evaluate the overpopulation hypothesis of cancer stem cells the association of percentage of CD133+ tumor cells with clinicopathological parameters in colon cancer was investigated since CD133 is a putative cancer stem cell marker shared by multiple solid tumors. Patients and methods Tumor tissues matched with adjacent normal tissues were collected from 104 stage IIIB colon cancer patients who were subject to radical resection between January, 1999 to July, 2003 in this center. The CD133 expression was examined with immunohistochemical staining. The correlation of the percentage of CD133+ cell with clinicopathological parameters and patients' 5-year survival was analyzed. Results The CD133+ cells were infrequent and heterogeneous distribution in the cancer tissue. Staining of CD133 was localized not only on the glandular-luminal surface of cancer cells but also on the invasive budding and the poorly differentiated tumors with ductal structures. Both univariate and multivariate survival analysis revealed that the percentage of CD133+ cancer cells and the invasive depth of tumor were independently prognostic. The patients with a lower percentage of CD133+ cancer cells (less than 5% were strongly associated with a higher 5-year survival rate than those with a higher percentage of CD133+ cancer cells (greater than or equal to 55%. Additionally, no correlation was obtained between the percentage of CD133+ cancer cells and the other clinicopathological parameters including gender, age, site of primary mass, pathologic types, grades, and invasive depth. Conclusion The fact that a higher percentage CD133+ cells were strongly associated

  11. Expressions of ABCG2, CD133, and Podoplanin in Salivary Adenoid Cystic Carcinoma

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    Wuwei Li

    2014-01-01

    Full Text Available Adenoid cystic carcinoma (ACC is one of the most common salivary gland malignant tumors with a high risk of recurrence and metastasis. Current studies on cancer stem cells (CSCs have verified that CSCs are the driving force behind tumor initiation and progression, suggesting that new cancer therapies may be established by effectively targeting and killing the CSCs. The primary goal of this study is to investigate the expression patterns of ABCG2, CD133, and podoplanin in ACC of minor salivary glands by immunohistochemistry analysis. We found that ABCG2 was weakly expressed in normal looking salivary gland tissues. A significant upregulation of ABCG2 expression in ACC was observed with a similar expression pattern of Ki-67. CD133 was detected in apical membrane of epithelial cells and podoplanin was expressed positively in myoepithelial cells of both normal looking tissue and ACC. However, no significant difference was found of the expression pattern of CD133 and podoplanin between normal looking tissues and ACC. Our observations suggest that CSCs may exist in quiescent cells with ABCG2 positive staining, which are surrounded by cells with positive expression of ABCG2 and Ki-67 in ACC, and costaining with ABCG2 and Ki-67 may help predict the location of CSCs.

  12. CD133 protein N-glycosylation processing contributes to cell surface recognition of the primitive cell marker AC133 epitope.

    Science.gov (United States)

    Mak, Anthony B; Blakely, Kim M; Williams, Rashida A; Penttilä, Pier-Andrée; Shukalyuk, Andrey I; Osman, Khan T; Kasimer, Dahlia; Ketela, Troy; Moffat, Jason

    2011-11-25

    The AC133 epitope expressed on the CD133 glycoprotein has been widely used as a cell surface marker of numerous stem cell and cancer stem cell types. It has been recently proposed that posttranslational modification and regulation of CD133 may govern cell surface AC133 recognition. Therefore, we performed a large scale pooled RNA interference (RNAi) screen to identify genes involved in cell surface AC133 expression. Gene hits could be validated at a rate of 70.5% in a secondary assay using an orthogonal RNAi system, demonstrating that our primary RNAi screen served as a powerful genetic screening approach. Within the list of hits from the primary screen, genes involved in N-glycan biosynthesis were significantly enriched as determined by Ingenuity Canonical Pathway analyses. Indeed, inhibiting biosynthesis of the N-glycan precursor using the small molecule tunicamycin or inhibiting its transfer to CD133 by generating N-glycan-deficient CD133 mutants resulted in undetectable cell surface AC133. Among the screen hits involved in N-glycosylation were genes involved in complex N-glycan processing, including the poorly characterized MGAT4C, which we demonstrate to be a positive regulator of cell surface AC133 expression. Our study identifies a set of genes involved in CD133 N-glycosylation as a direct contributing factor to cell surface AC133 recognition and provides biochemical evidence for the function and structure of CD133 N-glycans.

  13. Induced growth inhibition, cell cycle arrest and apoptosis in CD133+/CD44+ prostate cancer stem cells by flavopiridol

    Science.gov (United States)

    SONER, BURAK CEM; AKTUG, HUSEYIN; ACIKGOZ, EDA; DUZAGAC, FAHRIYE; GUVEN, UMMU; AYLA, SULE; CAL, CAG; OKTEM, GULPERI

    2014-01-01

    Flavopiridol is a flavone that inhibits several cyclin-dependent kinases and exhibits potent growth-inhibitory activity, apoptosis and G1-phase arrest in a number of human tumor cell lines. Flavopiridol is currently undergoing investigation in human clinical trials. The present study focused on the effect of flavopiridol in cell proliferation, cell cycle progression and apoptosis in prostate cancer stem cells (CSCs). Therefore, cluster of differentiation 133 (CD133)+high/CD44+high prostate CSCs were isolated from the DU145 human prostate cancer cell line. The cells were treated with flavopiridol in a dose- and time-dependent manner to determine the inhibitory effect. Cell viability and proliferation were analyzed and the efficiency of flavopiridol was assessed using the sphere-forming assay. Flavopiridol was applied to monolayer cultures of CD133high/CD44high human prostate CSCs at the following final concentrations: 100, 300, 500 and 1000 nM. The cultures were incubated for 24, 48 and 72 h. The half maximal inhibitory concentration (IC50) value of the drug was determined as 500 nM for monolayer cells. Dead cells were analyzed prior and subsequent to exposure to increasing flavopiridol doses. Annexin-V and immunofluorescence analyses were performed for the evaluation of apoptotic pathways. According to the results, flavopiridol treatment caused significant growth inhibition at 500 and 1000 nM when compared to the control at 24 h. G0/G1 analysis showed a statistically significant difference between 100 and 500 nM (P<0.005), 100 and 1000 nM (P<0.001), 300 and 1000 nM (P<0.001), and 500 and 1000 nM (P<0.001). Flavopiridol also significantly influenced the cells in the G2/M phase, particularly at high-dose treatments. Flavopiridol induced growth inhibition and apoptosis at the IC50 dose (500 nM), resulting in a significant increase in immunofluorescence staining of caspase-3, caspase-8 and p53. In conclusion, the present results indicated that flavopiridol could be a

  14. Quantum dots affect expression of CD133 surface antigen in melanoma cells

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    Steponkiene S

    2011-10-01

    Full Text Available Simona Steponkiene1-3, Simona Kavaliauskiene1, Rasa Purviniene4, Ricardas Rotomskis3,5, Petras Juzenas11Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, Radiumhospital, Oslo, Norway; 2Faculty of Natural Sciences, Vilnius University, Vilnius, Lithuania; 3Biomedical Physics Laboratory of Oncology Institute, Vilnius University, Vilnius, Lithuania; 4Immunology Laboratory of Oncology Institute, Vilnius University, Vilnius, Lithuania; 5Biophotonics Laboratory, Laser Research Center, Vilnius University, Vilnius, LithuaniaBackground: In novel treatment approaches, therapeutics should be designed to target cancer stem cells (CSCs. Quantum dots (QDs are a promising new tool in fighting against cancer. However, little is known about accumulation and cytotoxicity of QDs in CSCs.Methods: Accumulation and cytotoxicity of CdTe-MPA (mercaptopropionic acid QDs in CSCs were assessed using flow cytometry and fluorescence-activated cell sorting techniques as well as a colorimetric cell viability assay.Results: We investigated the expression of two cell surface-associated glycoproteins, CD44 and CD133, in four different cancer cell lines (glioblastoma, melanoma, pancreatic, and prostate adenocarcinoma. Only the melanoma cells were positive to both markers of CD44 and CD133, whereas the other cells were only CD44-positive. The QDs accumulated to a similar extent in all subpopulations of the melanoma cells. The phenotypical response after QD treatment was compared with the response after ionizing radiation treatment. The percentage of the CD44high-CD133high subpopulation decreased from 72% to 55%–58% for both treatments. The stem-like subpopulation CD44highCD133low/- increased from 26%–28% in the untreated melanoma cells to 36%–40% for both treatments.Conclusion: Treatment of melanoma cells with QDs results in an increase of stem-like cell subpopulations. The changes in phenotype distribution of the melanoma cells after

  15. 肿瘤干细胞表面标志物CD133单链抗体基因的分离与鉴定%Identification and characterization of single chain antibody of cancer stem cell surface marker CD133 scFv CD133

    Institute of Scientific and Technical Information of China (English)

    韩瑜; 周炳喜; 张延瑞; 韩双印

    2011-01-01

    Objective To identify,construct and express scFv CD133,verify its biological function.Methods VL and VH were isolated from hybridoma of mAb CD133 by using antibody engineering technology.Its DNA sequencing and CDR were determined.scFv CD133 was then cloned into pET32a,transformed into Origami,induced by IPTG,purified by Ni2+-NTA His resin.Its affinity and specificity were tested by NH4SCN elution and ELISA.Results The size of VL and VH of scFv CD133 was 339 bp and 342 bp,which coded 113 and 114 amino acid separately.Its VL belonged to mouse Igκ chain and VH belonged to mouse IgG heavy chain subtype I.The molecular weight of scFv CD133 was about 27 × 103 which was testified by SDSPAGE and Western blot.Its affinity and specificity were also verified.Conclusion scFv CD133 has been successfully constructed and expressed in Origami,which could supply basis for target therapy of CD+133 cancer stem cell.%目的 分离、构建和表达抗人CD133单链抗体(scFvCD133),测定其生物学活性.方法 用抗体工程技术从抗人CD133单克隆抗体(mAb)杂交瘤细胞中分离抗体可变区基因(VL和VH),测定DNA序列并确定抗体互补决定区(CDR);将scFv CD133基因克隆至pET32a中,转化Origami菌株,IPTG诱导,Ni2+-NTA His树脂纯化单链抗体,梯度硫氰酸盐洗脱法和酶联免疫吸附(ELISA)法检测其亲和性和特异性.结果 scFvCD133基因VL和VH长度分别为339 bp和342 bp,各编码113和114个氨基酸,其中VL隶属于小鼠Ig κ轻链,VH隶属于小鼠Ig重链Ⅰ亚类.scFvCD133经SDS-PAGE和Western blot 分析证明相对分子质量为27 × 103,体外实验具有一定的亲和性和特异性.结论 获得scFv CD133基因,为CD+133肿瘤干细胞的靶向治疗奠定了基础.

  16. 磁珠细胞分选CD133+/CD44+前列腺癌干细胞的初步鉴定%Initial identification of CD133+/CD44+ prostate cancer stem cell through magnetic bead cell sorting

    Institute of Scientific and Technical Information of China (English)

    盛夏; 王德林; 李文宾; 罗照

    2013-01-01

    目的:通过磁珠细胞分选(magnetic bead cell sorting,MACS)方法从人前列腺癌细胞系PC-3中分选CD133+/CD44+干细胞,为进一步功能性研究奠定基础.方法:运用流式细胞仪(flow cytometry,FCM)检测MACS分选前后PC-3细胞膜上CD133和CD44表达情况;观察无血清培养成球情况,免疫荧光(immunofluorescenee,IF)表达情况;比较细胞在分选前后的形态学、增殖能力方面的差异;免疫组化(immunohistochemistry,IHC)和Western blot检测诱导分化前后前列腺酸性磷酸酶(prostatic acid phosphatase,PAP)蛋白情况.结果:FCM检测PC-3细胞CD133和CD44的阳性表达分别是(1.33±0.05)%和(0.87±0.06)%,而MACS分选后PAP分别为(84.82±0.07)%和(99.91±0.03)%;IF检测CD133+/CD44+细胞培养后继续呈阳性表达;CD133+/CD44+细胞增殖能力高于PC-3细胞(t=11.0,P=0.008)以及高于诱导后的CD133+/CD44+细胞(t=40.1,P=0.001);CD133+/CD44+细胞经过转化生长因子-β诱导后IHC和Western blot检测PAP表达呈阳性,而未诱导的CD133+/CD44+细胞表达呈阴性.结论:MACS从PC-3细胞株中分选的CD 133+/CD44+细胞经过初步功能性鉴定具有干细胞的某些特性,可为前列腺癌干细胞的进一步探索充当铺垫.

  17. Expression status of CD44 and CD133 as a prognostic marker in esophageal squamous cell carcinoma treated with neoadjuvant chemotherapy followed by radical esophagectomy.

    Science.gov (United States)

    Okamoto, Koichi; Ninomiya, Itasu; Ohbatake, Yoshinao; Hirose, Atsushi; Tsukada, Tomoya; Nakanuma, Shinichi; Sakai, Seisho; Kinoshita, Jun; Makino, Isamu; Nakamura, Keishi; Hayashi, Hironori; Oyama, Katsunobu; Inokuchi, Masafumi; Nakagawara, Hisatoshi; Miyashita, Tomoharu; Hidehiro, Tajima; Takamura, Hiroyuki; Fushida, Sachio; Ohta, Tetsuo

    2016-12-01

    Cancer stem cells (CSCs) have self-renewal and pluripotency capabilities and contribute to cancer progression and chemoresistance. It has been proposed that the treatment resistance and heterogeneity of CSCs are deeply involved in the prognosis of patients with esophageal squamous cell carcinoma (ESCC). The objective of this study was to identify the influence of the expression status of the CSC markers CD44 and CD133 on chemotherapeutic efficacy and prognosis in ESCC patients who underwent radical esophagectomy after neoadjuvant chemotherapy (NAC). Endoscopically biopsied specimens taken before NAC and surgically resected specimens after NAC were immunohistochemically assessed for CD44 and CD133 expression for 47 ESCC patients who underwent NAC followed by radical esophagectomy. The correlation between CD44 and CD133 expression status and clinicopathological findings and the prognosis of ESCC patients after NAC followed by esophagectomy were analyzed. The percentages of CD44-positive cells and CD133-positive cells in specimens were increased after NAC. CD44 and CD133 expression status before NAC did not correlate with the degree of tumor progression and had no impact on the chemotherapeutic effect. However, strong expression of CD44 or CD133 and a high proportion of CD133-expressing cells before NAC were significantly associated with poorer esophageal cancer-specific survival. Patients with strong expression of CD44 or CD133 and those with a high ratio of CD133-positive tumor cells showed significantly poor prognosis regardless of the effect of chemotherapy. Multivariate analysis showed that simultaneous strong expression of CD44 and CD133 before NAC, a high rate of CD133-positive tumor cells before NAC, and primary tumor remission assessed by preoperative endoscopy were significant independent prognostic factors for ESCC. Our data indicate that CD44 and CD133 expression status prior to treatment dictates the malignant potential of ESCC and may be a novel

  18. Biology and clinical implications of CD133{sup +} liver cancer stem cells

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    Ma, Stephanie, E-mail: stefma@hku.hk [Department of Clinical Oncology, State Key Laboratory for Liver Research, LKS Faculty of Medicine, The University of Hong Kong (Hong Kong)

    2013-01-15

    Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver, accounting for 80%–90% of all liver cancers. The disease ranks as the fifth most common cancer worldwide and is the third leading cause of all cancer-associated deaths. Although advances in HCC detection and treatment have increased the likelihood of a cure at early stages of the disease, HCC remains largely incurable because of late presentation and tumor recurrence. Only 25% of HCC patients are deemed suitable for curative treatment, with the overall survival at just a few months for inoperable patients. Apart from surgical resection, loco-regional ablation and liver transplantation, current treatment protocols include conventional cytotoxic chemotherapy. But due to the highly resistant nature of the disease, the efficacy of the latter regimen is limited. The recent emergence of the cancer stem cell (CSC) concept lends insight into the explanation of why treatment with chemotherapy often may seem to be initially successful but results in not only a failure to eradicate the tumor but also possibly tumor relapse. Commonly used anti-cancer drugs in HCC work by targeting the rapidly proliferating and differentiated liver cancer cells that constitute the bulk of the tumor. However, a subset of CSCs exists within the tumor, which are more resistant and are able to survive and maintain residence after treatment, thus, growing and self-renewing to generate the development and spread of recurrent tumors in HCC. In the past few years, compelling evidence has emerged in support of the hierarchic CSC model for solid tumors, including HCC. And in particular, CD133 has drawn significant attention as a critical liver CSC marker. Understanding the characteristics and function of CD133{sup +} liver CSCs has also shed light on HCC management and treatment, including the implications for prognosis, prediction and treatment resistance. In this review, a detailed summary of the recent progress

  19. Effects of temozolomide chemotherapy on CD133+ cells in glioma U251%替莫唑胺化疗对胶质瘤U251中CD133+细胞的影响

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    杨伟现; 向定朝; 王存祖; 周月鹏; 欧阳琦; 张志坚; 许慧中; 陆晓峰

    2012-01-01

    目的 探讨化疗耐药与肿瘤干细胞之间的关系.方法 分析替莫唑胺(TMZ)作用前后胶质瘤U251中CD133蛋白表达及阳性细胞数量的变化.通过MTT实验评估U251细胞对TMZ的敏感性.利用免疫荧光染色和Western blot检测TMZ作用前后U251细胞中CD133、巢蛋白(nestin)、神经元特异性烯醇化酶(NSE)、神经胶质细胞纤维酸性蛋白(GFAP)等蛋白表达.结果 U251细胞中未加入TMZ组的CD133+细胞比例约为0.060±0.003,明显低于加入TMZ组的0.337±0.012(P<0.05).在TMZ作用后,CD133和nestin表达明显增加,而GFAP和NSE表达明显减少(P<0.05).结论 胶质瘤U251细胞株中存在CD133+细胞,大部分具有脑肿瘤干细胞特性;在TMZ作用后,这类细胞比例增加,提示其与化疗耐药有关.%Objective To explore the relationship between chemotherapy resistance and cancer stem cells. Methods The changes of the number and protein expression were analyzed before and after the CD133+ cells in glioma U251 were intervented by temozolomide(TMZ). The expressions of CD133,nestin,neuron-specific enolase(NSE),glial fibrillary acidic protein(GFAP) and other proteins in glioma U251 cells were detected by immunofluorescence and Western blot. Results U251 cells in the TMZ group did not join The proportion of CD133+ cells was significantly greater in the U251 cells with TMZ intervention than that without(0. 337±0.012 vs. 0. 060 + 0.003) (P<0. 05). After TMZ intervention, the proportion of nestin-positive cells in the CD133+ cells were increased,but GFAP and NSE-positive cells were decreased(P<0. 05). Conclusion There CD133+cells in the U251 glioma cell lines,majority of which have the properties of brain tumor stem cells. After TMZ intervention, an increased proportion of the CD133+ cells suggests its relationship with chemotherapy-related drug resistance.

  20. Prominin-1 (CD133 defines both stem and non-stem cell populations in CNS development and gliomas.

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    Karl Holmberg Olausson

    Full Text Available Prominin-1 (CD133 is a commonly used cancer stem cell marker in central nervous system (CNS tumors including glioblastoma (GBM. Expression of Prom1 in cancer is thought to parallel expression and function in normal stem cells. Using RNA in situ hybridization and antibody tools capable of detecting multiple isoforms of Prom1, we find evidence for two distinct Prom1 cell populations in mouse brain. Prom1 RNA is first expressed in stem/progenitor cells of the ventricular zone in embryonic brain. Conversely, in adult mouse brain Prom1 RNA is low in SVZ/SGZ stem cell zones but high in a rare but widely distributed cell population (Prom1(hi. Lineage marker analysis reveals Prom1(hi cells are Olig2+Sox2+ glia but Olig1/2 knockout mice lacking oligodendroglia retain Prom1(hi cells. Bromodeoxyuridine labeling identifies Prom1(hi as slow-dividing distributed progenitors distinct from NG2+Olig2+ oligodendrocyte progenitors. In adult human brain, PROM1 cells are rarely positive for OLIG2, but express astroglial markers GFAP and SOX2. Variability of PROM1 expression levels in human GBM and patient-derived xenografts (PDX - from no expression to strong, uniform expression--highlights that PROM1 may not always be associated with or restricted to cancer stem cells. TCGA and PDX data show that high expression of PROM1 correlates with poor overall survival. Within proneural subclass tumors, high PROM1 expression correlates inversely with IDH1 (R132H mutation. These findings support PROM1 as a tumor cell-intrinsic marker related to GBM survival, independent of its stem cell properties, and highlight potentially divergent roles for this protein in normal mouse and human glia.

  1. Specific detection of CD133-positive tumor cells with iron oxide nanoparticles labeling using noninvasive molecular magnetic resonance imaging

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    Chen YW

    2015-11-01

    Full Text Available Ya-Wen Chen,1,2 Gunn-Guang Liou,3 Huay-Ben Pan,4,5 Hui-Hwa Tseng,5,6 Yu-Ting Hung,4 Chen-Pin Chou4,5,7,8 1National Institute of Cancer Research, National Health Research Institutes, Miaoli, 2Graduate Institute of Basic Medical Science, China Medical University, Taichung, 3Institute of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli, 4Department of Radiology, Kaohsiung Veterans General Hospital, Kaohsiung, 5School of Medicine, National Yang-Ming University, Taipei, 6Department of Pathology, Kaohsiung Veterans General Hospital, Kaohsiung, 7Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, 8School of Medicine, National Defense Medical Center, Taipei, Taiwan Background: The use of ultrasmall superparamagnetic iron oxide (USPIO nanoparticles to visualize cells has been applied clinically, showing the potential for monitoring cells in vivo with magnetic resonance imaging (MRI. USPIO conjugated with anti-CD133 antibodies (USPIO-CD133 Ab that recognize the CD133 molecule, a cancer stem cell marker in a variety of cancers, was studied as a novel and potent agent for MRI contrast enhancement of tumor cells. Materials and methods: Anti-CD133 antibodies were used to conjugate with USPIO via interaction of streptavidin and biotin for in vivo labeling of CD133-positive cells in xenografted tumors and N-ethyl-N-nitrosourea (ENU-induced brain tumors. The specific binding of USPIO-CD133 Ab to CD133-positive tumor cells was subsequently detected by Prussian blue staining and MRI with T2-weighted, gradient echo and multiple echo recombined gradient echo images. In addition, the cellular toxicity of USPIO-CD133 Ab was determined by analyzing cell proliferation, apoptosis, and reactive oxygen species production. Results: USPIO-CD133 Ab specifically recognizes in vitro and labels CD133-positive cells, as validated using Prussian blue staining and MRI. The assays of cell proliferation, apoptosis, and

  2. Cancer stem cell markers CD133 and CD24 correlate with invasiveness and differentiation in colorectal adenocarcinoma

    Institute of Scientific and Technical Information of China (English)

    Dongho Choi; Hyo Won Lee; Kyung Yul Hur; Jae Joon Kim; Gyeong-Sin Park; Si-Hyong Jang; Young Soo Song; Ki-Seok Jang; Seung Sam Paik

    2009-01-01

    AIM: To verify that CD markers are available for detecting cancer stem cell populations and to evaluate their clinical significance in colon cancer. METHODS: Immunohistochemistry for CD133, CD24 and CD44 was performed on the tissue microarray of 523 colorectal adenocarcinomas. Medical records were reviewed and clinicopathological analysis was performed. RESULTS: In colorectal adenocarcinoma, 128 of 523 cases (24.5%) were positive and 395 cases (75.5%) were negative for CD133 expression. Two hundred and sixty-four of 523 cases (50.5%) were positive and 259 cases (49.5%) were negative for CD24 expression. Five hundred and two of 523 cases (96%) were negative and 21 cases (4%) were positive for CD44 expression. Upon clinicopathological analysis, CD133 expression as present more in male patients ( P = 0.002) and in advanced T stage cancer ( P = 0.024). Correlation between CD24 expression and clinicopathological factors was seen in the degree of differentiation ( P = 0.006). Correlation between CD44 expression and clinicopathological factors was seen in the tumor size ( P = 0.001). Survival was not significantly related to CD133, CD24 and CD44 expression. CONCLUSION: CD marker s were related t o invasiveness and differentiation of colorectal adenocarcinoma. However, CD expression was not closely related to survival.

  3. Identification and analysis of CD133(+) melanoma stem-like cells conferring resistance to taxol: An insight into the mechanisms of their resistance and response.

    Science.gov (United States)

    El-Khattouti, Abdelouahid; Selimovic, Denis; Haïkel, Youssef; Megahed, Mosaad; Gomez, Christian R; Hassan, Mohamed

    2014-02-01

    The presence and the involvement of cancer stem-like cells (CSCs) in tumor initiation and progression, and chemo-resistance are documented. Herein, we functionally analyzed melanoma stem-like cells (MSC)/CD133(+) cells on their resistance and response to taxol-induced apoptosis. Besides being taxol resistant, the CD133(+) cells demonstrated a growth advantage over the CD133(-) subpopulation. Taxol induced apoptosis on CD133(-) cells, but not on CD133(+) cells. In the CD133(-) subpopulation, the exposure to taxol induced the activation of apoptosis signal-regulating kinase1 (ASK1)/c-jun-N-terminal kinase (JNK), p38, extracellular signal regulated kinase (ERK) pathways and Bax expression, while in CD133(+) cells taxol was able only to enhance the activity of the ERK pathway. In CD133(+) cells, the direct gene transfer of Bax overcame the acquired resistance to taxol. Taken together, our data provide an insight into the mechanistic cascade of melanoma resistance to taxol and suggest Bax gene transfer as a complementary approach to chemotherapy.

  4. Decreased Number of Circulating Endothelial Progenitor Cells (CD133+/KDR+) in Patients with Psoriatic Arthritis.

    Science.gov (United States)

    Batycka-Baran, Aleksandra; Paprocka, Maria; Baran, Wojciech; Szepietowski, Jacek C

    2016-08-23

    Cardiovascular diseases are a major cause of mortality in patients with psoriatic arthritis (PsA), but the precise mechanism of increased cardiovascular risk is unknown. Endothelial dysfunction plays a crucial role in the development of atherosclerosis. Circulating endothelial progenitor cells (CEPCs) contribute to endothelial regeneration and their level may be affected by chronic inflammation. The aim of this study was to evaluate the number of CEPCs in patients with PsA (n = 24) compared with controls (n = 26). CEPCs were identified as CD133+/ KDR+ cells in peripheral blood, using flow cytometry. A significantly decreased number of CEPCs was observed in patients with PsA (p number of these cells was significantly, inversely correlated with the severity of skin and joint involvement (Psoriasis Area and Severity Index (PASI), DAS28) and the level of C-reactive protein. We hypothesize that the reduced number of CEPCs may indicate and contribute to the increased cardiovascular risk in patients with PsA.

  5. Biological Characteristics of CD133+CD44+ Cancer Stem Cells Sorting from Laryngeal Carcinoma Cell Line TU177%喉癌TU177细胞系中CD133+CD44+肿瘤干细胞分选及特性分析

    Institute of Scientific and Technical Information of China (English)

    杨俊岭; 高伟; 王珏; 付荣; 陈波; 李伟艳; 温树信; 王斌全

    2016-01-01

    Objective :Magnetic activated cell sorting was used to separate CD133+CD44+ cancer cells from laryngeal car-cinoma TU177 cell line. Analysis the biological characteristics of these subpopulations .Methods :TU177 cells were subjected to magnetic activated cell sorting to obtain CD133+CD44+、CD133+CD44-、CD133-CD44+、CD133-CD44-cells. Evaluate the efficiency of magnetic separation by flow cytometry . Test cell proliferation,migration,invasion,adhesion,colony forming ability of the cells.Results: CD133+CD44+ cells show higher proliferation,migration,invasion,adhesion,clone ability than other group(P<0.0001).Conclusions:TU177 cells can be serparated by Magnetic activated cell sorting effectively. CD133 is more powerful than CD44.Our study may provide evidence for target treatment of laryngeal cancer.%目的:免疫磁珠分选喉癌TU177细胞系中的CD133+CD44+细胞,探讨CD133+CD44+细胞作为肿瘤干细胞的生物学特性。方法:培养喉癌TU177细胞,采用免疫磁珠分选技术分选CD133+CD44+、CD133+CD44-、CD133-CD44+、CD133-CD44-细胞,流式检测分选效率,检测各组细胞的增殖、侵袭、迁移、粘附、克隆形成能力。结果:CD133+CD44+细胞的增殖、迁移、侵袭、粘附、克隆能力均明显高于其他组,差异有统计学意义(P<0.0001)。结论:免疫磁珠技术能有效进行TU177细胞系的分选,CD133+CD44+细胞亚群具有强增殖、侵袭、迁移、粘附、克隆形成能力,具有肿瘤干细胞特征,CD133作为干细胞标志物,其干细胞特性强于CD44,可为喉癌的进一步靶向治疗提供依据。

  6. Activation of the sonic hedgehog signaling pathway occurs in the CD133 positive cells of mouse liver cancer Hepa 1–6 cells

    Directory of Open Access Journals (Sweden)

    Jeng KS

    2013-08-01

    Full Text Available Kuo-Shyang Jeng,1 I-Shyan Sheen,2 Wen-Juei Jeng,2 Ming-Che Yu,3 Hsin-I Hsiau,3 Fang-Yu Chang,3 Hsin-Hua Tsai31Department of Surgery, Far Eastern Memorial Hospital, Taipei, 2Department of Hepato-Gastroenterology, Chang Gung Memorial Hospital, Linkou Medical Center, Chang Gung University, 3Department of Medical Research, Far Eastern Memorial Hospital, Taipei, Taiwan, Republic of ChinaBackground: The important role of cancer stem cells in carcinogenesis has been emphasized in research. CD133+ cells have been mentioned as liver cancer stem cells in hepatocellular carcinoma (HCC. Some researchers have proposed that the sonic hedgehog (Shh pathway contributes to hepatocarcinogenesis and that the pathway activation occurs mainly in cancer stem cells. We investigated whether the activation of the Shh pathway occurs in CD133+ cells from liver cancer.Materials and methods: We used magnetic sorting to isolate CD133+ cells from mouse cancer Hepa 1–6 cells. To examine the clonogenicity, cell culture and soft agar colony formation assay were performed between CD133+ and CD133- cells. To study the activation of the Shh pathway, we examined the mRNA expressions of Shh, patched homolog 1 (Ptch-1, glioma-associated oncogene homolog 1 (Gli-1, and smoothened homolog (Smoh by real-time polymerase chain reaction of both CD133+ and CD133- cells.Results: The number (mean ± standard deviation of colonies of CD133+ cells and CD133- cells was 1,031.0 ± 104.7 and 119.7 ± 17.6 respectively. This difference was statistically significant (P < 0.001. Their clonogenicity was 13.7% ± 1.4% and 1.6% ± 0.2% respectively with a statistically significant difference found (P < 0.001. CD133+ cells and CD133– cells were found to have statistically significant differences in Shh mRNA and Smoh mRNA (P = 0.005 and P = 0.043 respectively.Conclusion: CD133+ Hepa 1–6 cells have a significantly higher colony proliferation and clonogenicity. The Shh pathway is activated in these

  7. Ultrasound-targeted microbubble destruction-mediated downregulation of CD133 inhibits epithelial-mesenchymal transition, stemness and migratory ability of liver cancer stem cells.

    Science.gov (United States)

    Liu, Yan-Min; Li, Xuan-Fei; Liu, Hao; Wu, Xiao-Ling

    2015-12-01

    Hepatocellular carcinoma (HCC) is an aggressive disease with a poor outcome due to the high incidence of metastasis. Cancer stem cells (CSCs) have been identified to be responsible for tumor progression and may be generated by epithelial-mesenchymal transition (EMT) characteristics. CD133 is a specific surface marker for liver cancer stem cells (LCSCs), which is also considered as an important functional factor for tumorigenesis and overall survival in HCC. Ultrasound-targeted microbubble destruction (UTMD) has recently been used as a novel, safe and effective gene transfection technology. The aim of the present study was to elucidate the regulatory mechanism of CD133 and EMT in LCSCs and whether the UTMD-based shRNA delivery system facilitated gene delivery in LCSCs. In the present study, CD133+ cells were isolated from the SMMC-7721 HCC cell line and then transfected with shCD133 mediated by UTMD and liposomes, respectively. Compared to the liposomes group, the UTMD group resulted in significantly improved transfection efficiency. The downregulation of CD133 reversed the EMT program, attenuated self-renewal, proliferation and migration of CD133+ LCSCs and suppressed the growth of CSC tumor xenografts. Additionally, the downregulation of CD133 led to downregulation of the nuclear factor-κB (NF-κB) pathway. The present study demonstrated that CD133 plays a critical role in the regulation of the EMT process, tumor-initiating properties and migratory ability of LCSCs. The UTMD technique targeted for CD133 downregulation may be examined as a potential therapeutic strategy for HCC.

  8. A cross sectional study of p504s, CD133, and Twist expression in the esophageal metaplasia dysplasia adenocarcinoma sequence.

    Science.gov (United States)

    Ahmad, J; Arthur, K; Maxwell, P; Kennedy, A; Johnston, B T; Murray, L; McManus, D T

    2015-04-01

    The incidence of esophageal adenocarcinoma has increased dramatically over recent years and Barrett's esophagus is considered the most established risk factor for its development. Endoscopic surveillance of Barrett's esophagus is therefore recommended but hinges on histological interpretation of randomly taken biopsies which is poorly reproducible. The use of biomarkers presents an opportunity to improve our ability to risk-stratify these patients.We examined three biomarkers namely p504s, CD133, and Twist in the setting of Barrett's esophagus, low-grade dysplasia, and esophageal adenocarcinoma to evaluate differential expression between benign, dysplastic, and malignant Barrett's tissue in an exploratory cross-sectional study. Twenty-five cases each of Barrett's esophagus, low-grade dysplasia, and esophageal adenocarcinoma were included along-with 25 cases of esophagectomy resections for Barrett's adenocarcinoma. The biomarkers were immunostained on automated Ventana(®) immunostainer. The biopsies were assessed for biomarker expression by two independent observers. Granular cytoplasmic staining of p504s was observed in dysplastic Barrett's biopsies and esophageal adenocarcinoma but not in Barrett's esophagus. Apical and membranous CD133 expression was also observed in dysplastic Barrett's and esophageal adenocarcinoma. Nuclear Twist expression was seen predominantly in stromal cells. There was increased p504s expression in dysplastic Barrett's esophagus and esophageal adenocarcinoma compared with controls. CD133 expression was detected for the first time in esophageal adenocarcinoma and dysplastic Barrett's esophagus. Twist expression was not convincing enough to be labeled as Barrett's biomarker. p504s and CD133 have the potential to differentiate benign from malignant Barrett's tissue in this exploratory study. Their validity should be established in prospective longitudinal studies.

  9. Copper-64-diacetyl-bis (N{sup 4}-methylthiosemicarbazone) accumulates in rich regions of CD133{sup +} highly tumorigenic cells in mouse colon carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Yoshii, Yukie [Biomedical Imaging Research Center, University of Fukui, Eiheiji, Fukui 910-1193 (Japan); Furukawa, Takako [Molecular Imaging Center, National Institute of Radiological Sciences, Inage, Chiba 263-8555 (Japan); Kiyono, Yasushi [Biomedical Imaging Research Center, University of Fukui, Eiheiji, Fukui 910-1193 (Japan); Watanabe, Ryo [Faculty of Engineering, University of Fukui, Eiheiji, Fukui 910-8507 (Japan); Waki, Atsuo [Biomedical Imaging Research Center, University of Fukui, Eiheiji, Fukui 910-1193 (Japan); Mori, Tetsuya [Biomedical Imaging Research Center, University of Fukui, Eiheiji, Fukui 910-1193 (Japan); Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, Missouri 63110 (United States); Yoshii, Hiroshi; Oh, Myungmi [Faculty of Medical Sciences, University of Fukui, Eiheiji, Fukui 910-1193 (Japan); Asai, Tatsuya [Biomedical Imaging Research Center, University of Fukui, Eiheiji, Fukui 910-1193 (Japan); Faculty of Engineering, University of Fukui, Eiheiji, Fukui 910-8507 (Japan); Okazawa, Hidehiko [Biomedical Imaging Research Center, University of Fukui, Eiheiji, Fukui 910-1193 (Japan); Welch, Michael J. [Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, Missouri 63110 (United States); Fujibayashi, Yasuhisa [Biomedical Imaging Research Center, University of Fukui, Eiheiji, Fukui 910-1193 (Japan); Molecular Imaging Center, National Institute of Radiological Sciences, Inage, Chiba 263-8555 (Japan)], E-mail: yfuji@u-fukui.ac.jp

    2010-05-15

    Introduction: {sup 64}Cu-diacetyl-bis (N{sup 4}-methylthiosemicarbazone) ({sup 64}Cu-ATSM) is a potential imaging agent of hypoxic tumor for use with PET. Recent literature demonstrated that cancer cells expressing CD133, which is a frequently used marker for so-called cancer stem cells or cancer stem cell-like cells (collectively referred to here as CSCs), contribute to tumor's therapeutic resistance and metastasis ability. Culturing under hypoxia is also reported to enlarge the proportion of CD133{sup +} cells, which would indicate survival advantage of CD133{sup +} cells under hypoxia. Here, we investigated the relationships between {sup 64}Cu-ATSM accumulation and existence of CD133{sup +} cells using mouse colon carcinoma (colon-26) tumor. Methods: Intratumor distribution of {sup 64}Cu-ATSM and {sup 18}F-fluorodeoxyglucose ({sup 18}FDG) was compared with immunohistochemical staining for CD133 with a colon-26 model. In vitro characterization of CD133{sup +} colon-26 cells was also performed. Results: In colon-26 tumors, {sup 64}Cu-ATSM localized preferentially in regions with a high density of CD133{sup +} cells. The percentage of CD133{sup +} cells was 11-fold higher in {sup 64}Cu-ATSM high-uptake regions compared with {sup 18}FDG high- (but {sup 64}Cu-ATSM low-) uptake regions. CD133{sup +} colon-26 cells showed characteristics previously linked with CSCs in other cancer cell lines, such as high colony-forming ability, high tumor-initiating ability and enrichment under hypoxic cultivation. The proportion of CD133{sup +} cells was enlarged by culturing under glucose starvation as well as hypoxia, and {sup 64}Cu-ATSM uptake was increased under such conditions. Conclusions: Our findings showed that, in colon-26 tumors, {sup 64}Cu-ATSM accumulates in rich regions of CD133{sup +} cells with characteristics of CSCs. Therefore {sup 64}Cu-ATSM could be a potential imaging agent for rich regions of CD133{sup +} cells, associated with CSCs, within tumors.

  10. Distinct and conserved prominin-1/CD133-positive retinal cell populations identified across species.

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    József Jászai

    Full Text Available Besides being a marker of various somatic stem cells in mammals, prominin-1 (CD133 plays a role in maintaining the photoreceptor integrity since mutations in the PROM1 gene are linked with retinal degeneration. In spite of that, little information is available regarding its distribution in eyes of non-mammalian vertebrates endowed with high regenerative abilities. To address this subject, prominin-1 cognates were isolated from axolotl, zebrafish and chicken, and their retinal compartmentalization was investigated and compared to that of their mammalian orthologue. Interestingly, prominin-1 transcripts--except for the axolotl--were not strictly restricted to the outer nuclear layer (i.e., photoreceptor cells, but they also marked distinct subdivisions of the inner nuclear layer (INL. In zebrafish, where the prominin-1 gene is duplicated (i.e., prominin-1a and prominin-1b, a differential expression was noted for both paralogues within the INL being localized either to its vitreal or scleral subdivision, respectively. Interestingly, expression of prominin-1a within the former domain coincided with Pax-6-positive cells that are known to act as progenitors upon injury-induced retino-neurogenesis. A similar, but minute population of prominin-1-positive cells located at the vitreal side of the INL was also detected in developing and adult mice. In chicken, however, prominin-1-positive cells appeared to be aligned along the scleral side of the INL reminiscent of zebrafish prominin-1b. Taken together our data indicate that in addition to conserved expression of prominin-1 in photoreceptors, significant prominin-1-expressing non-photoreceptor retinal cell populations are present in the vertebrate eye that might represent potential sources of stem/progenitor cells for regenerative therapies.

  11. Spatial distribution of prominin-1 (CD133-positive cells within germinative zones of the vertebrate brain.

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    József Jászai

    Full Text Available BACKGROUND: In mammals, embryonic neural progenitors as well as adult neural stem cells can be prospectively isolated based on the cell surface expression of prominin-1 (CD133, a plasma membrane glycoprotein. In contrast, characterization of neural progenitors in non-mammalian vertebrates endowed with significant constitutive neurogenesis and inherent self-repair ability is hampered by the lack of suitable cell surface markers. Here, we have investigated whether prominin-1-orthologues of the major non-mammalian vertebrate model organisms show any degree of conservation as for their association with neurogenic geminative zones within the central nervous system (CNS as they do in mammals or associated with activated neural progenitors during provoked neurogenesis in the regenerating CNS. METHODS: We have recently identified prominin-1 orthologues from zebrafish, axolotl and chicken. The spatial distribution of prominin-1-positive cells--in comparison to those of mice--was mapped in the intact brain in these organisms by non-radioactive in situ hybridization combined with detection of proliferating neural progenitors, marked either by proliferating cell nuclear antigen or 5-bromo-deoxyuridine. Furthermore, distribution of prominin-1 transcripts was investigated in the regenerating spinal cord of injured axolotl. RESULTS: Remarkably, a conserved association of prominin-1 with germinative zones of the CNS was uncovered as manifested in a significant co-localization with cell proliferation markers during normal constitutive neurogenesis in all species investigated. Moreover, an enhanced expression of prominin-1 became evident associated with provoked, compensatory neurogenesis during the epimorphic regeneration of the axolotl spinal cord. Interestingly, significant prominin-1-expressing cell populations were also detected at distinct extraventricular (parenchymal locations in the CNS of all vertebrate species being suggestive of further, non

  12. CD133 selected stem cells from proliferating infantile hemangioma and establishment of an in vivo mice model of hemangioma

    Institute of Scientific and Technical Information of China (English)

    MAI Hua-ming; ZHENG Jia-wei; WANG Yan-an; YANG Xiu-juan; ZHOU Qin; QIN Zhong-ping; LI Ke-lei

    2013-01-01

    Background Infantile hemangioma (IH) is the most common benign tumor in children with prevalence in the face and neck.Various treatment options including oral propranolol have been described for IH,but the mechanism of drugs remains enigmatic.The aim of this study was to investigate the pathogenesis and establish a reliable in vivo model of IH which can provide platform for drug exploration.Methods Stem cells from the proliferating hemangiomas (HemSCs) were isolated by CD133-tagged immunomagnetic beads.Their phenotype and angiogenic property were investigated by flow cytometry,culturing on Matrigel,real-time polymerase chain reaction (PCR),immunofluorescent staining and injection into BALB/c-nu mice.Results HemSCs had robust ability of proliferating and cloning.The time of cells doubling in proliferative phase was 16 hours.Flow cytometry showed that HemSCs expressed mesenchymal markers CD29,CD44,but not endothelial/hematopoietic marker of CD34 and hematopoietic marker CD45.The expression of CD105 was much lower than that of the reported hemangioma derived or normal mesenchymal stem cell (MSC).Real-time PCR showed that the mRNA levels of vascular endothelial growth factor (VEGF),basic fibroblast growth factor (bFGF) and matrix metalloproteinase-1 (MMP-1) of HemSCs were higher than that of neonatal human dermal fibroblasts (NHDFs) and human umbilical vein endothelial cells (HUVECs).After HemSCs were cultured on Matrigel in vitro,they formed tube-like structure in a short time (16 hours) and differentiated into endothelial cells in 7 days.After 1-2 weeks of implantation into immunodeficient mice,HemSCs generated glucose transporter 1 positive blood vessels.When co-injected with HUVECs,the vascularization of HemSCs was greatly enhanced.However,the single implantation of HUVECs hardly formed blood vessels in BALB/c-nu mice (P <0.05).Conclusions HemSCs may be some kinds of primitive mesoderm derived stem cells with powerful angiogenic ability,which can recapitulate

  13. Self-renewal of CD133hi cells by IL6/Notch3 signalling regulates endocrine resistance in metastatic breast cancer

    OpenAIRE

    2016-01-01

    The mechanisms of metastatic progression from hormonal therapy (HT) are largely unknown in luminal breast cancer. Here we demonstrate the enrichment of CD133hi/ERlo cancer cells in clinical specimens following neoadjuvant endocrine therapy and in HT refractory metastatic disease. We develop experimental models of metastatic luminal breast cancer and demonstrate that HT can promote the generation of HT-resistant, self-renewing CD133hi/ERlo/IL6hi cancer stem cells (CSCs). HT initially abrogates...

  14. MGMT promoter methylation status and MGMT and CD133 immunohistochemical expression as prognostic markers in glioblastoma patients treated with temozolomide plus radiotherapy

    OpenAIRE

    Melguizo Consolación; Prados Jose; González Beatriz; Ortiz Raul; Concha Angel; Alvarez Pablo; Madeddu Roberto; Perazzoli Gloria; Oliver Jaime; López Rodrigo; Rodríguez-Serrano Fernando; Aránega Antonia

    2012-01-01

    Abstract Background The CD133 antigen is a marker of radio- and chemo-resistant stem cell populations in glioblastoma (GBM). The O6-methylguanine DNA methyltransferase (MGMT) enzyme is related with temozolomide (TMZ) resistance. Our propose is to analyze the prognostic significance of the CD133 antigen and promoter methylation and protein expression of MGMT in a homogenous group of GBM patients uniformly treated with radiotherapy and TMZ. The possible connection between these GBM markers was ...

  15. Internal radiotherapy with copper-64-diacetyl-bis (N{sup 4}-methylthiosemicarbazone) reduces CD133{sup +} highly tumorigenic cells and metastatic ability of mouse colon carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Yoshii, Yukie [Biomedical Imaging Research Center, University of Fukui, Eiheiji, Fukui 910-1193 (Japan); Furukawa, Takako [Molecular Imaging Center, National Institute of Radiological Sciences, Inage, Chiba 263-8555 (Japan); Kiyono, Yasushi [Biomedical Imaging Research Center, University of Fukui, Eiheiji, Fukui 910-1193 (Japan); Watanabe, Ryo [Faculty of Engineering, University of Fukui, Fukui 910-8507 (Japan); Mori, Tetsuya [Biomedical Imaging Research Center, University of Fukui, Eiheiji, Fukui 910-1193 (Japan); Yoshii, Hiroshi [Faculty of Medical Sciences, University of Fukui, Eiheiji, Fukui 910-1193 (Japan); Asai, Tatsuya [Biomedical Imaging Research Center, University of Fukui, Eiheiji, Fukui 910-1193 (Japan); Faculty of Engineering, University of Fukui, Fukui 910-8507 (Japan); Okazawa, Hidehiko [Biomedical Imaging Research Center, University of Fukui, Eiheiji, Fukui 910-1193 (Japan); Welch, Michael J. [Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, MO 63110 (United States); Fujibayashi, Yasuhisa, E-mail: yfuji@nirs.go.j [Biomedical Imaging Research Center, University of Fukui, Eiheiji, Fukui 910-1193 (Japan); Molecular Imaging Center, National Institute of Radiological Sciences, Inage, Chiba 263-8555 (Japan)

    2011-02-15

    Introduction: {sup 64}Cu-diacetyl-bis (N{sup 4}-methylthiosemicarbazone) ({sup 64}Cu-ATSM) is an imaging agent for positron emission tomography (PET) that targets hypoxic tumors. {sup 64}Cu-ATSM is also reported to be a potential agent for internal radiotherapy. In a mouse colon carcinoma (Colon-26) model, we have shown that {sup 64}Cu-ATSM preferentially localizes in intratumoral regions with a high density of CD133{sup +} cells, which show characteristics of cancer stem cells or cancer stem cell-like cells (collectively referred here as CSCs). In this study, we evaluated the therapeutic effect of {sup 64}Cu-ATSM in relation to CD133 expression using this model. Methods: Systemic administration of 37 MBq {sup 64}Cu-ATSM or saline was conducted twice within a 1-week interval to mice bearing 1-week-old Colon-26 tumors (days 0-7). At day 19, tumor size measurement, flow cytometry analysis and experimental lung metastatic assay were performed. The therapeutic effect of {sup 64}Cu-ATSM on sorted CD133{sup +} and CD133{sup -} Colon-26 cells was also examined in vitro. Results: In vivo studies showed that {sup 64}Cu-ATSM treatment inhibited tumor growth. The percentage of CD133{sup +} cells and metastatic ability in {sup 64}Cu-ATSM treated tumors was decreased compared with that in control animals. In vitro studies demonstrated that {sup 64}Cu-ATSM accumulated in cells under hypoxic conditions and incorporation of {sup 64}Cu-ATSM under hypoxia caused cell death in both CD133{sup +} and CD133{sup -} cells in a similar extent. Conclusions: {sup 64}Cu-ATSM administration reduced tumor volume as well as the percentage of CD133{sup +} cells and the metastatic ability of Colon-26 tumors. Together with our data, it is suggested that {sup 64}Cu-ATSM accumulates in regions high in CD133{sup +} highly tumorigenic cells and kills such regions by radiation, resulting in a decrease of the percentage of CD133{sup +} cells.

  16. 免疫磁珠法分离膀胱癌CD133+细胞及生物学行为研究%Isolation of CD133 positive bladder cancer cells with magnetic cell sorting system and research on the biological characteristics

    Institute of Scientific and Technical Information of China (English)

    李法平; 陈帅奇; 王艳波; 郭辉; 刘二鹏; 侯宇川

    2014-01-01

    目的 探讨免疫磁珠法分离膀胱癌5637细胞株中CD133+细胞的方法,观察CD133阳性、阴性细胞间生物学行为的差异.方法 采用免疫磁珠法分选出膀胱癌5637细胞株中CD133+细胞,流式细胞仪检测分选纯度,通过噻唑蓝(MTT)实验、平板克隆形成实验、细胞划痕实验、分化能力检测研究其生物学行为.结果 流式细胞仪检测CD133+细胞在膀胱癌5637细胞中比例为1.45%,经免疫磁珠法分选所得CD133+细胞比例为93.45%;CD133+细胞的增殖、迁移能力明显强于CD133-、未分选的肿瘤细胞(P<0.05);平板克隆形成实验结果示CD133+组[(89.333±4.530)%]细胞克隆形成率明显高于CD133-组[(22.667±4.041)%,P<0.05].流式细胞仪检测CD133+细胞培养2d后其纯度降至48.19%,4d后其纯度与分选前无明显差异.结论 免疫磁珠分选技术可高效快捷获得CD133+细胞,CD133+细胞具有自我更新、迁徙、生成其他表型肿瘤细胞等干细胞样特性.%Objective To isolate CD133 positive cell from bladder cancer cell line 5637 with magnetic cell sorting system (MACS) and to study the different biological characteristics of CD133+ and CD133 cells.Methods Flow cytometry was used to determine the proportion of CD133 + cells sorted by magnetic-activated cell sorting in the bladder cancer cell line 5637.The biological characteristics of CD133 + and CD133-cells was studied by methyl thiazol tetrazolium (MTT) assay,Flat colony formation,Wound healing assay and differentiation ability.Results The proportion of CD133 + cells in the bladder cancer cell line 5637 was 1.45%,which was determined by flow cytometry.CD133 + cells purified by MACS were in a considerable purity of 93.42%.The proliferation and migration capacity of CD133 + cells display stronger than that of CD133-cells and non-separated tumor cells (P <0.05).The formation rate of colony sphere in CD133+ group [(89.333 ± 4.530)%] was higher than that in CD133-group

  17. Placental perfusion - a human alternative

    DEFF Research Database (Denmark)

    Mose, Tina; Knudsen, Lisbeth E

    2006-01-01

    Foetal exposures to environmental and medicinal products have impact on the growth of the foetus (e.g. cigarette smoke) and development of organs (e.g. methylmercury and Thalidomide). Perfusion studies of the human term placenta enable investigation of placental transport of chemical substances...... and represents a supplement and alternative to animal testing, bypassing the animal to human extrapolation. Placentas are readily obtainable from most births upon informed consent from the mothers and are considered a promising tissue alternative/supplement to animal experiments. The system is validated...

  18. Silencing BMI1 eliminates tumor formation of pediatric glioma CD133+ cells not by affecting known targets but by down-regulating a novel set of core genes.

    Science.gov (United States)

    Baxter, Patricia A; Lin, Qi; Mao, Hua; Kogiso, Mari; Zhao, Xiumei; Liu, Zhigang; Huang, Yulun; Voicu, Horatiu; Gurusiddappa, Sivashankarappa; Su, Jack M; Adesina, Adekunle M; Perlaky, Laszlo; Dauser, Robert C; Leung, Hon-chiu Eastwood; Muraszko, Karin M; Heth, Jason A; Fan, Xing; Lau, Ching C; Man, Tsz-Kwong; Chintagumpala, Murali; Li, Xiao-Nan

    2014-01-01

    Clinical outcome of children with malignant glioma remains dismal. Here, we examined the role of over-expressed BMI1, a regulator of stem cell self-renewal, in sustaining tumor formation in pediatric glioma stem cells. Our investigation revealed BMI1 over-expression in 29 of 54 (53.7%) pediatric gliomas, 8 of 8 (100%) patient derived orthotopic xenograft (PDOX) mouse models, and in both CD133+ and CD133- glioma cells. We demonstrated that lentiviral-shRNA mediated silencing of suppressed cell proliferation in vitro in cells derived from 3 independent PDOX models and eliminated tumor-forming capacity of CD133+ and CD133- cells derived from 2 PDOX models in mouse brains. Gene expression profiling showed that most of the molecular targets of BMI1 ablation in CD133+ cells were different from that in CD133- cells. Importantly, we found that silencing BMI1 in CD133+ cells derived from 3 PDOX models did not affect most of the known genes previously associated with the activated BMI1, but modulated a novel set of core genes, including RPS6KA2, ALDH3A2, FMFB, DTL, API5, EIF4G2, KIF5c, LOC650152, C20ORF121, LOC203547, LOC653308, and LOC642489, to mediate the elimination of tumor formation. In summary, we identified the over-expressed BMI1 as a promising therapeutic target for glioma stem cells, and suggest that the signaling pathways associated with activated BMI1 in promoting tumor growth may be different from those induced by silencing BMI1 in blocking tumor formation. These findings highlighted the importance of careful re-analysis of the affected genes following the inhibition of abnormally activated oncogenic pathways to identify determinants that can potentially predict therapeutic efficacy.

  19. MGMT promoter methylation status and MGMT and CD133 immunohistochemical expression as prognostic markers in glioblastoma patients treated with temozolomide plus radiotherapy

    Directory of Open Access Journals (Sweden)

    Melguizo Consolación

    2012-12-01

    Full Text Available Abstract Background The CD133 antigen is a marker of radio- and chemo-resistant stem cell populations in glioblastoma (GBM. The O6-methylguanine DNA methyltransferase (MGMT enzyme is related with temozolomide (TMZ resistance. Our propose is to analyze the prognostic significance of the CD133 antigen and promoter methylation and protein expression of MGMT in a homogenous group of GBM patients uniformly treated with radiotherapy and TMZ. The possible connection between these GBM markers was also investigated. Methods Seventy-eight patients with GBM treated with radiotherapy combined with concomitant and adjuvant TMZ were analyzed for MGMT and CD133. MGMT gene promoter methylation was determined by methylation-specific polymerase chain reaction after bisulfite treatment. MGMT and CD133 expression was assessed immunohistochemically using an automatic quantification system. Overall and progression-free survival was calculated according to the Kaplan–Meier method. Results The MGMT gene promoter was found to be methylated in 34 patients (44.7% and unmethylated in 42 patients (55.3%. A significant correlation was observed between MGMT promoter methylation and patients’ survival. Among the unmethylated tumors, 52.4% showed low expression of MGMT and 47.6% showed high-expression. Among methylated tumors, 58.8% showed low-expression of MGMT and 41.2% showed high-expression. No correlation was found between MGMT promoter methylation and MGMT expression, or MGMT expression and survival. In contrast with recent results, CD133 expression was not a predictive marker in GBM patients. Analyses of possible correlation between CD133 expression and MGMT protein expression or MGMT promoter methylation were negative. Conclusions Our results support the hypothesis that MGMT promoter methylation status but not MGMT expression may be a predictive biomarker in the treatment of patients with GBM. In addition, CD133 should not be used for prognostic evaluation of these

  20. Distribution of CD133 reveals glioma stem cells self-renew through symmetric and asymmetric cell divisions.

    Science.gov (United States)

    Lathia, J D; Hitomi, M; Gallagher, J; Gadani, S P; Adkins, J; Vasanji, A; Liu, L; Eyler, C E; Heddleston, J M; Wu, Q; Minhas, S; Soeda, A; Hoeppner, D J; Ravin, R; McKay, R D G; McLendon, R E; Corbeil, D; Chenn, A; Hjelmeland, A B; Park, D M; Rich, J N

    2011-09-01

    Malignant gliomas contain a population of self-renewing tumorigenic stem-like cells; however, it remains unclear how these glioma stem cells (GSCs) self-renew or generate cellular diversity at the single-cell level. Asymmetric cell division is a proposed mechanism to maintain cancer stem cells, yet the modes of cell division that GSCs utilize remain undetermined. Here, we used single-cell analyses to evaluate the cell division behavior of GSCs. Lineage-tracing analysis revealed that the majority of GSCs were generated through expansive symmetric cell division and not through asymmetric cell division. The majority of differentiated progeny was generated through symmetric pro-commitment divisions under expansion conditions and in the absence of growth factors, occurred mainly through asymmetric cell divisions. Mitotic pair analysis detected asymmetric CD133 segregation and not any other GSC marker in a fraction of mitoses, some of which were associated with Numb asymmetry. Under growth factor withdrawal conditions, the proportion of asymmetric CD133 divisions increased, congruent with the increase in asymmetric cell divisions observed in the lineage-tracing studies. Using single-cell-based observation, we provide definitive evidence that GSCs are capable of different modes of cell division and that the generation of cellular diversity occurs mainly through symmetric cell division, not through asymmetric cell division.

  1. Evaluation of cancer stem cell markers CD133, CD44, CD24: association with AKT isoforms and radiation resistance in colon cancer cells.

    Directory of Open Access Journals (Sweden)

    Sara Häggblad Sahlberg

    Full Text Available The cell surface proteins CD133, CD24 and CD44 are putative markers for cancer stem cell populations in colon cancer, associated with aggressive cancer types and poor prognosis. It is important to understand how these markers may predict treatment outcomes, determined by factors such as radioresistance. The scope of this study was to assess the connection between EGFR, CD133, CD24, and CD44 (including isoforms expression levels and radiation sensitivity, and furthermore analyze the influence of AKT isoforms on the expression patterns of these markers, to better understand the underlying molecular mechanisms in the cell. Three colon cancer cell-lines were used, HT-29, DLD-1, and HCT116, together with DLD-1 isogenic AKT knock-out cell-lines. All three cell-lines (HT-29, HCT116 and DLD-1 expressed varying amounts of CD133, CD24 and CD44 and the top ten percent of CD133 and CD44 expressing cells (CD133high/CD44high were more resistant to gamma radiation than the ten percent with lowest expression (CD133low/CD44low. The AKT expression was lower in the fraction of cells with low CD133/CD44. Depletion of AKT1 or AKT2 using knock out cells showed for the first time that CD133 expression was associated with AKT1 but not AKT2, whereas the CD44 expression was influenced by the presence of either AKT1 or AKT2. There were several genes in the cell adhesion pathway which had significantly higher expression in the AKT2 KO cell-line compared to the AKT1 KO cell-line; however important genes in the epithelial to mesenchymal transition pathway (CDH1, VIM, TWIST1, SNAI1, SNAI2, ZEB1, ZEB2, FN1, FOXC2 and CDH2 did not differ. Our results demonstrate that CD133high/CD44high expressing colon cancer cells are associated with AKT and increased radiation resistance, and that different AKT isoforms have varying effects on the expression of cancer stem cell markers, which is an important consideration when targeting AKT in a clinical setting.

  2. Self-renewal of CD133hi cells by IL6/Notch3 signalling regulates endocrine resistance in metastatic breast cancer

    Science.gov (United States)

    Sansone, Pasquale; Ceccarelli, Claudio; Berishaj, Marjan; Chang, Qing; Rajasekhar, Vinagolu K.; Perna, Fabiana; Bowman, Robert L.; Vidone, Michele; Daly, Laura; Nnoli, Jennifer; Santini, Donatella; Taffurelli, Mario; Shih, Natalie N. C.; Feldman, Michael; Mao, Jun J.; Colameco , Christopher; Chen, Jinbo; DeMichele, Angela; Fabbri, Nicola; Healey, John H.; Cricca, Monica; Gasparre, Giuseppe; Lyden, David; Bonafé, Massimiliano; Bromberg, Jacqueline

    2016-01-01

    The mechanisms of metastatic progression from hormonal therapy (HT) are largely unknown in luminal breast cancer. Here we demonstrate the enrichment of CD133hi/ERlo cancer cells in clinical specimens following neoadjuvant endocrine therapy and in HT refractory metastatic disease. We develop experimental models of metastatic luminal breast cancer and demonstrate that HT can promote the generation of HT-resistant, self-renewing CD133hi/ERlo/IL6hi cancer stem cells (CSCs). HT initially abrogates oxidative phosphorylation (OXPHOS) generating self-renewal-deficient cancer cells, CD133hi/ERlo/OXPHOSlo. These cells exit metabolic dormancy via an IL6-driven feed-forward ERlo-IL6hi-Notchhi loop, activating OXPHOS, in the absence of ER activity. The inhibition of IL6R/IL6-Notch pathways switches the self-renewal of CD133hi CSCs, from an IL6/Notch-dependent one to an ER-dependent one, through the re-expression of ER. Thus, HT induces an OXPHOS metabolic editing of luminal breast cancers, paradoxically establishing HT-driven self-renewal of dormant CD133hi/ERlo cells mediating metastatic progression, which is sensitive to dual targeted therapy. PMID:26858125

  3. Self-renewal of CD133(hi) cells by IL6/Notch3 signalling regulates endocrine resistance in metastatic breast cancer.

    Science.gov (United States)

    Sansone, Pasquale; Ceccarelli, Claudio; Berishaj, Marjan; Chang, Qing; Rajasekhar, Vinagolu K; Perna, Fabiana; Bowman, Robert L; Vidone, Michele; Daly, Laura; Nnoli, Jennifer; Santini, Donatella; Taffurelli, Mario; Shih, Natalie N C; Feldman, Michael; Mao, Jun J; Colameco, Christopher; Chen, Jinbo; DeMichele, Angela; Fabbri, Nicola; Healey, John H; Cricca, Monica; Gasparre, Giuseppe; Lyden, David; Bonafé, Massimiliano; Bromberg, Jacqueline

    2016-02-09

    The mechanisms of metastatic progression from hormonal therapy (HT) are largely unknown in luminal breast cancer. Here we demonstrate the enrichment of CD133(hi)/ER(lo) cancer cells in clinical specimens following neoadjuvant endocrine therapy and in HT refractory metastatic disease. We develop experimental models of metastatic luminal breast cancer and demonstrate that HT can promote the generation of HT-resistant, self-renewing CD133(hi)/ER(lo)/IL6(hi) cancer stem cells (CSCs). HT initially abrogates oxidative phosphorylation (OXPHOS) generating self-renewal-deficient cancer cells, CD133(hi)/ER(lo)/OXPHOS(lo). These cells exit metabolic dormancy via an IL6-driven feed-forward ER(lo)-IL6(hi)-Notch(hi) loop, activating OXPHOS, in the absence of ER activity. The inhibition of IL6R/IL6-Notch pathways switches the self-renewal of CD133(hi) CSCs, from an IL6/Notch-dependent one to an ER-dependent one, through the re-expression of ER. Thus, HT induces an OXPHOS metabolic editing of luminal breast cancers, paradoxically establishing HT-driven self-renewal of dormant CD133(hi)/ER(lo) cells mediating metastatic progression, which is sensitive to dual targeted therapy.

  4. Molecular analysis of ex-vivo CD133+ GBM cells revealed a common invasive and angiogenic profile but different proliferative signatures among high grade gliomas

    Directory of Open Access Journals (Sweden)

    Garcia Juan L

    2010-08-01

    Full Text Available Abstract Background Gliomas are the most common type of primary brain tumours, and in this group glioblastomas (GBMs are the higher-grade gliomas with fast progression and unfortunate prognosis. Two major aspects of glioma biology that contributes to its awful prognosis are the formation of new blood vessels through the process of angiogenesis and the invasion of glioma cells. Despite of advances, two-year survival for GBM patients with optimal therapy is less than 30%. Even in those patients with low-grade gliomas, that imply a moderately good prognosis, treatment is almost never curative. Recent studies have demonstrated the existence of a small fraction of glioma cells with characteristics of neural stem cells which are able to grow in vitro forming neurospheres and that can be isolated in vivo using surface markers such as CD133. The aim of this study was to define the molecular signature of GBM cells expressing CD133 in comparison with non expressing CD133 cells. This molecular classification could lead to the finding of new potential therapeutic targets for the rationale treatment of high grade GBM. Methods Eight fresh, primary and non cultured GBMs were used in order to study the gene expression signatures from its CD133 positive and negative populations isolated by FACS-sorting. Dataset was generated with Affymetrix U133 Plus 2 arrays and analysed using the software of the Affymetrix Expression Console. In addition, genomic analysis of these tumours was carried out by CGH arrays, FISH studies and MLPA; Results Gene expression analysis of CD133+ vs. CD133- cell population from each tumour showed that CD133+ cells presented common characteristics in all glioblastoma samples (up-regulation of genes involved in angiogenesis, permeability and down-regulation of genes implicated in cell assembly, neural cell organization and neurological disorders. Furthermore, unsupervised clustering of gene expression led us to distinguish between two groups

  5. Synergistic Effect of Sodium Butyrate and Thalidomide in the Induction of Fetal Hemoglobin Expression in Erythroid Progenitors Derived from Cord Blood CD133 + Cells

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    Ali Dehghanifard

    2012-07-01

    Full Text Available Background: The use of drugs with the ability to induce production of fetal hemoglobin as a novel therapeutic approach in treating β-Hemoglobinopathies is considered. γ-globin gene expression inducer drugs including sodium butyrate and thalidomide can reduce additional α-globin chains accumulation in erythroid precursors. Materials and Methods: In this experimental study, MACS kit was used to isolate CD133+ cells of umbilical cord blood. Further, the effect of two drugs of thalidomide and sodium butyrate were separately and combined studied on the induction of quantitative expression of β-globin and γ-globin genes in erythroid precursor cells derived from CD133+ stem cells in-vitro. For this purpose, the technique SYBR green Real-time PCR was used.Results: Flow cytometry results showed that approximately 95% of purified cells were CD133+. Real-time PCR results also showed the increased levels of γ-globin mRNA in the cell groups treated with thalidomide, sodium butyrate and combination of drugs as 2.6 and 1.2 and 3.5 times respectively, and for β-globin gene, it is respectively 1.4 and 1.3 and 1.6 times compared with the control group (p<0.05.Conclusion: The study results showed that the mentioned drug combination can act as a pharmaceutical composition affecting the induction of fetal hemoglobin expression in erythroid precursor cells derived from CD133 + cells.

  6. Distinctive effects of CD34- and CD133-specific antibody-coated stents on re-endothelialization and in-stent restenosis at the early phase of vascular injury

    DEFF Research Database (Denmark)

    Wu, Xue; Yin, Tieying; Tian, Jie;

    2015-01-01

    It is not clear what effects of CD34- and CD133-specific antibody-coated stents have on re-endothelialization and in-stent restenosis (ISR) at the early phase of vascular injury. This study aims at determining the capabilities of different coatings on stents (e.g. gelatin, anti-CD133 and anti-CD34......-coated stents, the time of cells adhesion was longer and earlier present in the anti-CD133 antibody-coated stents and anti-CD133 antibody-coated stents have superiority in re-endothelialization and inhibition of ISR. In conclusion, this study demonstrated that anti-CD133 antibody as a stent coating...... for capturing EPCs is better than anti-CD34 antibody in promoting endothelialization and reducing ISR....

  7. 合并前列腺炎症的良性前列腺增生组织中Integrinα2β1/CD133表达及其意义%Expressions of Integrinα2β1 and CD133 in benign prostatic hyperplasia complicated by prostatitis and their significance

    Institute of Scientific and Technical Information of China (English)

    丁奇; 苗文隆; 刘硕; 畅继武; 杨宇明

    2011-01-01

    Objective: To study the expressions of Integrinα2β1 and CD133 in benign prostatic hyperplasia (BPH) complicated by prostatitis and their significance. Methods: Specimens were obtained from 56 BPH patients undergoing transvesical prostatectomy. Paraffin sections of the specimens were subjected to HE staining for pathological examination of inflammatory changes under the light microscope. Twenty-four patients with simple BPH were included in Group A, and the other 32 with BPH complicated with prostatitis in Croup B. The expressions of Integrincα2β1 and CD133 in the prostatic tissues of the two groups were determined by immunohisto-chemistry, Western blotting and IPP6.0 image analysis software. Results: The expressions of Integrinα2β1, and CD133 were significantly higher in Group B than in A ( P < 0.05 ) , and so were the mean relative value of the optical density of Integrinα2β1, (0.29 ± 0.18 vs 0.04 ± 0.03) and that of CD133 (0.08 ± 0.07 vs 0.002 0 ± 0.001 8) (P < 0.05). Conclusion: Inflammation can up-regulate the expressions of Integrinα2β1 and CD133 in BPH tissue.%目的:探讨合并前列腺炎症的良性前列腺增生(BPH)组织中Integrinα2β1/CD133表达及其意义.方法:收集56例行经膀胱前列腺摘除手术的BPH患者的前列腺标本,常规HE染色后观察其合并炎症情况,其中单纯BPH(24例)为A组,合并前列腺炎症的BPH(32例)为B组.采用免疫组化SP法及Western印迹法检测Integrinα2β1/CD133在两组前列腺组织中表达差异.Image-Pro@ Plus 6.0图像分析软件对结果进行分析.结果:免疫组化Mattern积分结果显示:两组中Integrinα2β1/CD133表达有显著差异(P<0.05).Western免疫印迹显示:B组Integrinαt2β1平均相对光密度值高于A组[(0.29±0.18)vs (0.04±0.03)],P<0.05;B组CD133平均相对光密度值高于A组[(0.08±0.07) vs(0.002 0±0.001 8)],P<0.05.提示B组中Integrinα2β1/CD133表达较A组显著升高.结论:炎症可以促进BPH组织中Integrinα2β1

  8. Expression of the zebrafish CD133/prominin1 genes in cellular proliferation zones in the embryonic central nervous system and sensory organs.

    Science.gov (United States)

    McGrail, Maura; Batz, Lindsey; Noack, Kristin; Pandey, Saumya; Huang, Yong; Gu, Xun; Essner, Jeffrey J

    2010-06-01

    The CD133/prominin1 gene encodes a pentamembrane glycoprotein cell surface marker that is expressed in stem cells from neuroepithelial, hematopoietic, and various organ tissues. Here we report the analysis of two zebrafish CD133/prominin1 orthologues, prominin1a and prominin1b. The expression patterns of the zebrafish prominin1a and b genes were analyzed during embryogenesis using whole mount in situ hybridization. prominin1a and b show novel complementary and overlapping patterns of expression in proliferating zones in the developing sensory organs and central nervous system. The expression patterns suggest functional conservation of the zebrafish prominin1 genes. Initial analyses of prominin1a and b in neoplastic tissue show increased expression of both genes in a subpopulation of cells in malignant peripheral nerve sheath tumors in tp53 mutants. Based on these analyses, the zebrafish prominin1 genes will be useful markers for examining proliferating cell populations in adult organs, tissues, and tumors.

  9. Long-term clinical results of autologous bone marrow CD 133+ cell transplantation in patients with ST-elevation myocardial infarction

    Science.gov (United States)

    Kirgizova, M. A.; Suslova, T. E.; Markov, V. A.; Karpov, R. S.; Ryabov, V. V.

    2015-11-01

    The aim of the study was investigate the long-term results of autologous bone marrow CD 133+ cell transplantation in patients with primary ST-Elevation Myocardial Infarction (STEMI). Methods and results: From 2006 to 2007, 26 patients with primary STEMI were included in an open randomized study. Patients were randomized to two groups: 1st - included patients underwent PCI and transplantation of autologous bone marrow CD 133+ cell (n = 10); 2nd - patients with only PCI (n = 16). Follow-up study was performed 7.70±0.42 years after STEMI and consisted in physical examination, 6-min walking test, Echo exam. Total and cardiovascular mortality in group 1 was lower (20% (n = 2) vs. 44% (n = 7), p = 0.1 and 22% (n = 2) vs. 25% (n = 4), (p=0.53), respectively). Analysis of cardiac volumetric parameters shows significant differences between groups: EDV of 100.7 ± 50.2 mL vs. 144.40±42.7 mL, ESV of 56.3 ± 37.8 mL vs. 89.7 ± 38.7 mL in 1st and 2nd groups, respectively. Data of the study showed positive effects of autologous bone marrow CD 133+ cell transplantation on the long-term survival of patients and structural status of the heart.

  10. Clinical significance of serum tumor stem cell marker CD133 and miR-145 in patients with ovarian cancer%卵巢癌患者血清肿瘤干细胞标志物CD133和miR-145的临床价值

    Institute of Scientific and Technical Information of China (English)

    王丹; 王莉; 王治洁

    2016-01-01

    目的 观察卵巢癌患者血清CD133和miR-145的表达及其对预后的影响.方法 选择2010年1月至2014年6月在同济大学医学院、上海市第八人民医院和抚顺中心医院手术的卵巢癌患者84例为卵巢癌组;良性肿瘤手术的患者30例为对照组.用qRT-PCP的方法测定血清CD133和miR-145相对表达量,分析CD133和miR-145表达量与年龄、肿瘤直径、病理类型、分化程度、CA125水平和是否转移分期的关系.结果 卵巢癌患者的血清CD133相对表达明显高于对照组(t=5.34,P0.05),血清CD133和miR-145表达量与肿瘤分化程度、CA125水平、淋巴转移和FIGO分期呈明显的相关性(t/F值为2.01~28.96,均P0.05), but they were obviously correlated with tumor differentiation, CA125 level, lymph node metastasis and FIGO stage (t/F value ranged 2.01 -28.96, all P<0.05).Postoperative follow-up revealed that the 1-year recurrence rate was 76.79%in patients with high expression of CD133, which was significantly higher than 42.86%in patients with low expression (χ2 =8.06,P<0.05).In patients with high expression of miR-1451-year recurrence rate was 43.59%, which was significantly lower than 84.44%in those with low expression (χ2 =13.63,P<0.05).Conclusion The serum expression of CD133 is high but miR-145 is low in patients with ovarian cancer.They both involve in the development and progression of ovarian cancer, and combining detection of them is of great clinical significance in diagnosis and assessment of prognosis.

  11. MiR-34a targeting of Notch ligand delta-like 1 impairs CD15+/CD133+ tumor-propagating cells and supports neural differentiation in medulloblastoma.

    Directory of Open Access Journals (Sweden)

    Pasqualino de Antonellis

    Full Text Available BACKGROUND: Through negative regulation of gene expression, microRNAs (miRNAs can function as oncosuppressors in cancers, and can themselves show altered expression in various tumor types. Here, we have investigated medulloblastoma tumors (MBs, which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many of the cell-fate-determining stages. Notch regulates a subset of MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulate these phenomena, and can be used in anti-cancer therapies. METHODOLOGY/PRINCIPAL FINDINGS: In a screening of potential targets within Notch signaling, miR-34a was seen to be a regulator of the Notch pathway through its targeting of Notch ligand Delta-like 1 (Dll1. Down-regulation of Dll1 expression by miR-34a negatively regulates cell proliferation, and induces apoptosis and neural differentiation in MB cells. Using an inducible tetracycline on-off model of miR-34a expression, we show that in Daoy MB cells, Dll1 is the first target that is regulated in MB, as compared to the other targets analyzed here: Cyclin D1, cMyc and CDK4. MiR-34a expression negatively affects CD133(+/CD15(+ tumor-propagating cells, then we assay through reverse-phase proteomic arrays, Akt and Stat3 signaling hypo-phosphorylation. Adenoviruses carrying the precursor miR-34a induce neurogenesis of tumor spheres derived from a genetic animal model of MB (Patch1(+/- p53(-/-, thus providing further evidence that the miR-34a/Dll1 axis controls both autonomous and non autonomous signaling of Notch. In vivo, miR-34a overexpression carried by adenoviruses reduces tumor burden in cerebellum xenografts of athymic mice, thus demonstrating an anti-tumorigenic role of miR-34a in vivo. CONCLUSIONS/SIGNIFICANCE: Despite advances in our understanding of the pathogenesis of MB, one-third of

  12. Gefitinib resistance in HCC mahlavu cells: upregulation of CD133 expression, activation of IGF-1R signaling pathway, and enhancement of IGF-1R nuclear translocation.

    Science.gov (United States)

    Bodzin, Adam S; Wei, Zhengyu; Hurtt, Reginald; Gu, Tina; Doria, Cataldo

    2012-07-01

    Hepatocellular carcinoma (HCC) is the major form of primary liver cancer which accounts for more than half million deaths annually worldwide. While the incidence of HCC is still on the rise, options of treatment are limited and the overall survival rate is poor. The acquisition of cancer drug resistance remains one of the key hurdles to successful treatment. Clearly, a thorough understanding of the underlying mechanisms is needed for new strategies to design novel treatments and/or to improve the current therapies. In the present study, we examined the expression of cancer stem cell (CSC) marker CD133, the activation of insulin-like growth factor 1 receptor (IGF-1R) signaling, and the nuclear translocation of IGF-1R in HCC Mahlavu cells under the treatment of gefitinib, a cancer drug that inhibits epidermal growth factor receptor (EGFR) pathway. Our results demonstrated that Mahlavu cells exhibited strong gefitinib resistance and the CD133 expression level was dramatically increased (from 3.88% to 32%) after drug treatment. In addition, the gefitinib treated cells displayed increased levels of phosphorylation in IGF-1R and Akt, indicating the intensified activation of this cancer-associated signaling pathway. Moreover, we revealed that IGF-1R underwent nuclear translocation in gefitinib treated cells using confocal microscopy. The IGF-1R nuclear translocation was enhanced under gefitinib treatment and appeared in a dose-dependent manner. Our findings suggest that increased IGF-1R nuclear translocation after gefitinib treatment may contribute to the drug resistance and IGF1-R activation, which might also associate with the upregulation of CD133 expression.

  13. Construction and identification of replication-competent adenovirus expressing siRNA targeting CD133 gene regulated by survivin promoter and its inhibition of liver cancer cell growth%survivin 启动子调控肿瘤干细胞标记 CD133基因 siRNA增殖型溶瘤腺病毒的构建及对肝癌细胞生长的抑制作用

    Institute of Scientific and Technical Information of China (English)

    牛坚; 王月; 刘斌; 王人颢; 朱志军; 申海莲

    2016-01-01

    目的:构建 survivin 启动子调控的靶向 CD133基因的 siRNA 增殖型溶瘤腺病毒,研究其对肝癌细胞生长的影响。方法RT-PCR 法扩增 survivin 启动子,测序鉴定,双酶切连接,获得 pH-XC2-survivin。酶切 pH-XC2-survivin、pZD55-CD133-siRNA 获得 survivin 启动子表达框的亚克隆和CD133-siRNA 基因表达框的亚克隆,连接获得 survivin 启动子调控的 siRNA 增殖型溶瘤腺病毒表达载体质粒 pT-ZD55-CD133-siRNA。增殖型溶瘤腺病毒 survivin-T-ZD55-CD133-siRNA 经 PCR 和测序鉴定。 qRT-PCR 法检测 CD133表达, Western blot 法检测 E1A,CCK-8法检测细胞生长,流式细胞术检测细胞凋亡。结果成功构建增殖型溶瘤腺病毒 sur-vivin-T-ZD55-CD133-siRNA。 qRT-PCR 法检测 CD133 mRNA明显下降, Western blot 证实 survivin-T-ZD55-CD133-siRNA在肿瘤细胞中表达 E1A 能抑制肝癌细胞 CD133表达及生长。结论构建的增殖型溶瘤腺病毒可有效降低肝癌细胞CD133的表达,用于肝癌基因治疗的进一步研究。%Objective To construct a replication-competent adenovirus expressing siRNA targeting CD133 gene regulated by survivin promoter and investigate its inhibitory effect on Hep 3B cells.Methods The fragment of the survivin promoter was amplified by PCR and inserted into pH -XC2 to reconstruct a recombinant plasmid pH -XC2-survivin.Complete digestion pH-XC2-survivin and pZD55-CD133-siRNA, combinational joining the subclones, then getting replication-competent adenovirus expressing short interference RNA targeting CD 133 gene regulated by survivin promoter, replication-competent adenovirus was constructed .The recombined adenoviruses ( T-ZD55-CD133-siRNA) were verified by PCR and sequencing .The effect of T-ZD55-CD133-siRNA on CD133 expression in Hep3B cells was detected by qRT-PCR.The expression of E1A was detected by Western blot.The antitumor po-tential of replication

  14. Convenient and efficient enrichment of the CD133+ liver cells from rat fetal liver as a source of liver stem/progenitor cells.

    Science.gov (United States)

    Liu, Weihui; You, Nan; Dou, Kefeng

    2012-01-01

    Although stem cells are commonly isolated by fluorescence-activated cell sorting or magnetic affinity cell sorting, they are very expensive, and they need known markers. However, there is no specific marker for liver stem/progenitor cells (LSPCs). Here, we describe a convenient and efficient method (three-step method) to enrich LSPCs. The fetal liver cells (FLCs) were firstly enriched by Percoll discontinuous gradient centrifugation from the rat fetal liver. Then the FLCs in culture were purified to be homogeneous in size by differential trypsinization and differential adherence. Finally, fetal liver stem/progenitor cells (FLSPCs) were enriched from purified FLCs by Percoll continuous gradient centrifugation. Flow cytometric analysis combining with marker CD133 was used to detect the purity of FLSPCs and evaluate the isolating effects of the three-step method.

  15. Biomarker screening of oral cancer cell lines revealed sub-populations of CD133-, CD44-, CD24- and ALDH1- positive cancer stem cells

    Directory of Open Access Journals (Sweden)

    Kendall K

    2013-05-01

    Full Text Available Head and neck squamous cell carcinoma (HNSCC ranks sixth worldwide for cancer-related mortality. For the past several decades the mainstay of treatment for HNSCC has been surgery and external beam radiation, although more recent trials combining chemotherapy and radiation have demonstrated improvements. However, cancer recurrence and treatment failures continue to occur in a significant percentage of patients. Recent advances in tumor biology have led to the discovery that many cancers, including HNSCC, may contain subpopulations of cells with stem cell-like properties that may explain relapse and recurrence. The objective of this study was to screen existing oral cancer cell lines for biomarkers specific for cells with stem cell-like properties. RNA was isolated for RT-PCR screening using primers for specific mRNA of the biomarkers: CD44, CD24, CD133, NANOG, Nestin, ALDH1, and ABCG2 in CAL27, SCC25 and SCC15 cells. This analysis revealed that some oral cancer cell lines (CAL27 and SCC25 may contain small subpopulations of adhesion- and contact-independent cells (AiDC that also express tumor stem cell markers, including CD44, CD133, and CD24. In addition, CAL27 cells also expressed the intracellular tumor stem cell markers, ALDH1 and ABCG2. Isolation and culture of the adhesion- and contact-independent cells from CAL27 and SCC25 populations revealed differential proliferation rates and more robust inhibition by the MEK inhibitor PD98059, as well as the chemotherapeutic agents Cisplatin and Paclitaxel, within the AiDC CAL27 cells. At least one oral cancer cell line (CAL27 contained subpopulations of cells that express specific biomarkers associated with tumor stem cells which were morphologically and phenotypically distinct from other cells within this cell line.

  16. CD133/CD15 defines distinct cell subpopulations with differential in vitro clonogenic activity and stem cell-related gene expression profile in in vitro propagated glioblastoma multiforme-derived cell line with a PNET-like component.

    Science.gov (United States)

    Kahlert, Ulf D; Bender, Noemi O; Maciaczyk, Donata; Bogiel, Tomasz; Bar, Eli E; Eberhart, Charles G; Nikkhah, Guido; Maciaczyk, Jarosław

    2012-01-01

    Glioblastoma multiforme (GBM), as many other solid tumours, contains a subpopulation of cells termed cancer stem-like cells responsible for the initiation and propagation of tumour growth. However, a unique immunophenotype/surface antigen composition for the clear identification of brain tumour stem cells (BTSC) has not yet been found. Here we report a novel code of cell surface markers for the identification of different cell subpopulations in neurospheres derived from a GBM with a primitive neuroectodermal tumour (PNET)-like component (GBM-PNET). These subgroups differ in their CD133/CD15 expression pattern and resemble cells with different stem-like genotype and developmental pathway activation levels. Strikingly, clonogenic analysis of cultures differentially expressing the investigated markers enabled the identification of distinct subpopulations of cells endowed with stem cell characteristics. High clonogenicity could be found in CD133(-)/CD15(-) and CD133(+)/CD15(+) but not in CD133(-)/CD15(+) cells. Moreover, cell subpopulations with pronounced clonogenic growth were characterized by high expression of stem cell-related genes. Interestingly, these observations were unique for GBM-PNET and differed from ordinary GBM cultures derived from tumours lacking a PNET component. This work elucidates the complex molecular heterogeneity of in vitro propagated glioblastoma-derived cells and potentially contributes to the development of novel diagnostic modalities aiming at the identification of the brain tumour stem-like cell population in a subgroup of GBMs.

  17. Isolation and Identification of Ovarian Cancer Stem Cells from HumanEpithelial Ovarian Carcinoma Cell Line 3AO%人卵巢肿瘤细胞系3AO中肿瘤干细胞的分离鉴定

    Institute of Scientific and Technical Information of China (English)

    李青; 唐良萏; 汪艳; 张曦; 朱慧芬

    2012-01-01

    Objective To isolate and identify cancer stem cells-like cells(CSC-LCs)from the human epithelial ovarian carcinoma cell line 3AO and define their biological characteristics through accessing the activity of the anti-apoptosis and drug resistance. Methods CD133 cells were isolated from 3AO by immunomagnetic beads(miniMACS). Cell counting was taken to reflect the proliferation ability. The self-renew capacity of CD133+ cells was detected by using flow cytometry(FCM). No more than 10' CD133+ cells were inoculated subcutaneously. Formation of xenografts in nude mice was taken to demonstrate the tu-morigenesis of these cells. MTT assay was also taken to characterize the sensitivity of CD133 cells against chemotherapeutic drug. Results CD133+ cancer stem cells were obtained from the human epithelial ovarian carcinoma cell line 3AO, which could self-renew, proliferate and differentiate to a number of CD133 cells. Tumors transplanted into nude mice showed that the tumor formation rate of CD133+ cells was 10/10 and the average tumor formation time was (58 + 6) days, and in CD133- group, tumor formation rate was 4/10 and the average tumor formation time was (145i8) days,suggesting the tumor formation ability of CD133+ cells was significantly stronger than CD133‐ cells. MTT results revealed that IC5q value of CD133+ was 2. 5 times higher than CD133‐ suggesting that CD133+ cells were not sensitive to cisplatin. Staining of cisplatin-treated cells with acridine orange displayed that a large number of CD133 cells showed apoptotic state, while most of the CD133 represented normal form. Further Annexin V-FITC and PI double staining results showed that CD133+ cells had stronger anti-apoptotic ability than CD133‐ cells after treatment with cisplatin. Conclusion CD133 could be further studied as a molecular marker for identification of ovary cancer stem cells, which also provides the basis for the isolation,culture and identification of cancer stem cells in epithelial ovarian

  18. CD133β-catenin和APC在结直肠癌组织中的表达及其与预后的关系%Expression and Prognostic Value of CD133,β-catenin, and APC Proteins in Colorectal Carcinoma Tissues

    Institute of Scientific and Technical Information of China (English)

    吴雪芳; 刘坤平; 罗枫; 伍世钢; 唐丽娟; 钟雪云

    2012-01-01

      目的:探讨CD133、β-catenin和APC蛋白在结直肠癌发生发展中的作用,分析三者的表达和临床病理特征与结直肠癌患者预后的关系.方法:应用组织芯片及免疫组织化学法检测74例结直肠腺瘤、135例结直肠癌及癌旁正常黏膜组织中CD133、β-catenin和APC蛋白的表达;结合随访资料进行单因素Kaplan-Meier生存分析及多因素Cox回归分析.结果:CD133在结直肠癌中阳性率为45.9%,高于腺瘤(9.5%)及癌旁正常黏膜(0,P<0.05),其表达与腺瘤不典型增生程度及结直肠癌组织分级具有相关性(P<0.05);β-catenin胞质/胞核阳性表达在结直肠癌(93.3%)和腺瘤(85.1%),高于癌旁正常黏膜(14.8%,P<0.05),β-catenin膜表达缺失率在结直肠癌(45.2%)高于腺瘤(4.1%)及癌旁正常黏膜(5.2%,P<0.05),且与淋巴结转移及Dukes'分期有关(P<0.05);APC阳性表达率在癌旁正常黏膜(100%)、腺瘤(90.5%)和结直肠癌(34.8%)呈逐级降低(P<0.05),在结直肠癌APC表达缺失组,β-catenin胞质/胞核表达与CD133表达呈正相关(P<0.05).单因素和多因素生存分析均筛选出Dukes'分期、β-catenin膜表达缺失为影响结直肠癌患者预后的高风险因素及独立预后影响因素(P<0.05).结论:CD133、β-catenin、APC参与了结直肠癌的发生发展,CD133表达与β-catenin及APC之间存在密切联系,三者的检测对结直肠癌的早期诊断、生物学行为及预后评估有一定意义.%10.3969/j.issn.1000-8179.2012.23.008

  19. Human cloning: three mistakes and an alternative.

    Science.gov (United States)

    Baylis, Françoise

    2002-06-01

    The current debate on the ethics of cloning humans is both uninspired and uninspiring. In large measure this is because of mistakes that permeate the discourse, including the mistake of thinking that cloning technology is strictly a reproductive technology when it is used to create whole beings. As a result, the challenge this technology represents regarding our understanding of ourselves and the species to which we belong typically is inappropriately downplayed or exaggerated. This has meant that important (albeit disquieting) societal issues and species-type concerns have not been fully explored. This paper, intended as a corrective, suggests that we take an alternate view of human cloning as both an enhancement and a reproductive technology. This proposed shift in the framework for analysis counters the current narrow framing of the issues and introduces new questions about the prospect of modifying the species.

  20. Tumorigenic lung tumorospheres exhibit stem-like features with significantly increased expression of CD133 and ABCG2

    OpenAIRE

    Zhao, Wensi; Luo, Yi; Li, Boyi; Zhang, Tao

    2016-01-01

    Accumulating evidence supports the existence of cancer stem cells (CSCs) in human tumors, and the successful certification of CSCs may lead to the identification of therapeutic targets, which are more effective for the treatment of cancer. The use of spherical cancer models has increased in popularity in cancer stem cell investigations. Tumorospheres, which are used as a model of CSCs and are established in serum-free medium supplemented with growth factors under non-adherent conditions, are ...

  1. A cancer/testis antigen, NY-SAR-35, induces EpCAM, CD44, and CD133, and activates ERK in HEK293 cells.

    Science.gov (United States)

    Song, Myung-Ha; Kim, Ye-Rin; Bae, Jae-Ho; Shin, Dong-Hoon; Lee, Sang-Yull

    2017-03-04

    The cancer/testis (CT) antigen NY-SAR-35 gene is located on the X chromosome and is aberrantly expressed in various cancers but not in normal tissues, other than testes. Previously, we reported the expression of NY-SAR-35 enhanced cell growth, proliferation, and invasion in HEK293 and cancer cells. To extend understanding of the NY-SAR-35 gene, we used a next generation sequencing (NGS) approach. NY-SAR-35 expression induced growth, proliferation, metastasis, and stemness genes, as indicated by the up-regulations of CXCR4, EpCAM, CD133, and CD44, at the mRNA and protein levels. The expression of NY-SAR-35 in HEK293 cells significantly increased ERK phosphorylation, but not the phosphorylation of AKT. In HEK293/NY-SAR-35 cells, the expressions of pro-apoptotic proteins, including p53, Bax, and p21, were reduced and that of cyclin E was increased. Also, NY-SAR-35 increased the expressions of pluripotency genes (Nanog, Oct-4, and Sox2) and the ability of HEK293 cells to form colonies. Taken together, the present study indicates NY-SAR-35 functions as a CT antigen that triggers oncogenesis and self-renewal.

  2. Human Stem Cell Derived Cardiomyocytes: An Alternative ...

    Science.gov (United States)

    Chemical spills and associated deaths in the US has increased 2.6-fold and 16-fold from 1983 to 2012, respectfully. In addition, the number of chemicals to which humans are exposed to in the environment has increased almost 10-fold from 2001 to 2013 within the US. Internationally, a WHO report on the global composite impact of chemicals on health reported that 16% of the total burden of cardiovascular disease was attributed to environmental chemical exposure with 2.5 million deaths per year. Clearly, the cardiovascular system, at all its various developmental and life stages, represents a critical target organ system that can be adversely affected by existing and emerging chemicals (e.g., engineered nanomaterials) in a variety of environmental media. The ability to assess chemical cardiac risk and safety is critically needed but extremely challenging due to the number and categories of chemicals in commerce, as indicated. This presentation\\session will evaluate the use of adult human stem cell derived cardiomyocytes, and existing platforms, as an alternative model to evaluate environmental chemical cardiac toxicity as well as provide key information for the development of predictive adverse outcomes pathways associated with environmental chemical exposures. (This abstract does not represent EPA policy) Rapid and translatable chemical safety screening models for cardiotoxicity current status for informing regulatory decisions, a workshop sponsored by the Society

  3. Osteopontin and the C-terminal peptide of thrombospondin-4 compete for CD44 binding and have opposite effects on CD133+ cell colony formation

    Directory of Open Access Journals (Sweden)

    Dobocan Monica C

    2009-10-01

    Full Text Available Abstract Background C21, the C-terminal peptide of thrombospondin-4, has growth promoting activity and was discovered as one of several erythropoietin-dependent endothelial proteins. C21 stimulates red cell formation in anemic mice and is a growth factor for CD34+ and CD36+ hematopoietic cells, skin fibroblasts and kidney epithelial cells. ROD1 has been identified as an intracellular mediator. Nothing is known about the existence of putative C21 receptors on plasma membranes of target cells. Findings We analyzed the nature of C21-binding proteins in cell lysates of skin fibroblasts using C21 affinity columns. The membrane receptor CD44 was identified as C21-binding protein by mass spectrometry. We were unable to demonstrate any direct involvement of CD44 on cell growth or the effect of C21 on cell proliferation. A soluble form of CD44 was synthesized in insect cells and purified from culture supernatants with a combination of PVDF filtration in the presence of ammonium sulphate and HPLC. Both osteopontin and hyaluronic acid competitively displaced Biotin-C21 binding to CD44. In a colony-forming assay using primitive CD133+ hematopoietic stem cells from cord blood, osteopontin and C21 had opposite effects and C21 reduced the inhibitory action of osteopontin. Conclusion CD44 is a C21-binding membrane protein. We could not demonstrate an involvement of CD44 in the proliferative action of C21. Nevertheless, based on the antagonism of C21 and osteopontin in hematopoietic precursors, we speculate that C21 could indirectly have a major impact on hematopoietic stem cell proliferation, by hindering osteopontin membrane binding at the level of the bone marrow niche.

  4. Cocktail treatment with EGFR-specific and CD133-specific chimeric antigen receptor-modified T cells in a patient with advanced cholangiocarcinoma

    Directory of Open Access Journals (Sweden)

    Kai-chao Feng

    2017-01-01

    Full Text Available Abstract Background Cholangiocarcinoma (CCA is one of the most fatal malignant tumors with increasing incidence, mortality, and insensitivity to traditional chemo-radiotherapy and targeted therapy. Chimeric antigen receptor-modified T cell (CART immunotherapy represents a novel strategy for the management of many malignancies. However, the potential of CART therapy in treating advanced unresectable/metastatic CCA is uncharted so far. Case presentation In this case, a 52-year-old female who was diagnosed as advanced unresectable/metastatic CCA and resistant to the following chemotherapy and radiotherapy was treated with CART cocktail immunotherapy, which was composed of successive infusions of CART cells targeting epidermal growth factor receptor (EGFR and CD133, respectively. The patient finally achieved an 8.5-month partial response (PR from the CART-EGFR therapy and a 4.5-month-lasting PR from the CART133 treatment. The CART-EGFR cells induced acute infusion-related toxicities such as mild chills, fever, fatigue, vomiting and muscle soreness, and a 9-day duration of delayed lower fever, accompanied by escalation of IL-6 and C reactive protein (CRP, acute increase of glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase, and grade 2 lichen striatus-like skin pathological changes. The CART133 cells induced an intermittent upper abdominal dull pain, chills, fever, and rapidly deteriorative grade 3 systemic subcutaneous hemorrhages and congestive rashes together with serum cytokine release, which needed emergent medical intervention including intravenous methylprednisolone. Conclusions This case suggests that CART cocktail immunotherapy may be feasible for the treatment of CCA as well as other solid malignancies; however, the toxicities, especially the epidermal/endothelial damages, require a further investigation. Trial registration ClinicalTrials.gov NCT01869166 and NCT02541370 .

  5. Alternative Control Technologies: Human Factors Issues

    Science.gov (United States)

    1998-10-01

    vertical vibration to the 29,4, 1980, pp 462-466. head and shoulders of seated men", Royal Aircraft Lee, J. M.; Chartier , V. L.; Hartmann, D. P.; Lee, G...Suarez, P. F., Rogers , S., K., Ruck, D. W., for Effective Human-Computer Interaction", 2nd edition, Arndt, C., and Kabrisky, M., "A facial feature

  6. Increased plasma microRNA and CD133/CK18-positive cancer cells in the pleural fluid of a pancreatic cancer patient with liver and pleural metastases and correlation with chemoresistance.

    Science.gov (United States)

    Ren, Chuanli; Chen, Hui; Han, Chongxu; Wang, Daxin; Fu, Deyuan

    2012-10-01

    We report a case of notably increased plasma levels of microRNA (miR)-21, miR-25, miR-103 and miR-151 in a pancreatic cancer patient with liver and pleural metastases. CD45-coated immunomagnetic beads detected an enrichment of malignant cancer cells in the pleural fluid, and CD133(+)CK18(+) cancer cells were identified. Using computer tomography (CT) combined with cancer cells stained in the pleural fluid, a previously healthy 60-year-old male was diagnosed with pancreatic cancer with multiple liver tumor metastases. Cancer antigen 19-9 (CA19-9), alkaline phosphatase (ALP) and γ-glutamate-transpeptidase (γ-GT) were notably increased in the serum, and carcinoembryonic antigen (CEA) was increased in the pleural fluid. The patient succumbed to the disease three months following standard chemotherapy. The increased levels of plasma miR-21, miR-25, miR-103 and miR-151, as well as the identification of CD133(+)CK18(+) cells in the pleural fluid of a pancreatic cancer patient with liver metastases, may regulate the molecular mechanisms involved in chemoresistance. The patient was insensitive to chemotherapy and succumbed 3 months later. Full elucidation of the molecular and pathological features of pancreatic cancer may be a novel strategy for diagnosis and tailored therapy.

  7. Quercetin induces cell cycle arrest and apoptosis in CD133+ cancer stem cells of human colorectal HT29 cancer cell line and enhances anticancer effects of doxorubicin

    Directory of Open Access Journals (Sweden)

    Shekoufeh Atashpour

    2015-07-01

    Conclusion:The CSCs were a minor population with a significantly high level of drug resistance within the HT29 cancer cells. Quercetin alone exhibited significant cytotoxic effects on HT29 cells and also increased cytoxicity of Dox in combination therapy. Altogether, our data showed that adding quercetin to Dox chemotherapy is an effective strategy for treatment of both CSCs and bulk tumor cells.

  8. Alternative Functional In Vitro Models of Human Intestinal Epithelia

    Directory of Open Access Journals (Sweden)

    Amanda L Kauffman

    2013-07-01

    Full Text Available Physiologically relevant sources of absorptive intestinal epithelial cells are crucial for human drug transport studies. Human adenocarcinoma-derived intestinal cell lines, such as Caco-2, offer conveniences of easy culture maintenance and scalability, but do not fully recapitulate in vivo intestinal phenotypes. Additional sources of renewable physiologically relevant human intestinal cells would provide a much needed tool for drug discovery and intestinal physiology. We sought to evaluate and compare two alternative sources of human intestinal cells, commercially available primary human intestinal epithelial cells (hInEpCs and induced pluripotent stem cell (iPSC-derived intestinal cells to Caco-2, for use in in vitro transwell monolayer intestinal transport assays. To achieve this for iPSC-derived cells, our previously described 3-dimensional intestinal organogenesis method was adapted to transwell differentiation. Intestinal cells were assessed by marker expression through immunocytochemical and mRNA expression analyses, monolayer integrity through Transepithelial Electrical Resistance (TEER measurements and molecule permeability, and functionality by taking advantage the well-characterized intestinal transport mechanisms. In most cases, marker expression for primary hInEpCs and iPSC-derived cells appeared to be as good as or better than Caco-2. Furthermore, transwell monolayers exhibited high TEER with low permeability. Primary hInEpCs showed molecule efflux indicative of P-glycoprotein transport. Primary hInEpCs and iPSC-derived cells also showed neonatal Fc receptor-dependent binding of immunoglobulin G variants. Primary hInEpCs and iPSC-derived intestinal cells exhibit expected marker expression and demonstrate basic functional monolayer formation, similar to or better than Caco-2. These cells could offer an alternative source of human intestinal cells for understanding normal intestinal epithelial physiology and drug transport.

  9. Coding potential of the products of alternative splicing in human.

    KAUST Repository

    Leoni, Guido

    2011-01-20

    BACKGROUND: Analysis of the human genome has revealed that as much as an order of magnitude more of the genomic sequence is transcribed than accounted for by the predicted and characterized genes. A number of these transcripts are alternatively spliced forms of known protein coding genes; however, it is becoming clear that many of them do not necessarily correspond to a functional protein. RESULTS: In this study we analyze alternative splicing isoforms of human gene products that are unambiguously identified by mass spectrometry and compare their properties with those of isoforms of the same genes for which no peptide was found in publicly available mass spectrometry datasets. We analyze them in detail for the presence of uninterrupted functional domains, active sites as well as the plausibility of their predicted structure. We report how well each of these strategies and their combination can correctly identify translated isoforms and derive a lower limit for their specificity, that is, their ability to correctly identify non-translated products. CONCLUSIONS: The most effective strategy for correctly identifying translated products relies on the conservation of active sites, but it can only be applied to a small fraction of isoforms, while a reasonably high coverage, sensitivity and specificity can be achieved by analyzing the presence of non-truncated functional domains. Combining the latter with an assessment of the plausibility of the modeled structure of the isoform increases both coverage and specificity with a moderate cost in terms of sensitivity.

  10. Enrichment of prostate cancer stem-like cells from human prostate cancer cell lines by culture in serum-free medium and chemoradiotherapy.

    Science.gov (United States)

    Wang, Lei; Huang, Xing; Zheng, Xinmin; Wang, Xinghuan; Li, Shiwen; Zhang, Lin; Yang, Zhonghua; Xia, Zhiping

    2013-01-01

    The discovery of rare subpopulations of cancer stem cells (CSCs) has created a new focus in cancer research. As CSCs demonstrate resistance to chemoradiation therapy relative to other cancer cells, this allows the enrichment of CSC populations by killing apoptosis-susceptible cancer cells. In this study, three commonly used human prostate cancer (PCa) cell lines (DU145, PC-3 and LNCaP) were examined for their expression of the putative stem cell markers CD133 and CD44 via flow cytometric analysis. Under normal culture conditions, CD133(+)/CD44(+) cells were only present in the DU145 cell line, and comprised only a minor percentage (0.1% ± 0.01%) of the total population. However, the proportion of these CD133(+)/CD44(+) prostate CSCs could be increased in these cell lines via culture in serum-free medium (SFM), or through chemotherapy or radiotherapy. Indeed, after culture in SFM, the proportion of CD133(+)/CD44(+) cells in DU145 and PC-3 had increased to 10.3% and 3.0%, respectively. Moreover, the proportion had increased to 9.8% enriched by chemotherapy and 3.5% by radiotherapy in DU145. Colony-formation tests, cell invasion assays, and tumor xenografts in BALB/c nude mice were used to evaluate the stem cell properties of CD133(+)/CD44(+) PCa cells that were isolated via fluorescence-activated cell sorting (FACS). CD133(+)/CD44(+) cells had an enhanced colony-formation capability and invasive ability in vitro, and displayed greater tumorigenic properties in vivo. These results demonstrate the presence of CD133(+)/CD44(+) prostate CSCs in established PCa cell lines and that populations of these cells can be enriched by culture in SFM or chemoradiotherapy. Finding novel therapies to override chemoradiation resistance in the prostate CSCs is the key to improve long-term results in PCa management.

  11. 76 FR 50221 - International Workshop on Alternative Methods for Human and Veterinary Rabies Vaccine Testing...

    Science.gov (United States)

    2011-08-12

    ... HUMAN SERVICES International Workshop on Alternative Methods for Human and Veterinary Rabies Vaccine... ``International Workshop on Alternative Methods for Human and Veterinary Rabies Vaccine Testing: State of the... approaches that may reduce, refine, or replace animal use in human and veterinary rabies vaccine...

  12. Molecular heterogeneity and alternative splicing of human lactoperoxidase.

    Science.gov (United States)

    Fragoso, Miryam A; Torbati, Aliza; Fregien, Nevis; Conner, Gregory E

    2009-02-01

    Human lactoperoxidase (LPO) exists as two distinct molecules independent of glycosylation. The N-terminus of one form is blocked and has not been identified while the other is proteolytically processed at the N-terminus similar to myeloperoxidase. Our analysis identified alternatively spliced human LPO mRNAs that may explain the observed molecular heterogeneity of LPO. Two mRNAs omit propeptide encoding exons while retaining the 5' exon encoding the secretion signal, consistent with the heterogeneity and suggesting a possible functional role for the propeptide. Two LPO forms were expressed using baculovirus and both showed similar enzyme activity. LC/MS/MS analysis of trypsin digested, partially purified, salivary LPO confirmed the larger unprocessed LPO is present in saliva. To compare variant expression patterns, antisera were raised against purified recombinant (rhLPO) as well as against an antigenic peptide sequence within the exons encoding the propeptide region. Immunohistochemistry demonstrated proLPO was differently localized within gland cells compared to other forms of LPO. The data suggested splice variants may contribute to LPO molecular heterogeneity and its regulation by intracellular compartmental localization.

  13. Two Domains of Vimentin Are Expressed on the Surface of Lymph Node, Bone and Brain Metastatic Prostate Cancer Lines along with the Putative Stem Cell Marker Proteins CD44 and CD133

    Energy Technology Data Exchange (ETDEWEB)

    Steinmetz, Nicole F. [Case Western Reserve University, Department of Biomedical Engineering, 10900 Euclid Ave, Cleveland, OH 44106 (United States); Maurer, Jochen [Sanford-Burnham, Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037 (United States); Sheng, Huiming [Torrey Pines Institute for Molecular Studies, Division of Immune Regulation, 3550 General Atomics Court, San Diego, CA 92121 (United States); Bensussan, Armand [INSERM U976, Hôpital Saint Louis, F-75475 Paris (France); Department of Immunology, Dermatology and Oncology, Univ Paris Diderot, Sorbonne Paris Cité, UMRS976 F-75475 Paris (France); Maricic, Igor; Kumar, Vipin [Torrey Pines Institute for Molecular Studies, Laboratory of Autoimmunity, 3550 General Atomics Court, San Diego, CA 92121 (United States); Braciak, Todd A., E-mail: tbraciak@tpims.org [Torrey Pines Institute for Molecular Studies, Division of Immune Regulation, 3550 General Atomics Court, San Diego, CA 92121 (United States)

    2011-07-13

    Vimentin was originally identified as an intermediate filament protein present only as an intracellular component in many cell types. However, this protein has now been detected on the surface of a number of different cancer cell types in a punctate distribution pattern. Increased vimentin expression has been indicated as an important step in epithelial-mesenchymal transition (EMT) required for the metastasis of prostate cancer. Here, using two vimentin-specific monoclonal antibodies (SC5 and V9 directed against the coil one rod domain and the C-terminus of the vimentin protein, respectively), we examined whether either of these domains would be displayed on the surface of three commonly studied prostate cancer cell lines isolated from different sites of metastases. Confocal analysis of LNCaP, PC3 and DU145 prostate cancer cell lines (derived from lymph node, bone or brain prostate metastases, respectively) demonstrated that both domains of vimentin are present on the surface of these metastatic cancer cell types. In addition, flow cytometric analysis revealed that vimentin expression was readily detected along with CD44 expression but only a small subpopulation of prostate cancer cells expressed vimentin and the putative stem cell marker CD133 along with CD44. Finally, Cowpea mosaic virus (CPMV) nanoparticles that target vimentin could bind and internalize into tested prostate cancer cell lines. These results demonstrate that at least two domains of vimentin are present on the surface of metastatic prostate cancer cells and suggest that vimentin could provide a useful target for nanoparticle- or antibody- cancer therapeutic agents directed against highly invasive cancer and/or stem cells.

  14. Complement alternative pathway activation in human nonalcoholic steatohepatitis.

    Directory of Open Access Journals (Sweden)

    Filip M Segers

    Full Text Available The innate immune system plays a major role in the pathogenesis of nonalcoholic steatohepatitis (NASH. Recently we reported complement activation in human NASH. However, it remained unclear whether the alternative pathway of complement, which amplifies C3 activation and which is frequently associated with pathological complement activation leading to disease, was involved. Here, alternative pathway components were investigated in liver biopsies of obese subjects with healthy livers (n = 10 or with NASH (n = 12 using quantitative PCR, Western blotting, and immunofluorescence staining. Properdin accumulated in areas where neutrophils surrounded steatotic hepatocytes, and colocalized with the C3 activation product C3c. C3 activation status as expressed by the C3c/native C3 ratio was 2.6-fold higher (p<0.01 in subjects with NASH despite reduced native C3 concentrations (0.94±0.12 vs. 0.57±0.09; p<0.01. Hepatic properdin levels positively correlated with levels of C3c (rs = 0.69; p<0.05 and C3c/C3 activation ratio (rs = 0.59; p<0.05. C3c, C3 activation status (C3c/C3 ratio and properdin levels increased with higher lobular inflammation scores as determined according to the Kleiner classification (C3c: p<0.01, C3c/C3 ratio: p<0.05, properdin: p<0.05. Hepatic mRNA expression of factor B and factor D did not differ between subjects with healthy livers and subjects with NASH (factor B: 1.00±0.19 vs. 0.71±0.07, p = 0.26; factor D: 1.00±0.21 vs. 0.66±0.14, p = 0.29;. Hepatic mRNA and protein levels of Decay Accelerating Factor tended to be increased in subjects with NASH (mRNA: 1.00±0.14 vs. 2.37±0.72; p = 0.22; protein: 0.51±0.11 vs. 1.97±0.67; p = 0.28. In contrast, factor H mRNA was downregulated in patients with NASH (1.00±0.09 vs. 0.71±0.06; p<0.05 and a similar trend was observed with hepatic protein levels (1.12±0.16 vs. 0.78±0.07; p = 0.08. Collectively, these data suggest a role for alternative

  15. Structures of alternatively spliced isoforms of human ketohexokinase.

    Science.gov (United States)

    Trinh, Chi H; Asipu, Aruna; Bonthron, David T; Phillips, Simon E V

    2009-03-01

    A molecular understanding of the unique aspects of dietary fructose metabolism may be the key to understanding and controlling the current epidemic of fructose-related obesity, diabetes and related adverse metabolic states in Western populations. Fructose catabolism is initiated by its phosphorylation to fructose 1-phosphate, which is performed by ketohexokinase (KHK). Here, the crystal structures of the two alternatively spliced isoforms of human ketohexokinase, hepatic KHK-C and the peripheral isoform KHK-A, and of the ternary complex of KHK-A with the substrate fructose and AMP-PNP are reported. The structure of the KHK-A ternary complex revealed an active site with both the substrate fructose and the ATP analogue in positions ready for phosphorylation following a reaction mechanism similar to that of the pfkB family of carbohydrate kinases. Hepatic KHK deficiency causes the benign disorder essential fructosuria. The effects of the disease-causing mutations (Gly40Arg and Ala43Thr) have been modelled in the context of the KHK structure.

  16. Expression and alternative splicing pattern of human telomerase reverse transcriptase in human lung cancer cells.

    Science.gov (United States)

    Fujiwara, Masachika; Kamma, Hiroshi; Wu, Wenwen; Hamasaki, Makoto; Kaneko, Setsuko; Horiguchi, Hisashi; Matsui-Horiguchi, Miwa; Satoh, Hiroaki

    2004-04-01

    Telomerase activity is generally considered to be necessary for cancer cells to avoid senescence. The expression of human telomerase reverse transcriptase (hTERT) is believed to be a rate-limiting step in telomerase activation. Recently, it has been proposed that the alternative splicing of hTERT is also involved in regulation of telomerase activity. However, the regulatory mechanism of telomerase in cancer cells has not been thoroughly investigated. To clarify it in lung cancer cells, we measured the expression of the hTERT transcript, analyzed its alternative splicing by RT-PCR, and compared it with telomerase activity and telomere length. The expression of the hTERT transcript was positively correlated with telomerase activity in lung cancer cells. Cancer cells with high telomerase activity contained 4 splicing variants of hTERT, and the full-length variant was 31.3-54.2% of the total transcripts. Cells of the TKB-20 cell line, which has extremely low telomerase activity, showed a different splicing pattern of hTERT in addition to low expression. The functional full-length variant was scarcely detected in TKB-20 cells, suggesting that the telomerase activity was repressed by alternative splicing of hTERT. Telomere length was not necessarily correlated with telomerase activity or hTERT expression in lung cancer cells. Cells of the TKB-4 cell line that also showed relatively low telomerase activity (as TKB-20 cells) had long telomeres. In conclusion, hTERT expression is regulated at both the transcriptional and post-transcriptional levels in lung cancer cells, and the alternative splicing of hTERT is involved in the control of telomerase activity.

  17. SSEA-1 is an enrichment marker for tumor-initiating cells in human glioblastoma.

    Science.gov (United States)

    Son, Myung Jin; Woolard, Kevin; Nam, Do-Hyun; Lee, Jeongwu; Fine, Howard A

    2009-05-08

    CD133+ populations of human glioblastoma multiforme (GBM) cells are reportedly enriched for tumor stem cells (TSCs) or tumor-initiating cells (TICs). Approximately 40% of freshly isolated GBM specimens, however, do not contain CD133+ tumor cells, raising the possibility that CD133 may not be a universal enrichment marker for GBM TSCs/TICs. Here we demonstrate that stage-specific embryonic antigen 1(SSEA-1/LeX)+ GBM cells fulfill the functional criteria for TSC/TIC, since (1) SSEA-1+ cells are highly tumorigenic in vivo, unlike SSEA-1- cells; (2) SSEA-1+ cells can give rise to both SSEA-1+ and SSEA-1- cells, thereby establishing a cellular hierarchy; and (3) SSEA-1+ cells have self-renewal and multilineage differentiation potentials. A distinct subpopulation of SSEA-1+ cells was present in all but one of the primary GBMs examined (n = 24), and most CD133+ tumor cells were also SSEA-1+, suggesting that SSEA-1 may be a general TSC/TIC enrichment marker in human GBMs.

  18. Alternative splice variants of the human PD-1 gene

    DEFF Research Database (Denmark)

    Nielsen, Christian; Ohm-Laursen, Line; Barington, Torben;

    2005-01-01

    PD-1 is an immunoregulatory receptor expressed on the surface of activated T cells, B cells, and monocytes. We describe four alternatively spliced PD-1 mRNA transcripts (PD-1Deltaex2, PD-1Deltaex3, PD-1Deltaex2,3, and PD-1Deltaex2,3,4) in addition to the full length isoform. PD-1Deltaex2 and PD-1......Deltaex3 are generated by alternative splicing where exon 2 (extracellular IgV-like domain) and exon 3 (transmembrane domain) respectively are spliced out. PD-1Deltaex3 is therefore likely to encode a soluble form of PD-1. PD-1Deltaex2,3 lacks exon 2 and 3. These three variants have unaffected open...

  19. Democratising Democracy, Humanising Human Rights. European Decolonial Movements and the “Alternative Thinking of Alternatives"

    DEFF Research Database (Denmark)

    Suárez-Krabbe, Julia

    2013-01-01

    des Indigènes de la République (PIR) in France, the Dutch Black Movement, the Islamic Human Rights Commission in the UK, and the Studies Group of the Andalusian Workers' Union (Grupo de Estudios - Sindicato Andaluz de Trabajadores; GE-SAT). These movements all point to two fundamental crises of longue...

  20. Alternative splicing of cyclooxygenase-1 mRNA in the human iris

    NARCIS (Netherlands)

    Dröge, M.J; van Sorge, A.A; van Haeringen, N.J; Quax, Wim; Zaagsma, Hans; Droge, MJ

    2003-01-01

    dIn homogenates of the human iris, the nonsteroidal antiinflammatory drug (NSAID) S(+)flurbiprofen has been reported to inhibit cyclooxygenase-1 (COX-1) 70-fold more potently than in human whole blood. We hypothesized that this difference may be due to alternative splicing of COX-1 mRNA in the human

  1. Alternative Responses to the Human Resource Challenge for CBR

    Directory of Open Access Journals (Sweden)

    Huib Cornielje

    2013-02-01

    Full Text Available This commentary outlines some ways of understanding CBR and offers corresponding suggestions for responding to the contemporary human resource challenge it is faced with. It is argued that CBR exists within an increasingly complex reality, characterised by new challenges, new approaches to development and numerous international principles and guidelines.  In response, the authors advocate the use of multiple research methods, participatory action and contextualised ways of addressing human resource issues.  They suggest that new understandings are required, for future CBR workers to be enablers of people with disabilities, agents of change in communities and societies, and champions of human rights.  The complex reality of CBR suggests the need for a CBR cadre which is capable of creative and reflective reasoning.  This might be achieved through the participatory development of contextualised training curricula, practical hands-on learning, the use of mentoring, and an emphasis on reflection and adaptability.

  2. Beyond membrane channelopathies: alternative mechanisms underlying complex human disease

    Institute of Scientific and Technical Information of China (English)

    Konstantinos Dean BOUDOULAS; Peter J MOHLER

    2011-01-01

    Over the past fifteen years, our understanding of the molecular mechanisms underlying human disease has flourished in large part due to the discovery of gene mutations linked with membrane ion channels and transporters. In fact, ion channel defects ("channelopathies" - the focus of this review series) have been associated with a spectrum of serious human disease phenotypes including cystic fibrosis, cardiac arrhythmia, diabetes, skeletal muscle defects, and neurological disorders. However, we now know that human disease, particularly excitable cell disease, may be caused by defects in non-ion channel polypeptides including in cellular components residing well beneath the plasma membrane. For example, over the past few years, a new class of potentially fatal cardiac arrhythmias has been linked with cytoplasmic proteins that include sub-membrane adapters such as ankyrin-B (ANK2),ankyrin-G (ANK3), and alpha-1 syntrophin, membrane coat proteins including caveolin-3 (CAV3), signaling platforms including yotiao (AKAPg), and cardiac enzymes (GPD1L). The focus of this review is to detail the exciting role of lamins, yet another class of gene products that have provided elegant new insight into human disease.

  3. Humanity at the crossroads: does sri aurobindo offer an alternative?

    Science.gov (United States)

    Singh, Shakuntala A; Singh, Ajai R

    2009-01-01

    In the light of Sri Aurobindo's philosophy, this paper looks into some of the problems of contemporary man as an individual, a member of society, a citizen of his country, a component of this world, and of nature itself. Concepts like Science; Nature,;Matter; Mental Being; Mana-purusa; Prana-purusa; Citta-purusa; Nation-ego and Nation-soul; True and False Subjectivism; World-state and World-union; Religion of Humanism are the focus of this paper. NATURE: Beneath the diversity and uniqueness of the different elements in Nature there is an essential unity that not only allows for this diversity but even supports it. Nature is both a benefactor and a force: a benefactor, because it acts to carry out the evolution of mankind; a force, because it also supplies the necessary energy and momentum to achieve it. NATURAL CALAMITIES: If mankind can quieten the tsunamis and cyclones and droughts and earthquakes that rage within, and behave with care and compassion towards Nature, not exploiting, denuding, or denigrating it, there is a strong possibility that Nature too will behave with equal care and compassion towards man and spare him the natural calamities than rend him asunder. SCIENCE: The limitation of science become obvious, according to Sri Aurobindo, when we realize that it has mastered knowledge of processes and helped in the creation of machinery but is ignorant of the foundations of being and, therefore, cannot perfect our nature or our life. SCIENCE AND PHILOSOPHY: The insights of philosophy could become heuristic and algorithmic models for scientific experimentation. MATTER: An interesting aspect of Sri Aurobindo's philosophy is his acceptance of the reality of matter even while highlighting its inadequacies; the ultimate goal, according to him, is the divination of matter itself. PURUSA: If the mana-purusa (mental being) were to log on to the genuine citta-purusa (psychic formation), without necessarily logging off from the prana-purusa (frontal formation), it

  4. Humanity at the Crossroads: Does Sri Aurobindo offer an alternative?

    Directory of Open Access Journals (Sweden)

    Singh Shakuntala

    2009-01-01

    Full Text Available In the light of Sri Aurobindo′s philosophy, this paper looks into some of the problems of contemporary man as an individual, a member of society, a citizen of his country, a component of this world, and of nature itself. Concepts like Science; Nature,;Matter; Mental Being; Mana-purusa; Prana-purusa; Citta-purusa; Nation-ego and Nation-soul; True and False Subjectivism; World-state and World-union; Religion of Humanism are the focus of this paper. Nature: Beneath the diversity and uniqueness of the different elements in Nature there is an essential unity that not only allows for this diversity but even supports it. Nature is both a benefactor and a force: a benefactor, because it acts to carry out the evolution of mankind; a force, because it also supplies the necessary energy and momentum to achieve it. Natural calamities: If mankind can quieten the tsunamis and cyclones and droughts and earthquakes that rage within, and behave with care and compassion towards Nature, not exploiting, denuding, or denigrating it, there is a strong possibility that Nature too will behave with equal care and compassion towards man and spare him the natural calamities than rend him asunder. Science: The limitation of science become obvious, according to Sri Aurobindo, when we realize that it has mastered knowledge of processes and helped in the creation of machinery but is ignorant of the foundations of being and, therefore, cannot perfect our nature or our life. Science and philosophy: The insights of philosophy could become heuristic and algorithmic models for scientific experimentation. Matter: An interesting aspect of Sri Aurobindo′s philosophy is his acceptance of the reality of matter even while highlighting its inadequacies; the ultimate goal, according to him, is the divination of matter itself. Purusa: If the mana-purusa (mental being were to log on to the genuine citta-purusa (psychic formation, without necessarily logging off from the prana

  5. Human cell lines: A promising alternative for recombinant FIX production.

    Science.gov (United States)

    de Sousa Bomfim, Aline; Cristina Corrêa de Freitas, Marcela; Picanço-Castro, Virgínia; de Abreu Soares Neto, Mário; Swiech, Kamilla; Tadeu Covas, Dimas; Maria de Sousa Russo, Elisa

    2016-05-01

    Factor IX (FIX) is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the Hemophilia B treatment. We evaluated the potential of recombinant human FIX (rhFIX) expression in 293T and SK-Hep-1 human cell lines. SK-Hep-1-FIX cells produced higher levels of biologically active protein. The growth profile of 293T-FIX cells was not influenced by lentiviral integration number into the cellular genome. SK-Hep-1-FIX cells showed a significantly lower growth rate than SK-Hep-1 cells. γ-carboxylation process is significant to FIX biological activity, thus we performed a expression analysis of genes involved in this process. The 293T gene expression suggests that this cell line could efficiently carboxylate FIX, however only 28% of the total secreted protein is active. SK-Hep-1 cells did not express high amounts of VKORC1 and carboxylase, but this cell line secreted large amounts of active protein. Enrichment of culture medium with Ca(+2) and Mg(+2) ions did not affect positively rhFIX expression in SK-Hep-1 cells. In 293T cells, the addition of 0.5 mM Ca(+2) and 1 mM Mg(+2) resulted in higher rhFIX concentration. SK-Hep-1 cell line proved to be very effective in rhFIX production, and it can be used as a novel biotechnological platform for the production of recombinant proteins.

  6. Genetic control of the alternative pathway of complement in humans and age-related macular degeneration.

    Science.gov (United States)

    Hecker, Laura A; Edwards, Albert O; Ryu, Euijung; Tosakulwong, Nirubol; Baratz, Keith H; Brown, William L; Charbel Issa, Peter; Scholl, Hendrik P; Pollok-Kopp, Beatrix; Schmid-Kubista, Katharina E; Bailey, Kent R; Oppermann, Martin

    2010-01-01

    Activation of the alternative pathway of complement is implicated in common neurodegenerative diseases including age-related macular degeneration (AMD). We explored the impact of common variation in genes encoding proteins of the alternative pathway on complement activation in human blood and in AMD. Genetic variation across the genes encoding complement factor H (CFH), factor B (CFB) and component 3 (C3) was determined. The influence of common haplotypes defining transcriptional and translational units on complement activation in blood was determined in a quantitative genomic association study. Individual haplotypes in CFH and CFB were associated with distinct and novel effects on plasma levels of precursors, regulators and activation products of the alternative pathway of complement in human blood. Further, genetic variation in CFH thought to influence cell surface regulation of complement did not alter plasma complement levels in human blood. Plasma markers of chronic activation (split-products Ba and C3d) and an activating enzyme (factor D) were elevated in AMD subjects. Most of the elevation in AMD was accounted for by the genetic variation controlling complement activation in human blood. Activation of the alternative pathway of complement in blood is under genetic control and increases with age. The genetic variation associated with increased activation of complement in human blood also increased the risk of AMD. Our data are consistent with a disease model in which genetic variation in the complement system increases the risk of AMD by a combination of systemic complement activation and abnormal regulation of complement activation in local tissues.

  7. Novel anti-apoptotic microRNAs 582-5p and 363 promote human glioblastoma stem cell survival via direct inhibition of caspase 3, caspase 9, and Bim.

    Directory of Open Access Journals (Sweden)

    Desiree Hunt Floyd

    Full Text Available Glioblastoma is the most common and lethal primary brain tumor. Tumor initiation and recurrence are likely caused by a sub-population of glioblastoma stem cells, which may derive from mutated neural stem and precursor cells. Since CD133 is a stem cell marker for both normal brain and glioblastoma, and to better understand glioblastoma formation and recurrence, we looked for dys-regulated microRNAs in human CD133+ glioblastoma stem cells as opposed to CD133+ neural stem cells isolated from normal human brain. Using FACS sorting of low-passage cell samples followed by microRNA microarray analysis, we found 43 microRNAs that were dys-regulated in common in three separate CD133+ human glioblastomas compared to CD133+ normal neural stem cells. Among these were several microRNAs not previously associated with cancer. We then verified the microRNAs dys-regulated in glioblastoma using quantitative real time PCR and Taqman analysis of the original samples, as well as human GBM stem cell and established cell lines and many human specimens. We show that two candidate oncogenic microRNAs, miR-363 and miR-582-5p, can positively influence glioblastoma survival, as shown by forced expression of the microRNAs and their inhibitors followed by cell number assay, Caspase 3/7 assay, Annexin V apoptosis/fluorescence activated cell sorting, siRNA rescue of microRNA inhibitor treatment, as well as 3'UTR mutagenesis to show luciferase reporter rescue of the most successful targets. miR-582-5p and miR-363 are shown to directly target Caspase 3, Caspase 9, and Bim.

  8. Cat dissection and human cadaver prosection versus sculpting human structures from clay: A comparison of alternate approaches to human anatomy laboratory education

    Science.gov (United States)

    Waters, John R.

    Dissection and vivisection are traditional approaches to biology laboratory education. In the case of human anatomy teaching laboratories, there is a long tradition of using human and animal cadaver specimens in the classroom. In a review of the literature comparing traditional dissection and vivisection lessons to alternative lessons designed to reduce the time spent dissecting or the numbers of animals used, we conclude that it is difficult to come to any conclusion regarding the efficacy of different approaches. An analysis of the literature is confounded because many studies have very low statistical power or other methodological weaknesses, and investigators rely on a wide variety of testing instruments to measure an equally varied number of course objectives. Additional well designed studies are necessary before educators can reach any informed conclusions about the efficacy of traditional versus alternative approaches to laboratory education. In our experiments, we compared a traditional cat dissection based undergraduate human anatomy lesson to an alternative where students sculpted human muscles onto plastic human skeletons. Students in the alternative treatment performed significantly better than their peers in the traditional treatment when answering both lower and higher order human anatomy questions. In a subsequent experiment with a similar design, we concluded that the superior performance of the students in the alternative treatment on anatomy exams was likely due to the similarity between the human anatomy representation studied in lab, and the human anatomy questions asked on the exams. When the anatomy questions were presented in the context of a cat specimen, students in the traditional cat dissection treatment outperformed their peers in the alternative treatment. In a final experiment where student performance on a human anatomy exam was compared between a traditional prosected human cadaver treatment and the alternative clay sculpting

  9. Investigation of tissue-specific human orthologous alternative splice events in pig

    DEFF Research Database (Denmark)

    Hillig, Ann-Britt Nygaard; Jørgensen, Claus Bøttcher; Salicio, Susanna Cirera;

    2010-01-01

    Alternative splicing of pre-mRNA can contribute to differences between tissues or cells either by regulating gene expression or creating proteins with various functions encoded by one gene. The number of investigated alternative splice events in pig has so far been limited. In this study we have...... investigated alternative splice events detected in humans, in orthologous pig genes. A total of 17 genes with predicted exon skipping events were selected for further studies. The splice events for the selected genes were experimentally verified using real-time quantitative PCR analysis (qPCR) with splice......-specific primers in 19 different tissues. The same splice variants as reported in humans were detected in 15 orthologous pig genes, however, the expression pattern predicted in the in silico analyses was only experimentally verified in a few cases. The results support the findings that splice events resulting...

  10. A subtle alternative splicing event gives rise to a widely expressed human RNase k isoform.

    Directory of Open Access Journals (Sweden)

    Evangelos D Karousis

    Full Text Available Subtle alternative splicing leads to the formation of RNA variants lacking or including a small number of nucleotides. To date, the impact of subtle alternative splicing phenomena on protein biosynthesis has been studied in frame-preserving incidents. On the contrary, mRNA isoforms derived from frame-shifting events were poorly studied and generally characterized as non-coding. This work provides evidence for a frame-shifting subtle alternative splicing event which results in the production of a novel protein isoform. We applied a combined molecular approach for the cloning and expression analysis of a human RNase κ transcript (RNase κ-02 which lacks four consecutive bases compared to the previously isolated RNase κ isoform. RNase κ-02 mRNA is expressed in all human cell lines tested end encodes the synthesis of a 134-amino-acid protein by utilizing an alternative initiation codon. The expression of RNase κ-02 in the cytoplasm of human cells was verified by Western blot and immunofluorescence analysis using a specific polyclonal antibody developed on the basis of the amino-acid sequence difference between the two protein isoforms. The results presented here show that subtle changes during mRNA splicing can lead to the expression of significantly altered protein isoforms.

  11. Divergent Expression and Metabolic Functions of Human Glucuronosyltransferases through Alternative Splicing.

    Science.gov (United States)

    Rouleau, Michèle; Tourancheau, Alan; Girard-Bock, Camille; Villeneuve, Lyne; Vaucher, Jonathan; Duperré, Anne-Marie; Audet-Delage, Yannick; Gilbert, Isabelle; Popa, Ion; Droit, Arnaud; Guillemette, Chantal

    2016-09-27

    Maintenance of cellular homeostasis and xenobiotic detoxification is mediated by 19 human UDP-glucuronosyltransferase enzymes (UGTs) encoded by ten genes that comprise the glucuronidation pathway. Deep RNA sequencing of major metabolic organs exposes a substantial expansion of the UGT transcriptome by alternative splicing, with variants representing 20% to 60% of canonical transcript expression. Nearly a fifth of expressed variants comprise in-frame sequences that may create distinct structural and functional features. Follow-up cell-based assays reveal biological functions for these alternative UGT proteins. Some isoforms were found to inhibit or induce inactivation of drugs and steroids in addition to perturbing global cell metabolism (energy, amino acids, nucleotides), cell adhesion, and proliferation. This work highlights the biological relevance of alternative UGT expression, which we propose increases protein diversity through the evolution of metabolic regulators from specific enzymes.

  12. An Alternate Splicing Variant of the Human Telomerase Catalytic Subunit Inhibits Telomerase Activity

    Directory of Open Access Journals (Sweden)

    Xiaoming Yi

    2000-09-01

    Full Text Available Telomerase, a cellular reverse transcriptase, adds telomeric repeats to chromosome ends. In normal human somatic cells, telomerase is repressed and telomeres progressively shorten, leading to proliferative senescence. Introduction of the telomerase (hTERT cDNA is sufficient to produce telomerase activity and immortalize normal human cells, suggesting that the repression of telomerase activity is transcriptional. The telomerase transcript has been shown to have at least six alternate splicing sites (four insertion sites and two deletion sites, and variants containing both or either of the deletion sites are present during development and in a panel of cancer cell lines we surveyed. One deletion (β site and all four insertions cause premature translation terminations, whereas the other deletion (α site is 36 by and lies within reverse transcriptase (RT motif A, suggesting that this deletion variant may be a candidate as a dominant-negative inhibitor of telomerase. We have cloned three alternately spliced hTERT variants that contain the α,β or both α and,β deletion sites. These alternate splicing variants along with empty vector and wild-type hTERT were introduced into normal human fibroblasts and several telomerase-positive immortal and tumor cell lines. Expression of the α site deletion variant (hTERT α− construct was confirmed by Western blotting. We found that none of the three alternate splicing variants reconstitutes telomerase activity in fibroblasts. However, hTERT α− inhibits telomerase activities in telomerase-positive cells, causes telomere shortening and eventually cell death. This alternately spliced dominant-negative variant may be important in understanding telomerase regulation during development, differentiation and in cancer progression.

  13. Gene expression in human skeletal muscle: alternative normalization method and effect of repeated biopsies

    DEFF Research Database (Denmark)

    Lundby, Carsten; Nordsborg, Nikolai; Kusuhara, K.

    2005-01-01

    . Specifically, we investigated (1) a new normalization method based on determining the cDNA content by the flourophores PicoGreen and OliGreen, (2) effect of repeated muscle biopsies on mRNA gene expression, and (3) the spatial heterogeneity in mRNA expression across the muscle. Standard curves using oligo.......99. The present determination of cDNA content in reversed transcribed human skeletal muscle RNA samples by both PicoGreen and OliGreen analyses suggests that these fluorophores provide a potential alternative normalization procedure for human gene expression studies. In addition, the present study shows...

  14. The Real Problem (Humans) and Some Potentially Effective Alternatives and New Tools

    Energy Technology Data Exchange (ETDEWEB)

    Hanlon, Edward; Capece, John

    2011-01-07

    The presentation offers a wider perspective, where on the example of Everglades’ restoration efforts, the real problem – humans – is exposed. Some potentially effective alternatives and new tools are offered and discussed. Everglades’s problems are primarily caused to human activity, whether these are related to poor water quality, land use changes or cheap short-sighted fixes. Ground rules need to be set in order to have a real discussion and seek real solutions to such problems. The difference between facts and opinions is explained, so is the difference between interests and positions. These terms are often partly coinciding in human minds, thus causing further misunderstandings in pursuing real solutions. Some confounding factors in the Everglades restoration efforts are listed, with one of the main examples being the assignment of a monetary value to all factors. Some of these factors are priceless, but not being valued easily and properly (such as water). The proposed alternative to monetary value is assigning energy as the unit of measurement. The two main methods for such alternate approach are (1) Embodied energy (Emergy) and (2) Life Cycle Analysis. Agriculturally-based ecosystems services are different from natural ecosystems services. Pursuing agriculturally-based ecosystems services (such as e.g. water storage on farms, reducing nutrients in water, using flood-tolerant crops cultivars, etc.) can develop eco-services into agricultural operations and systems, addressing everyone’s interest and benefiting the society.

  15. The effect of opponent type on human performance in a three-alternative choice task.

    Science.gov (United States)

    Lie, Celia; Baxter, Jennifer; Alsop, Brent

    2013-10-01

    Adult participants played computerised games of "Paper Scissors Rock". Participants in one group were told that they were playing against the computer, and those in the other group were told that they were playing against another participant in the adjacent room. The participant who won the most games would receive a $50 prize. For both groups however, the opponent's responses (paper, scissors, or rock) were generated by the computer, and the distribution of these responses was varied across four blocks of 126 trials. Results were analysed using the generalised matching law for the three possible pairs of alternatives (paper vs. scissors, paper vs. rock, and scissors vs. rock) across all participants in each group. Overall, significantly higher estimates of sensitivity to the distribution of opponent's responses were obtained from participants who were told their opponent was a computer compared to participants who were told their opponent was another participant. While adding to the existing literature showing that the generalised matching law is an adequate descriptor of human three-alternative choice behaviour, these findings show that external factors such as perceived opponent type can affect the efficacy of reinforcer contingencies on human behaviour. This suggests that generalising the results from tasks performed against a computer to real-life human-to-human interactions warrants some caution.

  16. Alternative methods for the use of non-human primates in biomedical research.

    Science.gov (United States)

    Burm, Saskia M; Prins, Jan-Bas; Langermans, Jan; Bajramovic, Jeffrey J

    2014-01-01

    The experimental use of non-human primates (NHP) in Europe is tightly regulated and is only permitted when there are no alternatives available. As a result, NHP are most often used in late, pre-clinical phases of biomedical research. Although the impetus for scientists, politicians and the general public to replace, reduce and refine NHP in biomedical research is strong, the development of 3Rs technology for NHP poses specific challenges. In February 2014 a workshop on "Alternative methods for the use of NHP in biomedical research" was organized within the international exchange program of EUPRIM-Net II, a European infrastructure initiative that links biomedical primate research centers. The workshop included lectures by key scientists in the field of alternatives as well as by experts from governmental and non-governmental organizations. Furthermore, parallel sessions were organized to stimulate discussion on the challenges of advancing the use of alternative methods for NHP. Subgroups voted on four statements and together composed a list with opportunities and priorities. This report summarizes the presentations that were held, the content of the discussion sessions and concludes with recommendations on 3Rs development for NHP specifically. These include technical, conceptual as well as political topics.

  17. Comprehensive polyadenylation site maps in yeast and human reveal pervasive alternative polyadenylation.

    Science.gov (United States)

    Ozsolak, Fatih; Kapranov, Philipp; Foissac, Sylvain; Kim, Sang Woo; Fishilevich, Elane; Monaghan, A Paula; John, Bino; Milos, Patrice M

    2010-12-10

    The emerging discoveries on the link between polyadenylation and disease states underline the need to fully characterize genome-wide polyadenylation states. Here, we report comprehensive maps of global polyadenylation events in human and yeast generated using refinements to the Direct RNA Sequencing technology. This direct approach provides a quantitative view of genome-wide polyadenylation states in a strand-specific manner and requires only attomole RNA quantities. The polyadenylation profiles revealed an abundance of unannotated polyadenylation sites, alternative polyadenylation patterns, and regulatory element-associated poly(A)(+) RNAs. We observed differences in sequence composition surrounding canonical and noncanonical human polyadenylation sites, suggesting novel noncoding RNA-specific polyadenylation mechanisms in humans. Furthermore, we observed the correlation level between sense and antisense transcripts to depend on gene expression levels, supporting the view that overlapping transcription from opposite strands may play a regulatory role. Our data provide a comprehensive view of the polyadenylation state and overlapping transcription.

  18. Alternative spliced variants in the pantetheinase family of genes expressed in human neutrophils.

    Science.gov (United States)

    Nitto, Takeaki; Inoue, Teruo; Node, Koichi

    2008-12-15

    Pantetheinase (EC 3.5.1.92) is an enzyme that hydrolyzes pantetheine, an intermediate metabolite of coenzyme A, into pantothenic acid (vitamin B(5)) and cysteamine, a potent antioxidant. The pantetheinase gene family consists of three independent genes, pantetheinase/vanin-1/VNN1, GPI-80/VNN2 and vanin-3/VNN3 that are each composed of seven exons. We herein report that human neutrophils express transcripts encoding at least nine splice variants of VNN3 and four splice variants of GPI-80/VNN2. Analysis of the DNA sequence of the human VNN3 gene demonstrated that the VNN3 locus in the human genome as well as the sequence of cDNA clones obtained in this study does not encode the complete VNN3 protein, as previously reported due to a frame shift caused by lack of one nucleotide. Moreover, the VNN3 locus indeed encodes smaller peptides compared to the proteins encoded by the mouse orthologous gene, vanin-3. The anti-GPI-80 monoclonal antibody 3H9 recognized amino acids 120-179 of the GPI-80/VNN2 protein as shown by the results of immunoblotting with recombinant GPI-80/VNN2 variant proteins. Immunoblotting with human neutrophil lysate suggests that the GPI-80/VNN2 variants exist in human neutrophils. The existence of splice variants in the pantetheinase gene family suggests the possibility of alternative roles in addition to canonical enzymatic activity in human neutrophils.

  19. Histopathological Analogies in Chronic Pulmonary Lesions between Cattle and Humans: Basis for an Alternative Animal Model

    Science.gov (United States)

    Ramírez-Romero, Rafael; Nevárez-Garza, Alicia M.; Rodríguez-Tovar, Luis E.; Wong-González, Alfredo; Ledezma-Torres, Rogelio A.; Hernández-Vidal, Gustavo

    2012-01-01

    Most of the natural cases of pneumonia in feedlot cattle are characterized by a longer clinical course due to chronic lung lesions. Microscopically, these lesions include interstitial fibroplasia, bronchitis, bronchiectasis, bronchiolitis obliterans, and epithelial metaplasia of the airways. Herein, the aim was to review, under a medical perspective, the pathologic mechanisms operating in these chronic pneumonic lesions in calves. Based on the similarities of these changes to those reported in bronchiolitis obliterans/organising pneumonia (BO/OP) and chronic obstructive pulmonary disease (COPD) in human beings, calves are proposed as an alternative animal model. PMID:22629176

  20. Histopathological Analogies in Chronic Pulmonary Lesions between Cattle and Humans: Basis for an Alternative Animal Model

    Directory of Open Access Journals (Sweden)

    Rafael Ramírez-Romero

    2012-01-01

    Full Text Available Most of the natural cases of pneumonia in feedlot cattle are characterized by a longer clinical course due to chronic lung lesions. Microscopically, these lesions include interstitial fibroplasia, bronchitis, bronchiectasis, bronchiolitis obliterans, and epithelial metaplasia of the airways. Herein, the aim was to review, under a medical perspective, the pathologic mechanisms operating in these chronic pneumonic lesions in calves. Based on the similarities of these changes to those reported in bronchiolitis obliterans/organising pneumonia (BO/OP and chronic obstructive pulmonary disease (COPD in human beings, calves are proposed as an alternative animal model.

  1. Genome-wide analysis of alternative splicing during human heart development

    Science.gov (United States)

    Wang, He; Chen, Yanmei; Li, Xinzhong; Chen, Guojun; Zhong, Lintao; Chen, Gangbing; Liao, Yulin; Liao, Wangjun; Bin, Jianping

    2016-10-01

    Alternative splicing (AS) drives determinative changes during mouse heart development. Recent high-throughput technological advancements have facilitated genome-wide AS, while its analysis in human foetal heart transition to the adult stage has not been reported. Here, we present a high-resolution global analysis of AS transitions between human foetal and adult hearts. RNA-sequencing data showed extensive AS transitions occurred between human foetal and adult hearts, and AS events occurred more frequently in protein-coding genes than in long non-coding RNA (lncRNA). A significant difference of AS patterns was found between foetal and adult hearts. The predicted difference in AS events was further confirmed using quantitative reverse transcription-polymerase chain reaction analysis of human heart samples. Functional foetal-specific AS event analysis showed enrichment associated with cell proliferation-related pathways including cell cycle, whereas adult-specific AS events were associated with protein synthesis. Furthermore, 42.6% of foetal-specific AS events showed significant changes in gene expression levels between foetal and adult hearts. Genes exhibiting both foetal-specific AS and differential expression were highly enriched in cell cycle-associated functions. In conclusion, we provided a genome-wide profiling of AS transitions between foetal and adult hearts and proposed that AS transitions and deferential gene expression may play determinative roles in human heart development.

  2. Genome-wide analysis of alternative splicing during human heart development

    Science.gov (United States)

    Wang, He; Chen, Yanmei; Li, Xinzhong; Chen, Guojun; Zhong, Lintao; Chen, Gangbing; Liao, Yulin; Liao, Wangjun; Bin, Jianping

    2016-01-01

    Alternative splicing (AS) drives determinative changes during mouse heart development. Recent high-throughput technological advancements have facilitated genome-wide AS, while its analysis in human foetal heart transition to the adult stage has not been reported. Here, we present a high-resolution global analysis of AS transitions between human foetal and adult hearts. RNA-sequencing data showed extensive AS transitions occurred between human foetal and adult hearts, and AS events occurred more frequently in protein-coding genes than in long non-coding RNA (lncRNA). A significant difference of AS patterns was found between foetal and adult hearts. The predicted difference in AS events was further confirmed using quantitative reverse transcription-polymerase chain reaction analysis of human heart samples. Functional foetal-specific AS event analysis showed enrichment associated with cell proliferation-related pathways including cell cycle, whereas adult-specific AS events were associated with protein synthesis. Furthermore, 42.6% of foetal-specific AS events showed significant changes in gene expression levels between foetal and adult hearts. Genes exhibiting both foetal-specific AS and differential expression were highly enriched in cell cycle-associated functions. In conclusion, we provided a genome-wide profiling of AS transitions between foetal and adult hearts and proposed that AS transitions and deferential gene expression may play determinative roles in human heart development. PMID:27752099

  3. Losses of Humanity in Times of War: The Actions of Alternative Subjects of Justice

    Directory of Open Access Journals (Sweden)

    Julia Estela Monárrez

    2014-12-01

    Full Text Available This article discusses loss of humanity due to violence in Ciudad Juarez (2008–2014 and the actions of alternative subjects of justice – the organized civil society – seeking to address it. This paper resonates with theoretical currents of feminism and humanism, both of which have created a critical apparatus for thinking about social inequality in the context of life, death, and injustice. The discussion draws on the theoretical concepts of discourse societies, necropolitics, private government and actions. With this theoretical structure, the paper seeks to understand the political actions of eight civil society organizations aiming to recover the right to the body, to space and to be a political subject for a community shattered by violence. The paper argues that, through these actions, they helped to prevent crime, enhance public safety and stabilise a society suffering from continued violence due in large part to the war on drugs.

  4. Modulation of Bcl-x Alternative Splicing Induces Apoptosis of Human Hepatic Stellate Cells

    Directory of Open Access Journals (Sweden)

    Lin Wu

    2016-01-01

    Full Text Available Liver fibrosis is a major cause of morbidity and mortality worldwide due to chronic viral hepatitis and, more recently, from fatty liver diseases. Activation and proliferation of hepatic stellate cells (HSCs represent a key aspect of fibrogenesis and are associated with progressive reduction of HSC apoptosis. Bcl-x, an antiapoptotic member of Bcl-2 gene family, plays a role in apoptosis regulation in mammalian cells. Through alternative splicing, the Bcl-x gene yields two major protein isoforms with opposing functions, antiapoptotic Bcl-xL and proapoptotic Bcl-xS. This study aimed to investigate the role of Bcl-x and its alternate splicing in HSC apoptosis. The results indicated that the expression of Bcl-xL was dramatically higher than Bcl-2 in activated human HSCs. The relative expression of Bcl-xL over Bcl-xS increased gradually when HSCs were activated in cell culture, which was consistent with the increase in apoptosis resistance of activated HSCs. Redirection of Bcl-x splicing by an antisense oligonucleotide from the antiapoptotic isoform to the proapoptotic isoform induced death of HSCs without other apoptosis stimuli. We conclude that Bcl-x plays a role in regulation of HSC apoptosis and modulation of Bcl-x alternative splicing may become a novel molecular therapy for liver fibrosis.

  5. Utilization of reconstructed cultured human skin models as an alternative skin for permeation studies of chemical compounds

    OpenAIRE

    Kano, Satoshi; 藤堂, 浩明; 杉江, 謙一; 藤本, 英哲; 中田, 圭一; 徳留, 嘉寛; 橋本, フミ惠; 杉林, 堅次

    2010-01-01

    Two reconstructed human skin models, EpiskinSM and EpiDermTM, have been approved as alternative membranes for skin corrosive/irritation experiments due to their close correlation with animal skin. Such reconstructed human skin models were evaluated as alternative membranes for skin permeation experiments. Seven drugs with different lipophilicities and almost the same molecular weight were used as test penetrants. Relationships were investigated between permeability coefficients (P values) of ...

  6. Evaluation of alternatives for human lysozyme purification from transgenic rice: impact of phytic acid and buffer.

    Science.gov (United States)

    Wilken, Lisa R; Nikolov, Zivko L

    2010-01-01

    Producing economically competitive recombinant human lysozyme from transgenic rice demands an inexpensive purification process for nonpharmaceutical applications. Human lysozyme is a basic protein, and thus, cation exchange chromatography was the selected method for lysozyme purification. Similar to other protein production systems, the identification of critical impurities in the rice extract was important for the development of an efficient purification process. Previous adsorption data indicated that phytic acid was probably responsible for an unacceptably low cation exchange adsorption capacity. In this study, we confirm that reducing phytic acid concentration improves lysozyme binding capacity and investigate alternative process conditions that reduce phytic acid interference. Compared with the previous best process, the adsorption capacity of human lysozyme was increased from 8.6 to 19.7 mg/mL when rice extract was treated with phytase to degrade phytic acid. Using tris buffer to adjust pH 4.5 extract to pH 6 before adsorption reduced phytic acid interference by minimizing phytic acid-lysozyme interactions, eliminated the need for phytase treatment, and increased the binding capacity to 25 mg/mL. Another method of reducing phytic acid concentration was to extract human lysozyme from rice flour at pH 10 with 50 mM NaCl in 50 mM sodium carbonate buffer. A similar binding capacity (25.5 mg/mL) was achieved from pH 10 extract that was clarified by acidic precipitation and adjusted to pH 6 for adsorption. Lysozyme purities ranged from 95 to 98% for all three processing methods. The tris-mediated purification was the most efficient of the alternatives considered.

  7. Genome-wide analysis of alternative transcripts in human breast cancer

    Science.gov (United States)

    Wen, Ji; Toomer, Kevin H.

    2016-01-01

    Transcript variants play a critical role in diversifying gene expression. Alternative splicing is a major mechanism for generating transcript variants. A number of genes have been implicated in breast cancer pathogenesis with their aberrant expression of alternative transcripts. In this study, we performed genome-wide analyses of transcript variant expression in breast cancer. With RNA-Seq data from 105 patients, we characterized the transcriptome of breast tumors, by pairwise comparison of gene expression in the breast tumor versus matched healthy tissue from each patient. We identified 2839 genes, ~10 % of protein-coding genes in the human genome, that had differential expression of transcript variants between tumors and healthy tissues. The validity of the computational analysis was confirmed by quantitative RT-PCR assessment of transcript variant expression from four top candidate genes. The alternative transcript profiling led to classification of breast cancer into two subgroups and yielded a novel molecular signature that could be prognostic of patients’ tumor burden and survival. We uncovered nine splicing factors (FOX2, MBNL1, QKI, PTBP1, ELAVL1, HNRNPC, KHDRBS1, SFRS2, and TIAR) that were involved in aberrant splicing in breast cancer. Network analyses for the coordinative patterns of transcript variant expression identified twelve “hub” genes that differentiated the cancerous and normal transcriptomes. Dysregulated expression of alternative transcripts may reveal novel biomarkers for tumor development. It may also suggest new therapeutic targets, such as the “hub” genes identified through the network analyses of transcript variant expression, or splicing factors implicated in the formation of the tumor transcriptome. PMID:25913416

  8. GAD2 Alternative Transcripts in the Human Prefrontal Cortex, and in Schizophrenia and Affective Disorders.

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    Kasey N Davis

    Full Text Available Genetic variation and early adverse environmental events work together to increase risk for schizophrenia. γ-aminobutyric acid (GABA, the major inhibitory neurotransmitter in adult mammalian brain, plays a major role in normal brain development, and has been strongly implicated in the pathobiology of schizophrenia. GABA synthesis is controlled by two glutamic acid decarboxylase (GAD genes, GAD1 and GAD2, both of which produce a number of alternative transcripts. Genetic variants in the GAD1 gene are associated with increased risk for schizophrenia, and reduced expression of its major transcript in the human dorsolateral prefrontal cortex (DLPFC. No consistent changes in GAD2 expression have been found in brains from patients with schizophrenia. In this work, with the use of RNA sequencing and PCR technologies, we confirmed and tracked the expression of an alternative truncated transcript of GAD2 (ENST00000428517 in human control DLPFC homogenates across lifespan besides the well-known full length transcript of GAD2. In addition, using quantitative RT-PCR, expression of GAD2 full length and truncated transcripts were measured in the DLPFC of patients with schizophrenia, bipolar disorder and major depression. The expression of GAD2 full length transcript is decreased in the DLPFC of schizophrenia and bipolar disorder patients, while GAD2 truncated transcript is increased in bipolar disorder patients but decreased in schizophrenia patients. Moreover, the patients with schizophrenia with completed suicide or positive nicotine exposure showed significantly higher expression of GAD2 full length transcript. Alternative transcripts of GAD2 may be important in the growth and development of GABA-synthesizing neurons as well as abnormal GABA signaling in the DLPFC of patients with schizophrenia and affective disorders.

  9. Isolation and characterization of connective tissue progenitor cells derived from human fracture-induced hemarthrosis in vitro.

    Science.gov (United States)

    Lee, Sang Yang; Miwa, Masahiko; Sakai, Yoshitada; Kuroda, Ryosuke; Oe, Keisuke; Niikura, Takahiro; Matsumoto, Tomoyuki; Fujioka, Hiroyuki; Doita, Minoru; Kurosaka, Masahiro

    2008-02-01

    In our search for alternative sources of connective tissue progenitor cells that can be obtained with minimal invasion, we studied human intraarticular fracture-induced hemarthrosis of the knee and attempted to isolate connective tissue progenitors from the hemarthrosis. Hemarthrosis was aspirated from the knee joints of 13 patients suffering from intraarticular osteochondral fractures of the knee. Mononuclear cells were isolated from the aspirated hemarthrosis by density gradient separation, and cultured. We were able to obtain fibroblastic adherent cells from the mononuclear cell fractions. Flow cytometry analysis after in vitro expansion on tissue culture plastic revealed that the fibroblastic cells were positive for CD29, CD44, CD105, and CD166, and negative for CD14, CD34, CD45, and CD133. These cells could differentiate in vitro into osteogenic, chondrogenic, and adipogenic cells in the presence of lineage-specific induction factors. These results demonstrate that human intraarticular fracture-induced knee hemarthrosis contains connective tissue progenitor cells with morphologic features, immunophenotypic markers, and differentiation potential that are similar to bone marrow stromal cells. This suggests that hemarthrosis, which is easy to harvest without unnecessary invasion to the patient, has possible future clinical applications such as in tissue-engineered therapies for severe osteochondral defects, posttraumatic osteoarthritis, and delayed fracture unions or nonunions.

  10. Human factors evaluation of teletherapy: Identification of problems and alternative approaches. Volume 1

    Energy Technology Data Exchange (ETDEWEB)

    Henriksen, K.; Kaye, R.D.; Jones, R. [Hughes Training, Inc., Falls Church, VA (United States); Morisseau, D.S.; Serig, D.I. [Nuclear Regulatory Commission, Washington, DC (United States). Div. of Systems Technology

    1995-07-01

    A series of human factors evaluations was undertaken to better understand the contributing factors to human error in the teletherapy environment. Teletherapy is a multi-disciplinary methodology for treating cancerous tissue through selective exposure to an external beam of ionizing radiation. The principal sources of radiation are a radioactive isotope, typically cobalt60 (Co-60), or a linear accelerator device capable of producing very high energy x-ray and electron beams. A team of human factors specialists, assisted by a panel of radiation oncologists, medical physicists, and radiation technologists, conducted site visits to radiation oncology departments at community hospitals, university centers, and free-standing clinics. A function and task analysis was initially performed to guide subsequent evaluations in the areas of user-system interfaces, procedures, training and qualifications, and organizational policies and practices. The final phase of the project focused on identification of the most significant human factors problems with respect to safe and effective operation of the teletherapy system and an identification and assessment of alternative approaches for resolving the problems. This report presents the findings of this final phase.

  11. Investigation of properties of human epidermal membrane under constant conductance alternating current iontophoresis.

    Science.gov (United States)

    Zhu, Honggang; Peck, Kendall D; Miller, David J; Liddell, Mark R; Yan, Guang; Higuchi, William I; Li, S Kevin

    2003-04-14

    Previous studies in our laboratory have shown that enhanced, constant permeant fluxes across human skin can be achieved by applying an alternating current (AC) to maintain skin electrical conductance at a constant level. Relative to conventional direct current (DC) iontophoresis, for which current is maintained at a constant level, this newly developed constant conductance alternating current (CCAC) method achieves constant fluxes with less inter- and intra-sample variability. The present study focused upon further investigating the permeability properties of human skin during CCAC iontophoresis at a variety of target resistance/conductance values. A three-stage experimental protocol was used with flux measurements determined on 3 consecutive days. Stage I was an AC only protocol (symmetrical AC square-wave signal), stage II was an AC plus DC protocol (AC square-wave with DC offset voltage), and stage III was a repeat of stage I. During this three-stage protocol, the skin electrical resistance was maintained at a constant target value by manually adjusting the applied AC voltage. Radiolabeled mannitol and urea were model permeants in all experiments. Their fluxes were determined and used to characterize the permeability properties of human skin. The results from the present study established that: (i) the CCAC protocol made it possible to reduce HEM electrical resistance to different target levels as low as 0.8 kOmega cm(2) and maintain the specific resistance level throughout the flux experiment, (ii) permeant fluxes are proportional to skin electrical conductance, (iii) under the studied CCAC passive conditions, membrane pore size tends to increase as skin resistance decreases, and (iv) as the membrane breaks down, its pore sizes become larger.

  12. Transcranial alternating current stimulation enhances individual alpha activity in human EEG.

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    Tino Zaehle

    Full Text Available Non-invasive electrical stimulation of the human cortex by means of transcranial direct current stimulation (tDCS has been instrumental in a number of important discoveries in the field of human cortical function and has become a well-established method for evaluating brain function in healthy human participants. Recently, transcranial alternating current stimulation (tACS has been introduced to directly modulate the ongoing rhythmic brain activity by the application of oscillatory currents on the human scalp. Until now the efficiency of tACS in modulating rhythmic brain activity has been indicated only by inference from perceptual and behavioural consequences of electrical stimulation. No direct electrophysiological evidence of tACS has been reported. We delivered tACS over the occipital cortex of 10 healthy participants to entrain the neuronal oscillatory activity in their individual alpha frequency range and compared results with those from a separate group of participants receiving sham stimulation. The tACS but not the sham stimulation elevated the endogenous alpha power in parieto-central electrodes of the electroencephalogram. Additionally, in a network of spiking neurons, we simulated how tACS can be affected even after the end of stimulation. The results show that spike-timing-dependent plasticity (STDP selectively modulates synapses depending on the resonance frequencies of the neural circuits that they belong to. Thus, tACS influences STDP which in turn results in aftereffects upon neural activity.The present findings are the first direct electrophysiological evidence of an interaction of tACS and ongoing oscillatory activity in the human cortex. The data demonstrate the ability of tACS to specifically modulate oscillatory brain activity and show its potential both at fostering knowledge on the functional significance of brain oscillations and for therapeutic application.

  13. Human Serum-Specific Activation of Alternative Sigma Factors, the Stress Responders in Aggregatibacter actinomycetemcomitans

    Science.gov (United States)

    Tang-Siegel, Gaoyan; Bumgarner, Roger; Ruiz, Teresa; Kittichotirat, Weerayuth; Chen, Weizhen; Chen, Casey

    2016-01-01

    Aggregatibacter actinomycetemcomitans, a known pathogen causing periodontal disease and infective endocarditis, is a survivor in the periodontal pocket and blood stream; both environments contain serum as a nutrient source. To screen for unknown virulence factors associated with this microorganism, A. actinomycetemcomitans was grown in serum-based media to simulate its in vivo environment. Different strains of A. actinomycetemcomitans showed distinct growth phenotypes only in the presence of human serum, and they were grouped into high- and low-responder groups. High-responders comprised mainly serotype c strains, and showed an unusual growth phenomenon, featuring a second, rapid increase in turbidity after 9-h incubation that reached a final optical density 2- to 7-fold higher than low-responders. Upon further investigation, the second increase in turbidity was not caused by cell multiplication, but by cell death. Whole transcriptomic analysis via RNA-seq identified 35 genes that were up-regulated by human serum, but not horse serum, in high-responders but not in low-responders, including prominently an alternative sigma factor rpoE (σE). A lacZ reporter construct driven by the 132-bp rpoE promoter sequence of A. actinomycetemcomitans responded dramatically to human serum within 90 min of incubation only when the construct was carried by a high responder strain. The rpoE promoter is 100% identical among high- and low-responder strains. Proteomic investigation showed potential interactions between human serum protein, e.g. apolipoprotein A1 (ApoA1) and A. actinomycetemcomitans. The data clearly indicated a different activation process for rpoE in high- versus low-responder strains. This differential human serum-specific activation of rpoE, a putative extra-cytoplasmic stress responder and global regulator, suggests distinct in vivo adaptations among different strains of A. actinomycetemcomitans. PMID:27490177

  14. Utilization of Alternative Polyadenylation Signals in the Novel Human Apoptosis-Inducing Gene hap

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    hap, a novel human apoptosis-inducing gene which can interact with another newly discovered apoptosis-inducing gene ASY, was identified by cloning its cDNAs from human lung cell line (WI-38) cDNA library. Two major mRNA species (1.8 and 2. 7 kb in length, respectively) were previously identified by Northern blot analysis of poly(A)+ RNA from human multiple tissues using partial hap cDNA as a probe. In the present work, the molecular mechanism accounting for the generation of the two hap transcripts were investigated. The rapid amplification of cDNA 3'-ends (3'-RACE) technique and the sequential Southern blot analysis, in conjunction with the sequencing analysis demonstrated that the two hap transcripts derive from the alternative polyadenylation site selection: a AATAAA signal at position 1 528 -1 533 nt for the 1.8 kb hap mRNA; and a AATAAA signal at position 2 375 -2 380 nt for the 2. 7 kb hap mRNA. Furthermore, a number of regulatory elements within hap 3'-untranslated region (3'-UTR) were also examined.

  15. Alternative splicing and extensive RNA editing of human TPH2 transcripts.

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    Maik Grohmann

    Full Text Available Brain serotonin (5-HT neurotransmission plays a key role in the regulation of mood and has been implicated in a variety of neuropsychiatric conditions. Tryptophan hydroxylase (TPH is the rate-limiting enzyme in the biosynthesis of 5-HT. Recently, we discovered a second TPH isoform (TPH2 in vertebrates, including man, which is predominantly expressed in brain, while the previously known TPH isoform (TPH1 is primarly a non-neuronal enzyme. Overwhelming evidence now points to TPH2 as a candidate gene for 5-HT-related psychiatric disorders. To assess the role of TPH2 gene variability in the etiology of psychiatric diseases we performed cDNA sequence analysis of TPH2 transcripts from human post mortem amygdala samples obtained from individuals with psychiatric disorders (drug abuse, schizophrenia, suicide and controls. Here we show that TPH2 exists in two alternatively spliced variants in the coding region, denoted TPH2a and TPH2b. Moreover, we found evidence that the pre-mRNAs of both splice variants are dynamically RNA-edited in a mutually exclusive manner. Kinetic studies with cell lines expressing recombinant TPH2 variants revealed a higher activity of the novel TPH2B protein compared with the previously known TPH2A, whereas RNA editing was shown to inhibit the enzymatic activity of both TPH2 splice variants. Therefore, our results strongly suggest a complex fine-tuning of central nervous system 5-HT biosynthesis by TPH2 alternative splicing and RNA editing. Finally, we present molecular and large-scale linkage data evidencing that deregulated alternative splicing and RNA editing is involved in the etiology of psychiatric diseases, such as suicidal behaviour.

  16. Alternative Ultrasound Gel for a Sustainable Ultrasound Program: Application of Human Centered Design.

    Science.gov (United States)

    Salmon, Margaret; Salmon, Christian; Bissinger, Alexa; Muller, Mundenga Mutendi; Gebreyesus, Alegnta; Geremew, Haimanot; Wendel, Sarah K; Wendell, Sarah; Azaza, Aklilu; Salumu, Maurice; Benfield, Nerys

    2015-01-01

    This paper describes design of a low cost, ultrasound gel from local products applying aspects of Human Centered Design methodology. A multidisciplinary team worked with clinicians who use ultrasound where commercial gel is cost prohibitive and scarce. The team followed the format outlined in the Ideo Took Kit. Research began by defining the challenge "how to create locally available alternative ultrasound gel for a low-resourced environment? The "End-Users," were identified as clinicians who use ultrasound in Democratic Republic of the Congo and Ethiopia. An expert group was identified and queried for possible alternatives to commercial gel. Responses included shampoo, oils, water and cornstarch. Cornstarch, while a reasonable solution, was either not available or too expensive. We then sought deeper knowledge of locally sources materials from local experts, market vendors, to develop a similar product. Suggested solutions gleaned from these interviews were collected and used to create ultrasound gel accounting for cost, image quality, manufacturing capability. Initial prototypes used cassava root flour from Great Lakes Region (DRC, Rwanda, Uganda, Tanzania) and West Africa, and bula from Ethiopia. Prototypes were tested in the field and resulting images evaluated by our user group. A final prototype was then selected. Cassava and bula at a 32 part water, 8 part flour and 4 part salt, heated, mixed then cooled was the product design of choice.

  17. Alternative splicing in the differentiation of human embryonic stem cells into cardiac precursors.

    Directory of Open Access Journals (Sweden)

    Nathan Salomonis

    2009-11-01

    Full Text Available The role of alternative splicing in self-renewal, pluripotency and tissue lineage specification of human embryonic stem cells (hESCs is largely unknown. To better define these regulatory cues, we modified the H9 hESC line to allow selection of pluripotent hESCs by neomycin resistance and cardiac progenitors by puromycin resistance. Exon-level microarray expression data from undifferentiated hESCs and cardiac and neural precursors were used to identify splice isoforms with cardiac-restricted or common cardiac/neural differentiation expression patterns. Splice events for these groups corresponded to the pathways of cytoskeletal remodeling, RNA splicing, muscle specification, and cell cycle checkpoint control as well as genes with serine/threonine kinase and helicase activity. Using a new program named AltAnalyze (http://www.AltAnalyze.org, we identified novel changes in protein domain and microRNA binding site architecture that were predicted to affect protein function and expression. These included an enrichment of splice isoforms that oppose cell-cycle arrest in hESCs and that promote calcium signaling and cardiac development in cardiac precursors. By combining genome-wide predictions of alternative splicing with new functional annotations, our data suggest potential mechanisms that may influence lineage commitment and hESC maintenance at the level of specific splice isoforms and microRNA regulation.

  18. Alternative Ultrasound Gel for a Sustainable Ultrasound Program: Application of Human Centered Design.

    Directory of Open Access Journals (Sweden)

    Margaret Salmon

    Full Text Available This paper describes design of a low cost, ultrasound gel from local products applying aspects of Human Centered Design methodology. A multidisciplinary team worked with clinicians who use ultrasound where commercial gel is cost prohibitive and scarce. The team followed the format outlined in the Ideo Took Kit. Research began by defining the challenge "how to create locally available alternative ultrasound gel for a low-resourced environment? The "End-Users," were identified as clinicians who use ultrasound in Democratic Republic of the Congo and Ethiopia. An expert group was identified and queried for possible alternatives to commercial gel. Responses included shampoo, oils, water and cornstarch. Cornstarch, while a reasonable solution, was either not available or too expensive. We then sought deeper knowledge of locally sources materials from local experts, market vendors, to develop a similar product. Suggested solutions gleaned from these interviews were collected and used to create ultrasound gel accounting for cost, image quality, manufacturing capability. Initial prototypes used cassava root flour from Great Lakes Region (DRC, Rwanda, Uganda, Tanzania and West Africa, and bula from Ethiopia. Prototypes were tested in the field and resulting images evaluated by our user group. A final prototype was then selected. Cassava and bula at a 32 part water, 8 part flour and 4 part salt, heated, mixed then cooled was the product design of choice.

  19. Human-in-the-Loop Assessment of Alternative Clearances in Interval Management Arrival Operations

    Science.gov (United States)

    Baxley, Brian T.; Wilson, Sara R.; Swieringa, Kurt A.; Johnson, William C.; Roper, Roy D.; Hubbs, Clay E.; Goess, Paul A.; Shay, Richard F.

    2016-01-01

    Interval Management Alternative Clearances (IMAC) was a human-in-the-loop simulation experiment conducted to explore the Air Traffic Management (ATM) Technology Demonstration (ATD-1) Concept of Operations (ConOps), which combines advanced arrival scheduling, controller decision support tools, and aircraft avionics to enable multiple time deconflicted, efficient arrival streams into a high-density terminal airspace. Interval Management (IM) is designed to support the ATD-1 concept by having an "Ownship" (IM-capable) aircraft achieve or maintain a specific time or distance behind a "Target" (preceding) aircraft. The IM software uses IM clearance information and the Ownship data (route of flight, current location, and wind) entered by the flight crew, and the Target aircraft's Automatic Dependent Surveillance-Broadcast state data, to calculate the airspeed necessary for the IM-equipped aircraft to achieve or maintain the assigned spacing goal.

  20. Alternative spliced CD1d transcripts in human bronchial epithelial cells.

    Directory of Open Access Journals (Sweden)

    Kambez Hajipouran Benam

    Full Text Available CD1d is a MHC I like molecule which presents glycolipid to natural killer T (NKT cells, a group of cells with diverse but critical immune regulatory functions in the immune system. These cells are required for optimal defence against bacterial, viral, protozoan, and fungal infections, and control of immune-pathology and autoimmune diseases. CD1d is expressed on antigen presenting cells but also found on some non-haematopoietic cells. However, it has not been observed on bronchial epithelium, a site of active host defence in the lungs. Here, we identify for the first time, CD1D mRNA variants and CD1d protein expression on human bronchial epithelial cells, describe six alternatively spliced transcripts of this gene in these cells; and show that these variants are specific to epithelial cells. These findings provide the basis for investigations into a role for CD1d in lung mucosal immunity.

  1. Disabled-1 alternative splicing in human fetal retina and neural tumors.

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    Sachin Katyal

    Full Text Available BACKGROUND: The Reelin-Dab1 signaling pathway plays a critical role in the positioning of migrating neurons, dendrite formation and lamination in the developing central nervous system. We have previously identified two alternatively spliced forms of Dab1 in the developing chick retina: an early form, Dab1-E, expressed in retinal progenitor cells, and a late form, Dab1 or Dab1-L, expressed in amacrine and ganglion cells. Compared to Dab1-L, Dab1-E lacks two exons that encode two Src family kinase (SFK phosphorylation sites. PRINCIPAL FINDINGS: Both Dab1-L and Dab1-E-like transcripts were identified in human fetal retina. Expression of human Dab1-L in primary chick retinal cultures resulted in Reelin-mediated induction of SFK phosphorylation and formation of neurite-like processes. In contrast, human Dab1-E-expressing cells retained an undifferentiated morphology. The human Dab1 gene is located within a common fragile site, and it has been postulated that it may function as a tumor suppressor. Analysis of Dab1 splice forms in retinoblastoma and neuroblastoma tumor cells revealed relative enrichment of Dab1-L-like (includes exons 7 and 8 and Dab1-E-like (excludes exons 7 and 8 transcripts in retinoblastoma and neuroblastoma, respectively. Treatment of retinoblastoma cell line RB522A with Reelin resulted in increased tyrosine phosphorylation of Dab1. As Nova2 has previously been implicated in the exclusion of exons 9B and 9C in Dab1, we examined the expression of this splicing factor in neuroblastoma and retinoblastoma cell lines. Nova2 was only detected in neuroblastoma cells, suggesting a correlation between Nova2 expression and increased levels of Dab1-E-like splice forms in neuroblastoma. CONCLUSIONS: These results indicate that alternative splicing of Dab1 is conserved in avian and mammalian species, with Dab1-L driving SFK phosphorylation in both species. Dab1-E- and Dab-L-like isoforms are also expressed in childhood neural tumors, with

  2. Disabled-1 Alternative Splicing in Human Fetal Retina and Neural Tumors

    Science.gov (United States)

    Katyal, Sachin; Glubrecht, Darryl D.; Li, Lei; Gao, Zhihua; Godbout, Roseline

    2011-01-01

    Background The Reelin-Dab1 signaling pathway plays a critical role in the positioning of migrating neurons, dendrite formation and lamination in the developing central nervous system. We have previously identified two alternatively spliced forms of Dab1 in the developing chick retina: an early form, Dab1-E, expressed in retinal progenitor cells, and a late form, Dab1 or Dab1-L, expressed in amacrine and ganglion cells. Compared to Dab1-L, Dab1-E lacks two exons that encode two Src family kinase (SFK) phosphorylation sites. Principal Findings Both Dab1-L and Dab1-E-like transcripts were identified in human fetal retina. Expression of human Dab1-L in primary chick retinal cultures resulted in Reelin-mediated induction of SFK phosphorylation and formation of neurite-like processes. In contrast, human Dab1-E-expressing cells retained an undifferentiated morphology. The human Dab1 gene is located within a common fragile site, and it has been postulated that it may function as a tumor suppressor. Analysis of Dab1 splice forms in retinoblastoma and neuroblastoma tumor cells revealed relative enrichment of Dab1-L-like (includes exons 7 and 8) and Dab1-E-like (excludes exons 7 and 8) transcripts in retinoblastoma and neuroblastoma, respectively. Treatment of retinoblastoma cell line RB522A with Reelin resulted in increased tyrosine phosphorylation of Dab1. As Nova2 has previously been implicated in the exclusion of exons 9B and 9C in Dab1, we examined the expression of this splicing factor in neuroblastoma and retinoblastoma cell lines. Nova2 was only detected in neuroblastoma cells, suggesting a correlation between Nova2 expression and increased levels of Dab1-E-like splice forms in neuroblastoma. Conclusions These results indicate that alternative splicing of Dab1 is conserved in avian and mammalian species, with Dab1-L driving SFK phosphorylation in both species. Dab1-E- and Dab-L-like isoforms are also expressed in childhood neural tumors, with preferential enrichment

  3. Alternate-day fasting and chronic disease prevention: a review of human and animal trials.

    Science.gov (United States)

    Varady, Krista A; Hellerstein, Marc K

    2007-07-01

    Calorie restriction (CR) and alternate-day fasting (ADF) represent 2 different forms of dietary restriction. Although the effects of CR on chronic disease prevention were reviewed previously, the effects of ADF on chronic disease risk have yet to be summarized. Accordingly, we review here animal and human evidence concerning ADF and the risk of certain chronic diseases, such as type 2 diabetes, cardiovascular disease, and cancer. We also compare the magnitude of risk reduction resulting from ADF with that resulting from CR. In terms of diabetes risk, animal studies of ADF find lower diabetes incidence and lower fasting glucose and insulin concentrations, effects that are comparable to those of CR. Human trials to date have reported greater insulin-mediated glucose uptake but no effect on fasting glucose or insulin concentrations. In terms of cardiovascular disease risk, animal ADF data show lower total cholesterol and triacylglycerol concentrations, a lower heart rate, improved cardiac response to myocardial infarction, and lower blood pressure. The limited human evidence suggests higher HDL-cholesterol concentrations and lower triacylglycerol concentrations but no effect on blood pressure. In terms of cancer risk, there is no human evidence to date, yet animal studies found decreases in lymphoma incidence, longer survival after tumor inoculation, and lower rates of proliferation of several cell types. The findings in animals suggest that ADF may effectively modulate several risk factors, thereby preventing chronic disease, and that ADF may modulate disease risk to an extent similar to that of CR. More research is required to establish definitively the consequences of ADF.

  4. Human uroporphyrinogen-III synthase: genomic organization, alternative promoters, and erythroid-specific expression.

    Science.gov (United States)

    Aizencang, G; Solis, C; Bishop, D F; Warner, C; Desnick, R J

    2000-12-01

    Uroporphyrinogen-III (URO) synthase is the heme biosynthetic enzyme defective in congenital erythropoietic porphyria. The approximately 34-kb human URO-synthase gene (UROS) was isolated, and its organization and tissue-specific expression were determined. The gene had two promoters that generated housekeeping and erythroid-specific transcripts with unique 5'-untranslated sequences (exons 1 and 2A) followed by nine common coding exons (2B to 10). Expression arrays revealed that the housekeeping transcript was present in all tissues, while the erythroid transcript was only in erythropoietic tissues. The housekeeping promoter lacked TATA and SP1 sites, consistent with the observed low level expression in most cells, whereas the erythroid promoter contained GATA1 and NF-E2 sites for erythroid specificity. Luciferase reporter assays demonstrated that the housekeeping promoter was active in both erythroid K562 and HeLa cells, while the erythroid promoter was active only in erythroid cells and its activity was increased during hemin-induced erythroid differentiation. Thus, human URO-synthase expression is regulated during erythropoiesis by an erythroid-specific alternative promoter.

  5. High resolution analysis of the human transcriptome: detection of extensive alternative splicing independent of transcriptional activity

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    Rouet Fabien

    2009-10-01

    Full Text Available Abstract Background Commercially available microarrays have been used in many settings to generate expression profiles for a variety of applications, including target selection for disease detection, classification, profiling for pharmacogenomic response to therapeutics, and potential disease staging. However, many commercially available microarray platforms fail to capture transcript diversity produced by alternative splicing, a major mechanism for driving proteomic diversity through transcript heterogeneity. Results The human Genome-Wide SpliceArray™ (GWSA, a novel microarray platform, utilizes an existing probe design concept to monitor such transcript diversity on a genome scale. The human GWSA allows the detection of alternatively spliced events within the human genome through the use of exon body and exon junction probes to provide a direct measure of each transcript, through simple calculations derived from expression data. This report focuses on the performance and validation of the array when measured against standards recently published by the Microarray Quality Control (MAQC Project. The array was shown to be highly quantitative, and displayed greater than 85% correlation with the HG-U133 Plus 2.0 array at the gene level while providing more extensive coverage of each gene. Almost 60% of splice events among genes demonstrating differential expression of greater than 3 fold also contained extensive splicing alterations. Importantly, almost 10% of splice events within the gene set displaying constant overall expression values had evidence of transcript diversity. Two examples illustrate the types of events identified: LIM domain 7 showed no differential expression at the gene level, but demonstrated deregulation of an exon skip event, while erythrocyte membrane protein band 4.1 -like 3 was differentially expressed and also displayed deregulation of a skipped exon isoform. Conclusion Significant changes were detected independent of

  6. Transcriptomic analysis of human polarized macrophages: more than one role of alternative activation?

    Directory of Open Access Journals (Sweden)

    Eleonora Derlindati

    Full Text Available Macrophages are a heterogeneous cell population which in response to the cytokine milieu polarize in either classically activated macrophages (M1 or alternatively activated macrophages (M2. This plasticity makes macrophages essential in regulating inflammation, immune response and tissue remodeling and a novel therapeutic target in inflammatory diseases such as atherosclerosis. The aim of the study was to describe the transcriptomic profiles of differently polarized human macrophages to generate new hypotheses on the biological function of the different macrophage subtypes.Polarization of circulating monocytes/macrophages of blood donors was induced in vitro by IFN-γ and LPS (M1, by IL-4 (M2a, and by IL-10 (M2c. Unstimulated cells (RM served as time controls. Gene expression profile of M1, M2a, M2c and RM was assessed at 6, 12 and 24h after polarization with Whole Human Genome Agilent Microarray technique. When compared to RM, M1 significantly upregulated pathways involved in immunity and inflammation, whereas M2a did the opposite. Conversely, decreased and increased expression of mitochondrial metabolism, consistent with insulin resistant and insulin sensitive patterns, was seen in M1 and M2a, respectively. The time sequence in the expression of some pathways appeared to have some specific bearing on M1 function. Finally, canonical and non-canonical Wnt genes and gene groups, promoting inflammation and tissue remodeling, were upregulated in M2a compared to RM.Our data in in vitro polarized human macrophages: 1. confirm and extend known inflammatory and anti-inflammatory gene expression patterns; 2. demonstrate changes in mitochondrial metabolism associated to insulin resistance and insulin sensitivity in M1 and M2a, respectively; 3. highlight the potential relevance of gene expression timing in M1 function; 4. unveil enhanced expression of Wnt pathways in M2a suggesting a potential dual (pro-inflammatory and anti-inflammatory role of M2a in

  7. Toenails as an alternative source material for the extraction of DNA from decomposed human remains.

    Science.gov (United States)

    Schlenker, Andrew; Grimble, Katelyn; Azim, Arani; Owen, Rebecca; Hartman, Dadna

    2016-01-01

    The DNA identification of decomposed human remains for coronial investigations at the Victorian Institute of Forensic Medicine routinely requires the retrieval and processing of a bone sample obtained from the deceased. Bone is a difficult sample type to work with as it requires surgical removal from the deceased, refrigerated storage, and additional processing steps prior to DNA analysis in comparison to other samples types such as buccal swabs or blood stains. In an attempt to overcome the issues posed by bone, a DNA extraction method utilising toenails as an alternate source material was optimised and trialled. Two DNA extraction methods were optimised for digestion of toenail material, with the method utilising the QIAGEN DNA Investigator Kit selected for a casework trial. Single source DNA profiles, matching those of the conventional samples taken, were obtained for toenail samples collected from 28 of 30 coronial cases available for this study. Of these, 26 toenail samples produced full profiles. Although the overall DNA profile quality from the toenails was less than that of the conventional sample, the profiles from toenails met the reporting requirements for identification. Based on the results obtained, the Victorian Institute of Forensic Medicine will be implementing toenails as the primary sample type for collection from decomposed remains when blood is not a suitable sample type.

  8. Modulation of alternative splicing with chemical compounds in new therapeutics for human diseases.

    Science.gov (United States)

    Ohe, Kenji; Hagiwara, Masatoshi

    2015-04-17

    Alternative splicing is a critical step where a limited number of human genes generate a complex and diverse proteome. Various diseases, including inherited diseases with abnormalities in the "genome code," have been found to result in an aberrant mis-spliced "transcript code" with correlation to the resulting phenotype. Chemical compound-based and nucleic acid-based strategies are trying to target this mis-spliced "transcript code". We will briefly mention about how to obtain splicing-modifying-compounds by high-throughput screening and overview of what is known about compounds that modify splicing pathways. The main focus will be on RNA-binding protein kinase inhibitors. In the main text, we will refer to diseases where splicing-modifying-compounds have been intensively investigated, with comparison to nucleic acid-based strategies. The information on their involvement in mis-splicing as well as nonsplicing events will be helpful in finding better compounds with less off-target effects for future implications in mis-splicing therapy.

  9. Alternative Fuel Implementation in a Cement Plant: Human Health Risks and Economical Valuation.

    Science.gov (United States)

    Rovira, Joaquim; Nadal, Martí; Schuhmacher, Marta; Domingo, José L

    2016-11-01

    In June 2010, the cement plant of Montcada i Reixac (MR) (Catalonia, Spain) began a gradual implementation of alternative fuel to replace fossil fuel. Between December 2010 and November 2014, we conducted three monitoring surveys to evaluate the state of the environment around the facility. Data were compared with results from three monitoring surveys performed in 2008-2009. In all these studies, samples of soil, vegetation, and air were collected, being the content of a number of trace elements and polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) in these matrices determined. In general terms, a decrease of metal and PCDD/F concentrations was found. Human health risks followed a similar temporal trend, being acceptable according to national and international standards and independent on the fuel used. The Disability-Adjusted Life Year and the costs of cancer cases were also estimated for the population living around the MR cement plant, accounting for 4 years and 31,000 €/year, respectively.

  10. Exploring Codon Usage Patterns of Alternatively Spliced Genes in Human Chromosome 1

    Institute of Scientific and Technical Information of China (English)

    马飞; 庄永龙; 黄颖; 李衍达

    2004-01-01

    In this study, 414 whole protein-coding sequences (238 004 codons) of alternatively spliced genes of human chromosome 1 have been employed to explore the patterns of codon usage bias among genes. Overall codon usage data analysis indicates that G- and C-ending codons are predominant in the genes. The base usage in all three codon positions suggests a selection-mutation balance. Multivariate statistical analysis reveals that the codon usage variation has a strong positive correlation with the expressivities of the genes (r=0.5790, P<0.0001). All 27 codons identified as optimal are G- and C-ending codons.Correlation analysis shows a strong negative correlation between the gene length and codon adaptation index value (r=-0.2252, P<0.0001), and a significantly positive correlation between the gene length and Nc values (r=0.1876, P<0.0001). These results suggest that the comparatively shorter genes in the genes have higher codon usage bias to maximize translational efficiency, and selection may also contribute to the reduction of highly expressed proteins.

  11. Subcellular localization of human heparanase and its alternative splice variant in COS-7 cells.

    Science.gov (United States)

    Sato, Mayumi; Amemiya, Kana; Hayakawa, Sumio; Munakata, Hiroshi

    2008-08-01

    Heparanase, the enzyme that degrades heparan sulfate, has been implicated to play important and characteristic roles in organogenesis, tissue organization, cell migration, and tumor metastasis. Clarification of its expression, its intracellular sorting, and its secretion is, therefore, of much importance to understand its role in cell biology. In addition to the 1.7 Kb transcript previously reported, we detected a 1.5 Kb transcript of human heparanase by RT-PCR. The smaller transcript was shown to be an alternatively spliced variant lacking exon 5, which contains the essential glutamic acid residue required for enzyme activity. When expressed in COS-7 cells this variant did not show any heparanase activity. Full-length heparanase and the exon 5-deleted splice variant were expressed in COS-7 cells and examined by confocal laser scanning microscopy. Both proteins co-localized with calnexin, a marker protein for the endoplasmic reticulum, and they co-immunoprecipitated with calnexin. Both proteins were postulated to be precursors based upon the results of SDS-PAGE analyses. Treatment with endoglycosidases revealed that all potential N-glycosylation sites in the proteins were glycosylated. Tunicamycin treatment of transfected COS-7 cells inhibited N-glycosylation but did not change the subcellular localization. These results indicate that overexpressed heparanase and its splice variant localize to the endoplasmic reticulum independent of glycosylation in COS-7 cells.

  12. Expression of a novel alternative transcript of the novel retinal pigment epithelial cell gene NORPEG in human testes

    Institute of Scientific and Technical Information of China (English)

    Wa Yuan; Ying Zheng; Ran Huo; Li Lu; Xiao-Yan Huang; Lan-Lan Yin; Jian-Min Li; Zuo-Min Zhou; Jia-Hao Sha

    2005-01-01

    Aim: To identify a novel alternative transcript of the novel retinal pigment epithelial cell gene (NORPEG) expressed in the human testis. Methods: A human testis cDNA microarray was established and hybridized with cDNA probes from human fetal testes, adult testes and human spermatozoa. Differentially expressed clones were sequenced and analyzed. One of these clones was a short transcript of NORPEG which we proceeded to analyze by RT-PCR.Results: The novel short alternative transcript of NORPEG was isolated and named sNORPEG. It was 3486 bp in length and contained a 2952-bp open reading frame, encoding a 110.4-kDa protein of 983 amino acids. Amino acid sequence analysis showed that the sNORPEG protein contains six ankyrin repeats and two coiled-coil domains. It shares a high homology with the NORPEG and ankycorbin proteins in both its sequence and motifs. Blasting the human genome database localized sNORPEG to human chromosome 5p13.2-13.3. Expression profiles showed that sNORPEG was expressed in human fetal testes, adult testes and spermatozoa. Moreover, sNORPEG was found to be ubiquitously expressed in human tissues. Conclusion: sNORPEG is expressed in different developmental stages of the testis and encodes a protein that may have roles in human testis development and spermatogenesis.

  13. Immunophenotypic comparison of heterogenous non-sorted versus sorted mononuclear cells from human umbilical cord blood: a novel cell enrichment approach.

    Science.gov (United States)

    Indumathi, S; Harikrishnan, R; Rajkumar, J S; Dhanasekaran, M

    2015-01-01

    Human umbilical cord blood (hUCB) has been the preferred source of stem cells for the treatment of haematological malignancies and genetic disorders. This is primarily due to its non-invasiveness, high accessibility with relative ease of isolation. Still failures do prevail due to its heterogeneity and lesser frequency of MSC identified in UCB. This study, thus, employs a cell enrichment technology to improve its therapeutic efficacy. This was achieved by immunophenotypic comparison of stem cells isolated from the heterogenous non-sorted mononuclear cells (MNCs), linage depleted (Lin+ and Lin-) fractions obtained from magnetic activated cell sorter (MACS) and sorted MNCs obtained by fluorescent activated cell sorter (FACS). The markers under consideration were CD29, CD44, CD34, CD45, CD133, CD90 and CD117. FACS sorted MNCs were rich in naive stem cell population, whereas non-sorted MNCs and lineage depleted fractions were found to be rich in progenitors. Thus, we suggest that a combination therapy of both sorted population might serve as an alternative valuable tool in treating haematologic/genetic disorders. However, further research on cell enrichment technology might give a clue for improved cell based therapy in regenerative medicine.

  14. Human amniotic epithelial cell transplantation induces markers of alternative macrophage activation and reduces established hepatic fibrosis.

    Directory of Open Access Journals (Sweden)

    Ursula Manuelpillai

    Full Text Available Chronic hepatic inflammation from multiple etiologies leads to a fibrogenic response that can progress to cirrhosis and liver failure. Transplantation of human amniotic epithelial cells (hAEC from term delivered placenta has been shown to decrease mild to moderate hepatic fibrosis in a murine model. To model advanced human liver disease and assess the efficacy of hAEC therapy, we transplanted hAEC in mice with advanced hepatic fibrosis. Immunocompetent C57BL/6 mice were administered carbon tetrachloride (CCl(4 twice weekly resulting in bridging fibrosis by 12 weeks. hAEC (2 × 10(6 were infused via the tail vein at week 8 or weeks 8 and 10 (single and double dose, respectively. Human cells were detected in mouse liver four weeks after transplantation showing hAEC engraftment. CCl(4 treated mice receiving single or double hAEC doses showed a significant but similar decrease in liver fibrosis area associated with decreased activation of collagen-producing hepatic stellate cells and decreased hepatic protein levels of the pro-fibrogenic cytokine, transforming growth factor-beta1. CCl(4 administration caused hepatic T cell infiltration that decreased significantly following hAEC transplantation. Hepatic macrophages play a crucial role in both fibrogenesis and fibrosis resolution. Mice exposed to CCl(4 demonstrated increased numbers of hepatic macrophages compared to normal mice; the number of macrophages decreased significantly in CCl(4 treated mice given hAEC. These mice had significantly lower hepatic protein levels of the chemokine monocyte chemoattractant protein-1 than mice given CCl(4 alone. Alternatively activated M2 macrophages are associated with fibrosis resolution. CCl(4 treated mice given hAEC showed increased expression of genes associated with M2 macrophages including YM-1, IL-10 and CD206. We provide novel data showing that hAEC transplantation induces a wound healing M2 macrophage phenotype associated with reduction of established

  15. Identification, characterization and expression of novel Sex Hormone Binding Globulin alternative first exons in the human prostate

    Directory of Open Access Journals (Sweden)

    de Torres Inés

    2009-06-01

    Full Text Available Abstract Background The human Sex Hormone Binding Globulin (SHBG gene, located at 17p13.1, comprises, at least, two different transcription units regulated by two different promoters. The first transcription unit begins with the exon 1 sequence and is responsible for the production of plasma SHBG by the hepatocytes, while the second begins with an alternative exon 1 sequence, which replaces the exon 1 present in liver transcripts. Alternative exon 1 transcription and translation has only been demonstrated in the testis of transgenic mice containing an 11-kb human SHBG transgene and in the human testis. Our goal has been to further characterize the 5' end of the SHBG gene and analyze the presence of the SHBG alternative transcripts in human prostate tissue and derived cell lines. Results Using a combination of in silico and in vitro studies, we have demonstrated that the SHBG gene, along with exon 1 and alternative exon 1 (renamed here exon 1A, contains four additional alternative first exons: the novel exons 1B, 1C, and 1E, and a previously identified exon 1N, which has been further characterized and renamed as exon 1D. We have shown that these four alternative first exons are all spliced to the same 3' splice site of SHBG exon 2, and that exon 1A and the novel exon 1B can be spliced to exon 1. We have also demonstrated the presence of SHBG transcripts beginning with exons 1B, 1C and 1D in prostate tissues and cell lines, as well as in several non-prostatic cell lines. Finally, the alignment of the SHBG mammalian sequences revealed that, while exons 1C, 1D and 1E are very well conserved phylogenetically through non-primate mammal species, exon 1B probably aroused in apes due to a single nucleotide change that generated a new 5' splice site in exon 1B. Conclusion The identification of multiple transcription start sites (TSS upstream of the annotated first exon of human SHBG, and the detection of the alternative transcripts in human prostate

  16. Effects of electrophoresis and electroosmosis during alternating current iontophoresis across human epidermal membrane.

    Science.gov (United States)

    Yan, Guang; Peck, Kendall D; Zhu, Honggang; Higuchi, William I; Li, S Kevin

    2005-03-01

    Previous studies in our laboratory have demonstrated that skin electrical resistance can be controlled by an alternating current (AC) electric field. By maintaining constant skin resistance, AC iontophoresis has been shown to reduce the iontophoretic flux variability of neutral permeants. Recently, it was found that symmetric square-wave AC could enhance iontophoretic transport of both neutral and ionic permeants by means of electrophoresis and/or electroosmosis in a synthetic membrane system, and a model was presented to describe the experimental results. The objective of the present study was to assess the effects of AC voltage and frequency and direct current (DC) offset on the flux of neutral and ionic model permeants with human epidermal membrane (HEM). Experiments were conducted under two different conditions: constant AC voltage iontophoresis and iontophoresis using constant HEM resistance with DC offset voltage. The following are the main findings in these experiments. In the constant AC voltage study, when the permeability data were compared at the same HEM electrical resistance, it was demonstrated that AC even at high frequency (approximately 1 kHz) could enhance the transport of the ionic permeant (tetraethylammonium ion) across HEM, but no enhancement was observed for the neutral permeant (arabinose). For the ionic permeant flux enhancement, the higher the applied AC voltage, the greater the flux enhancement. There was little or no AC frequency dependence of the flux enhancement in the frequency range of 50-1000 Hz. In the constant HEM resistance study of AC with DC offset, approximately linear relationships were observed between flux enhancement and the DC offset voltage for both the neutral and ionic permeants, and these results were found to be consistent with predictions of the modified Nernst-Planck model for conventional constant voltage DC iontophoresis. When the DC offset voltage was increased, the AC component of the flux enhancement for the

  17. Alternative splicing isoforms of synaptotagmin VII in the mouse, rat and human.

    Science.gov (United States)

    Fukuda, Mitsunori; Ogata, Yukie; Saegusa, Chika; Kanno, Eiko; Mikoshiba, Katsuhiko

    2002-07-01

    Synaptotagmin VII (Syt VII) has been proposed to regulate several different types of Ca2+-dependent exocytosis, but its subcellular localization (lysosome or plasma membrane) and the number of alternative splicing isoforms of Syt VII (single or multiple forms) are matters of controversy. In the present study, we show by reverse transcriptase-PCR analysis that mouse Syt VII has one major isoform (Syt VIIalpha), the original Syt VII, and two minor isoforms (Syt VIIbeta and Syt VIIgamma), which contain unique insertions (of 44 and 116 amino acids respectively) in the spacer domain between the transmembrane and C2 domains of Syt VIIalpha. Similar results were obtained with respect to rat and human Syt VII mRNA expression. An antibody against the N-terminal domain of mouse Syt VII [anti-(Syt VII-N)], which specifically recognized recombinant Syt VII but not other Syt isoforms expressed in COS-7 cells, recognized two major, closely co-migrating bands (p58 and p60) and minor bands of approx. 65 kDa in mouse brain. Immunoaffinity purification of proteins that bind the anti-(Syt VII-N) antibody, and peptide sequence analysis revealed that: (i) the major p58 and p60 bands are identified as adenylate cyclase-associated protein 2; (ii) actin-binding protein is localized at the plasma membrane; and (iii) Syt VIIalpha (65 kDa) is the major Syt VII isoform, but with a much lower expression level than previously thought. It was also shown that FLAG-Syt VII-green-fluorescence-protein fusion protein stably expressed in PC12 cells is localized in the perinuclear region (co-localization with TGN38 protein, even after brefeldin A treatment) and in the tips of neurites (co-localization with Syt I), and not in the plasma membrane.

  18. Israel’s Associated Regime: Exceptionalism, Human Rights and Alternative Legality

    Directory of Open Access Journals (Sweden)

    Federica D’Alessandra

    2014-08-01

    Full Text Available In the context of Israel’s declared permanent state of exception, this article focuses on the legal protection awarded to the Palestinian populations under Israeli control. To broaden the discussion over Palestinian people’s rights, which generally focuses on the confiscation of land and the right to return, the author consciously focuses on anti-terrorism and security measures, which contribute to the creation of what the International Court of Justice has defined as an ‘associated regime’ of occupation. The article is divided into three parts. In the first part, the author discusses Israel’s domestic obligations towards Palestinians (arguing the case of both Palestinian citizens of Israel, and Palestinian residents and their de jure and de facto discrimination. The second part discusses the applicability of humanitarian law, specifically the applicability of the Fourth Geneva Convention. This section discusses the applicability of the Convention to both territories and people under Israeli control. The third part discusses the applicability of international human rights law to all territories under Israeli control and delves into the issue of the mutual relationship between the two international legal regimes in the territories under occupation. The article posits that Israel’s rationale for the non-applicability of such legislation to the Palestinian territories and populations it controls constitutes a form of ‘alternative legality’. The article concludes that Israel’s disproportionate application of security practices and anti-terrorism measures to the Palestinian segment of its population violates Palestinian rights protected under Israel’s domestic and international legal obligations.

  19. Alternative Routes to Induce Naïve Pluripotency in Human Embryonic Stem Cells.

    Science.gov (United States)

    Duggal, Galbha; Warrier, Sharat; Ghimire, Sabitri; Broekaert, Dorien; Van der Jeught, Margot; Lierman, Sylvie; Deroo, Tom; Peelman, Luc; Van Soom, Ann; Cornelissen, Ria; Menten, Björn; Mestdagh, Pieter; Vandesompele, Jo; Roost, Matthias; Slieker, Roderick C; Heijmans, Bastiaan T; Deforce, Dieter; De Sutter, Petra; De Sousa Lopes, Susana Chuva; Heindryckx, Björn

    2015-09-01

    Human embryonic stem cells (hESCs) closely resemble mouse epiblast stem cells exhibiting primed pluripotency unlike mouse ESCs (mESCs), which acquire a naïve pluripotent state. Efforts have been made to trigger naïve pluripotency in hESCs for subsequent unbiased lineage-specific differentiation, a common conundrum faced by primed pluripotent hESCs due to heterogeneity in gene expression existing within and between hESC lines. This required either ectopic expression of naïve genes such as NANOG and KLF2 or inclusion of multiple pluripotency-associated factors. We report here a novel combination of small molecules and growth factors in culture medium (2i/LIF/basic fibroblast growth factor + Ascorbic Acid + Forskolin) facilitating rapid induction of transgene-free naïve pluripotency in hESCs, as well as in mESCs, which has not been shown earlier. The converted naïve hESCs survived long-term single-cell passaging, maintained a normal karyotype, upregulated naïve pluripotency genes, and exhibited dependence on signaling pathways similar to naïve mESCs. Moreover, they undergo global DNA demethylation and show a distinctive long noncoding RNA profile. We propose that in our medium, the FGF signaling pathway via PI3K/AKT/mTORC induced the conversion of primed hESCs toward naïve pluripotency. Collectively, we demonstrate an alternate route to capture naïve pluripotency in hESCs that is fast, reproducible, supports naïve mESC derivation, and allows efficient differentiation.

  20. Alternative tools to mass production and human performance indicators in sheltered work centers of Valencian community (Spain

    Directory of Open Access Journals (Sweden)

    Juan A. Marin-Garcia

    2011-10-01

    Full Text Available Purpose: The most popular alternative systems to mass production at an academic level (lean manufacturing, agile manufacturing, flexible customization, mass customization... share many characteristics. Our article identifies an extensive set of alternative practices to mass production; analyzes the classification of practices in categories (Flow, TQM, TPM, Customer Relations, Supplier Relations and Human Resources Practices and analyzes the impact on several human performance indicators such as satisfaction, absenteeism, voluntary turnover, permanent contracts, knowledge, personal & social adjustment activities and integration of workers into ordinary companies. Design/methodology/approach: Survey in sheltered work centers. We use regression analysis in order to prove relations between explicative and criterion variables. Findings: The results of our research allow us to identify that human resource management and customer relationship practices have significant effects on job satisfaction, knowledge, integration into ordinary companies and personal and social adjustment. Research limitations/implications: Data came only from one industry; therefore the results would not be directly generalized to other contexts. Practical implications: Managers in Sheltered work centers can estimate the impact of the deployment of alternative tools to mass production. Originality/value: There are few papers relating lean manufacturing tools and human resources performance indicators. At the same time, there are very few research carried out in sheltered work centers context.

  1. CR2-mediated activation of the complement alternative pathway results in formation of membrane attack complexes on human B lymphocytes

    DEFF Research Database (Denmark)

    Nielsen, C H; Marquart, H V; Prodinger, W M;

    2001-01-01

    Normal human B lymphocytes activate the alternative pathway of complement via complement receptor type 2 (CR2, CD21), that binds hydrolysed C3 (iC3) and thereby promotes the formation of a membrane-bound C3 convertase. We have investigated whether this might lead to the generation of a C5...... convertase and consequent formation of membrane attack complexes (MAC). Deposition of C3 fragments and MAC was assessed on human peripheral B lymphocytes in the presence of 30% autologous serum containing 4.4 mM MgCl2/20 mM EGTA, which abrogates the classical pathway of complement without affecting...... the alternative pathway. Blockade of the CR2 ligand-binding site with the monoclonal antibody FE8 resulted in 56 +/- 13% and 71 +/- 9% inhibition of the C3-fragment and MAC deposition, respectively, whereas the monoclonal antibody HB135, directed against an irrelevant CR2 epitope, had no effect. Blockade...

  2. Two Human ACAT2 mRNA Variants Produced by Alternative Splicing and Coding for Novel Isoenzymes

    Institute of Scientific and Technical Information of China (English)

    Xiao-Min YAO; Bo-Liang LI; Can-Hua WANG; Bao-Liang SONG; Xin-Ying YANG; Zhen-Zhen WANG; Wei QI; Zhi-Xin LIN; Catherine C. Y. CHANG; Ta-Yuan CHANG

    2005-01-01

    Acyl coenzyme A:cholesterol acyltransferase 2 (ACAT2) plays an important role in cholesterol absorption. Human ACAT2 is highly expressed in small intestine and fetal liver, but its expression is greatly diminished in adult liver. The full-length human ACAT2 mRNA encodes a protein, designated ACAT2a, with 522 amino acids. We have previously reported the organization of the human ACAT2 gene and the differentiation-dependent promoter activity in intestinal Caco-2 cells. In the current work, two human ACAT2 mRNA variants produced by alternative splicing are cloned and predicted to encode two novel ACAT2 isoforms,named ACAT2b and ACAT2c, with 502 and 379 amino acids, respectively. These mRNA variants differ from ACAT2a mRNA by lack of the exon 4 (ACAT2b mRNA) and exons 4-5 plus 8-9-10 (ACAT2c mRNA).Significantly, comparable amounts of the alternatively spliced ACAT2 mRNA variants were detected by RTPCR, and Western blot analysis confirmed the presence of their corresponding proteins in human liver and intestine cells. Furthermore, phosphorylation and enzymatic activity analyses demonstrated that the novel isoenzymes ACAT2b and ACAT2c lacked the phosphorylatable site SLLD, and their enzymatic activities reduced to 25%-35% of that of ACAT2a. These evidences indicate that alternative splicing produces two human ACAT2 mRNA variants that encode the novel ACAT2 isoenzymes. Our findings might help to understand the regulation of the ACAT2 gene expression under certain physiological and pathological conditions.

  3. Validating determinants for an alternate foot placement selection algorithm during human locomotion in cluttered terrain.

    Science.gov (United States)

    Moraes, Renato; Allard, Fran; Patla, Aftab E

    2007-10-01

    The goal of this study was to validate dynamic stability and forward progression determinants for the alternate foot placement selection algorithm. Participants were asked to walk on level ground and avoid stepping, when present, on a virtual white planar obstacle. They had a one-step duration to select an alternate foot placement, with the task performed under two conditions: free (participants chose the alternate foot placement that was appropriate) and forced (a green arrow projected over the white planar obstacle cued the alternate foot placement). To validate the dynamic stability determinant, the distance between the extrapolated center of mass (COM) position, which incorporates the dynamics of the body, and the limits of the base of support was calculated in both anteroposterior (AP) and mediolateral (ML) directions in the double support phase. To address the second determinant, COM deviation from straight ahead was measured between adaptive and subsequent steps. The results of this study showed that long and lateral choices were dominant in the free condition, and these adjustments did not compromise stability in both adaptive and subsequent steps compared with the short and medial adjustments, which were infrequent and adversely affected stability. Therefore stability is critical when selecting an alternate foot placement in a cluttered terrain. In addition, changes in the plane of progression resulted in small deviations of COM from the endpoint goal. Forward progression of COM was maintained even for foot placement changes in the frontal plane, validating this determinant as part of the selection algorithm.

  4. HDL does not influence the polarization of human monocytes toward an alternative phenotype

    NARCIS (Netherlands)

    Colin, Sophie; Fanchon, Melanie; Belloy, Loic; Bochem, Andrea E.; Copin, Corinne; Derudas, Bruno; Stroes, Erik S. G.; Hovingh, G. Kees; Kuivenhoven, Jan A.; Dallinga-Thie, Geesje M.; Staels, Bart; Chinetti-Gbaguidi, Giulia

    2014-01-01

    BACKGROUND: Macrophages are crucial cells in the pathogenesis of atherosclerosis. Macrophages are plastic cells which can switch from a classical pro-inflammatory M1 to an alternative anti-inflammatory M2 macrophage phenotype, depending on the environmental stimuli. Because high-density lipoprotein

  5. Safe Thinking and Affect Regulation (STAR): Human Immunodeficiency Virus Prevention in Alternative/Therapeutic Schools

    Science.gov (United States)

    Brown, Larry K.; Nugent, Nicole R.; Houck, Christopher D.; Lescano, Celia M.; Whiteley, Laura B.; Barker, David; Viau, Lisa; Zlotnick, Caron

    2011-01-01

    Objective: To evaluate the effectiveness of Safe Thinking and Affect Regulation (STAR), a 14-session HIV-prevention program for adolescents at alternative/therapeutic schools. Because these youth frequently have difficulties with emotions and cognitions, it was designed to improve sexuality-specific affect management and cognitive monitoring, as…

  6. Integrative analyses of RNA editing, alternative splicing, and expression of young genes in human brain transcriptome by deep RNA sequencing.

    Science.gov (United States)

    Wu, Dong-Dong; Ye, Ling-Qun; Li, Yan; Sun, Yan-Bo; Shao, Yi; Chen, Chunyan; Zhu, Zhu; Zhong, Li; Wang, Lu; Irwin, David M; Zhang, Yong E; Zhang, Ya-Ping

    2015-08-01

    Next-generation RNA sequencing has been successfully used for identification of transcript assembly, evaluation of gene expression levels, and detection of post-transcriptional modifications. Despite these large-scale studies, additional comprehensive RNA-seq data from different subregions of the human brain are required to fully evaluate the evolutionary patterns experienced by the human brain transcriptome. Here, we provide a total of 6.5 billion RNA-seq reads from different subregions of the human brain. A significant correlation was observed between the levels of alternative splicing and RNA editing, which might be explained by a competition between the molecular machineries responsible for the splicing and editing of RNA. Young human protein-coding genes demonstrate biased expression to the neocortical and non-neocortical regions during evolution on the lineage leading to humans. We also found that a significantly greater number of young human protein-coding genes are expressed in the putamen, a tissue that was also observed to have the highest level of RNA-editing activity. The putamen, which previously received little attention, plays an important role in cognitive ability, and our data suggest a potential contribution of the putamen to human evolution.

  7. Creating Clay Models of a Human Torso as an Alternative to Dissection

    Science.gov (United States)

    Shipley, Gwendolyn

    2010-01-01

    Instead of dissecting animals, students create small clay models of human internal organs to demonstrate their understanding of the positioning and interlocking shapes of the organs. Not only is this approach more environmentally friendly, it also forces them to learn human anatomy--which is more relevant to them than the anatomy of other…

  8. Recent developments in microbial oils production: a possible alternative to vegetable oils for biodiesel without competition with human food?

    Directory of Open Access Journals (Sweden)

    Gwendoline Christophe

    2012-02-01

    Full Text Available Since centuries vegetable oils are consumed as human food but it also finds applications in biodiesel production which is attracting more attention. But due to being in competition with food it could not be sustainable and leads the need to search for alternative. Nowdays microbes-derived oils (single cell oils seem to be alternatives for biodiesel production due to their similar composition to that of vegetable oils. However, the cold flow properties of the biodiesel produced from microbial oils are unacceptable and have to be modified by an efficient transesterification. Glycerol which is by product of transesterification can be valorised into some more useful products so that it can also be utilised along with biodiesel to simplify the downstream processing. The review paper discusses about various potent microorganisms for biodiesel production, enzymes involved in the lipid accumulation, lipid quantification methods, catalysts used in transesterification (including enzymatic catalyst and valorisation of glycerol.

  9. A DNMT3B alternatively spliced exon and encoded peptide are novel biomarkers of human pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Sailesh Gopalakrishna-Pillai

    Full Text Available A major obstacle in human stem cell research is the limited number of reagents capable of distinguishing pluripotent stem cells from partially differentiated or incompletely reprogrammed derivatives. Although human embryonic stem cells (hESCs and induced pluripotent stem cells (iPSCs express numerous alternatively spliced transcripts, little attention has been directed at developing splice variant-encoded protein isoforms as reagents for stem cell research. In this study, several genes encoding proteins involved in important signaling pathways were screened to detect alternatively spliced transcripts that exhibited differential expression in pluripotent stem cells (PSCs relative to spontaneously differentiated cells (SDCs. Transcripts containing the alternatively spliced exon 10 of the de novo DNA methyltransferase gene, DNMT3B, were identified that are expressed in PSCs. To demonstrate the utility and superiority of splice variant specific reagents for stem cell research, a peptide encoded by DNMT3B exon 10 was used to generate an antibody, SG1. The SG1 antibody detects a single DNMT3B protein isoform that is expressed only in PSCs but not in SDCs. The SG1 antibody is also demonstrably superior to other antibodies at distinguishing PSCs from SDCs in mixed cultures containing both pluripotent stem cells and partially differentiated derivatives. The tightly controlled down regulation of DNMT3B exon 10 containing transcripts (and exon 10 encoded peptide upon spontaneous differentiation of PSCs suggests that this DNMT3B splice isoform is characteristic of the pluripotent state. Alternatively spliced exons, and the proteins they encode, represent a vast untapped reservoir of novel biomarkers that can be used to develop superior reagents for stem cell research and to gain further insight into mechanisms controlling stem cell pluripotency.

  10. Aquaporin-10 represents an alternative pathway for glycerol efflux from human adipocytes.

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    Umberto Laforenza

    Full Text Available BACKGROUND: Glycerol outflow from adipocytes has been considered for a decade to be mediated by aquaporin-7, an aquaglyceroporin highly expressed in the adipose tissue. Its involvement in glycerol metabolism has been widely studied also in humans. Recent studies in different aquaporin-7 KO mice models pose two different questions 1 the exact localization of aquaporin-7 in human white adipose tissue; 2 the existence of other aquaglyceroporins that work with aquaporin-7 to guarantee glycerol efflux and thus a normal adiposity in humans. To this purpose we investigated the expression, the localization and the functioning of aquaglyceroporin-10 in subcutaneous white adipose tissue, in isolated and cultured differentiated adipocytes. METHODOLOGY/PRINCIPAL FINDINGS: Aquaporin-7 and -10 were expressed in the white adipose tissue both at mRNA and at protein level. Immunofluorescence revealed aquaporin-7 and -10 labelling in the human adipose tissue both to the plasma membrane and to a thin rim of cytoplasm of adipocytes. Aquaporin-7, but not aquaporin-10, colocalized with the endothelial marker CD34. Human cultured differentiated adipocytes showed an aquaporin-7 and -10 labelling mainly in the cytoplasm and in the lipid droplets with insulin reinforcing the lipid droplets staining and isoproterenol inducing its translocation to the plasma membrane compartment. Water and glycerol permeability measurements using adipocytes and adipose membrane vesicles confirmed the presence of functioning aquaglyceroporins. Aquaporin-10 silencing in human differentiated adipocytes resulted in a 50% decrease of glycerol and osmotic water permeability. CONCLUSIONS/SIGNIFICANCE: The results indicate that aquaporin-7, differently from mice, is present in both adipocyte and capillary plasma membranes of human adipose tissue. Aquaporin-10, on the contrary, is expressed exclusively in the adipocytes. The expression of two aquaglyceroporins in human adipose tissue is

  11. Cloning and characterization of the mouse Mcoln1 gene reveals an alternatively spliced transcript not seen in humans

    Directory of Open Access Journals (Sweden)

    Stahl Stefanie

    2002-02-01

    Full Text Available Abstract Background Mucolipidosis type IV (MLIV is an autosomal recessive lysosomal storage disorder characterized by severe neurologic and ophthalmologic abnormalities. Recently the MLIV gene, MCOLN1, has been identified as a new member of the transient receptor potential (TRP cation channel superfamily. Here we report the cloning and characterization of the mouse homologue, Mcoln1, and report a novel splice variant that is not seen in humans. Results The human and mouse genes display a high degree of synteny. Mcoln1 shows 91% amino acid and 86% nucleotide identity to MCOLN1. Also, Mcoln1 maps to chromosome 8 and contains an open reading frame of 580 amino acids, with a transcript length of approximately 2 kb encoded by 14 exons, similar to its human counterpart. The transcript that results from murine specific alternative splicing encodes a 611 amino acid protein that differs at the c-terminus. Conclusions Mcoln1 is highly similar to MCOLN1, especially in the transmembrane domains and ion pore region. Also, the late endosomal/lysosomal targeting signal is conserved, supporting the hypothesis that the protein is localized to these vesicle membranes. To date, there are very few reports describing species-specific splice variants. While identification of Mcoln1 is crucial to the development of mouse models for MLIV, the fact that there are two transcripts in mice suggests an additional or alternate function of the gene that may complicate phenotypic assessment.

  12. Putative alternative polyadenylation (APA) events in the early interaction of Salmonella enterica Typhimurium and human host cells.

    Science.gov (United States)

    Afonso-Grunz, Fabian

    2015-12-01

    The immune response of epithelial cells upon infection is mediated by changing activity levels of a variety of proteins along with changes in mRNA, and also ncRNA abundance. Alternative polyadenylation (APA) represents a mechanism that diversifies gene expression similar to alternative splicing. T-cell activation, neuronal activity, development and several human diseases including viral infections involve APA, but at present it remains unclear if this mechanism is also implicated in the response to bacterial infections. Our recently published study of interacting Salmonella enterica Typhimurium and human host cells includes genome-wide expression profiles of human epithelial cells prior and subsequent to infection with the invasive pathogen. The generated dataset (GEO accession number: GSE61730) covers several points of time post infection, and one of these interaction stages was additionally profiled with MACE-based dual 3'Seq, which allows for identification of polyadenylation (PA) sites. The present study features the polyadenylation landscape in early interacting cells based on this data, and provides a comparison of the identified PA sites with those of a corresponding 3P-Seq dataset of non-interacting cells. Differential PA site usage of FTL, PRDX1 and VAPA results in transcription of mRNA isoforms with distinct sets of miRNA and protein binding sites that influence processing, localization, stability, and translation of the respective mRNA. APA of these candidate genes consequently harbors the potential to modulate the host cell response to bacterial infection.

  13. Alternative splice variants of the human centrosome kinase Nek2 exhibit distinct patterns of expression in mitosis.

    Science.gov (United States)

    Hames, Rebecca S; Fry, Andrew M

    2002-01-01

    Nek2 is a cell-cycle-regulated protein kinase that localizes to the centrosome and is likely to be involved in regulating centrosome structure at the G(2)/M transition. Here, we localize the functional human Nek2 gene to chromosome 1 and show that alternative polyadenylation signals provide a mechanism for generating two distinct isoforms. Sequencing of products generated by reverse transcriptase PCR, immunoblotting of cell extracts and transfection of antisense oligonucleotides together demonstrate that human Nek2 is expressed as two splice variants. These isoforms, designated Nek2A and Nek2B, are detected in primary blood lymphocytes as well as adult transformed cells. Nek2A and Nek2B, which can form homo- and hetero-dimers, both localize to the centrosome, although only Nek2A can induce centrosome splitting upon overexpression. Importantly, Nek2A and Nek2B exhibit distinct patterns of cell-cycle-dependent expression. Both are present in low amounts in the G(1) phase and exhibit increased abundance in the S and G(2) phases. However, Nek2A disappears in prometaphase-arrested cells, whereas Nek2B remains elevated. These results demonstrate that two alternative splice variants of the human centrosomal kinase Nek2 exist that differ in their expression patterns during mitosis. This has important implications for our understanding of both Nek2 protein kinase regulation and the control of centrosome structure during mitosis.

  14. Unravelling the mystery of stem/progenitor cells in human breast milk.

    Directory of Open Access Journals (Sweden)

    Yiping Fan

    Full Text Available BACKGROUND: Mammary stem cells have been extensively studied as a system to delineate the pathogenesis and treatment of breast cancer. However, research on mammary stem cells requires tissue biopsies which limit the quantity of samples available. We have previously identified putative mammary stem cells in human breast milk, and here, we further characterised the cellular component of human breast milk. METHODOLOGY/PRINCIPAL FINDINGS: We identified markers associated with haemopoietic, mesenchymal and neuro-epithelial lineages in the cellular component of human breast milk. We found 2.6 ± 0.8% (mean ± SEM and 0.7 ± 0.2% of the whole cell population (WCP were found to be CD133+ and CD34+ respectively, 27.8 ± 9.1% of the WCP to be positive for Stro-1 through flow-cytometry. Expressions of neuro-ectodermal stem cell markers such as nestin and cytokeratin 5 were found through reverse-transcription polymerase chain reaction (RT-PCR, and in 4.17 ± 0.2% and 0.9 ± 0.2% of the WCP on flow-cytometry. We also established the presence of a side-population (SP (1.8 ± 0.4% of WCP as well as CD133+ cells (1.7 ± 0.5% of the WCP. Characterisation of the sorted SP and non-SP, CD133+ and CD133- cells carried out showed enrichment of CD326 (EPCAM in the SP cells (50.6 ± 8.6 vs 18.1 ± 6.0, P-value  = 0.02. However, culture in a wide range of in vitro conditions revealed the atypical behaviour of stem/progenitor cells in human breast milk; in that if they are present, they do not respond to established culture protocols of stem/progenitor cells. CONCLUSIONS/SIGNIFICANCE: The identification of primitive cell types within human breast milk may provide a non-invasive source of relevant mammary cells for a wide-range of applications; even the possibility of banking one's own stem cell for every breastfeeding woman.

  15. Argonaute-1 binds transcriptional enhancers and controls constitutive and alternative splicing in human cells

    Science.gov (United States)

    Alló, Mariano; Agirre, Eneritz; Bessonov, Sergey; Bertucci, Paola; Gómez Acuña, Luciana; Buggiano, Valeria; Bellora, Nicolás; Singh, Babita; Petrillo, Ezequiel; Blaustein, Matías; Miñana, Belén; Dujardin, Gwendal; Pozzi, Berta; Pelisch, Federico; Bechara, Elías; Agafonov, Dmitry E.; Srebrow, Anabella; Lührmann, Reinhard; Valcárcel, Juan; Eyras, Eduardo; Kornblihtt, Alberto R.

    2014-01-01

    The roles of Argonaute proteins in cytoplasmic microRNA and RNAi pathways are well established. However, their implication in small RNA-mediated transcriptional gene silencing in the mammalian cell nucleus is less understood. We have recently shown that intronic siRNAs cause chromatin modifications that inhibit RNA polymerase II elongation and modulate alternative splicing in an Argonaute-1 (AGO1)-dependent manner. Here we used chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) to investigate the genome-wide distribution of AGO1 nuclear targets. Unexpectedly, we found that about 80% of AGO1 clusters are associated with cell-type-specific transcriptional enhancers, most of them (73%) overlapping active enhancers. This association seems to be mediated by long, rather than short, enhancer RNAs and to be more prominent in intragenic, rather than intergenic, enhancers. Paradoxically, crossing ChIP-seq with RNA-seq data upon AGO1 depletion revealed that enhancer-bound AGO1 is not linked to the global regulation of gene transcription but to the control of constitutive and alternative splicing, which was confirmed by an individual gene analysis explaining how AGO1 controls inclusion levels of the cassette exon 107 in the SYNE2 gene. PMID:25313066

  16. Insects feeding on cadavers as an alternative source of human genetic material

    Directory of Open Access Journals (Sweden)

    Rafał Skowronek

    2015-03-01

    Full Text Available In some criminal cases, the use of classical sources of human genetic material is difficult or even impossible. One solution may be the use of insects, especially blowfly larvae which feed on corpses. A recent review of case reports and experimental studies available in biomedical databases has shown that insects can be a valuable source of human mitochondrial and genomic deoxyribonucleic acid (DNA, allowing for an effective analysis of hypervariable region (HVR sequences and short tandem repeat (STR profiles, respectively. The optimal source of human DNA is the crop (a part of the gut of active third-instar blowfly larvae. Pupae and insect faeces can be also used in forensic genetic practice instead of the contents of the alimentary tract.

  17. Can Human-made Saltpans Represent an Alternative Habitat for Shorebirds? Implications for a Predictable Loss of Estuarine Sediment Flats

    Science.gov (United States)

    Dias, Maria P.; Lecoq, Miguel; Moniz, Filipe; Rabaça, João E.

    2014-01-01

    Estuarine areas worldwide are under intense pressure due to human activities such as upstream dam building. Shorebirds strongly depend on estuarine intertidal flats during migration and wintering periods and so are particularly vulnerable to such impacts, whose magnitude will depend on the availability of alternative feeding habitats. In this study we analyze if man-made saltpans can represent an alternative habitat for wintering and migrating shorebirds in the Guadiana estuary, a wetland that is already experiencing environmental changes due to the building of the Alqueva reservoir, the largest in Western Europe. We compared the use of mudflats and saltpans as feeding areas by several shorebird species before the construction of the dam. A dataset with 26 years of counts data was also analyzed in order to detect any long-term trend in shorebirds abundance. We concluded that saltpans, in particular the fully mechanized, can be used as an alternative habitat by larger species during winter and southward migration, thus playing a major role in minimizing the possible effects of sediment loss due to dam building. In contrast, smaller species were particularly dependent on mudflats to feed. A significant change in population trends, from positive to negative, was detected for two species. Although we still have no evidence that this is directly linked to dam building, this result and documented changes that limit primary productivity justifies the implementation of a long-term monitoring scheme of shorebird populations in this estuary. We also reinforce the need to manage the saltpans as key habitats for shorebirds.

  18. Space station crew safety alternatives study. Volume 3: Safety impact of human factors

    Science.gov (United States)

    Rockoff, L. A.; Raasch, R. F.; Peercy, R. L., Jr.

    1985-01-01

    The first 15 years of accumulated space station concepts for Initial Operational Capability (IOC) during the early 1990's was considered. Twenty-five threats to the space station are identified and selected threats addressed as impacting safety criteria, escape and rescue, and human factors safety concerns. Of the 25 threats identified, eight are discussed including strategy options for threat control: fire, biological or toxic contamination, injury/illness, explosion, loss of pressurization, radiation, meteoroid penetration and debris. Of particular interest here is volume three (of five volumes) pertaining to the safety impact of human factors.

  19. An Alternative to the Human Hemoglobin Test in the Investigation of Bloodstains Treated with Active Oxygen: The Human Glycophorin A Test

    Directory of Open Access Journals (Sweden)

    Ana Castelló

    2011-01-01

    Full Text Available In criminal investigations, there are three stages involved when studying bloodstains: search and orientation, confirmation, and individualization. Confirmatory tests have two aims: to show that the stain contains a human biological fluid and to confirm the type of biological fluid. The need to determine the nature of the evidence is reflected in the latest bibliography, where the possibility of employing mRNA and miRNA markers for this purpose is proposed. While these new proposals are being investigated, the kits for determining human hemoglobin currently provide a simple solution for resolving this issue. With these kits, the possibility of obtaining false positives and false negatives is well known. However, recently, a new problem has been detected. This involves the interference caused by new cleaning products that contain sodium percarbonate (or active oxygen when determining human hemoglobin. With the aim to resolve this problem, this work studied the ability of the human glycophorin A test to determine human blood in samples that have been treated with active oxygen. Our results show that the human glycophorin A test has a greater resistance to the destructive effect of the new detergents containing active oxygen; consequently, it provides an alternative to be taken into consideration in the confirmatory diagnoses of bloodstains.

  20. Confirmed low prevalence of Listeria mastitis in she-camel milk delivers a safe, alternative milk for human consumption.

    Science.gov (United States)

    Osman, Kamelia M; Samir, Ahmed; Orabi, Ahmed; Zolnikov, Tara Rava

    2014-02-01

    She-camel milk is an alternative solution for people allergic to milk; unfortunately, potential harmful bacteria have not been tested in she-camel milk. Listeria monocytogenes is one harmful bacterium that causes adverse health effects if chronically or acutely ingested by humans. The purpose of this study was to estimate the prevalence, characterize the phenotypic, genetic characterization, virulence factors, and antibiopotential harmful bacteria resistance profile of Listeria isolated from the milk of she-camel. Udder milk samples were collected from 100 she-camels and screened for mastitis using the California mastitis test (46 healthy female camels, 24 subclinical mastitic animals and 30 clinical mastitic animals). Samples were then examined for the presence of pathogenic Listeria spp; if located, the isolation of Listeria was completed using the International Organization for Standards technique to test for pathogenicity. The isolates were subjected to PCR assay for virulence-associated genes. Listeria spp. were isolated from 4% of samples and only 1.0% was confirmed as L. monocytogenes. The results of this study provide evidence for the low prevalence of intramammary Listeria infection; additionally, this study concludes she-camel milk in healthy camels milked and harvested in proper hygienic conditions may be used as alternative milk for human consumption.

  1. A genome wide analysis of alternative splicing events during the osteogenic differentiation of human cartilage endplate-derived stem cells.

    Science.gov (United States)

    Shang, Jin; Wang, Honggang; Fan, Xin; Shangguan, Lei; Liu, Huan

    2016-08-01

    Low back pain is a prevalent disease, which leads to suffering and disabilities in a vast number of individuals. Degenerative disc diseases are usually the underlying causes of low back pain. However, the pathogenesis of degenerative disc diseases is highly complex and difficult to determine. Current therapies for degenerative disc diseases are various. In particular, cell-based therapies have proven to be effective and promising. Our research group has previously isolated and identified the cartilage endplate‑derived stem cells. In addition, alternative splicing is a sophisticated regulatory mechanism, which greatly increases cellular complexity and phenotypic diversity of eukaryotic organisms. The present study continued to investigate alternative splicing events in osteogenic differentiation of cartilage endplate‑derived stem cells. An Affymetrix Human Transcriptome Array 2.0 was used to detect splicing changes between the control and differentiated samples. Additionally, molecular function and pathway analysis were also performed. Following rigorous bioinformatics analysis of the data, 3,802 alternatively spliced genes were identified, and 10 of these were selected for validation by reverse transcription‑polymerase chain reaction. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway analysis also revealed numerous enriched GO terms and signaling pathways. To the best of our knowledge, the present study is the first to investigate alternative splicing mechanisms in osteogenic differentiation of stem cells on a genome‑wide scale. The illumination of molecular mechanisms of stem cell osteogenic differentiation may assist the development novel bioengineered methods to treat degenerative disc diseases.

  2. A novel antibody against human properdin inhibits the alternative complement system and specifically detects properdin from blood samples.

    Directory of Open Access Journals (Sweden)

    Diana Pauly

    Full Text Available The complement system is an essential part of the innate immune system by acting as a first line of defense which is stabilized by properdin, the sole known positive regulator of the alternative complement pathway. Dysregulation of complement can promote a diversity of human inflammatory diseases which are treated by complement inhibitors. Here, we generated a novel blocking monoclonal antibody (mAb against properdin and devised a new diagnostic assay for this important complement regulator. Mouse mAb 1340 specifically detected native properdin from human samples with high avidity. MAb 1340 inhibited specifically the alternative complement mediated cell lysis within a concentration range of 1-10 µg/mL. Thus, in vitro anti-properdin mAb 1340 was up to fifteen times more efficient in blocking the complement system as compared to anti-C5 or anti-Ba antibodies. Computer-assisted modelling suggested a three-dimensional binding epitope in a properdin-C3(H2O-clusterin complex to be responsible for the inhibition. Recovery of properdin in a newly established sandwich ELISA using mAb 1340 was determined at 80-125% for blood sample dilutions above 1∶50. Reproducibility assays showed a variation below 25% at dilutions less than 1∶1,000. Systemic properdin concentrations of healthy controls and patients with age-related macular degeneration or rheumatic diseases were all in the range of 13-30 µg/mL and did not reveal significant differences. These initial results encourage further investigation into the functional role of properdin in the development, progression and treatment of diseases related to the alternative complement pathway. Thus, mAb 1340 represents a potent properdin inhibitor suitable for further research to understand the exact mechanisms how properdin activates the complement C3-convertase and to determine quantitative levels of properdin in biological samples.

  3. Haemophilus ducreyi Seeks Alternative Carbon Sources and Adapts to Nutrient Stress and Anaerobiosis during Experimental Infection of Human Volunteers.

    Science.gov (United States)

    Gangaiah, Dharanesh; Zhang, Xinjun; Baker, Beth; Fortney, Kate R; Gao, Hongyu; Holley, Concerta L; Munson, Robert S; Liu, Yunlong; Spinola, Stanley M

    2016-05-01

    Haemophilus ducreyi causes the sexually transmitted disease chancroid in adults and cutaneous ulcers in children. In humans, H. ducreyi resides in an abscess and must adapt to a variety of stresses. Previous studies (D. Gangaiah, M. Labandeira-Rey, X. Zhang, K. R. Fortney, S. Ellinger, B. Zwickl, B. Baker, Y. Liu, D. M. Janowicz, B. P. Katz, C. A. Brautigam, R. S. Munson, Jr., E. J. Hansen, and S. M. Spinola, mBio 5:e01081-13, 2014, http://dx.doi.org/10.1128/mBio.01081-13) suggested that H. ducreyi encounters growth conditions in human lesions resembling those found in stationary phase. However, how H. ducreyi transcriptionally responds to stress during human infection is unknown. Here, we determined the H. ducreyi transcriptome in biopsy specimens of human lesions and compared it to the transcriptomes of bacteria grown to mid-log, transition, and stationary phases. Multidimensional scaling showed that the in vivo transcriptome is distinct from those of in vitro growth. Compared to the inoculum (mid-log-phase bacteria), H. ducreyi harvested from pustules differentially expressed ∼93 genes, of which 62 were upregulated. The upregulated genes encode homologs of proteins involved in nutrient transport, alternative carbon pathways (l-ascorbate utilization and metabolism), growth arrest response, heat shock response, DNA recombination, and anaerobiosis. H. ducreyi upregulated few genes (hgbA, flp-tad, and lspB-lspA2) encoding virulence determinants required for human infection. Most genes regulated by CpxRA, RpoE, Hfq, (p)ppGpp, and DksA, which control the expression of virulence determinants and adaptation to a variety of stresses, were not differentially expressed in vivo, suggesting that these systems are cycling on and off during infection. Taken together, these data suggest that the in vivo transcriptome is distinct from those of in vitro growth and that adaptation to nutrient stress and anaerobiosis is crucial for H. ducreyi survival in humans.

  4. Calpains: potential targets for alternative chemotherapeutic intervention against human pathogenic trypanosomatids.

    Science.gov (United States)

    Branquinha, M H; Marinho, F A; Sangenito, L S; Oliveira, S S C; Goncalves, K C; Ennes-Vidal, V; d'Avila-Levy, C M; Santos, A L S

    2013-01-01

    The treatment for both leishmaniasis and trypanosomiasis, which are severe human infections caused by trypanosomatids belonging to Leishmania and Trypanosoma genera, respectively, is extremely limited because of concerns of toxicity and efficacy with the available anti-protozoan drugs, as well as the emergence of drug resistance. Consequently, the urgency for the discovery of new trypanosomatid targets and novel bioactive compounds is particularly necessary. In this context, the investigation of changes in parasite gene expression between drug resistant/sensitive strains and in the up-regulation of virulence-related genes in infective forms has brought to the fore the involvement of calpain-like proteins in several crucial pathophysiological processes performed by trypanosomatids. These studies were encouraged by the publication of the complete genome sequences of three human pathogenic trypanosomatids, Trypanosoma brucei, Trypanosoma cruzi and Leishmania major, which allowed in silico analyses that in turn directed the identification of numerous genes with interesting chemotherapeutic characteristics, including a large family of calpain-related proteins, in which to date 23 genes were assigned as calpains in T. brucei, 40 in T. cruzi and 33 in L. braziliensis. In the present review, we intend to add to these biochemical/biological reports the investigations performed upon the inhibitory capability of calpain inhibitors against human pathogenic trypanosomatids.

  5. Investing in human and natural capital. An alternative paradigm for sustainable development in Awassa, Ethiopia

    Energy Technology Data Exchange (ETDEWEB)

    Reynolds, Travis W. [Evans School of Public Affairs, University of Washington, Seattle, Washington, 98195 (United States); Farley, Joshua [Gund Institute for Ecological Economics and Department of Community Development and Applied Economics, University of Vermont, Burlington, Vermont, 05405 (United States); Huber, Candice [UVM Agricultural Extension Service, University of Vermont, Burlington, Vermont, 05405 (United States)

    2010-09-15

    Ethiopia remains underdeveloped due to limitations in natural, human, social and built capital. A 2006 scientific atelier conducted in the city of Awassa, Ethiopia investigated investments in human and natural capital as a sustainable development strategy. Local stakeholders identified firewood shortages, degradation of croplands, rising lake levels encroaching on croplands and poor water quality as major impediments to development. They further identified ecological degradation as a key component of these problems, and they acknowledged multiple vicious cycles compounding the environmental and economic threats to the Awassa community. Proposed solutions included investment in natural capital in the form of reforestation activities, investment in human capital in the form of promoting more efficient wood stoves along with increasing public awareness of environmental threats, and investments in social capital in the form of inter-institutional coordination to address environmental problems. All recommended investments rely primarily on national resources, in distinct contrast to the extensive imports required for most built capital investments. Unfortunately, Awassa lacks the surplus necessary for major capital investments of any kind. The atelier therefore helped local participants identify potential funders and write grant proposals for various projects, though none have been funded so far. Reversing the ecological degradation on the scale necessary for sustained economic development in Ethiopia however will require a steady flow of substantial investments, and cannot rely solely on the short term generosity of funders. International payments for carbon sequestration and other ecosystem services could help provide the necessary resources. (author)

  6. Prevention of adenosine A2A receptor activation diminishes beat-to-beat alternation in human atrial myocytes.

    Science.gov (United States)

    Molina, Cristina E; Llach, Anna; Herraiz-Martínez, Adela; Tarifa, Carmen; Barriga, Montserrat; Wiegerinck, Rob F; Fernandes, Jacqueline; Cabello, Nuria; Vallmitjana, Alex; Benitéz, Raúl; Montiel, José; Cinca, Juan; Hove-Madsen, Leif

    2016-01-01

    Atrial fibrillation (AF) has been associated with increased spontaneous calcium release from the sarcoplasmic reticulum and linked to increased adenosine A2A receptor (A2AR) expression and activation. Here we tested whether this may favor atrial arrhythmogenesis by promoting beat-to-beat alternation and irregularity. Patch-clamp and confocal calcium imaging was used to measure the beat-to-beat response of the calcium current and transient in human atrial myocytes. Responses were classified as uniform, alternating or irregular and stimulation of Gs-protein coupled receptors decreased the frequency where a uniform response could be maintained from 1.0 ± 0.1 to 0.6 ± 0.1 Hz; p < 0.01 for beta-adrenergic receptors and from 1.4 ± 0.1 to 0.5 ± 0.1 Hz; p < 0.05 for A2ARs. The latter was linked to increased spontaneous calcium release and after-depolarizations. Moreover, A2AR activation increased the fraction of non-uniformly responding cells in HL-1 myocyte cultures from 19 ± 3 to 51 ± 9 %; p < 0.02, and electrical mapping in perfused porcine atria revealed that adenosine induced electrical alternans at longer cycle lengths, doubled the fraction of electrodes showing alternation, and increased the amplitude of alternations. Importantly, protein kinase A inhibition increased the highest frequency where uniform responses could be maintained from 0.84 ± 0.12 to 1.86 ± 0.11 Hz; p < 0.001 and prevention of A2AR-activation with exogenous adenosine deaminase selectively increased the threshold from 0.8 ± 0.1 to 1.2 ± 0.1 Hz; p = 0.001 in myocytes from patients with AF. In conclusion, A2AR-activation promotes beat-to-beat irregularities in the calcium transient in human atrial myocytes, and prevention of A2AR activation may be a novel means to maintain uniform beat-to-beat responses at higher beating frequencies in patients with atrial fibrillation.

  7. Real-time sweat analysis via alternating current conductivity of artificial and human sweat

    Science.gov (United States)

    Liu, Gengchen; Alomari, Mahmoud; Sahin, Bunyamin; Snelgrove, Samuel E.; Edwards, Jeffrey; Mellinger, Axel; Kaya, Tolga

    2015-03-01

    Dehydration is one of the most profound physiological challenges that significantly affects athletes and soldiers if not detected early. Recently, a few groups have focused on dehydration detection using sweat as the main biomarker. Although there are some proposed devices, the electrical and chemical characteristics of sweat have yet to be incorporated into the validations. In this work, we have developed a simple test setup to analyze artificial sweat that is comprised the main components of human sweat. We provide theoretical and experimental details on the electrical and chemical behavior of the artificial sweat for various concentration values within a temperature range of 5 °C to 50 °C. We have also developed an efficient sweat collecting and detection system based on 3D printing. Human studies were conducted and this particular protocol has shown that dehydration starts to take effect as early as 40 min into the physical activity if there is no fluid intake during the exercise. We believe that our device will lead to developing viable real-time sweat analysis systems.

  8. The role of the alternative coreceptor GPR15 in SIV tropism for human cells.

    Science.gov (United States)

    Kiene, Miriam; Marzi, Andrea; Urbanczyk, Andreas; Bertram, Stephanie; Fisch, Tanja; Nehlmeier, Inga; Gnirss, Kerstin; Karsten, Christina B; Palesch, David; Münch, Jan; Chiodi, Francesca; Pöhlmann, Stefan; Steffen, Imke

    2012-11-10

    Many SIV isolates can employ the orphan receptor GPR15 as coreceptor for efficient entry into transfected cell lines, but the role of endogenously expressed GPR15 in SIV cell tropism is largely unclear. Here, we show that several human B and T cell lines express GPR15 on the cell surface, including the T/B cell hybrid cell line CEMx174, and that GPR15 expression is essential for SIV infection of CEMx174 cells. In addition, GPR15 expression was detected on subsets of primary human CD4(+), CD8(+) and CD19(+) peripheral blood mononuclear cells (PBMCs), respectively. However, GPR15(+) PBMCs were not efficiently infected by HIV and SIV, including cells from individuals homozygous for the defective Δ32 ccr5 allele. These results suggest that GPR15 is coexpressed with CD4 on PBMCs but that infection of CD4(+), GPR15(+) cells is not responsible for the well documented ability of SIV to infect CCR5(-) blood cells.

  9. Similarity of recombinant human perlecan domain 1 by alternative expression systems bioactive heterogenous recombinant human perlecan D1

    DEFF Research Database (Denmark)

    Ellis, April L; Pan, Wensheng; Yang, Guang

    2010-01-01

    BACKGROUND: Heparan sulfate glycosaminoglycans are diverse components of certain proteoglycans and are known to interact with growth factors as a co-receptor necessary to induce signalling and growth factor activity. In this report we characterize heterogeneously glycosylated recombinant human...... perlecan domain 1 (HSPG2 abbreviated as rhPln.D1) synthesized in either HEK 293 cells or HUVECs by transient gene delivery using either adenoviral or expression plasmid technology. RESULTS: By SDS-PAGE analysis following anion exchange chromatography, the recombinant proteoglycans appeared to possess...... glycosaminoglycan chains ranging, in total, from 6 kDa to >90 kDa per recombinant. Immunoblot analysis of enzyme-digested high Mr rhPln.D1 demonstrated that the rhPln.D1 was synthesized as either a chondroitin sulfate or heparan sulfate proteoglycan, in an approximately 2:1 ratio, with negligible hybrids. Secondary...

  10. Hair breakage index: an alternative tool for damage assessment of human hair.

    Science.gov (United States)

    Mhaskar, Sudhakar; Kalghatgi, Bhargavi; Chavan, Madhavi; Rout, Suryamani; Gode, Vaishali

    2011-01-01

    Improper hair care, mechanical abrasion, sun damage and chemical treatment changes the physical and morphological characteristics of hair. Several methods involving microscopic techniques, protein loss and assessment of tensile properties of the hair are generally used to evaluate the extent of damage caused. These are also used to determine the protective effect of hair care products. In the present investigation, the hair breakage index (HBI) was used as an alternative tool to determine the change in the properties of hair on weathering. HBI is a measure of the diameter of hair in a given cross sectional area of a marked region of hair on the scalp. The hair diameter changes as we progress towards the tip of the hair due to breakage. The ratio of the diameter of hair bundle in the distal region to the diameter of hair bundle in the proximal region from the scalp is used as an indicator of hair breakage. Higher HBI value is an indicator of hair damage.A study was conducted for duration of 16 weeks to assess the effect of weathering due to grooming practices on HBI values. The HBI and break stress for a group of 30 subjects were measured at baseline and at the end of 16 weeks (NU). Since Coconut oil (CNO) is known to have a positive benefit on tensile properties of hair, another matched group of 30 subjects who oiled their hair daily with CNO was used as a positive control (CNO). The HBI and break stress for this group were also measured at the baseline and after 16 weeks. It was observed that the HBI significantly increased in the NU group versus the CNO user group. The break stress also significantly decreased in the NU group suggesting its correlation with the HBI data. This study demonstrates the usefulness of HBI as a simple and effective tool for determining hair damage and its protection by different hair care products.

  11. Paired associative transcranial alternating current stimulation increases the excitability of corticospinal projections in humans.

    Science.gov (United States)

    McNickle, Emmet; Carson, Richard G

    2015-04-01

    Many types of non-invasive brain stimulation alter corticospinal excitability (CSE). Paired associative stimulation (PAS) has attracted particular attention as its effects ostensibly adhere to Hebbian principles of neural plasticity. In prototypical form, a single electrical stimulus is directed to a peripheral nerve in close temporal contiguity with transcranial magnetic stimulation delivered to the contralateral primary motor cortex (M1). Repeated pairing of the two discrete stimulus events (i.e. association) over an extended period either increases or decreases the excitability of corticospinal projections from M1, contingent on the interstimulus interval. We studied a novel form of associative stimulation, consisting of brief trains of peripheral afferent stimulation paired with short bursts of high frequency (≥80 Hz) transcranial alternating current stimulation (tACS) over contralateral M1. Elevations in the excitability of corticospinal projections to the forearm were observed for a range of tACS frequency (80, 140 and 250 Hz), current (1, 2 and 3 mA) and duration (500 and 1000 ms) parameters. The effects were at least as reliable as those brought about by PAS or transcranial direct current stimulation. When paired with tACS, muscle tendon vibration also induced elevations of CSE. No such changes were brought about by the tACS or peripheral afferent stimulation alone. In demonstrating that associative effects are expressed when the timing of the peripheral and cortical events is not precisely circumscribed, these findings suggest that multiple cellular pathways may contribute to a long term potentiation-type response. Their relative contributions will differ depending on the nature of the induction protocol that is used.

  12. Differential Cytotoxic Potential of Silver Nanoparticles in Human Ovarian Cancer Cells and Ovarian Cancer Stem Cells

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    Yun-Jung Choi

    2016-12-01

    Full Text Available The cancer stem cell (CSC hypothesis postulates that cancer cells are composed of hierarchically-organized subpopulations of cells with distinct phenotypes and tumorigenic capacities. As a result, CSCs have been suggested as a source of disease recurrence. Recently, silver nanoparticles (AgNPs have been used as antimicrobial, disinfectant, and antitumor agents. However, there is no study reporting the effects of AgNPs on ovarian cancer stem cells (OvCSCs. In this study, we investigated the cytotoxic effects of AgNPs and their mechanism of causing cell death in A2780 (human ovarian cancer cells and OvCSCs derived from A2780. In order to examine these effects, OvCSCs were isolated and characterized using positive CSC markers including aldehyde dehydrogenase (ALDH and CD133 by fluorescence-activated cell sorting (FACS. The anticancer properties of the AgNPs were evaluated by assessing cell viability, leakage of lactate dehydrogenase (LDH, reactive oxygen species (ROS, and mitochondrial membrane potential (mt-MP. The inhibitory effect of AgNPs on the growth of ovarian cancer cells and OvCSCs was evaluated using a clonogenic assay. Following 1–2 weeks of incubation with the AgNPs, the numbers of A2780 (bulk cells and ALDH+/CD133+ colonies were significantly reduced. The expression of apoptotic and anti-apoptotic genes was measured by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR. Our observations showed that treatment with AgNPs resulted in severe cytotoxicity in both ovarian cancer cells and OvCSCs. In particular, AgNPs showed significant cytotoxic potential in ALDH+/CD133+ subpopulations of cells compared with other subpopulation of cells and also human ovarian cancer cells (bulk cells. These findings suggest that AgNPs can be utilized in the development of novel nanotherapeutic molecules for the treatment of ovarian cancers by specific targeting of the ALDH+/CD133+ subpopulation of cells.

  13. Alternative uses of didactics scripts and anatomy models in the teaching-learning in practical human anatomy

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    Gleidially Nayara Bezerra Moraes

    2016-04-01

    Full Text Available The teaching and learning process is complex and difficult presented with respect to the human anatomy. Thus, the improvement of teaching resources applied to the teaching of this discipline, shows up as a satisfactory trend and encourages student participation as an active subject in the search for new informations, giving essential support teaching-learning process. The aim of the study was to verify the existence and utilization of teaching scripts and anatomical models in practicals classes of Human Anatomy. The study was a descriptive systematic review, developed with scientific production indexed in electronic databases LILACS, MEDLINE, GOOGLE ACADEMICO and SciELO; as well as Brazilian proceedings. Among the 17 articles found, 9 showed the use of anatomical models, 7 showed other methods used, and only 1 on the use of didactic manual on classroom practice of this discipline. From the study, it can be observed that the use of teaching scripts for teaching in practical classes of Human Anatomy is an innovative method and the use of anatomical models alternative has shown positive results in the teaching-learning process. However, these methods, ever can replace the use of the corpse in the teaching of this discipline.

  14. Probiotics as an alternative strategy for prevention and treatment of human diseases: a review.

    Science.gov (United States)

    Khani, Soghra; Hosseini, Hamideh M; Taheri, Mohammad; Nourani, Mohammad R; Imani Fooladi, Abbas A

    2012-04-01

    Probiotics are live microbial food supplements or their components, which have been shown to have beneficial effects on human health. Probiotics can be bacteria, molds, or yeasts, but most of them fall into the group known as lactic acid bacteria and are normally consumed in the form of yogurt, fermented milk, or other fermented foods. Data from clinical trials have shown contrasting effects and should be interpreted with caution. A large variety of potential beneficial effects have been reported including improvement of intestinal tract health, enhancing the immune system, reducing symptoms of lactose intolerance, decreasing the prevalence of allergy in susceptible individuals, reducing risk of certain cancers, treating colitis, lowering serum cholesterol concentrations, reducing blood pressure in hypertensives, and improving female urogenital infections and Helicobacter pylori infections. The aim of this article is to present a review of the current expanding knowledge of applications of utilizing probiotic microorganisms in the prevention and treatment of several diseases.

  15. Alternative nucleophilic substrates for the endonuclease activities of human immunodeficiency virus type 1 integrase

    Energy Technology Data Exchange (ETDEWEB)

    Ealy, Julie B. [Department of Medicine, Penn State College of Medicine, Milton S. Hershey Medical Center, 500 University Drive, PO Box 850, Mail Services H036, Hershey, PA 17033 (United States); Department of Chemistry, Penn State Lehigh Valley, 2809 E. Saucon Valley Road, Center Valley, PA 18034 (United States); Sudol, Malgorzata [Department of Medicine, Penn State College of Medicine, Milton S. Hershey Medical Center, 500 University Drive, PO Box 850, Mail Services H036, Hershey, PA 17033 (United States); Krzeminski, Jacek; Amin, Shantu [Department of Pharmacology, Penn State College of Medicine, Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033 (United States); Katzman, Michael, E-mail: mkatzman@psu.edu [Department of Medicine, Penn State College of Medicine, Milton S. Hershey Medical Center, 500 University Drive, PO Box 850, Mail Services H036, Hershey, PA 17033 (United States); Department of Microbiology and Immunology, Penn State College of Medicine, Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033 (United States)

    2012-11-10

    Retroviral integrase can use water or some small alcohols as the attacking nucleophile to nick DNA. To characterize the range of compounds that human immunodeficiency virus type 1 integrase can accommodate for its endonuclease activities, we tested 45 potential electron donors (having varied size and number or spacing of nucleophilic groups) as substrates during site-specific nicking at viral DNA ends and during nonspecific nicking reactions. We found that integrase used 22 of the 45 compounds to nick DNA, but not all active compounds were used for both activities. In particular, 13 compounds were used for site-specific and nonspecific nicking, 5 only for site-specific nicking, and 4 only for nonspecific nicking; 23 other compounds were not used for either activity. Thus, integrase can accommodate a large number of nucleophilic substrates but has selective requirements for its different activities, underscoring its dynamic properties and providing new information for modeling and understanding integrase.

  16. Reference gene alternatives to Gapdh in rodent and human heart failure gene expression studies

    Directory of Open Access Journals (Sweden)

    Levy Finn Olav

    2010-03-01

    Full Text Available Abstract Background Quantitative real-time RT-PCR (RT-qPCR is a highly sensitive method for mRNA quantification, but requires invariant expression of the chosen reference gene(s. In pathological myocardium, there is limited information on suitable reference genes other than the commonly used Gapdh mRNA and 18S ribosomal RNA. Our aim was to evaluate and identify suitable reference genes in human failing myocardium, in rat and mouse post-myocardial infarction (post-MI heart failure and across developmental stages in fetal and neonatal rat myocardium. Results The abundance of Arbp, Rpl32, Rpl4, Tbp, Polr2a, Hprt1, Pgk1, Ppia and Gapdh mRNA and 18S ribosomal RNA in myocardial samples was quantified by RT-qPCR. The expression variability of these transcripts was evaluated by the geNorm and Normfinder algorithms and by a variance component analysis method. Biological variability was a greater contributor to sample variability than either repeated reverse transcription or PCR reactions. Conclusions The most stable reference genes were Rpl32, Gapdh and Polr2a in mouse post-infarction heart failure, Polr2a, Rpl32 and Tbp in rat post-infarction heart failure and Rpl32 and Pgk1 in human heart failure (ischemic disease and cardiomyopathy. The overall most stable reference genes across all three species was Rpl32 and Polr2a. In rat myocardium, all reference genes tested showed substantial variation with developmental stage, with Rpl4 as was most stable among the tested genes.

  17. Creation of Alternate Stable States in Tropical Dry Forests as a Consequence of Human Disturbance

    Science.gov (United States)

    Van Bloem, S. J.; Whitmire, S. L.; Thaxton, J.

    2015-12-01

    Anthropogenic disturbances in Caribbean subtropical dry forests of the Caribbean lead to either novel species assemblages in post-disturbance forest, or stable degraded grassland states. Among these disturbances are fire and clearing. Short-term responses to both disturbances include sharp increases in nutrient fluxes, shifts in soil moisture availability, and the spread of invasive grass species if seed sources are nearby. In Guánica Forest, Puerto Rico, our studies in sites that were frequently burned and a chronosequence of once-burned sites beginning 30 years ago showed that fire is associated with invasion by exotic grass species and that once invaded, grass dominated sites do not revert to forest. After 30 years, single burn sites were still ≥65% lower in basal area and species richness than native forest. Frequently burned sites had 60-75% lower soil N, 50-65% lower S, and 56-68% lower C, as well as elevated Fe and pH. A single burn in a previously unburned forest elevated NO3 flux ~6-fold for at least the next year. Once dominated by grass, tree seed rain is extremely low (7 seeds/m2/y). Low dispersal rates combined with low germination rates makes forest regeneration unlikely. In contrast, sites that cleared or plowed without grass invasion regain structural characteristics of native forests in approximately 40 years. However, these sites are typically dominated by Leucaena leucocephala, a naturalized tree introduced to the island in the 1700s. The proportion of native-species saplings in the understory of Leucaena-dominated forests increased by 0.85%/yr in stands >35 years old until reaching ~95% in 76 yr old stands, suggesting that Leucaena facilitated the reestablishment of native species. We conclude that when anthropogenic disturbance creates grass-dominated sites, these areas will remain stable degraded states. Leucaena can serve as an alternate pathway to forest restoration, facilitating the return of forest cover with a novel combination of

  18. Neprilysin is a Mediator of Alternative Renin-Angiotensin-System Activation in the Murine and Human Kidney

    Science.gov (United States)

    Domenig, Oliver; Manzel, Arndt; Grobe, Nadja; Königshausen, Eva; Kaltenecker, Christopher C.; Kovarik, Johannes J.; Stegbauer, Johannes; Gurley, Susan B.; van Oyen, Dunja; Antlanger, Marlies; Bader, Michael; Motta-Santos, Daisy; Santos, Robson A.; Elased, Khalid M.; Säemann, Marcus D.; Linker, Ralf A.; Poglitsch, Marko

    2016-01-01

    Cardiovascular and renal pathologies are frequently associated with an activated renin-angiotensin-system (RAS) and increased levels of its main effector and vasoconstrictor hormone angiotensin II (Ang II). Angiotensin-converting-enzyme-2 (ACE2) has been described as a crucial enzymatic player in shifting the RAS towards its so-called alternative vasodilative and reno-protective axis by enzymatically converting Ang II to angiotensin-(1-7) (Ang-(1-7)). Yet, the relative contribution of ACE2 to Ang-(1-7) formation in vivo has not been elucidated. Mass spectrometry based quantification of angiotensin metabolites in the kidney and plasma of ACE2 KO mice surprisingly revealed an increase in Ang-(1-7), suggesting additional pathways to be responsible for alternative RAS activation in vivo. Following assessment of angiotensin metabolism in kidney homogenates, we identified neprilysin (NEP) to be a major source of renal Ang-(1-7) in mice and humans. These findings were supported by MALDI imaging, showing NEP mediated Ang-(1-7) formation in whole kidney cryo-sections in mice. Finally, pharmacologic inhibition of NEP resulted in strongly decreased Ang-(1-7) levels in murine kidneys. This unexpected new role of NEP may have implications for the combination therapy with NEP-inhibitors and angiotensin-receptor-blockade, which has been shown being a promising therapeutic approach for heart failure therapy. PMID:27649628

  19. An alternative promoter of the human plakophilin-3 gene controls the expression of the new isoform PKP3b.

    Science.gov (United States)

    Mühmer, Mario; Ditthardt, Denise; Jäkel, Jörg; Wischmann, Viktoria; Moll, Roland; Schmidt, Ansgar

    2014-01-01

    The plakophilin family (PKP1 to PKP3) is an essential component of the desmosomal adhesion complex with differentiation-dependent and partially overlapping expression and possible participation of the corresponding genes in malignant transformation. Here, we describe a new protein variant of the human PKP3 gene, namely PKP3b, which differs from the published PKP3a only at the amino-terminus by the splicing in of the newly identified exon 1b. Specific antibodies have demonstrated differential expression patterns of the two variants. Whereas PKP3a is broadly expressed among epithelial cells, PKP3b is abundant in the desmosomes of stratified epithelial cells, such as HaCaT but absent or heterogeneous in simple epithelial cells such as CaCo2 or MCF7. The differential expression of the PKP3 variants has been observed in a similar manner in selected normal human tissues and carcinomas derived thereof. Both variants are localized to the desmosomes of all cells of stratified tissues, whereas the new PKP3b is heterogeneously expressed in the colon and its tumors. Therefore, we assume that both variants are controlled by alternative promoters. Reporter gene assays have confirmed that a fragment upstream of exon 1b exhibits transcriptional activity only in HaCaT cells but not in CaCo2 cells and thus has been identified as an alternative promoter driving the expression of PKP3b. Finally, by using electromobility shift assays, we found a potential binding site in the PKP3b promoter for transcription factor C/EBP regulating keratinocyte differentiation and probably also PKP3b expression. We discuss the properties of the new variant PKP3b as a possible marker protein for the analyses of differentiation and malignant transformation.

  20. Three new alternative splicing variants of human cytochrome P450 2D6 mRNA in human extratumoral liver tissue

    Institute of Scientific and Technical Information of China (English)

    Jian Zhuge; Ying-Nian Yu

    2004-01-01

    AIM: To identify the new alternative splicing variants of human CYP2D6 in human extratumoral liver tissue with RT-PCR and sequencing.METHODS: Full length of human CYP2D6 cDNAs was amplificated by reverse transcription-polymerase chain reaction (RT-PCR) from a human extratumoral liver tissue and cloned into pGEM-T vector. The cDNA was sequenced.Exons from 1 to 4 of human CYP2D6 cDNAs were also amplificated by RT-PCR from extratumoral liver tissues of17 human hepatocellular carcinomas. Some RT-PCR products were sequenced. Exons 1 to 4 of CYP2D6 gene were amplified by PCR from extratumoral liver tissue DNA.Two PCR products from extratumoral liver tissues expressing skipped mRNA were partially sequenced.RESULTS: One of the CYP2D6cDNAs had 470 nucleotides from 79 to 548 (3' portion of exons 1 to 5' portion of exon 4),and was skipped. Exons 1 to 4 of CYP2D6 cDNA were assayed with RT-PCR in 17 extratumoral liver tissues. Both wild type and skipped mRNAs were expressed in 4 samples,only wild type mRNA was expressed in 5 samples, and only skipped mRNA was expressed in 8 samples. Two more variants were identified by sequencing the RT-PCR products of exons 1 to 4 of CYP2D6cDNA. The second variant skipped 411 nucleotides from 175 to 585. This variant was identified in 4 different liver tissues by sequencing the RT-PCR products. We sequenced partially 2 of the PCR products amplified of CYP2D6 exon 1 to exon 4 from extratumoral liver tissue genomic DNA that only expressed skipped mRNA by RT-PCR. No point mutations around exon 1, intron 1, and exon 4, and no deletion in CYP2D6gene were detected. The third variant was the skipped exon 3, and 153 bp was lost.CONCLUSION: Three new alternative splicing variants of CYP2D6 mRNA have been identified. They may not be caused by gene mutation and may lose CYP2D6 activity and act as a down-regulator of CYP2D6.

  1. Osteoblastic differentiation and stress response of human mesenchymal stem cells exposed to alternating current electric fields

    Directory of Open Access Journals (Sweden)

    Kaplan David L

    2011-01-01

    Full Text Available Abstract Background Electric fields are integral to many biological events, from maintaining cellular homeostasis to embryonic development to healing. The application of electric fields offers substantial therapeutic potential, while optimal dosing regimens and the underlying mechanisms responsible for the positive clinical impact are poorly understood. Methods The purpose of this study was to track the differentiation profile and stress response of human bone marrow derived mesenchymal stem cells (hMSCs undergoing osteogenic differentiation during exposure to a 20 mV/cm, 60 kHz electric field. Morphological and biochemical changes were imaged using endogenous two-photon excited fluorescence (TPEF and quantitatively assessed through eccentricity calculations and extraction of the redox ratio from NADH, FAD and lipofuscin contributions. Real time reverse transcriptase-polymerase chain reactions (RT-PCR were used to track osteogenic differentiation markers, namely alkaline phosphatase (ALP and collagen type 1 (col1, and stress response markers, such as heat shock protein 27 (hsp27 and heat shock protein 70 (hsp70. Comparisons of collagen deposition between the stimulated hMSCs and controls were examined through second harmonic generation (SHG imaging. Results Quantitative differences in cell morphology, as described through an eccentricity ratio, were found on days 2 and days 5 (p Conclusions Electrical stimulation is a useful tool to improve hMSC osteogenic differentiation, while heat shock proteins may reveal underlying mechanisms, and optical non-invasive imaging may be used to monitor the induced morphological and biochemical changes.

  2. Bioacoustics of human whistled languages: an alternative approach to the cognitive processes of language.

    Science.gov (United States)

    Meyer, Julien

    2004-06-01

    Whistled languages are a valuable heritage of human culture. This paper gives a first survey about a new multidisciplinary approach to these languages. Previous studies on whistled equivalents of languages have already documented that they can provide significant information about the role of rhythm and melody in language. To substantiate this, most whistles are represented by modulations of frequency, centered around 2000 Hz (+/- 1000 Hz) and often reach a loudness of about 130 dB (measured at 1m from the source). Their transmission range can reach up to 10 km (as verified in La Gomera, Canary Island), and the messages can remain understandable, even if the signal is deteriorated. In some cultures the use of whistled language is associated with some "talking musical instruments" (e.g. flutes, guitars, harps, gongs, drums, khens). Finally, whistles as a means of conveying information have some analogues in the animal kingdom (e.g. some birds, cetaceans, primates), providing opportunities to compare the acoustic characteristics of the respective signals. With such properties as a reference, the project reported here has two major tasks: to further elucidate the many facets of whistled language and, above all, help to immediately stop the process of its gradual disappearance.

  3. Bioacoustics of human whistled languages: an alternative approach to the cognitive processes of language

    Directory of Open Access Journals (Sweden)

    Meyer Julien

    2004-01-01

    Full Text Available Whistled languages are a valuable heritage of human culture. This paper gives a first survey about a new multidisciplinary approach to these languages. Previous studies on whistled equivalents of languages have already documented that they can provide significant information about the role of rhythm and melody in language. To substantiate this, most whistles are represented by modulations of frequency, centered around 2000 Hz (±1000 Hz and often reach a loudness of about 130 dB (measured at 1m from the source. Their transmission range can reach up to 10 km (as verified in La Gomera, Canary Island, and the messages can remain understandable, even if the signal is deteriorated. In some cultures the use of whistled language is associated with some "talking musical instruments" (e.g. flutes, guitars, harps, gongs, drums, khens. Finally, whistles as a means of conveying information have some analogues in the animal kingdom (e.g. some birds, cetaceans, primates, providing opportunities to compare the acoustic characteristics of the respective signals. With such properties as a reference, the project reported here has two major tasks: to further elucidate the many facets of whistled language and, above all, help to immediately stop the process of its gradual disappearance.

  4. Identification and characterization of an alternative promoter of the human PGC-1{alpha} gene

    Energy Technology Data Exchange (ETDEWEB)

    Yoshioka, Toyo; Inagaki, Kenjiro [Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Noguchi, Tetsuya, E-mail: noguchi@med.kobe-u.ac.jp [Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Sakai, Mashito; Ogawa, Wataru; Hosooka, Tetsuya [Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Iguchi, Haruhisa; Watanabe, Eijiro; Matsuki, Yasushi; Hiramatsu, Ryuji [Genomic Science Laboratories, DainipponSumitomo Pharma Co. Ltd., 4-2-1 Takatsukasa, Takarazuka 665-8555 (Japan); Kasuga, Masato [Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655 (Japan)

    2009-04-17

    The transcriptional regulator peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha} (PGC-1{alpha}) controls mitochondrial biogenesis and energy homeostasis. Although physical exercise induces PGC-1{alpha} expression in muscle, the underlying mechanism of this effect has remained incompletely understood. We recently identified a novel muscle-enriched isoform of PGC-1{alpha} transcript (designated PGC-1{alpha}-b) that is derived from a previously unidentified first exon. We have now cloned and characterized the human PGC-1{alpha}-b promoter. The muscle-specific transcription factors MyoD and MRF4 transactivated this promoter through interaction with a proximal E-box motif. Furthermore, either forced expression of Ca{sup 2+}- and calmodulin-dependent protein kinase IV (CaMKIV), calcineurin A, or the p38 mitogen-activated protein kinase (p38 MAPK) kinase MKK6 or the intracellular accumulation of cAMP activated the PGC-1{alpha}-b promoter in cultured myoblasts through recruitment of cAMP response element (CRE)-binding protein (CREB) to a putative CRE located downstream of the E-box. Our results thus reveal a potential molecular basis for isoform-specific regulation of PGC-1{alpha} expression in contracting muscle.

  5. Alternative Polyadenylation of Human Bocavirus at Its 3′ End Is Regulated by Multiple Elements and Affects Capsid Expression

    Science.gov (United States)

    Hao, Sujuan; Zhang, Junmei; Chen, Zhen; Xu, Huanzhou; Wang, Hanzhong

    2016-01-01

    ABSTRACT Alternative processing of human bocavirus (HBoV) P5 promoter-transcribed RNA is critical for generating the structural and nonstructural protein-encoding mRNA transcripts. The regulatory mechanism by which HBoV RNA transcripts are polyadenylated at proximal [(pA)p] or distal [(pA)d] polyadenylation sites is still unclear. We constructed a recombinant HBoV infectious clone to study the alternative polyadenylation regulation of HBoV. Surprisingly, in addition to the reported distal polyadenylation site, (pA)d, a novel distal polyadenylation site, (pA)d2, which is located in the right-end hairpin (REH), was identified during infectious clone transfection or recombinant virus infection. (pA)d2 does not contain typical hexanucleotide polyadenylation signal, upstream elements (USE), or downstream elements (DSE) according to sequence analysis. Further study showed that HBoV nonstructural protein NS1, REH, and cis elements of (pA)d were necessary and sufficient for efficient polyadenylation at (pA)d2. The distance and sequences between (pA)d and (pA)d2 also played a key role in the regulation of polyadenylation at (pA)d2. Finally, we demonstrated that efficient polyadenylation at (pA)d2 resulted in increased HBoV capsid mRNA transcripts and protein translation. Thus, our study revealed that all the bocaviruses have distal poly(A) signals on the right-end palindromic terminus, and alternative polyadenylation at the HBoV 3′ end regulates its capsid expression. IMPORTANCE The distal polyadenylation site, (pA)d, of HBoV is located about 400 nucleotides (nt) from the right-end palindromic terminus, which is different from those of bovine parvovirus (BPV) and canine minute virus (MVC) in the same genus whose distal polyadenylation is located in the right-end stem-loop structure. A novel polyadenylation site, (pA)d2, was identified in the right-end hairpin of HBoV during infectious clone transfection or recombinant virus infection. Sequence analysis showed that (pA)d2

  6. Reflections on human Pavlovian decelerative heart-rate conditioning with negative tilt as US: alternative approaches.

    Science.gov (United States)

    Furedy, J J

    1992-01-01

    The negative-tilt preparation that has been reported since the late seventies is a specific form of Pavlovian conditioning that is of scientific interest and has potential applications. In this paper I reflect on the usefulness, to the development of this preparation, of two approaches to Pavlovian conditioning. One approach is the older S-R learning, stimulus-substitution paradigm exemplified by learning texts of the sixties. The other is the modern, Tolman-like view, according to which the phenomenon of Pavlovian conditioning is "now described as the learning of relations among events so as to allow the organism to represent its environment." The three assumptions encapsulated by this approach are: (a) that only CS-US contingency relations are learned; (b) that teleological modes of explanations are adequate; (c) that the representational theory of knowledge is sound. Concerning Pavlovian conditioning in general, questions been raised in the literature for all three assumptions; they have not been adequately answered. Regarding the specific problem of developing the human Pavlovian heart-rate decelerative conditioning with negative tilt as the US, I suggest that the cognitive approach has been much less helpful than the older, S-R, stimulus-substitution paradigm. Nevertheless, other literature clearly indicates that the cognitive, S-S approach has generated considerable interest and research, especially in preparations like the conditioned emotional response (CER), which are CS-IR ones in the sense that the effects on the CR are assessed indirectly through measuring an indicator or instrumental response (IR).(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Direct differentiation of atrial and ventricular myocytes from human embryonic stem cells by alternating retinoid signals

    Institute of Scientific and Technical Information of China (English)

    Qiangzhe Zhang; Li Chen; Tian Tian; Xin Wang; Pu Li; Jurgen Hescheler; Guangju Ji; Yue Ma; Junjie Jiang; Pengcheng Han; Qi Yuan; Jing Zhang; Xiaoqian Zhang; Yanyan Xu; Henghua Cao; Qingzhang Meng

    2011-01-01

    Although myocyte cell transplantation studies have suggested a promising therapeutic potential for myocardial infarction, a major obstacle to the development of clinical therapies for myocardial repair is the difficulties associated with obtaining relatively homogeneous ventricular myocytes for transplantation. Human embryonic stem cells (hESCs)are a promising source of cardiomyocytes. Here we report that retinoid signaling regulates the fate specification of atrial versus ventricular myocytes during cardiac differentiation of hESCs. We found that both Noggin and the panretinoic acid receptor antagonist BMS-189453 (RAi) significantly increased the cardiac differentiation efficiency of hESCs. To investigate retinoid functions, we compared Noggin+RAi-treated cultures with Noggin+RA-treated cultures. Our results showed that the expression levels of the ventricular-specific gene IRX-4 were radically elevated in Noggin+RAi-treated cultures. MLC-2V, another ventricular-specific marker, was expressed in the majority of the cardiomyocytes in Noggin+RAi-treated cultures, hut not in the cardiomyocytes of Noggin+RA-treated cultures. Flow cytometry analysis and electrophysiologicai studies indicated that with 64.7 ± 0.88% (mean ± s.e.m) cardiac differentiation efficiency, 83% of the cardiomyocytes in Noggin+RAi-treated cultures had embryonic ventricular-like action potentials (APs). With 50.7 ± 1.76% cardiac differentiation efficiency, 94% of the cardiomyocytes in Noggin+RA-treated cultures had embryonic atrial-like APs. These results were further confirmed by imaging studies that assessed the patterns and properties of the Ca2+ sparks of the cardiomyocytes from the two cultures. These findings demonstrate that retinoid signaling specifies the atrial versus ventricular differentiation of hESCs. This study also shows that relatively homogeneous embryonic atrial- and ventricular-like myocyte populations can be efficiently derived from hESCs by specifically regulating Noggin

  8. Haemophilus ducreyi Seeks Alternative Carbon Sources and Adapts to Nutrient Stress and Anaerobiosis during Experimental Infection of Human Volunteers

    Science.gov (United States)

    Gangaiah, Dharanesh; Zhang, Xinjun; Baker, Beth; Fortney, Kate R.; Gao, Hongyu; Holley, Concerta L.; Munson, Robert S.; Liu, Yunlong

    2016-01-01

    Haemophilus ducreyi causes the sexually transmitted disease chancroid in adults and cutaneous ulcers in children. In humans, H. ducreyi resides in an abscess and must adapt to a variety of stresses. Previous studies (D. Gangaiah, M. Labandeira-Rey, X. Zhang, K. R. Fortney, S. Ellinger, B. Zwickl, B. Baker, Y. Liu, D. M. Janowicz, B. P. Katz, C. A. Brautigam, R. S. Munson, Jr., E. J. Hansen, and S. M. Spinola, mBio 5:e01081-13, 2014, http://dx.doi.org/10.1128/mBio.01081-13) suggested that H. ducreyi encounters growth conditions in human lesions resembling those found in stationary phase. However, how H. ducreyi transcriptionally responds to stress during human infection is unknown. Here, we determined the H. ducreyi transcriptome in biopsy specimens of human lesions and compared it to the transcriptomes of bacteria grown to mid-log, transition, and stationary phases. Multidimensional scaling showed that the in vivo transcriptome is distinct from those of in vitro growth. Compared to the inoculum (mid-log-phase bacteria), H. ducreyi harvested from pustules differentially expressed ∼93 genes, of which 62 were upregulated. The upregulated genes encode homologs of proteins involved in nutrient transport, alternative carbon pathways (l-ascorbate utilization and metabolism), growth arrest response, heat shock response, DNA recombination, and anaerobiosis. H. ducreyi upregulated few genes (hgbA, flp-tad, and lspB-lspA2) encoding virulence determinants required for human infection. Most genes regulated by CpxRA, RpoE, Hfq, (p)ppGpp, and DksA, which control the expression of virulence determinants and adaptation to a variety of stresses, were not differentially expressed in vivo, suggesting that these systems are cycling on and off during infection. Taken together, these data suggest that the in vivo transcriptome is distinct from those of in vitro growth and that adaptation to nutrient stress and anaerobiosis is crucial for H. ducreyi survival in humans. PMID:26930707

  9. Human pregnancy-specific glycoprotein 1a (PSG1a) induces alternative activation in human and mouse monocytes and suppresses the accessory cell-dependent T cell proliferation.

    Science.gov (United States)

    Motrán, Claudia Cristina; Díaz, Fernando López; Gruppi, Adriana; Slavin, Daniela; Chatton, Bruno; Bocco, José Luis

    2002-09-01

    It has been proposed that pregnancy-specific factors induce the suppression of a specific arm of the maternal response accompanied by activation of the nonspecific, innate immune system. The aim of this study was to determine whether pregnancy-specific glycoprotein 1a (PSG1a), the major variant of PSG polypeptides, is able to modulate the monocyte/macrophage (Mo) metabolism to regulate T cell activation and proliferation. Using the recombinant form of this glycoprotein (rec-PSG1a), expressed in mammalian cells with a vaccinia-based expression vector, we have demonstrated that human PSG1a induces arginase activity in peripheral blood human Mo and human and murine Mo cell lines. In addition, rec-PSG1a is able to induce alternative activation because it up-regulates the arginase activity and inhibits the nitric oxide production in Mo activated by lipopolysaccharides. We also observed that rec-PSG1a is an important accessory cells-dependent T cell suppressor factor that causes partial growth arrest at the S/G2/M phase of the cell cycle. Additionally, an impaired T cell proliferative response induced by mitogens and specific antigen was observed in BALB/c mice upon in vivo expression of PSG1a. Our results suggest that PSG1a function contributes to the immunomodulation during pregnancy, having opposite effects on maternal innate and adaptative systems.

  10. Characterization of novel elongated Parvulin isoforms that are ubiquitously expressed in human tissues and originate from alternative transcription initiation

    Directory of Open Access Journals (Sweden)

    Hartmann-Fatu Cristina

    2006-03-01

    Full Text Available Abstract Background The peptidyl prolyl cis/trans isomerase (PPIase Parvulin (Par14/PIN4 is highly conserved in all metazoans and is assumed to play a role in cell cycle progression and chromatin remodeling. It is predominantly localized to the nucleus and binds to chromosomal DNA as well as bent oligonucleotides in vitro. Results In this study we confirm by RT-PCR the existence of a longer Parvulin isoform expressed in all tissues examined so far. This isoform contains a 5' extension including a 75 bp extended open reading frame with two coupled SNPs leading to amino acid substitutions Q16R and R18S. About 1% of all Parvulin mRNAs include the novel extension as quantified by real-time PCR. The human Parvulin promoter is TATA-less and situated in a CpG island typical for house keeping genes. Thus, different Parvulin mRNAs seem to arise by alternative transcription initiation. N-terminally extended Parvulin is protected from rapid proteinaseK degradation. In HeLa and HepG2 cell lysates two protein species of about 17 and 28 KDa are detected by an antibody against an epitope within the N-terminal extension. These two bands are also recognized by an antibody towards the PPIase domain of Parvulin. The longer Parvulin protein is encoded by the human genome but absent from rodent, bovine and non-mammalian genomes. Conclusion Due to its molecular weight of 16.6 KDa we denote the novel Parvulin isoform as Par17 following the E. coli Par10 and human Par14 nomenclature. The N-terminal elongation of Par17-QR and Par17-RS suggests these isoforms to perform divergent functions within the eukaryotic cell than the well characterized Par14.

  11. Near-microsecond human aquaporin 4 gating dynamics in static and alternating external electric fields: Non-equilibrium molecular dynamics.

    Science.gov (United States)

    English, Niall J; Garate, José-A

    2016-08-28

    An extensive suite of non-equilibrium molecular-dynamics simulation has been performed for ∼0.85-0.9 μs of human aquaporin 4 in the absence and presence of externally applied static and alternating electric fields applied along the channels (in both axial directions in the static case, taken as the laboratory z-axis). These external fields were of 0.0065 V/Å (r.m.s.) intensity (of the same order as physiological electrical potentials); alternating fields ranged in frequency from 2.45 to 500 GHz. In-pore gating dynamics was studied, particularly of the relative propensities for "open" and "closed" states of the conserved arginines in the arginine/aromatic area (itself governed in no small part by external-field response of the dipolar alignment of the histidine-201 residue in the selectivity filter). In such a manner, the intimate connection of field-response governing "two-state" histidine states was established statistically and mechanistically. Given the appreciable size of the energy barriers for histidine-201 alignment, we have also performed non-equilibrium metadynamics/local-elevation of static fields applied along both directions to construct the free-energy landscape thereof in terms of external-field direction, elucidating the importance of field direction on energetics. We conclude from direct measurement of deterministic molecular dynamics in conjunction with applied-field metadynamics that the intrinsic electric field within the channel points along the +z-axis, such that externally applied static fields in this direction serve to "open" the channel in the selectivity-filter and the asparagine-proline-alanine region.

  12. Near-microsecond human aquaporin 4 gating dynamics in static and alternating external electric fields: Non-equilibrium molecular dynamics

    Science.gov (United States)

    English, Niall J.; Garate, José-A.

    2016-08-01

    An extensive suite of non-equilibrium molecular-dynamics simulation has been performed for ˜0.85-0.9 μs of human aquaporin 4 in the absence and presence of externally applied static and alternating electric fields applied along the channels (in both axial directions in the static case, taken as the laboratory z-axis). These external fields were of 0.0065 V/Å (r.m.s.) intensity (of the same order as physiological electrical potentials); alternating fields ranged in frequency from 2.45 to 500 GHz. In-pore gating dynamics was studied, particularly of the relative propensities for "open" and "closed" states of the conserved arginines in the arginine/aromatic area (itself governed in no small part by external-field response of the dipolar alignment of the histidine-201 residue in the selectivity filter). In such a manner, the intimate connection of field-response governing "two-state" histidine states was established statistically and mechanistically. Given the appreciable size of the energy barriers for histidine-201 alignment, we have also performed non-equilibrium metadynamics/local-elevation of static fields applied along both directions to construct the free-energy landscape thereof in terms of external-field direction, elucidating the importance of field direction on energetics. We conclude from direct measurement of deterministic molecular dynamics in conjunction with applied-field metadynamics that the intrinsic electric field within the channel points along the +z-axis, such that externally applied static fields in this direction serve to "open" the channel in the selectivity-filter and the asparagine-proline-alanine region.

  13. Revised genomic structure of the human ghrelin gene and identification of novel exons, alternative splice variants and natural antisense transcripts

    Directory of Open Access Journals (Sweden)

    Herington Adrian C

    2007-08-01

    Full Text Available Abstract Background Ghrelin is a multifunctional peptide hormone expressed in a range of normal tissues and pathologies. It has been reported that the human ghrelin gene consists of five exons which span 5 kb of genomic DNA on chromosome 3 and includes a 20 bp non-coding first exon (20 bp exon 0. The availability of bioinformatic tools enabling comparative analysis and the finalisation of the human genome prompted us to re-examine the genomic structure of the ghrelin locus. Results We have demonstrated the presence of an additional novel exon (exon -1 and 5' extensions to exon 0 and 1 using comparative in silico analysis and have demonstrated their existence experimentally using RT-PCR and 5' RACE. A revised exon-intron structure demonstrates that the human ghrelin gene spans 7.2 kb and consists of six rather than five exons. Several ghrelin gene-derived splice forms were detected in a range of human tissues and cell lines. We have demonstrated ghrelin gene-derived mRNA transcripts that do not code for ghrelin, but instead may encode the C-terminal region of full-length preproghrelin (C-ghrelin, which contains the coding region for obestatin and a transcript encoding obestatin-only. Splice variants that differed in their 5' untranslated regions were also found, suggesting a role of these regions in the post-transcriptional regulation of preproghrelin translation. Finally, several natural antisense transcripts, termed ghrelinOS (ghrelin opposite strand transcripts, were demonstrated via orientation-specific RT-PCR, 5' RACE and in silico analysis of ESTs and cloned amplicons. Conclusion The sense and antisense alternative transcripts demonstrated in this study may function as non-coding regulatory RNA, or code for novel protein isoforms. This is the first demonstration of putative obestatin and C-ghrelin specific transcripts and these findings suggest that these ghrelin gene-derived peptides may also be produced independently of preproghrelin

  14. PTCH2, a novel human patched gene, undergoing alternative splicing and up-regulated in basal cell carcinomas.

    Science.gov (United States)

    Zaphiropoulos, P G; Undén, A B; Rahnama, F; Hollingsworth, R E; Toftgård, R

    1999-02-15

    By a combination of cDNA library screening, rapid amplification of cDNA ends analysis, and BAC sequencing, a novel human patched-like gene (PTCH2) has been cloned and sequenced. The genomic organization is similar to PTCH1 with 22 exons and, by radiation hybrid mapping, PTCH2 has been localized to chromosome 1p33-34, a region often lost in a variety of tumors. Several alternatively spliced mRNA forms of PTCH2 were identified, including transcripts lacking segments thought to be involved in sonic hedgehog binding and mRNAs with differentially defined 3' terminal exons. In situ hybridization revealed high expression of PTCH2 transcripts in both familial and sporadic basal cell carcinomas in similarity to what has been observed for PTCH1, suggesting a negative regulation of PTCH2 by PTCH1. This finding tightly links PTCH2 with the sonic hedgehog/PTCH signaling pathway, implying that PTCH2 has related, but yet distinct, functions than PTCH1.

  15. Isolation and characterization of cancer stem-like cells from MHCC97H Cell Lines

    Institute of Scientific and Technical Information of China (English)

    Shanyong Yi; Kejun Nan; Aihua Yuan; Chuangxin Lu

    2009-01-01

    Objective:To identify and isolate CD133 positive cancer stem-like cells (CD133+ cells) from the highly invasive human hepatocellular carcinoma cell line(MHCC97H), and examine their potential for clonogenicity and tumorigenicity. Methods: CD133+ and CD133- cells were isolated from MHCC97H cell line by magnetic bead cell sorting(MACS), and the potentials of CD133+ cells for colony formation and tumorigenicity were evaluated by soft agar cloning and tumor formation following nude mice inoculation. Results:CD133+ cells represent a minority(0.5-2.0%) of the tumor cell population with a greater colony-forming efficiency and greater tumor production ability. The colony-forming efficiency of CD133+ cells in soft agar was significantly higher than CD133- cells(36.8±1.4 vs 12.9±0.8, P<0.05).After 6 weeks, 3/5 mice inoculated with 1 × 103 CD133+ cells, 4/5 with 1 × 104 CD133+ cells and 5/5 with 1 × 105 CD133+ cells developed detectable tumors at the injection site, while only one tumor was found in mice treated with same numbers of CD133- cells. Conclusion: CD133 may be a hallmark of liver cancer stem cells (CSC) in human hepatocellular carcinoma(HCC), because the CD133+ cells identified and isolated with anti-CD133 labeled magnetic beads from MHCC97H cell line exhibit high potentials for clonogenicity and tumorigenicity. These CD133+ cells might contribute to hepatocarcinogenesis, as well as the growth and recurrence of human HCC, and therefore may be a useful target for anti-cancer therapy.

  16. Alternative energies; Energies alternatives

    Energy Technology Data Exchange (ETDEWEB)

    Bonal, J.; Rossetti, P

    2007-07-01

    The earth took millions years to made the petroleum, the gas the coal and the uranium. Only a few centuries will be needed to exhaust these fossil fuels and some years to reach expensive prices. Will the wold continue on this way of energy compulsive consumption? The renewable energies and some citizen attitudes are sufficient to break this spiral. This book proposes to discuss these alternative energies. It shows that this attitude must be supported by the government. It takes stock on the more recent information concerning the renewable energies. it develops three main points: the electricity storage, the housing and the transports. (A.L.B.)

  17. To Be "as" Not to Be: In Search of an Alternative Humanism in the Light of Early Daoism and Deconstruction

    Science.gov (United States)

    Hung, Ruyu

    2015-01-01

    Humanism and humanistic education have been recognised as an issue of the utmost importance, whether in the East or in the West. Underpinning the Eastern and Western humanism is a common belief that there is an essence or essences of humanness. In the Confucian tradition, the core of humanity lies in the idea of "ren"; in the Platonic…

  18. Progress on research of the alternative splicing of human cytochrome P450 pre-mRNA%人细胞色素P450前mRNA的可变剪接研究进展

    Institute of Scientific and Technical Information of China (English)

    诸葛坚; 余应年

    2005-01-01

    Human genes typically contain multiple introns, and in many cases the exons can be joined more than one way to generate multiple rnRNAs, encoding distinct protein isoforms. This process is called alternative splicing. The article summarized the human cytochrome P450 pre-mRNA alternative splicing and their regulatory mechanism and impacts on biological functions.

  19. Human Platelet Lysate as a Xeno Free Alternative of Fetal Bovine Serum for the In Vitro Expansion of Human Mesenchymal Stromal Cells

    Science.gov (United States)

    Mohammadi, Saeed; Nikbakht, Mohsen; Malek Mohammadi, Ashraf; Zahed Panah, Mahdi; Ostadali, Mohammad Reza; Nasiri, Hajar; Ghavamzadeh, Ardeshir

    2016-01-01

    Background: Mesenchymal stromal cells (MSCs) are employed in various different clinical settings in order to modulate immune response. Human autologous and allogeneic supplements including platelet derivatives such as platelet lysate (PL), platelet-released factors (PRF) and serum are assessed in clinical studies to replace fetal bovine serum (FBS). The immunosuppressive activity and multi-potential characteristic of MSCs appear to be maintained when the cells are expanded in platelet derivatives. Materials and Methods: Platelet-rich plasma was collected from umbrical cord blood (UCB). Platelet-derived growth factors obtained by freeze and thaw methods. CD62P expression was determined by flow cytometry. The concentration of PDGF-BB and PDGF-AB was detemined by ELISA. We tested the ability of a different concentration of PL-supplemented medium to support the ex vivo expansion of Wharton's jelly derived MSCs. We also investigated the biological/functional properties of expanded MSCs in presence of different concentration of PL. The conventional karyotyping was performed in order to study the chromosomal stability. The gene expression of Collagen I and II aggrecan and SOX-9 in the presence of different concentrations of PL was evaluated by Real-time PCR. Results: We observed 5% and 10% PL, causing greater effects on proliferation of MSCs .These cells exhibited typical morphology, immunophenotype and differentiation capacity. The genetic stability of these derivative cells from Wharton's jelly was demonstrated by a normal karyotype. Furthermore, the results of Real-time PCR analysis showed that the expression of chondrocyte specific genes was higher in MSCs in the presence of 5% and 10% PL, compared with FBS supplement. Conclusions: We demonstrated that PL could be used as an alternative safe source of growth factors for expansion of MSCs and also maintained similar growing potential and phenotype without any effect on chromosomal stability. PMID:27489592

  20. Alternative additives; Alternative additiver

    Energy Technology Data Exchange (ETDEWEB)

    2007-08-15

    In this project a number of industrial and agricultural waste products have been characterised and evaluated in terms of alkali-getter performance. The intended use is for biomass-fired power stations aiming at reducing corrosion or slagging related problems. The following products have been obtained, characterised and evaluated: 1) Brewery draff 2) Danish de-gassed manure 3) Paper sludge 4) Moulding sand 5) Spent bleaching earth 6) Anorthosite 7) Sand 8) Clay-sludge. Most of the above alternative additive candidates are deemed unsuitable due to insufficient chemical effect and/or expensive requirements for pre-treatment (such as drying and transportation). 3 products were selected for full-scale testing: de-gassed manure, spent bleaching earth and clay slugde. The full scale tests were undertaken at the biomass-fired power stations in Koege, Slagelse and Ensted. Spent bleaching earth (SBE) and clay sludge were the only tested additive candidates that had a proven ability to react with KCl, to thereby reduce Cl-concentrations in deposits, and reduce the deposit flux to superheater tubes. Their performance was shown to nearly as good as commercial additives. De-gassed manure, however, did not evaluate positively due to inhibiting effects of Ca in the manure. Furthermore, de-gassed manure has a high concentration of heavy metals, which imposes a financial burden with regard to proper disposal of the ash by-products. Clay-sludge is a wet clay slurring, and drying and transportation of this product entails substantial costs. Spent bleaching does not require much pre-treatment and is therefore the most promising alternative additive. On the other hand, bleaching earth contains residual plant oil which means that a range of legislation relating to waste combustion comes into play. Not least a waste combustion fee of 330 DKK/tonne. For all alternative (and commercial) additives disposal costs of the increase ash by-products represents a significant cost. This is

  1. A Subjective Assessment of Alternative Mission Architecture Operations Concepts for the Human Exploration of Mars at NASA Using a Three-Dimensional Multi-Criteria Decision Making Model

    Science.gov (United States)

    Tavana, Madjid

    2003-01-01

    The primary driver for developing missions to send humans to other planets is to generate significant scientific return. NASA plans human planetary explorations with an acceptable level of risk consistent with other manned operations. Space exploration risks can not be completely eliminated. Therefore, an acceptable level of cost, technical, safety, schedule, and political risks and benefits must be established for exploratory missions. This study uses a three-dimensional multi-criteria decision making model to identify the risks and benefits associated with three alternative mission architecture operations concepts for the human exploration of Mars identified by the Mission Operations Directorate at Johnson Space Center. The three alternatives considered in this study include split, combo lander, and dual scenarios. The model considers the seven phases of the mission including: 1) Earth Vicinity/Departure; 2) Mars Transfer; 3) Mars Arrival; 4) Planetary Surface; 5) Mars Vicinity/Departure; 6) Earth Transfer; and 7) Earth Arrival. Analytic Hierarchy Process (AHP) and subjective probability estimation are used to captures the experts belief concerning the risks and benefits of the three alternative scenarios through a series of sequential, rational, and analytical processes.

  2. Alternative messenger RNA splicing of autophagic gene Beclin 1 in human B-cell acute lymphoblastic leukemia cells.

    Science.gov (United States)

    Niu, Yu-Na; Liu, Qing-Qing; Zhang, Su-Ping; Yuan, Na; Cao, Yan; Cai, Jin-Yang; Lin, Wei-Wei; Xu, Fei; Wang, Zhi-Jian; Chen, Bo; Wang, Jian-Rong

    2014-01-01

    Beclin 1 is a key factor for initiation and regulation of autophagy, which is a cellular catabolic process involved in tumorigenesis. To investigate the role of alternative splicing of Beclin1 in the regulation of autophagy in leukemia cells, Beclin1 mRNA from 6 different types of cell lines and peripheral blood mononuclear cells from 2 healthy volunteers was reversely transcribed, subcloned, and screened for alternative splicing. New transcript variants were analyzed by DNA sequencing. A transcript variant of Beclin 1 gene carrying a deletion of exon 11, which encoded a C-terminal truncation of Beclin 1 isoform, was found. The alternative isoform was assessed by bioinformatics, immunoblotting and subcellular localization. The results showed that this variable transcript is generated by alternative 3' splicing, and its translational product displayed a reduced activity in induction of autophagy by starvation, indicating that the spliced isoform might function as a dominant negative modulator of autophagy. Our findings suggest that the alternative splicing of Beclin 1 might play important roles in leukemogenesis regulated by autophagy.

  3. Translational regulation of human neuronal nitric-oxide synthase by an alternatively spliced 5'-untranslated region leader exon.

    Science.gov (United States)

    Newton, Derek C; Bevan, Sian C; Choi, Stephen; Robb, G Brett; Millar, Adam; Wang, Yang; Marsden, Philip A

    2003-01-03

    Expression of the neuronal nitric-oxide synthase (nNOS) mRNA is subject to complex cell-specific transcriptional regulation, which is mediated by alternative promoters. Unexpectedly, we identified a 89-nucleotide alternatively spliced exon located in the 5'-untranslated region between exon 1 variants and a common exon 2 that contains the translational initiation codon. Alternative splicing events that do not affect the open reading frame are distinctly uncommon in mammals; therefore, we assessed its functional relevance. Transient transfection of reporter RNAs performed in a variety of cell types revealed that this alternatively spliced exon acts as a potent translational repressor. Stably transfected cell lines confirmed that the alternatively spliced exon inhibited translation of the native nNOS open reading frame. Reverse transcription-PCR and RNase protection assays indicated that nNOS mRNAs containing this exon are common and expressed in both a promoter-specific and tissue-restricted fashion. Mutational analysis identified the functional cis-element within this novel exon, and a secondary structure prediction revealed that it forms a putative stem-loop. RNA electrophoretic mobility shift assay techniques revealed that a specific cytoplasmic RNA-binding complex interacts with this motif. Hence, a unique splicing event within a 5'-untranslated region is demonstrated to introduce a translational control element. This represents a newer model for the translational control of a mammalian mRNA.

  4. Effects of alternating and direct electrical current application on the odontoblastic layer in human teeth : an in vitro study

    NARCIS (Netherlands)

    Alwas-Danowska, HM; Huysmans, MCDNJM; Verdonschot, EH

    1999-01-01

    Aim The aim of this study was to investigate the influence of a low intensity alternating current on the odontoblasts and odontoblast layer and compare this with the effects of a direct current. Methodology Teeth extracted for orthodontic were immersed in physiological saline stabilized with thymol

  5. Unlike PPAR{gamma}, PPAR{alpha} or PPAR{beta}/{delta} activation does not promote human monocyte differentiation toward alternative macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Bouhlel, Mohamed Amine [Univ Lille Nord de France, F-59000 Lille (France); Inserm U545, F-59000 Lille (France); UDSL, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France); Brozek, John [Genfit, Loos (France); Derudas, Bruno [Univ Lille Nord de France, F-59000 Lille (France); Inserm U545, F-59000 Lille (France); UDSL, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France); Zawadzki, Christophe; Jude, Brigitte [Inserm ERI-9 and Equipe d' Accueil 2693, IFR114, Universite de Lille, Lille (France); Staels, Bart, E-mail: bart.staels@pasteur-lille.fr [Univ Lille Nord de France, F-59000 Lille (France); Inserm U545, F-59000 Lille (France); UDSL, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France); Chinetti-Gbaguidi, Giulia [Univ Lille Nord de France, F-59000 Lille (France); Inserm U545, F-59000 Lille (France); UDSL, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France)

    2009-08-28

    Macrophages adapt their response to micro-environmental signals. While Th1 cytokines promote pro-inflammatory M1 macrophages, Th2 cytokines promote an 'alternative' anti-inflammatory M2 macrophage phenotype. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors expressed in macrophages where they control the inflammatory response. It has been shown that PPAR{gamma} promotes the differentiation of monocytes into anti-inflammatory M2 macrophages in humans and mice, while a role for PPAR{beta}/{delta} in this process has been reported only in mice and no data are available for PPAR{alpha}. Here, we show that in contrast to PPAR{gamma}, expression of PPAR{alpha} and PPAR{beta}/{delta} overall does not correlate with the expression of M2 markers in human atherosclerotic lesions, whereas a positive correlation with genes of lipid metabolism exists. Moreover, unlike PPAR{gamma}, PPAR{alpha} or PPAR{beta}/{delta} activation does not influence human monocyte differentiation into M2 macrophages in vitro. Thus, PPAR{alpha} and PPAR{beta}/{delta} do not appear to modulate the alternative differentiation of human macrophages.

  6. Human retina-specific amine oxidase: genomic structure of the gene (AOC2), alternatively spliced variant, and mRNA expression in retina.

    Science.gov (United States)

    Imamura, Y; Noda, S; Mashima, Y; Kudoh, J; Oguchi, Y; Shimizu, N

    1998-07-15

    Previously, we reported the isolation of cDNA for human retina-specific amine oxidase (RAO) and the expression of RAO exclusively in retina. Bacterial artificial chromosome clones containing the human RAO gene (AOC2) were mapped to human chromosome 17q21 (Imamura et al., 1997, Genomics 40: 277-283). Here, we report the complete genomic structure of the RAO gene, including 5' flanking sequence, and mRNA expression in retina. The human RAO gene spans 6 kb and is composed of four exons corresponding to the amino acid sequence 1-530, 530-598, 598-641, and 642-729 separated by three introns of 3000, 310, and 351 bp. Screening of a human retina cDNA library revealed the existence of an alternatively spliced cDNA variant with an additional 81 bp at the end of exon 2. The sizes of exons and the locations of exon/intron boundaries in the human RAO gene showed remarkable similarity to those of the human kidney diamine oxidase gene (AOC1). In situ hybridization revealed that mRNA coding for RAO is expressed preferentially in the ganglion cell layer of the mouse retina. We designed four sets of PCR primers to amplify four exons, which will be valuable for analyzing mutations in patients with ocular diseases affecting the retinal ganglion cell layer.

  7. Ex vivo differentiation of human bone marrow-derived stem cells into neuronal cell-like lineages

    Directory of Open Access Journals (Sweden)

    Al-Zoubi A

    2016-06-01

    Full Text Available Adeeb Al-Zoubi,1,2 Feras Altwal,3 Farah Khalifeh,2 Jamil Hermas,4 Ziad Al-Zoubi,5 Emad Jafar,5 Mohammed El-Khateeb,6,7 1Department of Surgery, University of Illinois College of Medicine at Peoria, Peoria, IL, USA; 2Stem Cells of Arabia, Amman, Jordan; 3Department of Neuroscience, School of Graduate and Postdoctoral Studies, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA; 4Stem Cell Division, Al-Yamama Company, 5Jordan Orthopedic and Spinal Center, 6National Center for Diabetes, Endocrinology and Genetics, 7Department of Pathology, Faculty of Medicine, University of Jordan, Amman, Jordan Background: Methods to obtain safe and practical populations of stem cells (SCs at a clinical grade that are able to differentiate into neuronal cell lineages are yet to be developed. In a previous study, we showed that mouse bone marrow-derived SCs (BM-SCs differentiated into neuronal cell-like lineages when put in a neuronal-like environment, which is a special media supplemented with the necessary growth factors needed for the differentiation of SCs into neuronal cell-like lineages. Aim: In this study, we aim to assess the potentials of adult human CD34+ and CD133+ SCs to differentiate into neuronal cell-like lineages ex vivo when placed in a neuronal-like microenvironment. Methods: The neuronal-like microenvironment was created by culturing cells in nonhematopoietic expansion media (NHEM supplemented with growth factors that favor differentiation into neuronal cell lineages (low-affinity nerve growth factor [LNGF], mouse spinal cord extract [mSpE], or both. Cultured cells were assessed for neuronal differentiation by cell morphologies and by expression of GFAP. Results: Our results show that culturing unpurified human BM-derived mononuclear cells (hBM-MNCs in NHEM+LNGF+mSpE did not lead to neuronal differentiation. In contrast, culturing of purified CD34+ hBM-SCs in NHEM+LNGF+mSpE favored their differentiation into astrocyte

  8. Teste alternativo para detecção de coliformes em leite humano ordenhado Alternative test for detection of coliforms bacteria in manually expressed human milk

    Directory of Open Access Journals (Sweden)

    Franz R. Novak

    2002-01-01

    Full Text Available Objetivo: comparar um método alternativo com o teste do número mais provável (NMP para detecção de coliformes totais em leite humano ordenhado. Métodos: 343 amostras de leite humano ordenhado, obtidas a partir de frascos oriundos de coleta domiciliar, recebidas pelo Banco de Leite Humano do Instituto Fernandes Figueira - IFF, por doadoras previamente orientadas, foram encaminhadas ao laboratório de controle de alimentos do IFF e empregadas na comparação de dois métodos: 1 - técnica do número mais provável, conforme descrito no Standard methods for the examination of dairy products; 2 - método alternativo proposto. Resultados: os microorganismos do grupo coliformes foram detectados em 31,2% das amostras analisadas, com populações variando de 3,0 x 100 a 1,1 x 104 coliformes totais N.M.P/ml. A comparação do teste clássico com o alternativo revelou resultados semelhantes quanto à recuperação de microorganismos coliformes em amostras de leite humano ordenhado. O método alternativo detectou a presença de coliformes totais em todas as amostras contaminadas e em quatro amostras não contaminadas, segundo o teste de NMP. Conclusão: o teste alternativo permite constatar a presença ou ausência de coliformes, tornando-se útil no controle de qualidade dos frascos de leite humano ordenhado pasteurizados, manipulados nos bancos de leite humano. Portanto, o teste de NMP pode ser substituído pelo teste alternativo, que poderá ser empregado como rotina nos bancos de leite humano, já que seu custo equivale a 1/7 do tradicional.Objective: To compare an alternative method to the most probable number (MPN test for the detection of total coliform present in manually expressed human milk. Methods: 343 samples of manually expressed human milk from flasks donated to the Human Milk Bank of Instituto Fernandes Figueira - IFF were sent to the Laboratory of Food Control of IFF. The samples were used for comparing both methods, i.e., the most

  9. 21 CFR 201.26 - Exceptions or alternatives to labeling requirements for human drug products held by the Strategic...

    Science.gov (United States)

    2010-04-01

    ... requirements for human drug products held by the Strategic National Stockpile. 201.26 Section 201.26 Food and... drug products held by the Strategic National Stockpile. (a) The appropriate FDA Center Director may... lots, batches, or other units of a human drug product that are or will be held in the...

  10. Functional Recombinant Extra Membrane Loop of Human CD20, an Alternative of the Full Length CD20 Antigen

    OpenAIRE

    Anbouhi, Mahdi Habibi; Baraz, Aida Feiz; Bouzari, Saeid; Abolhassani,Mohsen; Khanahmad, Hossein; Golkar, Majid; Aghasadeghi, Mohammad Reza; Behdani, Mahdi; Najafabadi, Ali Jahanian; Shokrgozar, Mohammad Ali

    2012-01-01

    Background: Targeting of CD20 antigen with monoclonal antibodies has become the mainstay in the treatment of non-Hodgkin's lymphomas and immunotherapeutic depletion of malignant B cells. Accessibility of antigen is one of the crucial factors in development of monoclonal antibodies against this antigen. One major problem in expression of full length CD20 is aggregation and misfolding. Therefore, production of an alternative polypeptide is easer and favorable comparing to that of a full length ...

  11. The classical and alternative pathways of complement activation play distinct roles in spontaneous C3 fragment deposition and membrane attack complex (MAC) formation on human B lymphocytes

    DEFF Research Database (Denmark)

    Leslie, Robert Graham Quinton; Nielsen, Claus Henrik

    2004-01-01

    The contributions of the classical (CP) and alternative (AP) pathways of complement activation to the spontaneous deposition of C3 fragments and the formation of membrane attack complexes (MAC) on human B lymphocytes, were assessed by incubating peripheral blood mononuclear cells with autologous ...... of MAC formation was also found to be highly pathway dependent, with the AP being about 15-fold more efficient at initiating this process than the CP. A model accounting for the effectiveness of the AP in both preserving C3 fragment integrity and initiating MAC is presented....

  12. Functional diversity of human basic helix-loop-helix transcription factor TCF4 isoforms generated by alternative 5' exon usage and splicing.

    Directory of Open Access Journals (Sweden)

    Mari Sepp

    Full Text Available BACKGROUND: Transcription factor 4 (TCF4 alias ITF2, E2-2, ME2 or SEF2 is a ubiquitous class A basic helix-loop-helix protein that binds to E-box DNA sequences (CANNTG. While involved in the development and functioning of many different cell types, recent studies point to important roles for TCF4 in the nervous system. Specifically, human TCF4 gene is implicated in susceptibility to schizophrenia and TCF4 haploinsufficiency is the cause of the Pitt-Hopkins mental retardation syndrome. However, the structure, expression and coding potential of the human TCF4 gene have not been described in detail. PRINCIPAL FINDINGS: In the present study we used human tissue samples to characterize human TCF4 gene structure and TCF4 expression at mRNA and protein level. We report that although widely expressed, human TCF4 mRNA expression is particularly high in the brain. We demonstrate that usage of numerous 5' exons of the human TCF4 gene potentially yields in TCF4 protein isoforms with 18 different N-termini. In addition, the diversity of isoforms is increased by alternative splicing of several internal exons. For functional characterization of TCF4 isoforms, we overexpressed individual isoforms in cultured human cells. Our analysis revealed that subcellular distribution of TCF4 isoforms is differentially regulated: Some isoforms contain a bipartite nuclear localization signal and are exclusively nuclear, whereas distribution of other isoforms relies on heterodimerization partners. Furthermore, the ability of different TCF4 isoforms to regulate E-box controlled reporter gene transcription is varied depending on whether one or both of the two TCF4 transcription activation domains are present in the protein. Both TCF4 activation domains are able to activate transcription independently, but act synergistically in combination. CONCLUSIONS: Altogether, in this study we have described the inter-tissue variability of TCF4 expression in human and provided evidence

  13. Functional Cross-Talking between Differentially Expressed and Alternatively Spliced Genes in Human Liver Cancer Cells Treated with Berberine.

    Directory of Open Access Journals (Sweden)

    Zhen Sheng

    Full Text Available Berberine has been identified with anti-proliferative effects on various cancer cells. Many researchers have been trying to elucidate the anti-cancer mechanisms of berberine based on differentially expressed genes. However, differentially alternative splicing genes induced by berberine might also contribute to its pharmacological actions and have not been reported yet. Moreover, the potential functional cross-talking between the two sets of genes deserves further exploration. In this study, RNA-seq technology was used to detect the differentially expressed genes and differentially alternative spliced genes in BEL-7402 cancer cells induced by berberine. Functional enrichment analysis indicated that these genes were mainly enriched in the p53 and cell cycle signalling pathway. In addition, it was statistically proven that the two sets of genes were locally co-enriched along chromosomes, closely connected to each other based on protein-protein interaction and functionally similar on Gene Ontology tree. These results suggested that the two sets of genes regulated by berberine might be functionally cross-talked and jointly contribute to its cell cycle arresting effect. It has provided new clues for further researches on the pharmacological mechanisms of berberine as well as the other botanical drugs.

  14. Functional Cross-Talking between Differentially Expressed and Alternatively Spliced Genes in Human Liver Cancer Cells Treated with Berberine.

    Science.gov (United States)

    Sheng, Zhen; Sun, Yi; Zhu, Ruixin; Jiao, Na; Tang, Kailin; Cao, Zhiwei; Ma, Chao

    2015-01-01

    Berberine has been identified with anti-proliferative effects on various cancer cells. Many researchers have been trying to elucidate the anti-cancer mechanisms of berberine based on differentially expressed genes. However, differentially alternative splicing genes induced by berberine might also contribute to its pharmacological actions and have not been reported yet. Moreover, the potential functional cross-talking between the two sets of genes deserves further exploration. In this study, RNA-seq technology was used to detect the differentially expressed genes and differentially alternative spliced genes in BEL-7402 cancer cells induced by berberine. Functional enrichment analysis indicated that these genes were mainly enriched in the p53 and cell cycle signalling pathway. In addition, it was statistically proven that the two sets of genes were locally co-enriched along chromosomes, closely connected to each other based on protein-protein interaction and functionally similar on Gene Ontology tree. These results suggested that the two sets of genes regulated by berberine might be functionally cross-talked and jointly contribute to its cell cycle arresting effect. It has provided new clues for further researches on the pharmacological mechanisms of berberine as well as the other botanical drugs.

  15. Targeting of a human iron-sulfur cluster assembly enzyme, nifs, to different subcellular compartments is regulated through alternative AUG utilization.

    Science.gov (United States)

    Land, T; Rouault, T A

    1998-12-01

    Iron-sulfur clusters are prosthetic groups that are required for the function of numerous enzymes in the cell, including enzymes important in respiration, photosynthesis, and nitrogen fixation. Here we report cloning of the human homolog of NifS, a cysteine desulfurase that is proposed to supply the inorganic sulfur in iron-sulfur clusters. In human cells, different forms of NifS that localize either to mitochondria or to the cytosol and nucleus are synthesized from a single transcript through initiation at alternative inframe AUGs, and initiation site selection varies according to the pH of the medium or cytosol. Thus, a novel form of translational regulation permits rapid redistribution of NifS proteins into different compartments of the cell in response to changes in metabolic status.

  16. Environmental levels of PCDD/Fs and metals around a cement plant in Catalonia, Spain, before and after alternative fuel implementation. Assessment of human health risks

    Energy Technology Data Exchange (ETDEWEB)

    Rovira, Joaquim [Laboratory of Toxicology and Environmental Health, School of Medicine, Universitat Rovira i Virgili, Sant Llorenç 21, 43201 Reus, Catalonia (Spain); Environmental Engineering Laboratory, Departament d' Enginyeria Quimica, Universitat Rovira i Virgili, Av. Països Catalans 26, 43007 Tarragona, Catalonia (Spain); Nadal, Martí, E-mail: marti.nadal@urv.cat [Laboratory of Toxicology and Environmental Health, School of Medicine, Universitat Rovira i Virgili, Sant Llorenç 21, 43201 Reus, Catalonia (Spain); Schuhmacher, Marta [Laboratory of Toxicology and Environmental Health, School of Medicine, Universitat Rovira i Virgili, Sant Llorenç 21, 43201 Reus, Catalonia (Spain); Environmental Engineering Laboratory, Departament d' Enginyeria Quimica, Universitat Rovira i Virgili, Av. Països Catalans 26, 43007 Tarragona, Catalonia (Spain); Domingo, José L. [Laboratory of Toxicology and Environmental Health, School of Medicine, Universitat Rovira i Virgili, Sant Llorenç 21, 43201 Reus, Catalonia (Spain)

    2014-07-01

    The concentrations of As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Sn, Tl, V, and Zn, and the levels of polychlorinated dibenzo-p-dioxins and dibenzofurans were determined in samples of soil, vegetation, and air, collected in the vicinity of a cement plant (Catalonia, Spain), before (January 2011 and July 2011) and after (January 2012 and June 2013) alternative fuel partial substitution (fossil fuels by sewage sludge). Seven sampling points were selected at different directions and distances to the facility including two background sampling points. The results were used to assess the health risk assessment for the population living near the facility. Only few significant differences were found before and after alternative fuel partial substitution (Mn in soils and Cd in vegetation). Non-carcinogenic risks were below the safety threshold (HQ < 1), while carcinogenic risks were below 10{sup −5}, or exceeding slightly that value, always in the range considered as assumable (10{sup −6}–10{sup −4}). - Highlights: • The environmental impact of a cement plant using alternative fuel was monitored. • No significant differences in most pollutants were noted after the fuel change. • Traffic has a notable influence on the environmental levels of PCDD/Fs and metals. • Human health risks were below safety thresholds regardless of the used fuel.

  17. Alternative splice variants in TIM barrel proteins from human genome correlate with the structural and evolutionary modularity of this versatile protein fold.

    Science.gov (United States)

    Ochoa-Leyva, Adrián; Montero-Morán, Gabriela; Saab-Rincón, Gloria; Brieba, Luis G; Soberón, Xavier

    2013-01-01

    After the surprisingly low number of genes identified in the human genome, alternative splicing emerged as a major mechanism to generate protein diversity in higher eukaryotes. However, it is still not known if its prevalence along the genome evolution has contributed to the overall functional protein diversity or if it simply reflects splicing noise. The (βα)8 barrel or TIM barrel is one of the most frequent, versatile, and ancient fold encountered among enzymes. Here, we analyze the structural modifications present in TIM barrel proteins from the human genome product of alternative splicing events. We found that 87% of all splicing events involved deletions; most of these events resulted in protein fragments that corresponded to the (βα)2, (βα)4, (βα)5, (βα)6, and (βα)7 subdomains of TIM barrels. Because approximately 7% of all the splicing events involved internal β-strand substitutions, we decided, based on the genomic data, to design β-strand and α-helix substitutions in a well-studied TIM barrel enzyme. The biochemical characterization of one of the chimeric variants suggests that some of the splice variants in the human genome with β-strand substitutions may be evolving novel functions via either the oligomeric state or substrate specificity. We provide results of how the splice variants represent subdomains that correlate with the independently folding and evolving structural units previously reported. This work is the first to observe a link between the structural features of the barrel and a recurrent genetic mechanism. Our results suggest that it is reasonable to expect that a sizeable fraction of splice variants found in the human genome represent structurally viable functional proteins. Our data provide additional support for the hypothesis of the origin of the TIM barrel fold through the assembly of smaller subdomains. We suggest a model of how nature explores new proteins through alternative splicing as a mechanism to diversify the

  18. Genetic programs expressed in resting and IL-4 alternatively activated mouse and human macrophages : similarities and differences

    NARCIS (Netherlands)

    Martinez, Fernando O.; Helming, Laura; Milde, Ronny; Varin, Audrey; Melgert, Barbro N.; Draijer, Christina; Thomas, Benjamin; Fabbri, Marco; Crawshaw, Anjali; Ho, Ling Pei; Ten Hacken, Nick H.; Jimenez, Viviana Cobos; Kootstra, Neeltje A.; Hamann, Jorg; Greaves, David R.; Locati, Massimo; Mantovani, Alberto; Gordon, Siamon

    2013-01-01

    The molecular repertoire of macrophages in health and disease can provide novel biomarkers for diagnosis, prognosis, and treatment. Th2-IL-4-activated macrophages (M2) have been associated with important diseases in mice, yet no specific markers are available for their detection in human tissues. Al

  19. Human platelet lysate is an alternative to fetal bovine serum for large-scale expansion of bone marrow-derived mesenchymal stromal cells.

    Science.gov (United States)

    Gottipamula, Sanjay; Sharma, Archana; Krishnamurthy, Sagar; Majumdar, Anish Sen; Seetharam, Raviraja N

    2012-07-01

    Human platelet lysate (HPL) was evaluated as an alternative to fetal bovine serum (FBS) in large-scale culturing of bone marrow-derived mesenchymal stromal cells (BM-MSCs) for therapeutic applications. Dulbecco's modified Eagle medium (DMEM)of low glucose (LG) and Knock Out (KO) were used with human platelet lysate (HPL) as LG-HPL and KO-HPL, and with FBS as LG-FBS and KO-FBS to culture the BM-MSCs. HPL at 10 % (v/v) supported BM-MSCs growth and subsequent isolation efficiency generated >90 × 10(6) MSCs in LG-HPL. Population doublings (PDs) and population doubling times of LG-HPL and KO-HPL (PDT) were not significantly different but LG-HPL showed a significant clonogenic potential and HPL cultures had an average PDT of 36.5 ± 6.5 h and an average PDs of 5 ± 0.7/passage. BM-MSCs cultured with LG-HPL had significantly higher immunosuppression compared to LG-FBS, but KO-HPL and KO-FBS-grown cultures were not significantly different. HPL is therefore alternative to FBS for large-scale production of BM-MSCs for therapeutic applications.

  20. AMPT-induced monoamine depletion in humans: evaluation of two alternative [{sup 123}I]IBZM SPECT procedures

    Energy Technology Data Exchange (ETDEWEB)

    Boot, Erik [Academic Medical Centre (AMC), University of Amsterdam, Department of Psychiatry, Amsterdam (Netherlands); De Bruggen, Centre for People with Intellectual Disability, Zwammerdam (Netherlands); Booij, Jan [AMC, Department of Nuclear Medicine, Amsterdam (Netherlands); Hasler, Gregor [University Hospital, Department of Psychiatry, Zuerich (Switzerland); Zinkstok, Janneke R.; Haan, Lieuwe de; Linszen, Don H.; Amelsvoort, Therese A. van [Academic Medical Centre (AMC), University of Amsterdam, Department of Psychiatry, Amsterdam (Netherlands)

    2008-07-15

    Acute monoamine depletion paradigms using alpha-methyl-para-tyrosine (AMPT) combined with single photon emission computed tomography (SPECT) have been used successfully to evaluate disturbances in central dopaminergic neurotransmission. However, severe side effects due to relatively high doses (4,500 to 8,000 mg) of AMPT have been reasons for study withdrawal. Thus, we assessed the effectiveness and tolerability of two alternative procedures, using lower doses of AMPT. Six healthy subjects underwent three measurements of striatal dopamine D{sub 2} receptor (D{sub 2}R)-binding potential (BP{sub ND}) with SPECT and the selective radiolabeled D{sub 2}R antagonist [{sup 123}I]IBZM. All subjects were scanned in the absence of pharmacological intervention (baseline) and after two different depletion procedures. In the first depletion session, over 6 h, subjects were administered 1,500 mg of AMPT before scanning. In the second depletion session, over 25 h, subjects were administered 40 mg AMPT/kg body weight. We also administered the Subjective Well-being Under Neuroleptic Treatment Scale, a self-report instrument designed to measure the subjective experience of patients on neuroleptic medication. We found no change of mean D{sub 2}R BP{sub ND} after the first and short AMPT challenge compared to the baseline. However, we found a significant increase in striatal D{sub 2}R BP{sub ND} binding after the AMPT challenge adjusted for bodyweight compared to both other regimen. Although subjective well-being worsened after the prolonged AMPT challenge, no severe side effects were reported. Our results imply a low-dosage, suitable alternative to the common AMPT procedure. The probability of side effects and study withdrawal can be reduced by this procedure. (orig.)

  1. Huh-7 cell line as an alternative cultural model for the production of human like erythropoietin (EPO

    Directory of Open Access Journals (Sweden)

    Kausar Humera

    2011-11-01

    Full Text Available Abstract Background and Aims Erythropoietin (EPO is a glycoprotein hormone which is required to regulate the production of red blood cells. Deficiency of EPO is known to cause anemia in chronically infected renal patients and they require regular blood transfusion. Availability of recombinant EPO has eliminated the need for blood transfusion and now it is extensively used for the treatment of anemia. Glycosylation of erythropoietin is essential for its secretion, stability, protein conformation and biological activity. However, maintenance of human like glycosylation pattern during manufacturing of EPO is a major challenge in biotechnology. Currently, Chinese hamster ovary (CHO cell line is used for the commercial production of erythropoietin but this cell line does not maintain glycosylation resembling human system. With the trend to eliminate non-human constituent from biopharmaceutical products, as a preliminary approach, we have investigated the potential of human hepatoma cell line (Huh-7 to produce recombinant EPO. Materials and methods Initially, the secretory signal and Kozak sequences was added before the EPO mature protein sequence using overlap extension PCR technique. PCR-amplified cDNA fragments of EPO was inserted into mammalian expression vector under the control of the cytomegalovirus (CMV promoter and transiently expressed in CHO and Huh-7 cell lines. After RT-PCR analysis, ELISA and Western blotting was performed to verify the immunochemical properties of secreted EPO. Results Addition of secretory signal and Kozak sequence facilitated the extra-cellular secretion and enhanced the expression of EPO protein. Significant expression (P Conclusion Huh-7 cell line has a great potential to produce glycosylated EPO, suggesting the use of this cell line to produce glycoproteins of the therapeutic importance resembling to the natural human system.

  2. The Number of Human Population, a Statistical and Economical Synthesis of Alternative Evolution towards Demographical Explosion or Implosion

    Directory of Open Access Journals (Sweden)

    Gheorghe Savoiu

    2006-10-01

    Full Text Available The number of the human population had reunited both in its quantitative side, and, especially, in the qualitative one, the most significant aspects of the various and detailed human evolution and dynamic. This demographic and synthetic indicator allowed a detailed and original process of population segmentation in the field of the contemporary analysis. Referring to a new process of demographic evolution and defining the population’s decline, demographers like Ehrlich and Longman have used the concept of demographic explosion but with the opposite demographic implosion too. World population through the accomplishment of the United Nations Population Revision or UNO prognosis seems to be forever somewhere between the limits of explosion and implosion.

  3. Elimination of Dog-Mediated Human Rabies Deaths by 2030: Needs Assessment and Alternatives for Progress Based on Dog Vaccination

    OpenAIRE

    Wallace, Ryan M.; Eduardo A Undurraga; Jesse D. Blanton; Cleaton, Julie; Franka, Richard

    2017-01-01

    Background Rabies imposes a substantial burden to about half of the world population. The World Health Organization (WHO), World Organization for Animal Health, and the Food and Agriculture Organization have set the goal of eliminating dog-mediated human rabies deaths by 2030. This could be achieved largely by massive administration of post-exposure prophylaxis—in perpetuity—, through elimination of dog rabies, or combining both. Here, we focused on the resources needed for the elimination of...

  4. An Alternative Humans to Mars Approach: Reducing Mission Mass with Multiple Mars Flyby Trajectories and Minimal Capability Investments

    Science.gov (United States)

    Whitley, Ryan J.; Jedrey, Richard; Landau, Damon; Ocampo, Cesar

    2015-01-01

    Mars flyby trajectories and Earth return trajectories have the potential to enable lower- cost and sustainable human exploration of Mars. Flyby and return trajectories are true minimum energy paths with low to zero post-Earth departure maneuvers. By emplacing the large crew vehicles required for human transit on these paths, the total fuel cost can be reduced. The traditional full-up repeating Earth-Mars-Earth cycler concept requires significant infrastructure, but a Mars only flyby approach minimizes mission mass and maximizes opportunities to build-up missions in a stepwise manner. In this paper multiple strategies for sending a crew of 4 to Mars orbit and back are examined. With pre-emplaced assets in Mars orbit, a transit habitat and a minimally functional Mars taxi, a complete Mars mission can be accomplished in 3 SLS launches and 2 Mars Flyby's, including Orion. While some years are better than others, ample opportunities exist within a given 15-year Earth-Mars alignment cycle. Building up a mission cadence over time, this approach can translate to Mars surface access. Risk reduction, which is always a concern for human missions, is mitigated by the use of flybys with Earth return (some of which are true free returns) capability.

  5. Evaluation of magnesium alloys with alternative surface finishing for the proliferation and chondro-differentiation of human mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Trinidad, J; Arruebarrena, G; De Argandona, E Saenz; De Eguino, G Ruiz; Infante, A; RodrIguez, C I, E-mail: jtrinidad@eps.mondragon.edu

    2010-11-01

    Articular cartilage has little capacity for self-repair. As a result, continuous mechanical stress can lead to the degradation of articular cartilage, culminating in progressive damage and joint degeneration. Tissue engineering has arisen as a promising therapeutic approach to cartilage repair. Magnesium alloys are one of the most important metallic biomaterials emerging in this area due to their biocompatibility, bio-absorbability and especially to their mechanical properties. These properties make magnesium alloys a promising biomaterial in the regeneration of cartilage tissue. Objective. This study was undertaken to analyze the influence of surface characteristics of magnesium alloys in the adhesion, proliferation and differentiation of human mesenchymal stem cells (MSCs). Methods. Two commercial magnesium alloys (AZ31B and ZM21) were subjected to different treatments in order to obtain four different surfaces in each alloy. Human MSCs were seeded into the magnesium alloys and analyzed for their proliferation and chondrogenesis differentiation ability. Results. Human MSCs showed a greater proliferation and chondro-differentiation when cultured in the ZM21 magnesium alloy with a surface finishing of fine sanding, polishing, and etching.

  6. Structural organization of the human and mouse laminin beta2 chain genes, and alternative splicing at the 5' end of the human transcript

    DEFF Research Database (Denmark)

    Durkin, M E; Gautam, M; Loechel, F;

    1996-01-01

    We have determined the structural organization of the human and mouse genes that encode the laminin beta2 chain (s-laminin), an essential component of the basement membranes of the neuromuscular synapse and the kidney glomerulus. The human and mouse genes have a nearly identical exon......-intron organization and are the smallest laminin chain genes characterized to date, due to the unusually small size of their introns. The laminin beta2 chain genes of both species consist of 33 exons that span...

  7. Towards Human-Robot Teams : Model-Based Analysis of Human Decision Making in Two-Alternative Choice Tasks With Social Feedback

    NARCIS (Netherlands)

    Stewart, Andrew; Cao, Ming; Nedic, Andrea; Tomlin, Damon; Leonard, Naomi Ehrich

    2012-01-01

    With a principled methodology for systematic design of human-robot decision-making teams as a motivating goal, we seek an analytic, model-based description of the influence of team and network design parameters on decision-making performance. Given that there are few reliably predictive models of hu

  8. Expression map of the human exome in CD34+ cells and blood cells: increased alternative splicing in cell motility and immune response genes.

    Directory of Open Access Journals (Sweden)

    Sylvie Tondeur

    Full Text Available BACKGROUND: Hematopoietic cells are endowed with very specific biological functions, including cell motility and immune response. These specific functions are dramatically altered during hematopoietic cell differentiation, whereby undifferentiated hematopoietic stem and progenitor cells (HSPC residing in bone marrow differentiate into platelets, red blood cells and immune cells that exit into the blood stream and eventually move into lymphoid organs or inflamed tissues. The contribution of alternative splicing (AS to these functions has long been minimized due to incomplete knowledge on AS events in hematopoietic cells. PRINCIPAL FINDINGS: Using Human Exon ST 1.0 microarrays, the entire exome expression profile of immature CD34+ HSPC and mature whole blood cells was mapped, compared to a collection of solid tissues and made freely available as an online exome expression atlas (Amazonia Exon! : http://amazonia.transcriptome.eu/exon.php. At a whole transcript level, HSPC strongly expressed EREG and the pluripotency marker DPPA4. Using a differential splicing index scheme (dsi, a list of 849 transcripts differentially expressed between hematopoietic cells and solid tissues was computed, that included NEDD9 and CD74. Some of these genes also underwent alternative splicing events during hematopoietic differentiation, such as INPP4B, PTPLA or COMMD6, with varied contribution of CD3+ T cells, CD19+ B cells, CD14+ or CD15+ myelomonocytic populations. Strikingly, these genes were significantly enriched for genes involved in cell motility, cell adhesion, response to wounding and immune processes. CONCLUSION: The relevance and the precision provided by this exon expression map highlights the contribution of alternative splicing to key feature of blood cells differentiation and function.

  9. The bifactor model of the Maslach Burnout Inventory-Human Services Survey (MBI-HSS)--an alternative measurement model of burnout.

    Science.gov (United States)

    Mészáros, V; Adám, Sz; Szabó, M; Szigeti, R; Urbán, R

    2014-02-01

    The purpose of the present study was to examine the construct validity of the Hungarian language version of the Maslach Burnout Inventory-Human Services Survey (MBI-HSS). A sample of 653 healthcare professionals (420 physicians and 233 nurses and nursing assistants) completed the MBI-HSS. A series of confirmatory factor analyses showed that a hierarchical bifactor model including a global burnout factor and three specific factors of emotional exhaustion, depersonalization and reduced personal accomplishment had the closest fit to the data, compared with an alternative second-order three-factor hierarchical model as well as to non-hierarchical one-factor, two-factor, three-factor, four-factor and five-factor models. However, only the global burnout factor and the specific personal accomplishment factor explained a considerable unique proportion of variance in observed scores. Our study confirms the validity of the MBI-HSS and suggests an alternative structural model, which may contribute to further understanding of the burnout construct.

  10. Alternative metrics

    Science.gov (United States)

    2012-11-01

    As the old 'publish or perish' adage is brought into question, additional research-impact indices, known as altmetrics, are offering new evaluation alternatives. But such metrics may need to adjust to the evolution of science publishing.

  11. Age-dependent decrease and alternative splicing of methionine synthase mRNA in human cerebral cortex and an accelerated decrease in autism.

    Directory of Open Access Journals (Sweden)

    Christina R Muratore

    Full Text Available The folate and vitamin B12-dependent enzyme methionine synthase (MS is highly sensitive to cellular oxidative status, and lower MS activity increases production of the antioxidant glutathione, while simultaneously decreasing more than 200 methylation reactions, broadly affecting metabolic activity. MS mRNA levels in postmortem human cortex from subjects across the lifespan were measured and a dramatic progressive biphasic decrease of more than 400-fold from 28 weeks of gestation to 84 years was observed. Further analysis revealed alternative splicing of MS mRNA, including deletion of folate-binding domain exons and age-dependent deletion of exons from the cap domain, which protects vitamin B12 (cobalamin from oxidation. Although three species of MS were evident at the protein level, corresponding to full-length and alternatively spliced mRNA transcripts, decreasing mRNA levels across the lifespan were not associated with significant changes in MS protein or methionine levels. MS mRNA levels were significantly lower in autistic subjects, especially at younger ages, and this decrease was replicated in cultured human neuronal cells by treatment with TNF-α, whose CSF levels are elevated in autism. These novel findings suggest that rather than serving as a housekeeping enzyme, MS has a broad and dynamic role in coordinating metabolism in the brain during development and aging. Factors adversely affecting MS activity, such as oxidative stress, can be a source of risk for neurological disorders across the lifespan via their impact on methylation reactions, including epigenetic regulation of gene expression.

  12. Alternative splicing in the human gene for the core protein A1 generates another hnRNP protein.

    Science.gov (United States)

    Buvoli, M; Cobianchi, F; Bestagno, M G; Mangiarotti, A; Bassi, M T; Biamonti, G; Riva, S

    1990-01-01

    The human hnRNP core protein A1 (34 kd) is encoded by a 4.6 kb gene split into 10 exons. Here we show that the A1 gene can be differentially spliced by the addition of an extra exon. The new transcript encodes a minor protein of the hnRNP complex, here defined A1B protein, with a calculated mol. wt of 38 kd, that coincides with a protein previously designated as B2 by some authors. In vitro translation of the mRNAs selected by hybridization with A1 cDNA produced two proteins of 34 and 38 kd; Northern blot analysis of poly(A)+ RNA from HeLa cells revealed that the abundance of the A1B mRNA was approximately 5% that of A1. The A1B protein was detected by Western blotting with an anti-A1 monoclonal antibody both in enriched preparations of basic hnRNP proteins and in 40S hnRNP particles. The A1B protein exhibits a significantly higher affinity than A1 for ssDNA. The recombinant A1B protein, expressed in Escherichia coli, shows the same electrophoretic mobility and charge as the cellular one. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:1691095

  13. First meeting "Cystic echinococcosis in Chile, update in alternatives for control and diagnostics in animals and humans".

    Science.gov (United States)

    Alvarez Rojas, Cristian A; Fredes, Fernando; Torres, Marisa; Acosta-Jamett, Gerardo; Alvarez, Juan Francisco; Pavletic, Carlos; Paredes, Rodolfo; Cortés, Sandra

    2016-09-13

    This report summarizes the outcomes of a meeting on cystic echinococcosis (CE) in animals and humans in Chile held in Santiago, Chile, between the 21st and 22nd of January 2016. The meeting participants included representatives of the Departamento de Zoonosis, Ministerio de Salud (Zoonotic Diseases Department, Ministry of Health), representatives of the Secretarias Regionales del Ministerio de Salud (Regional Department of Health, Ministry of Health), Instituto Nacional de Desarrollo Agropecuario (National Institute for the Development of Agriculture and Livestock, INDAP), Instituto de Salud Pública (National Institute for Public Health, ISP) and the Servicio Agrícola y Ganadero (Animal Health Department, SAG), academics from various universities, veterinarians and physicians. Current and future CE control activities were discussed. It was noted that the EG95 vaccine was being implemented for the first time in pilot control programmes, with the vaccine scheduled during 2016 in two different regions in the South of Chile. In relation to use of the vaccine, the need was highlighted for acquiring good quality data, based on CE findings at slaughterhouse, previous to initiation of vaccination so as to enable correct assessment of the efficacy of the vaccine in the following years. The current world's-best-practice concerning the use of ultrasound as a diagnostic tool for the screening population in highly endemic remote and poor areas was also discussed.

  14. Imprinting of IGF2 P0 transcript and novel alternatively spliced INS-IGF2 isoforms show differences between mouse and human.

    Science.gov (United States)

    Monk, D; Sanches, R; Arnaud, P; Apostolidou, S; Hills, F A; Abu-Amero, S; Murrell, A; Friess, H; Reik, W; Stanier, P; Constância, M; Moore, G E

    2006-04-15

    Genomic imprinting is limited to a subset of genes that play critical roles in fetal growth, development and behaviour. One of the most studied imprinted genes encodes insulin-like growth factor 2, and aberrant imprinting and DNA methylation of this gene is associated with the growth disorders Beckwith-Wiedemann and Silver-Russell syndromes and many human cancers. Specific isoforms of this gene have been shown to be essential for normal placental function, as mice carrying paternal null alleles for the Igf2-P0 transcript are growth restricted at birth. We report here the identification of three novel human transcripts from the IGF2 locus. One is equivalent to the mouse Igf2-P0 transcript, whereas the two others (INSIGF long and short) originate from the upstream INS gene that alternatively splices to downstream IGF2 exons. In order to elucidate the molecular mechanisms involved in the complex imprinting of these novel IGF2 transcripts, both the allele-specific expression and methylation for all the IGF2 promoters including P0 and the INSIGF transcripts were analysed in human tissues. Similar to the mouse, the human IGF2-P0 transcript is paternally expressed; however, its expression is not limited to placenta. This expression correlates with tissue-specific promoter methylation on the maternal allele. The two novel INSIGF transcripts reported here use the INS promoter and show highly restricted tissue expression profiles including the pancreas. As previously reported for INS in the yolk sac, we demonstrate complex, tissue-specific imprinting of these transcripts. The finding of additional transcripts within this locus will have important implications for IGF2 regulation in both cancer and metabolism.

  15. Human Mre11/human Rad50/Nbs1 and DNA ligase IIIalpha/XRCC1 protein complexes act together in an alternative nonhomologous end joining pathway.

    Science.gov (United States)

    Della-Maria, Julie; Zhou, Yi; Tsai, Miaw-Sheue; Kuhnlein, Jeff; Carney, James P; Paull, Tanya T; Tomkinson, Alan E

    2011-09-30

    Recent studies have implicated a poorly defined alternative pathway of nonhomologous end joining (alt-NHEJ) in the generation of large deletions and chromosomal translocations that are frequently observed in cancer cells. Here, we describe an interaction between two factors, hMre11/hRad50/Nbs1 (MRN) and DNA ligase IIIα/XRCC1, that have been linked with alt-NHEJ. Expression of DNA ligase IIIα and the association between MRN and DNA ligase IIIα/XRCC1 are altered in cell lines defective in the major NHEJ pathway. Most notably, DNA damage induced the association of these factors in DNA ligase IV-deficient cells. MRN interacts with DNA ligase IIIα/XRCC1, stimulating intermolecular ligation, and together these proteins join incompatible DNA ends in a reaction that mimics alt-NHEJ. Thus, our results provide novel mechanistic insights into the alt-NHEJ pathway that not only contributes to genome instability in cancer cells but may also be a therapeutic target.

  16. Timing of Peripheral Blood Stem Cell Yield: Comparison of Alternative Methods with the Classic Method for CD34+ Cell Determination

    Directory of Open Access Journals (Sweden)

    I. Fatorova

    2014-01-01

    Full Text Available Hematopoietic stem cells (HSCs, still represent a certain mystery in biology, have a unique property of dividing into equal cells and repopulating the hematopoietic tissue. This potential enables their use in transplantation treatments. The quality of the HSC grafts for transplantation is evaluated by flow cytometric determination of the CD34+ cells, which enables optimal timing of the first apheresis and the acquisition of maximal yield of the peripheral blood stem cells (PBSCs. To identify a more efficient method for evaluating CD34+ cells, we compared the following alternative methods with the reference method: hematopoietic progenitor cells (HPC enumeration (using the Sysmex XE-2100 analyser, detection of CD133+ cells, and quantification of aldehyde dehydrogenase activity in the PBSCs. 266 aphereses (84 patients were evaluated. In the preapheretic blood, the new methods produced data that were in agreement with the reference method. The ROC curves have shown that for the first-day apheresis target, the optimal predictive cut-off value was 0.032 cells/mL for the HPC method (sensitivity 73.4%, specificity 69.3%. HPC method exhibited a definite practical superiority as compared to other methods tested. HPC enumeration could serve as a supplementary method for the optimal timing of the first apheresis; it is simple, rapid, and cheap.

  17. The digital aqueous humor outflow meter: an alternative tool for screening of the human eye outflow facility

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    Vassilios P Kozobolis

    2010-08-01

    Full Text Available Vassilios P Kozobolis, Eleftherios I Paschalis, Nikitas C Foudoulakis, Stavrenia C Koukoula, Georgios LabirisDepartment of Ophthalmology and Eye Institute of Thrace, Democritus University of Thrace, Alexandroupolis, GreecePurpose: To develop, characterize, and validate a prototype digital aqueous humor outflow tonographer (DAHOM.Material and methods: The DAHOM was developed, characterized, and validated in three phases. Phase 1 involved construction of the sensor. This was broadly based on the fundamental design of a typical Schiotz tonographer with a series of improvements, including corneal indentation, which was converted to an electrical signal via a linear variable differential transducer, an analog signal which was converted to digital via ADC circuitry, and digital data acquisition and processing which was made possible by a serial port interface. Phase 2 comprised development of software for automated assessment of the outflow facility. Automated outflow facility assessment incorporated a series of fundamental improvements in comparison with traditional techniques, including software-based filtering of ripple noise and extreme variations, rigidity impact analysis, and evaluation of the impact of patient age, central corneal thickness, and ocular axial length. Phase 3 comprised characterization and validation of DAHOM, for which we developed an experimental setup using porcine cadaver eyes. DAHOM’s repeatability was evaluated by means of Cronbach’s alpha and intraclass correlation coefficient. The level of agreement with a standard Schiotz tonographer was evaluated by means of paired t-tests and Bland-Altman analysis in human eyes.Results: The experimental setup provided the necessary data for the characterization of DAHOM. A fourth order polynomial equation provided excellent fit (R square >0.999. DAHOM demonstrated high repeatability (Cronbach’s alpha ≥0.997; intraclass correlation coefficient ≥0.987 and an adequate level of

  18. Oligophrenin-1 (OPHN1, a gene involved in X-linked intellectual disability, undergoes RNA editing and alternative splicing during human brain development.

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    Sabina Barresi

    Full Text Available Oligophrenin-1 (OPHN1 encodes for a Rho-GTPase-activating protein, important for dendritic morphogenesis and synaptic function. Mutations in this gene have been identified in patients with X-linked intellectual disability associated with cerebellar hypoplasia. ADAR enzymes are responsible for A-to-I RNA editing, an essential post-transcriptional RNA modification contributing to transcriptome and proteome diversification. Specifically, ADAR2 activity is essential for brain development and function. Herein, we show that the OPHN1 transcript undergoes post-transcriptional modifications such as A-to-I RNA editing and alternative splicing in human brain and other tissues. We found that OPHN1 editing is detectable already at the 18th week of gestation in human brain with a boost of editing at weeks 20 to 33, concomitantly with OPHN1 expression increase and the appearance of a novel OPHN1 splicing isoform. Our results demonstrate that multiple post-transcriptional events occur on OPHN1, a gene playing an important role in brain function and development.

  19. Damage-inducible intragenic demethylation of the human TP53 tumor suppressor gene is associated with transcription from an alternative intronic promoter.

    Science.gov (United States)

    Blackburn, James; Roden, Daniel L; Ng, Robert; Wu, Jianmin; Bosman, Alexis; Epstein, Richard J

    2016-12-01

    Wild-type TP53 exons 5-8 contain CpG dinucleotides that are prone to methylation-dependent mutation during carcinogenesis, but the regulatory effects of methylation affecting these CpG sites are unclear. To clarify this, we first assessed site-specific TP53 CpG methylation in normal and transformed cells. Both DNA damage and cell ageing were associated with site-specific CpG demethylation in exon 5 accompanied by induction of a truncated TP53 isoform regulated by an adjacent intronic promoter (P2). We then synthesized novel synonymous TP53 alleles with divergent CpG content but stable encodement of the wild-type polypeptide. Expression of CpG-enriched TP53 constructs selectively reduced production of the full-length transcript (P1), consistent with a causal relationship between intragenic demethylation and transcription. 450K methylation comparison of normal (TP53-wildtype) and cancerous (TP53-mutant) human cells and tissues revealed focal cancer-associated declines in CpG methylation near the P1 transcription start site, accompanied by rises near the alternate exon 5 start site. These data confirm that site-specific changes of intragenic TP53 CpG methylation are extrinsically inducible, and suggest that human cancer progression is mediated in part by dysregulation of damage-inducible intragenic CpG demethylation that alters TP53 P1/P2 isoform expression. © 2015 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc.

  20. Fluorescent quantification of terazosin hydrochloride content in human plasma and tablets using second-order calibration based on both parallel factor analysis and alternating penalty trilinear decomposition.

    Science.gov (United States)

    Zou, Hong-Yan; Wu, Hai-Long; OuYang, Li-Qun; Zhang, Yan; Nie, Jin-Fang; Fu, Hai-Yan; Yu, Ru-Qin

    2009-09-14

    Two second-order calibration methods based on the parallel factor analysis (PARAFAC) and the alternating penalty trilinear decomposition (APTLD) method, have been utilized for the direct determination of terazosin hydrochloride (THD) in human plasma samples, coupled with the excitation-emission matrix fluorescence spectroscopy. Meanwhile, the two algorithms combing with the standard addition procedures have been applied for the determination of terazosin hydrochloride in tablets and the results were validated by the high-performance liquid chromatography with fluorescence detection. These second-order calibrations all adequately exploited the second-order advantages. For human plasma samples, the average recoveries by the PARAFAC and APTLD algorithms with the factor number of 2 (N=2) were 100.4+/-2.7% and 99.2+/-2.4%, respectively. The accuracy of two algorithms was also evaluated through elliptical joint confidence region (EJCR) tests and t-test. It was found that both algorithms could give accurate results, and only the performance of APTLD was slightly better than that of PARAFAC. Figures of merit, such as sensitivity (SEN), selectivity (SEL) and limit of detection (LOD) were also calculated to compare the performances of the two strategies. For tablets, the average concentrations of THD in tablet were 63.5 and 63.2 ng mL(-1) by using the PARAFAC and APTLD algorithms, respectively. The accuracy was evaluated by t-test and both algorithms could give accurate results, too.

  1. Oligophrenin-1 (OPHN1), a gene involved in X-linked intellectual disability, undergoes RNA editing and alternative splicing during human brain development.

    Science.gov (United States)

    Barresi, Sabina; Tomaselli, Sara; Athanasiadis, Alekos; Galeano, Federica; Locatelli, Franco; Bertini, Enrico; Zanni, Ginevra; Gallo, Angela

    2014-01-01

    Oligophrenin-1 (OPHN1) encodes for a Rho-GTPase-activating protein, important for dendritic morphogenesis and synaptic function. Mutations in this gene have been identified in patients with X-linked intellectual disability associated with cerebellar hypoplasia. ADAR enzymes are responsible for A-to-I RNA editing, an essential post-transcriptional RNA modification contributing to transcriptome and proteome diversification. Specifically, ADAR2 activity is essential for brain development and function. Herein, we show that the OPHN1 transcript undergoes post-transcriptional modifications such as A-to-I RNA editing and alternative splicing in human brain and other tissues. We found that OPHN1 editing is detectable already at the 18th week of gestation in human brain with a boost of editing at weeks 20 to 33, concomitantly with OPHN1 expression increase and the appearance of a novel OPHN1 splicing isoform. Our results demonstrate that multiple post-transcriptional events occur on OPHN1, a gene playing an important role in brain function and development.

  2. OCT4 and downstream factors are expressed in human somatic urogenital epithelia and in culture of epididymal spheres

    DEFF Research Database (Denmark)

    Audouze, Karine Marie Laure; Brunak, Søren; Kristensen, DM;

    2010-01-01

    promoter. In culture, human primary epididymis cells formed spheres that continued to express the investigated genes for at least 20 days. Transcriptomic analysis of cultured cells showed up-regulation of CD29, CD44, and CD133 that are normally associated with sphere-forming cancer stem cells. Furthermore......The transcription factor OCT4 plays a crucial role in the earliest differentiation of the mammalian embryo and in self-renewal of embryonic stem cells. However, it remains controversial whether this gene is also expressed in somatic tissues. Here we use a combination of RT-PCR on whole......, stimulation with retinoic acid resulted in down-regulation of OCT4 expression, however, without multilineage differentiation. Our results show that OCT4 and associated genes are expressed in somatic epithelial cells from the urogenital tract and that these cells can form spheres, a general marker of stem cell...

  3. Magnetostrictive Alternator

    Science.gov (United States)

    Dyson, Rodger; Bruder, Geoffrey

    2013-01-01

    This innovation replaces the linear alternator presently used in Stirling engines with a continuous-gradient, impedance-matched, oscillating magnetostrictive transducer that eliminates all moving parts via compression, maintains high efficiency, costs less to manufacture, reduces mass, and eliminates the need for a bearing system. The key components of this new technology are the use of stacked magnetostrictive materials, such as Terfenol-D, under a biased magnetic and stress-induced compression, continuous-gradient impedance-matching material, coils, force-focusing metallic structure, and supports. The acoustic energy from the engine travels through an impedancematching layer that is physically connected to the magnetostrictive mass. Compression bolts keep the structure under compressive strain, allowing for the micron-scale compression of the magnetostrictive material and eliminating the need for bearings. The relatively large millimeter displacement of the pressure side of the impedance-matching material is reduced to micron motion, and undergoes stress amplification at the magnetostrictive interface. The alternating compression and expansion of the magnetostrictive material creates an alternating magnetic field that then induces an electric current in a coil that is wound around the stack. This produces electrical power from the acoustic pressure wave and, if the resonant frequency is tuned to match the engine, can replace the linear alternator that is commonly used.

  4. Growing Alternatives

    DEFF Research Database (Denmark)

    Bagger-Petersen, Mai Corlin

    2014-01-01

    From 2014, Anhui Province will pilot a reform of the residential land market in China, thus integrating rural Anhui in the national housing market. In contrast, artist and activist Ou Ning has proposed the Bishan time money currency, intending to establish an alternative economic circuit in Bishan...

  5. Alternative Treatments

    Science.gov (United States)

    ... triglyceride (fat) produced by processing coconut oil or palm kernel oil. The body breaks down caprylic acid into substances called “ketone bodies.” The theory behind Axona is that the ketone bodies derived from caprylic acid may provide an alternative energy source for brain cells that have lost ...

  6. Epigenetic regulation of pluripotent genes mediates stem cell features in human hepatocellular carcinoma and cancer cell lines.

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    Xiao Qi Wang

    Full Text Available Activation of the stem cell transcriptional circuitry is an important event in cancer development. Although cancer cells demonstrate a stem cell-like gene expression signature, the epigenetic regulation of pluripotency-associated genes in cancers remains poorly understood. In this study, we characterized the epigenetic regulation of the pluripotency-associated genes NANOG, OCT4, c-MYC, KLF4, and SOX2 in a variety of cancer cell lines and in primary tumor samples, and investigated the re-activation of pluripotency regulatory circuits in cancer progression. Differential patterns of DNA methylation, histone modifications, and gene expression of pluripotent genes were demonstrated in different types of cancers, which may reflect their tissue origins. NANOG promoter hypomethylation and gene upregulation were found in metastatic human liver cancer cells and human hepatocellular carcinoma (HCC primary tumor tissues. The upregulation of NANOG, together with p53 depletion, was significantly associated with clinical late stage of HCC. A pro-metastatic role of NANOG in colon cancer cells was also demonstrated, using a NANOG-overexpressing orthotopic tumor implantation mouse model. Demethylation of NANOG promoter was observed in CD133+(high cancer cells. In accordance, overexpression of NANOG resulted in an increase in the population of CD133+(high cells. In addition, we demonstrated a cross-regulation between OCT4 and NANOG in cancer cells via reprogramming of promoter methylation. Taken together, epigenetic reprogramming of NANOG can lead to the acquisition of stem cell-like properties. These results underscore the restoration of pluripotency circuits in cancer cells as a potential mechanism for cancer progression.

  7. Combined 5-FU and ChoKα inhibitors as a new alternative therapy of colorectal cancer: evidence in human tumor-derived cell lines and mouse xenografts.

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    Ana de la Cueva

    Full Text Available BACKGROUND: Colorectal cancer (CRC is the third major cause of cancer related deaths in the world. 5-fluorouracil (5-FU is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Choline kinase alpha (ChoKα, an enzyme that plays a role in cell proliferation and transformation, has been reported overexpressed in many different tumors, including colorectal tumors. ChoKα inhibitors have recently entered clinical trials as a novel antitumor strategy. METHODOLOGY/PRINCIPAL FINDINGS: ChoKα specific inhibitors, MN58b and TCD-717, have demonstrated a potent antitumoral activity both in vitro and in vivo against several tumor-derived cell line xenografts including CRC-derived cell lines. The effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors has been investigated both in vitro in CRC-tumour derived cell lines, and in vivo in mouse xenografts models. The effects on thymidilate synthase (TS and thymidine kinase (TK1 levels, two enzymes known to play an essential role in the mechanism of action of 5-FU, were analyzed by western blotting and quantitative PCR analysis. The combination of 5-FU with ChoKα inhibitors resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. ChoKα inhibitors modulate the expression levels of TS and TK1 through inhibition of E2F production, providing a rational for its mechanism of action. CONCLUSION/SIGNIFICANCE: Our data suggest that both drugs in combination display a synergistic antitumoral effect due to ChoKα inhibitors-driven modulation of the metabolization of 5-FU. The clinical relevance of these findings is strongly supported since TCD-717 has recently entered Phase I clinical trials against solid tumors.

  8. Transcranial alternating current stimulation at beta frequency: lack of immediate effects on excitation and interhemispheric inhibition of the human motor cortex

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    Viola Rjosk

    2016-11-01

    Full Text Available Transcranial alternating current stimulation (tACS is a form of noninvasive brain stimulation and is capable of influencing brain oscillations and cortical networks. In humans, the endogenous oscillation frequency in sensorimotor areas peaks at 20 Hz. This beta-band typically occurs during maintenance of tonic motor output and seems to play a role in interhemispheric coordination of movements. Previous studies showed that tACS applied in specific frequency bands over primary motor cortex (M1 or the visual cortex modulates cortical excitability within the stimulated hemisphere. However, the particular impact remains controversial because effects of tACS were shown to be frequency, duration and location specific. Furthermore, the potential of tACS to modulate cortical interhemispheric processing, like interhemispheric inhibition (IHI, remains elusive. Transcranial magnetic stimulation (TMS is a noninvasive and well-tolerated method of directly activating neurons in superficial areas of the human brain and thereby a useful tool for evaluating the functional state of motor pathways. The aim of the present study was to elucidate the immediate effect of 10 min tACS in the β-frequency band (20 Hz over left M1 on IHI between M1s in 19 young, healthy, right-handed participants. A series of TMS measurements (MEP size, RMT, IHI from left to right M1 and vice versa was performed before and immediately after tACS or sham using a double-blinded, cross-over design. We did not find any significant tACS-induced modulations of intracortical excitation (as assessed by MEP size and RMT and/or interhemispheric inhibition (IHI. These results indicate that 10 min of 20 Hz tACS over left M1 seems incapable of modulating immediate brain activity or inhibition. Further studies are needed to elucidate potential aftereffects of 20 Hz tACS as well as frequency-specific effects of tACS on intracortical excitation and interhemispheric inhibition.

  9. Transcranial Alternating Current Stimulation at Beta Frequency: Lack of Immediate Effects on Excitation and Interhemispheric Inhibition of the Human Motor Cortex.

    Science.gov (United States)

    Rjosk, Viola; Kaminski, Elisabeth; Hoff, Maike; Gundlach, Christopher; Villringer, Arno; Sehm, Bernhard; Ragert, Patrick

    2016-01-01

    Transcranial alternating current stimulation (tACS) is a form of noninvasive brain stimulation and is capable of influencing brain oscillations and cortical networks. In humans, the endogenous oscillation frequency in sensorimotor areas peaks at 20 Hz. This beta-band typically occurs during maintenance of tonic motor output and seems to play a role in interhemispheric coordination of movements. Previous studies showed that tACS applied in specific frequency bands over primary motor cortex (M1) or the visual cortex modulates cortical excitability within the stimulated hemisphere. However, the particular impact remains controversial because effects of tACS were shown to be frequency, duration and location specific. Furthermore, the potential of tACS to modulate cortical interhemispheric processing, like interhemispheric inhibition (IHI), remains elusive. Transcranial magnetic stimulation (TMS) is a noninvasive and well-tolerated method of directly activating neurons in superficial areas of the human brain and thereby a useful tool for evaluating the functional state of motor pathways. The aim of the present study was to elucidate the immediate effect of 10 min tACS in the β-frequency band (20 Hz) over left M1 on IHI between M1s in 19 young, healthy, right-handed participants. A series of TMS measurements (motor evoked potential (MEP) size, resting motor threshold (RMT), IHI from left to right M1 and vice versa) was performed before and immediately after tACS or sham using a double-blinded, cross-over design. We did not find any significant tACS-induced modulations of intracortical excitation (as assessed by MEP size and RMT) and/or IHI. These results indicate that 10 min of 20 Hz tACS over left M1 seems incapable of modulating immediate brain activity or inhibition. Further studies are needed to elucidate potential aftereffects of 20 Hz tACS as well as frequency-specific effects of tACS on intracortical excitation and IHI.

  10. Alternate Reading Frame Protein (F Protein) of Hepatitis C Virus: Paradoxical Effects of Activation and Apoptosis on Human Dendritic Cells Lead to Stimulation of T Cells

    Science.gov (United States)

    Samrat, Subodh Kumar; Li, Wen; Singh, Shakti; Kumar, Rakesh; Agrawal, Babita

    2014-01-01

    Hepatitis C virus (HCV) leads to chronic infection in the majority of infected individuals due to lack, failure, or inefficiency of generated adaptive immune responses. In a minority of patients, acute infection is followed by viral clearance. The immune correlates of viral clearance are not clear yet but have been extensively investigated, suggesting that multispecific and multifunctional cellular immunity is involved. The generation of cellular immunity is highly dependent upon how antigen presenting cells (APCs) process and present various viral antigens. Various structural and non-structural HCV proteins derived from the open reading frame (ORF) have been implicated in modulation of dendritic cells (DCs) and APCs. Besides the major ORF proteins, the HCV core region also encodes an alternate reading frame protein (ARFP or F), whose function in viral pathogenesis is not clear. In the current studies, we sought to determine the role of HCV-derived ARFP in modulating dendritic cells and stimulation of T cell responses. Recombinant adenovirus vectors containing F or core protein derived from HCV (genotype 1a) were prepared and used to endogenously express these proteins in dendritic cells. We made an intriguing observation that endogenous expression of F protein in human DCs leads to contrasting effects on activation and apoptosis of DCs, allowing activated DCs to efficiently internalize apoptotic DCs. These in turn result in efficient ability of DCs to process and present antigen and to prime and stimulate F protein derived peptide-specific T cells from HCV-naive individuals. Taken together, our findings suggest important aspects of F protein in modulating DC function and stimulating T cell responses in humans. PMID:24475147

  11. Identification and evolutionary analysis of novel exons and alternative splicing events using cross-species EST-to-genome comparisons in human, mouse and rat

    Directory of Open Access Journals (Sweden)

    Ho Jar-Yi

    2006-03-01

    Full Text Available Abstract Background Alternative splicing (AS is important for evolution and major biological functions in complex organisms. However, the extent of AS in mammals other than human and mouse is largely unknown, making it difficult to study AS evolution in mammals and its biomedical implications. Results Here we describe a cross-species EST-to-genome comparison algorithm (ENACE that can identify novel exons for EST-scanty species and distinguish conserved and lineage-specific exons. The identified exons represent not only novel exons but also evolutionarily meaningful AS events that are not previously annotated. A genome-wide AS analysis in human, mouse and rat using ENACE reveals a total of 758 novel cassette-on exons and 167 novel retained introns that have no EST evidence from the same species. RT-PCR-sequencing experiments validated ~50 ~80% of the tested exons, indicating high presence of exons predicted by ENACE. ENACE is particularly powerful when applied to closely related species. In addition, our analysis shows that the ENACE-identified AS exons tend not to pass the nonsynonymous-to-synonymous substitution ratio test and not to contain protein domain, implying that such exons may be under positive selection or relaxed negative selection. These AS exons may contribute to considerable inter-species functional divergence. Our analysis further indicates that a large number of exons may have been gained or lost during mammalian evolution. Moreover, a functional analysis shows that inter-species divergence of AS events may be substantial in protein carriers and receptor proteins in mammals. These exons may be of interest to studies of AS evolution. The ENACE programs and sequences of the ENACE-identified AS events are available for download. Conclusion ENACE can identify potential novel cassette exons and retained introns between closely related species using a comparative approach. It can also provide information regarding lineage- or species

  12. The human peripheral benzodiazepine receptor gene: cloning and characterization of alternative splicing in normal tissues and in a patient with congenital lipoid adrenal hyperplasia.

    Science.gov (United States)

    Lin, D; Chang, Y J; Strauss, J F; Miller, W L

    1993-12-01

    The mitochondrial benzodiazepine receptor (mBzR) appears to be a key factor in the flow of cholesterol into mitochondria to permit the initiation of steroid hormone synthesis. The mBzR consists of three components; the 18-kDa component on the outer mitochondrial membrane appears to contain the benzodiazepine binding site, and is hence often termed the peripheral benzodiazepine receptor (PBR). Using a cloned human PBR cDNA as probe, we have cloned the human PBR gene. The 13-kb gene is divided into four exons, with exon 1 encoding only a short 5' untranslated segment. The 5' flanking DNA lacks TATA and CAAT boxes but contains a cluster of SP-1 binding sites, typical of "house-keeping" genes. The encoded PBR mRNA is alternately spliced into two forms: "authentic" PBR mRNA retains all four exons, while a short form termed PBR-S lacks exon 2. While PBR-S contains a 102-codon open reading frame with a typical initiator sequence, the reading frame differs from that of PBR, so that the encoded protein is unrelated to PBR. RT-PCR and RNase protection experiments confirm that both PBR and PBR-S are expressed in all tissues examined and that expression PBR-S is about 10 times the level of PBR. Expression of PBR cDNA in pCMV5 vectors transfected into COS-1 cells resulted in increased binding of [3H]PK11195, but expression of PBR-S did not. It has been speculated that patients with congenital lipoid adrenal hyperplasia, who cannot make any steroids, might have a genetic lesion in mBzR. RT-PCR analysis of testicular RNA from such a patient, sequencing of the cDNA, and blotting analysis of genomic DNA all indicate that the gene and mRNA for the PBR component of mBzR are normal in this disease.

  13. Alternate reading frame protein (F protein of hepatitis C virus: paradoxical effects of activation and apoptosis on human dendritic cells lead to stimulation of T cells.

    Directory of Open Access Journals (Sweden)

    Subodh Kumar Samrat

    Full Text Available Hepatitis C virus (HCV leads to chronic infection in the majority of infected individuals due to lack, failure, or inefficiency of generated adaptive immune responses. In a minority of patients, acute infection is followed by viral clearance. The immune correlates of viral clearance are not clear yet but have been extensively investigated, suggesting that multispecific and multifunctional cellular immunity is involved. The generation of cellular immunity is highly dependent upon how antigen presenting cells (APCs process and present various viral antigens. Various structural and non-structural HCV proteins derived from the open reading frame (ORF have been implicated in modulation of dendritic cells (DCs and APCs. Besides the major ORF proteins, the HCV core region also encodes an alternate reading frame protein (ARFP or F, whose function in viral pathogenesis is not clear. In the current studies, we sought to determine the role of HCV-derived ARFP in modulating dendritic cells and stimulation of T cell responses. Recombinant adenovirus vectors containing F or core protein derived from HCV (genotype 1a were prepared and used to endogenously express these proteins in dendritic cells. We made an intriguing observation that endogenous expression of F protein in human DCs leads to contrasting effects on activation and apoptosis of DCs, allowing activated DCs to efficiently internalize apoptotic DCs. These in turn result in efficient ability of DCs to process and present antigen and to prime and stimulate F protein derived peptide-specific T cells from HCV-naive individuals. Taken together, our findings suggest important aspects of F protein in modulating DC function and stimulating T cell responses in humans.

  14. The latin-american alternative ecological model between the protection of the environment as a human right and the recognition of the rights of nature

    Directory of Open Access Journals (Sweden)

    David Fabio Esborraz

    2016-06-01

    Full Text Available The concern of Latin-American Law about the “environmental issue” is rooted in the “inclusion of natural resources among the public goods” supported by the “Social Constitutionalism”. Subsequently it has been revitalised thanks to the development of the “International Environmental Law” and further intensified with the rise of the “New Latin-American Constitutionalism” and the unfolding of a real “Environmental Constitutionalism” which is clearly recognising the human right to an adequate (for the development of the person and sustainable environment. Nonetheless, in particular due to the fact that this law is built on a strongly “anthropocentric” conception, this was not enough to avoid nature despoil. Therefore, the so-called “New Andean  Constitutionalism”, by reaffirming the visio mundi of the Latin-American indigenous peoples, proposes to directly recognize the nature as a subject of law and to complete such a paradigm shift by adopting an alternative development model based on the Amerindian ethic-moral principle of the “Good Living”/“Living Well” (“Buen Vivir”/“Vivir Bien”.

  15. Human mesenchymal stem cells: a bank perspective on the isolation, characterization and potential of alternative sources for the regeneration of musculoskeletal tissues.

    Science.gov (United States)

    Moroni, Lorenzo; Fornasari, Pier Maria

    2013-04-01

    The continuous discovery of human mesenchymal stem cells (hMSCs) in different tissues is stirring up a tremendous interest as a cell source for regenerative medicine therapies. Historically, hMSCs have been always considered a sub-population of mononuclear cells present in the bone marrow (BM). Although BM-hMSCs are still nowadays considered as the most promising mesenchymal stem cell population to reach the clinics due to their capacity to differentiate into multiple tissues, hMSCs derived from other adult and fetal tissues have also demonstrated to possess similar differentiation capacities. Furthermore, different reports have highlighted a higher recurrence of hMSCs in some of these tissues as compared to BM. This offer a fascinating panorama for cell banking, since the creation of a stem cell factory could be envisioned where hMSCs are stocked and used for ad hoc clinical applications. In this review, we summarize the main findings and state of the art in hMSCs isolation, characterization, and differentiation from alternative tissue sources and we attempt to compare their potency for musculoskeletal regeneration.

  16. Evaluation of the expression of stem cell markers in human breast cancer reveals a correlation with clinical progression and metastatic disease in ductal carcinoma.

    Science.gov (United States)

    Martin, Tracey Amanda; Jiang, Wen Guo

    2014-01-01

    The tumor stem cell theory could explain how patients with metastatic disease show clinical relapse several months after starting treatment due to the survival of a small group of cells with unique characteristics. We examined the distribution and expression of a panel of stem cell markers in human breast cancer primary tumors. Human breast tissues were processed for immunohistochemistry, and RNA was extracted for analysis by quantitative-PCR. Immunohistochemical assay revealed that CD44 was strongly expressed in background endothelia and epithelia. CD133 expression was lost in tumor-associated endothelial cells. Conversely, CD49b was strongly stained in the tumors, associated vessels and ducts but was weakly stained in the background epithelia. q-PCR analysis revealed that CD44 and PSCA were reduced in patients with poor outcome (metastatic disease and death from breast cancer), with a marked reduction in ductal carcinoma, particularly with metastasis to bone although these did not reach significant difference. CD133 was significantly reduced in patients with metastatic disease and was also significantly reduced in patients with ductal carcinoma/bone metastasis. Conversely, CD49F was increased in patients with a poor outcome and those with ductal cancer and bone metastases. This is the first study to determine the distribution and expression pattern of these stem cell markers in human breast cancer. There was a significant association between loss of expression and metastatic disease in patients with breast cancer. Such differential expression may play a part in breast cancer disease progression, and suggests that the current stem cell theory may not hold true for all cancer types.

  17. Human aquaporin 4 gating dynamics under and after nanosecond-scale static and alternating electric-field impulses: a molecular dynamics study of field effects and relaxation.

    Science.gov (United States)

    Reale, Riccardo; English, Niall J; Garate, José-Antonio; Marracino, Paolo; Liberti, Micaela; Apollonio, Francesca

    2013-11-28

    Water self-diffusion and the dipolar response of the selectivity filter within human aquaporin 4 have been studied using molecular dynamics (MD) simulations in the absence and presence of pulses of external static and alternating electric fields. The pulses were approximately 50 and 100 ns in duration and 0.0065 V/Å in (r.m.s.) intensity and were either static or else 2.45 or 100 GHz in frequency and applied both along and perpendicular to the channels. In addition, the relaxation of the aquaporin, water self-diffusion and gating dynamics following cessation of the impulses was studied. In previous work it was determined that switches in the dihedral angle of the selectivity filter led to boosting of water permeation events within the channels, in the presence of identical external static and alternating electric fields, although applied continuously. Here the application of field impulses (and subsequently, upon removal) has shown that it is the dipolar orientation of the histidine-201 residue in the selectivity filter which governs the dihedral angle, and hence influences water self-diffusion; this constitutes an appropriate order parameter. The dipolar response of this residue to the applied field leads to the adoption of four distinct states, which we modelled as time-homogeneous Markov jump processes, and may be distinguished in the potential of mean force (PMF) as a function of the dipolar orientation of histidine-201. The observations of enhanced "dipolar flipping" of H201 serve to explain increased levels of water self-diffusion within aquaporin channels during, and immediately following, field impulses, although the level of statistical certainty here is lower. Given the appreciable size of the energy barriers evident in PMFs computed directly from deterministic MD (whether in the absence or presence of external fields), metadynamics calculations were undertaken to explore the free-energy landscape of histidine-201 orientation with greater accuracy and

  18. A boost of BMP4 accelerates the commitment of human embryonic stem cells to the endothelial lineage.

    Science.gov (United States)

    Goldman, Orit; Feraud, Olivier; Boyer-Di Ponio, Julie; Driancourt, Catherine; Clay, Denis; Le Bousse-Kerdiles, Marie-Caroline; Bennaceur-Griscelli, Annelise; Uzan, Georges

    2009-08-01

    Embryoid bodies (EBs) generated during differentiation of human embryonic stem cells (hESCs) contain vascular-like structures, suggesting that commitment of mesoderm progenitors into endothelial cells occurs spontaneously. We showed that bone morphogenetic protein 4 (BMP4), an inducer of mesoderm, accelerates the peak expression of CD133/kinase insert domain-containing receptor (KDR) and CD144/KDR. Because the CD133(+)KDR(+) population could represent endothelial progenitors, we sorted them at day 7 and cultured them in endothelial medium. These cells were, however, unable to differentiate into endothelial cells. Under standard conditions, the CD144(+)KDR(+) population represents up to 10% of the total cells at day 12. In culture, these cells, if sorted, give rise to a homogeneous population with a morphology typical of endothelial cells and express endothelial markers. These endothelial cells derived from the day 12 sorted population were functional, as assessed by different in vitro assays. When EBs were stimulated by BMP4, the CD144(+)KDR(+) peak was shifted to day 7. Most of these cells, however, were CD31(-), becoming CD31(+) in culture. They then expressed von Willebrand factor and were functional. This suggests that, initially, the BMP4-boosted day 7, CD144(+)KDR(+)CD31(-) population represents immature endothelial cells that differentiate into mature endothelial cells in culture. The expression of OCT3/4, a marker of immaturity for hESCs decreases during EB differentiation, decreasing faster following BMP4 induction. We also show that BMP4 inhibits the global expression of GATA2 and RUNX1, two transcription factors involved in hemangioblast formation, at day 7 and day 12.

  19. Expression and its significance of embryonic stem cell-associated factor Nanog in human gliomas%胚胎干细胞关键因子Nanog在人胶质瘤中的表达及意义

    Institute of Scientific and Technical Information of China (English)

    郭雨霁; 邴鲁军; 刘尚明; 郝爱军

    2013-01-01

    Objective To explore the expression of embryonic stem cell-associated factor Nanog in human gliomas and its significance. Methods Forty cases of human gliomas were examined. The expression of Nanog was analysed by immunohistochemistry, reverse transcription polymerase chain reaction and Western blotting. Double immunofluorescence staining was used to investigate the coexpression of Nanog and markers of cancer stem cells or embryonic stem cell pluripotency-related gene Oct4. Results We found a positive correlation between the expression levels of Nanog and tumour malignancy. Immunohistochemistry showed that Nanogexpressed in both the nuclei and the cytoplasm of glioma cells. Double immunofluorescence staining revealed that more than 50% of Nanog-positive cells coexpressed the putative CSC markers CD133 and Nestin. More than 95% Nanog positive cells also expressed embryonic stem cell pluripotency-related gene Oct4. Conclusion The result suggests that Nanog is mainly expressed in cancer stem cells in gliomas and positively correlated with the malignancy of gliomas. Nanog may play an important role in the progress of gliomas.%目的 探讨胚胎干细胞关键因子Nanog在人胶质瘤中的表达及意义.方法 通过免疫荧光双标技术,分析40例胶质瘤组织内胚胎干细胞关键因子Nanog与肿瘤干细胞相关基因Nestin、CD133及胚胎干细胞全能性相关基因Oct4的共表达情况,并通过RT-PCR、Western blotting技术分析胶质瘤组织内Nanog与脑胶质瘤恶性程度之间的关系.结果 Nanog在胶质瘤组织中的表达随着胶质瘤病理级别增高而升高,而且超过50%的Nanog阳性细胞同时表达Nestin和CD133.95%以上的Nanog阳性细胞都同时表达胚胎干细胞全能性相关基因Oct4.结论 胶质瘤组织中Nanog多表达在肿瘤干细胞中,与胶质瘤的恶性程度呈正相关,在胶质瘤的发生、发展过程中起着重要作用,为研究胶质瘤的起源及胶质瘤的诊断和预后判断提供帮助.

  20. Human epidermal membrane constant conductance iontophoresis: alternating current to obtain reproducible enhanced permeation and reduced lag times of a nonionic polar permeant.

    Science.gov (United States)

    Song, Yang; Li, S Kevin; Peck, Kendall D; Zhu, Honggang; Ghanem, Abdel-Halim; Higuchi, William I

    2002-01-31

    An experimental protocol, using an initial 1 min direct current (DC) applied potential of 4 V followed by alternating current (AC), was established to: (a) increase conductance and permeability and decrease lag time for human epidermal membrane (HEM) relative to unaltered HEM and; (b) maintain constant conductance and permeability during flux studies. The protocol allowed specific permeation parameters of the membrane to be characterized under electrically enhanced, constant flux conditions. The permeability, lag time, and effective membrane thickness were determined using a nonionic polar permeant, urea, while the enhanced conductance was maintained at a constant level with AC. A tortuous pore pathway model was employed to analyze the data. The AC protocol increased membrane permeability, and decreased lag time and effective membrane thickness relative to similar parameters obtained in previous studies from unaltered HEM. Lag times ranged from 32.0 to 105.5 min, and permeability coefficients calculated from steady state fluxes ranged from 1.68 to 6.03x10(-7) cm/s for HEM samples with electrical resistance values during transport of 2.3-8.0 kOmega x cm2. Effective membrane thicknesses were calculated to range from 0.34 to 0.61 cm during AC iontophoresis. Significant additional results were obtained when the protocol was applied for two consecutive runs using the same HEM sample, with time for the HEM sample to recover between runs. During the second run, the applied potential was adjusted to reproduce the conductance obtained on the first run. Under these conditions, the consecutive runs yielded essentially the same lag time, permeability and effective membrane thickness values. These results suggest that constant fluxes can be achieved by keeping HEM electrical conductance constant during AC iontophoresis.

  1. Advanced flow cytometric analysis of nanoparticle targeting to rare leukemic stem cells in peripheral human blood in a defined model system

    Science.gov (United States)

    Cooper, Christy L.; Leary, James F.

    2015-03-01

    Leukemia stem cells are both stem-like and leukemic-like. This complicates their detection as rare circulating tumor cells in the peripheral blood of leukemia patients. Since leukemic stem cells are also resistant to standard chemotherapeutic regimens, new therapeutic strategies need to be designed to kill the leukemic stem cells without killing normal stem cells. In these initial targeting studies we utilized a bioinformatics approach to design an antibodyfluorescent nanoparticle conjugate for targeting to these leukemic stem cells and to minimize targeting to normal stemprogenitor cells. Multicolor flow cytometric analyses were performed on a BD FACS Aria III. Human leukemic stem cell-like cell RS4;11 (with putative immunophenotype CD133+/CD24+/-, CD34+/-, CD38+, CD10-/Flt3+) was spiked into normal hematopoietic stem-progenitor cells obtained from a "buffy coat" prep (with putative immunophenotype CD133- /CD34+/CD38-/CD10-/Flt-3-) to be used as a model human leukemia patient. To analyze the model system, digital data mixtures of the two cell types were first created and assigned classifiers in order to create truth sets. ROC (Receiver Operating Characteristic) and multidimensional cluster analyses were used to evaluate the specificity and sensitivity of the immunophenotyping panel and for automated cell population identification, respectively. Costs of misclassification (false targeting) were also accounted for by this analysis scheme. Ultimately, this analysis scheme will be applied to use of nanoparticle-antibody conjugates at therapeutic doses for targeted killing of leukemia stem cells preferentially to normal stem -progenitor cells.

  2. Characterization of a distinct population of circulating human non-adherent endothelial forming cells and their recruitment via intercellular adhesion molecule-3.

    Directory of Open Access Journals (Sweden)

    Sarah L Appleby

    Full Text Available Circulating vascular progenitor cells contribute to the pathological vasculogenesis of cancer whilst on the other hand offer much promise in therapeutic revascularization in post-occlusion intervention in cardiovascular disease. However, their characterization has been hampered by the many variables to produce them as well as their described phenotypic and functional heterogeneity. Herein we have isolated, enriched for and then characterized a human umbilical cord blood derived CD133(+ population of non-adherent endothelial forming cells (naEFCs which expressed the hematopoietic progenitor cell markers (CD133, CD34, CD117, CD90 and CD38 together with mature endothelial cell markers (VEGFR2, CD144 and CD31. These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8 or myeloid markers (CD11b and CD14 which distinguishes them from 'early' endothelial progenitor cells (EPCs. Functional studies demonstrated that these naEFCs (i bound Ulex europaeus lectin, (ii demonstrated acetylated-low density lipoprotein uptake, (iii increased vascular cell adhesion molecule (VCAM-1 surface expression in response to tumor necrosis factor and (iv in co-culture with mature endothelial cells increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human endothelial cells (ECs. Extensive genomic and proteomic analyses of the naEFCs showed that intercellular adhesion molecule (ICAM-3 is expressed on their cell surface but not on mature endothelial cells. Furthermore, functional analysis demonstrated that ICAM-3 mediated the rolling and adhesive events of the naEFCs under shear stress. We suggest that the distinct population of naEFCs identified and characterized here represents a new valuable therapeutic target to control aberrant vasculogenesis.

  3. Characterization of a distinct population of circulating human non-adherent endothelial forming cells and their recruitment via intercellular adhesion molecule-3.

    Science.gov (United States)

    Appleby, Sarah L; Cockshell, Michaelia P; Pippal, Jyotsna B; Thompson, Emma J; Barrett, Jeffrey M; Tooley, Katie; Sen, Shaundeep; Sun, Wai Yan; Grose, Randall; Nicholson, Ian; Levina, Vitalina; Cooke, Ira; Talbo, Gert; Lopez, Angel F; Bonder, Claudine S

    2012-01-01

    Circulating vascular progenitor cells contribute to the pathological vasculogenesis of cancer whilst on the other hand offer much promise in therapeutic revascularization in post-occlusion intervention in cardiovascular disease. However, their characterization has been hampered by the many variables to produce them as well as their described phenotypic and functional heterogeneity. Herein we have isolated, enriched for and then characterized a human umbilical cord blood derived CD133(+) population of non-adherent endothelial forming cells (naEFCs) which expressed the hematopoietic progenitor cell markers (CD133, CD34, CD117, CD90 and CD38) together with mature endothelial cell markers (VEGFR2, CD144 and CD31). These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8) or myeloid markers (CD11b and CD14) which distinguishes them from 'early' endothelial progenitor cells (EPCs). Functional studies demonstrated that these naEFCs (i) bound Ulex europaeus lectin, (ii) demonstrated acetylated-low density lipoprotein uptake, (iii) increased vascular cell adhesion molecule (VCAM-1) surface expression in response to tumor necrosis factor and (iv) in co-culture with mature endothelial cells increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human endothelial cells (ECs). Extensive genomic and proteomic analyses of the naEFCs showed that intercellular adhesion molecule (ICAM)-3 is expressed on their cell surface but not on mature endothelial cells. Furthermore, functional analysis demonstrated that ICAM-3 mediated the rolling and adhesive events of the naEFCs under shear stress. We suggest that the distinct population of naEFCs identified and characterized here represents a new valuable therapeutic target to control aberrant vasculogenesis.

  4. Alternative Energies

    Energy Technology Data Exchange (ETDEWEB)

    Planting, A.; De saint Jacob, Y.; Verwijs, H.; Belin, H.; Preesman, L.

    2009-03-15

    In two articles, one interview and one column attention is paid to alternative energies. The article 'A new light on saving energy' discusses the option to save energy by modernising lighting systems in urban areas. The column 'View from Paris' focuses on investment decisions in France with regard to renewable energy and energy savings. The article 'Europe turns a blind eye to big battery' discusses developments in batteries to store energy. The interview concerns fuel cell expert and formerly President of UTC Power Jan van Dokkum. The last article gives a brief overview of the European Energy Research Alliance (EERA) and the challenges this alliance will have to face with regard to climate change and energy security.

  5. Alternative Energy Sources

    CERN Document Server

    Michaelides, Efstathios E (Stathis)

    2012-01-01

    Alternative Energy Sources is designed to give the reader, a clear view of the role each form of alternative energy may play in supplying the energy needs of the human society in the near and intermediate future (20-50 years).   The two first chapters on energy demand and supply and environmental effects, set the tone as to why the widespread use of alternative energy is essential for the future of human society. The third chapter exposes the reader to the laws of energy conversion processes, as well as the limitations of converting one energy form to another. The sections on exergy give a succinct, quantitative background on the capability/potential of each energy source to produce power on a global scale. The fourth, fifth and sixth chapters are expositions of fission and fusion nuclear energy. The following five chapters (seventh to eleventh) include detailed descriptions of the most common renewable energy sources – wind, solar, geothermal, biomass, hydroelectric – and some of the less common sources...

  6. Looking for an Alternative.

    Science.gov (United States)

    Kennedy, Jack

    1999-01-01

    Argues that high school newspapers might do well to create stronger ties with alternative weeklies. Discusses issues of niche marketing, alternative content, and alternative presentation. Notes that high school papers could learn a lot from alternative newspapers. (SR)

  7. Redirection of Human Cancer Cells upon the Interaction with the Regenerating Mouse Mammary Gland Microenvironment

    Directory of Open Access Journals (Sweden)

    Sonia M. Rosenfield

    2013-01-01

    Full Text Available Tumorigenesis is often described as a result of accumulated mutations that lead to growth advantage and clonal expansion of mutated cells. There is evidence in the literature that cancer cells are influenced by the microenvironment. Our previous studies demonstrated that the mouse mammary gland is capable of redirecting mouse cells of non-mammary origins as well as Mouse Mammary Tumor Virus (MMTV-neu transformed cells toward normal mammary epithelial cell fate during gland regeneration. Interestingly, the malignant phenotype of MMTV-neu transformed cells was suppressed during serial transplantation experiments. Here, we discuss our studies that demonstrated the potential of the regenerating mouse mammary gland to redirect cancer cells of different species into a functional tumor-free mammary epithelial cell progeny. Immunochemistry for human specific CD133, mitochondria, cytokeratins as well as milk proteins and FISH for human specific probe identified human epithelial cell progeny in ducts, lobules, and secretory acini. Fluorescent In Situ Hybridization (FISH for human centromeric DNA and FACS analysis of propidium iodine staining excluded the possibility of mouse-human cell fusion. To our knowledge this is the first evidence that human cancer cells of embryonic or somatic origins respond to developmental signals generated by the mouse mammary gland microenvironment during gland regeneration in vivo.

  8. A Social Justice Alternative for Framing Post-Compulsory Education: A Human Development Perspective of VET in Times of Economic Dominance

    Science.gov (United States)

    Lopez-Fogues, Aurora

    2016-01-01

    The article provides an alternative theoretical framework for evaluating contemporary issues facing education, specifically vocational education and training (VET) in Europe. In order to accomplish this, it draws on the theoretical insights of the capability approach in the work of Amartya Sen; the concept of vulnerability as intrinsic to every…

  9. Limited variation during circulation of a polyomavirus in the human population involves the COCO-VA toggling site of Middle and Alternative T-antigen(s)

    NARCIS (Netherlands)

    S. Kazem (Siamaque); C. Lauber (Chris); E. van der Meijden (Els); S. Kooijman (Sander); A.A. Kravchenko (Alexander A.); M.C.W. Feltkamp (Mariet C.W.); A.E. Gorbalenya (Alexander); J.C. Browning (John C.); K. Busam (Klaus); S. Bialasiewicz (Seweryn); T. Benoit (Taylor); P. Fleckman (Philip); L.C. Hughey (Lauren C.); R.W.A. Janssens (René W.A.); F. Mechinaud (Francoise); E. Pope (Elena); A.S. Rosenberg (Arlene S.); E. Rácz (Emoke); G. Sadler (Genevieve); S.N. Tabrizi (Sepehr N.); E. de Vries (E.); R.C. Wang (Richard C.)

    2016-01-01

    textabstractWe have recently shown that the trichodysplasia spinulosa-associated polyomavirus (TSPyV) belongs to a large monophyletic group of mammalian polyomaviruses that experienced accelerated codon-constrained Val-Ala (COCO-VA) toggling at a protein site common to both Middle and Alternative T-

  10. Characterization of glioma stem-like cells from human glioblastomas.

    Science.gov (United States)

    Yamamuro, Shun; Okamoto, Yutaka; Sano, Emiko; Ochiai, Yushi; Ogino, Akiyoshi; Ohta, Takashi; Hara, Hiroyuki; Ueda, Takuya; Nakayama, Tomohiro; Yoshino, Atsuo; Katayama, Yoichi

    2015-07-01

    Glioma stem-like cells (GSCs) could have potential for tumorigenesis, treatment resistance, and tumor recurrence (GSC hypothesis). However, the mechanisms underlying such potential has remained elusive and few ultrastructural features of the cells have been reported in detail. We therefore undertook observations of the antigenic characteristics and ultrastructural features of GSCs isolated from human glioblastomas. Tumor spheres formed by variable numbers of cells, exhibiting a variable appearance in both their size and shape, were frequently seen in GSCs expressing the stem cell surface markers CD133 and CD15. Increased cell nucleus atypia, mitochondria, rough endoplasmic reticulum, coated vesicles, and microvilli, were noted in the GSCs. Furthermore, cells at division phases and different phases of the apoptotic process were occasionally observed. These findings could imply that GSCs have certain relations with human neural stem cells (NSCs) but are primitively different from undifferentiated NSCs. The data may provide support for the GSC hypothesis, and also facilitate the establishment of future glioblastoma treatments targeting GSCs.

  11. Environmental levels of PCDD/Fs and metals around a cement plant in Catalonia, Spain, before and after alternative fuel implementation. Assessment of human health risks.

    Science.gov (United States)

    Rovira, Joaquim; Nadal, Martí; Schuhmacher, Marta; Domingo, José L

    2014-07-01

    The concentrations of As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Sn, Tl, V, and Zn, and the levels of polychlorinated dibenzo-p-dioxins and dibenzofurans were determined in samples of soil, vegetation, and air, collected in the vicinity of a cement plant (Catalonia, Spain), before (January 2011 and July 2011) and after (January 2012 and June 2013) alternative fuel partial substitution (fossil fuels by sewage sludge). Seven sampling points were selected at different directions and distances to the facility including two background sampling points. The results were used to assess the health risk assessment for the population living near the facility. Only few significant differences were found before and after alternative fuel partial substitution (Mn in soils and Cd in vegetation). Non-carcinogenic risks were below the safety threshold (HQ<1), while carcinogenic risks were below 10(-5), or exceeding slightly that value, always in the range considered as assumable (10(-6)-10(-4)).

  12. Regulation of human adenovirus alternative RNA splicing by the adenoviral L4-33K and L4-22K proteins.

    Science.gov (United States)

    Biasiotto, Roberta; Akusjärvi, Göran

    2015-01-28

    Adenovirus makes extensive use of alternative RNA splicing to produce a complex set of spliced viral mRNAs. Studies aimed at characterizing the interactions between the virus and the host cell RNA splicing machinery have identified three viral proteins of special significance for the control of late viral gene expression: L4-33K, L4-22K, and E4-ORF4. L4-33K is a viral alternative RNA splicing factor that controls L1 alternative splicing via an interaction with the cellular protein kinases Protein Kinase A (PKA) and DNA-dependent protein kinase (DNA-PK). L4-22K is a viral transcription factor that also has been implicated in the splicing of a subset of late viral mRNAs. E4-ORF4 is a viral protein that binds the cellular protein phosphatase IIA (PP2A) and controls Serine/Arginine (SR)-rich protein activity by inducing SR protein dephosphorylation. The L4-33K, and most likely also the L4-22K protein, are highly phosphorylated in vivo. Here we will review the function of these viral proteins in the post-transcriptional control of adenoviral gene expression and further discuss the significance of potential protein kinases phosphorylating the L4-33K and/or L4-22K proteins.

  13. Alternative medicine - pain relief

    Science.gov (United States)

    Alternative medicine refers to treatments that are used instead of conventional (standard) ones. If you use an alternative ... with conventional medicine or therapy, it is considered complementary therapy. There are many forms of alternative medicine. Acupuncture ...

  14. An animal model of adult T-cell leukemia: humanized mice with HTLV-1-specific immunity.

    Science.gov (United States)

    Tezuka, Kenta; Xun, Runze; Tei, Mami; Ueno, Takaharu; Tanaka, Masakazu; Takenouchi, Norihiro; Fujisawa, Jun-ichi

    2014-01-16

    Human T-cell leukemia virus type 1 (HTLV-1) is causally associated with adult T-cell leukemia (ATL), an aggressive T-cell malignancy with a poor prognosis. To elucidate ATL pathogenesis in vivo, a variety of animal models have been established; however, the mechanisms driving this disorder remain poorly understood due to deficiencies in each of these animal models. Here, we report a novel HTLV-1-infected humanized mouse model generated by intra-bone marrow injection of human CD133(+) stem cells into NOD/Shi-scid/IL-2Rγc null (NOG) mice (IBMI-huNOG mice). Upon infection, the number of CD4(+) human T cells in the periphery increased rapidly, and atypical lymphocytes with lobulated nuclei resembling ATL-specific flower cells were observed 4 to 5 months after infection. Proliferation was seen in both CD25(-) and CD25(+) CD4 T cells with identical proviral integration sites; however, a limited number of CD25(+)-infected T-cell clones eventually dominated, indicating an association between clonal selection of infected T cells and expression of CD25. Additionally, HTLV-1-specific adaptive immune responses were induced in infected mice and might be involved in the control of HTLV-1-infected cells. Thus, the HTLV-1-infected IBMI-huNOG mouse model successfully recapitulated the development of ATL and may serve as an important tool for investigating in vivo mechanisms of ATL leukemogenesis and evaluating anti-ATL drug and vaccine candidates.

  15. High levels of PROM1 (CD133) transcript are a potential predictor of poor prognosis in medulloblastoma.

    Science.gov (United States)

    Raso, Alessandro; Mascelli, Samantha; Biassoni, Roberto; Nozza, Paolo; Kool, Marcel; Pistorio, Angela; Ugolotti, Elisabetta; Milanaccio, Claudia; Pignatelli, Sara; Ferraro, Manuela; Pavanello, Marco; Ravegnani, Marcello; Cama, Armando; Garrè, Maria Luisa; Capra, Valeria

    2011-05-01

    The surface marker PROM1 is considered one of the most important markers of tumor-initiating cells, and its expression is believed to be an adverse prognostic factor in gliomas and in other malignancies. To date, to our knowledge, no specific studies of its expression in medulloblastoma series have been performed. The aims of our study were to evaluate the expression profile of the PROM1 gene in medulloblastoma and to assess its possible role as a prognostic factor. The PROM1 gene expression was evaluated by quantitative- polymerase chain reaction on 45 medulloblastoma samples by using specific dye-labeled probe systems. A significantly higher expression of PROM1 was found both in patients with poorer prognosis (P= .007) and in those with metastasis (P= .03). Kaplan-Meier analysis showed that both overall survival (OS) and progression-free survival (PFS) were shorter in patients with higher PROM1 mRNA levels than in patients with lower expression, even when the desmoplastic cases were excluded (P= .0004 and P= .002, for OS and PFS for all cases, respectively; P= .002 and P= .008 for OS and PFS for nondesmoplastic cases, respectively). Cox regression model demonstrated that PROM1 expression is an independent prognostic factor (hazard ratio, 4.56; P= .008). The result was validated on an independent cohort of 42 cases by microarray-based analysis (P= .019). This work suggests that high mRNA levels of PROM1 are associated with poor outcome in pediatric medulloblastoma. Furthermore, high PROM1 expression levels seem to increase the likelihood of metastases. Such results need to be confirmed in larger prospective series to possibly incorporate PROM1 gene expression into risk classification systems to be used in the clinical setting.

  16. Persistence of Lactobacillus Reuteri DSM17938 in the Human Intestinal Tract: Response to Consecutive and Alternate-Day Consumption with Varying Storage Conditions

    Science.gov (United States)

    2011-08-25

    0.85% NaCl (1:5, w/v) and stored at -70oC until bacterial enumerations were performed. When ready to process, thawed and weighed samples were...the probiotic intervention, represents the volunteer’s natural microbiota and served as the control. The D1 fecal sample, taken on the first day of...physiological factors affecting responsiveness to L. reuteri consumption, for example, gut microbiota of the individual (5). If volunteers in the alternate-day

  17. HIF induces human embryonic stem cell markers in cancer cells.

    Science.gov (United States)

    Mathieu, Julie; Zhang, Zhan; Zhou, Wenyu; Wang, Amy J; Heddleston, John M; Pinna, Claudia M A; Hubaud, Alexis; Stadler, Bradford; Choi, Michael; Bar, Merav; Tewari, Muneesh; Liu, Alvin; Vessella, Robert; Rostomily, Robert; Born, Donald; Horwitz, Marshall; Ware, Carol; Blau, C Anthony; Cleary, Michele A; Rich, Jeremy N; Ruohola-Baker, Hannele

    2011-07-01

    Low oxygen levels have been shown to promote self-renewal in many stem cells. In tumors, hypoxia is associated with aggressive disease course and poor clinical outcomes. Furthermore, many aggressive tumors have been shown to display gene expression signatures characteristic of human embryonic stem cells (hESC). We now tested whether hypoxia might be responsible for the hESC signature observed in aggressive tumors. We show that hypoxia, through hypoxia-inducible factor (HIF), can induce an hESC-like transcriptional program, including the induced pluripotent stem cell (iPSC) inducers, OCT4, NANOG, SOX2, KLF4, cMYC, and microRNA-302 in 11 cancer cell lines (from prostate, brain, kidney, cervix, lung, colon, liver, and breast tumors). Furthermore, nondegradable forms of HIFα, combined with the traditional iPSC inducers, are highly efficient in generating A549 iPSC-like colonies that have high tumorigenic capacity. To test potential correlation between iPSC inducers and HIF expression in primary tumors, we analyzed primary prostate tumors and found a significant correlation between NANOG-, OCT4-, and HIF1α-positive regions. Furthermore, NANOG and OCT4 expressions positively correlated with increased prostate tumor Gleason score. In primary glioma-derived CD133 negative cells, hypoxia was able to induce neurospheres and hESC markers. Together, these findings suggest that HIF targets may act as key inducers of a dynamic state of stemness in pathologic conditions.

  18. In vitro analysis of neurospheres derived from glioblastoma primary culture: a novel methodology paradigm.

    Directory of Open Access Journals (Sweden)

    Lorena Favaro Pavon

    2014-01-01

    Full Text Available Glioblastomas are the most lethal primary brain tumour frequently relapse or progress as focal masses after radiation, suggesting that only a fraction of tumour cells are responsible for the tumor regrowth. The identification of a brain tumour cell subpopulation with potent tumorigenic activity supports the cancer stem cell hypothesis in solid tumours. The goal of this study was to determine a methodology for the establishment of primary human glioblastoma stem cell lines. Our aim was achieved by taking the following approaches: i the establishment of primary glioblastoma cell culture; ii isolation of neurospheres derived from glioblastoma primary culture and derived straight from the tumor; iii CD133 microbeads purified neurospheres by MACS, iv Formation of subspheres in the CD133+ population, v Study of the expression level of GFAP, CD133, Nestin, Nanog, CD34 and Sox2 markers on tumor subspheres. Here, we describe a successful method for isolation of CD133+ cell population and establishment of glioblastoma neurospheres from this primary culture, which are more robust than the ones derived straight from the tumor. Highlight that the neurospheres derived from glioblatoma primary culture showed 89% expression of CD133+ cells, whereas tumor-derived neurospheres showed a 60% expression of CD133+ cells. These results show a higher concentration of CD133+ cells in neurospheres derived from glioblastoma primary culture. These CD133+ fractions were able to further generate subspheres. The subspheres derived from glioblastoma primary culture presented a well defined morphology while the ones derived form the fresh tumor were sparce and less robust. The negative fraction of CD133 cells was unable to generate subspheres. The tumor subspheres expressed GFAP, CD133, Nestin and Nanog. The present study describes an optimization of isolation of neurospheres/subspheres derived from glioblastoma primary culture by process of selection of CD133+ adherent stem

  19. Multimedia communications: architectural alternatives

    Science.gov (United States)

    McGarty, Terrence P.; Treves, S. T.

    1992-03-01

    Multimedia communications systems are a combination of human interfaces and end users interacting with multimedia data bases and highly disparate but interconnected communications networks. This paper discusses several architectural alternatives and system requirements that will assist in the design and development of MMCS in actual environments. The approaches taken in this paper are based upon the development of such systems in both medical and printing and publishing environments. This paper develops several key concepts as how best to define and structure data in a multimedia environment, how best to integrate the communications elements, and how best to permit the maximum flexibility to the end user to utilize the system's capabilities in the context of a fully conversational environment.

  20. Human Stem Cell Derived Cardiomyocytes: An Alternative Model to Evaluate Environmental Chemical Cardiac Safety and Development of Predictive Adverse Outcome Pathways

    Science.gov (United States)

    Chemical spills and associated deaths in the US has increased 2.6-fold and 16-fold from 1983 to 2012, respectfully. In addition, the number of chemicals to which humans are exposed to in the environment has increased almost 10-fold from 2001 to 2013 within the US. Internationally...

  1. Pre- and Postexposure Use of Human Monoclonal Antibody against H5N1 and H1N1 Influenza Virus in Mice: Viable Alternative to Oseltamivir

    NARCIS (Netherlands)

    Koudstaal, W.; Koldijk, M.H.; Brakenhoff, J.P.J.; Cornelissen, A.H.M.; Weverling, G.J.; Friesen, R.H.E.; Goudsmit, J.

    2009-01-01

    New strategies to prevent and treat influenza virus infections are urgently needed. A recently discovered class of monoclonal antibodies (mAbs) neutralizing an unprecedented spectrum of influenza virus subtypes may have the potential for future use in humans. Here, we assess the efficacies of CR6261

  2. The application of mouse and human embryonic stem cells with transcriptomics in alternative developmental toxicity tests : A bridge from model species to man

    NARCIS (Netherlands)

    Schulpen, S.H.W.

    2015-01-01

    During life humans are exposed to diverse hazardous compounds, which can have toxicological effects. Reproductive and developmental toxicology are research areas dedicated to the study of the potential of a compound to affect male and female fertility, and development of the embryo and fetus during

  3. Quantification of strontium in human serum by ICP-MS using alternate analyte-free matrix and its application to a pilot bioequivalence study of two strontium ranelate oral formulations in healthy Chinese subjects.

    Science.gov (United States)

    Zhang, Dan; Wang, Xiaolin; Liu, Man; Zhang, Lina; Deng, Ming; Liu, Huichen

    2015-01-01

    A rapid, sensitive and accurate ICP-MS method using alternate analyte-free matrix for calibration standards preparation and a rapid direct dilution procedure for sample preparation was developed and validated for the quantification of exogenous strontium (Sr) from the drug in human serum. Serum was prepared by direct dilution (1:29, v/v) in an acidic solution consisting of nitric acid (0.1%) and germanium (Ge) added as internal standard (IS), to obtain simple and high-throughput preparation procedure with minimized matrix effect, and good repeatability. ICP-MS analysis was performed using collision cell technology (CCT) mode. Alternate matrix method by using distilled water as an alternate analyte-free matrix for the preparation of calibration standards (CS) was used to avoid the influence of endogenous Sr in serum on the quantification. The method was validated in terms of selectivity, carry-over, matrix effects, lower limit of quantification (LLOQ), linearity, precision and accuracy, and stability. Instrumental linearity was verified in the range of 1.00-500ng/mL, corresponding to a concentration range of 0.0300-15.0μg/mL in 50μL sample of serum matrix and alternate matrix. Intra- and inter-day precision as relative standard deviation (RSD) were less than 8.0% and accuracy as relative error (RE) was within ±3.0%. The method allowed a high sample throughput, and was sensitive and accurate enough for a pilot bioequivalence study in healthy male Chinese subjects following single oral administration of two strontium ranelate formulations containing 2g strontium ranelate.

  4. Distinct motifs in the intracellular domain of human CD30 differentially activate canonical and alternative transcription factor NF-κB signaling.

    Directory of Open Access Journals (Sweden)

    Sarah L Buchan

    Full Text Available The TNF-receptor superfamily member CD30 is expressed on normal and malignant lymphocytes, including anaplastic large cell lymphoma (ALCL cells. CD30 transmits multiple effects, including activation of NF-κB signaling, cell proliferation, growth arrest and apoptosis. How CD30 generates these pleiotropic effects is currently unknown. Herein we describe ALCL cells expressing truncated forms of the CD30 intracellular domain that allowed us to identify the key regions responsible for transmitting its biological effects in lymphocytes. The first region (CD30(519-537 activated both the alternative and canonical NF-κB pathways as detected by p100 and IκBα degradation, IKKβ-dependent transcription of both IκBα and the cyclin-dependent kinase inhibitor p21(WAF1/CIP1 and induction of cell cycle arrest. In contrast, the second region of CD30 (CD30(538-595 induced some aspects of canonical NF-κB activation, including transcription of IκBα, but failed to activate the alternative NF-κB pathway or drive p21(WAF1/CIP1-mediated cell-cycle arrest. Direct comparison of canonical NF-κB activation by the two motifs revealed 4-fold greater p65 nuclear translocation following CD30(519-537 engagement. These data reveal that independent regions of the CD30 cytoplasmic tail regulate the magnitude and type of NF-κB activation and additionally identify a short motif necessary for CD30-driven growth arrest signals in ALCL cells.

  5. All About Alternatives

    Science.gov (United States)

    Barr, Robert D.; And Others

    1972-01-01

    A primer on alternative schools. Described are existing programs in different areas, philosophy of the alternative schools, funding, student behavior, community relations, accountability, State regulations, management, and the environment of the alternative school. A list of sources of additional information on alternative schools is included.…

  6. Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

    Energy Technology Data Exchange (ETDEWEB)

    Varga, Nora [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary); Vereb, Zoltan; Rajnavoelgyi, Eva [Department of Immunology, Medical and Health Science Centre, University of Debrecen, Debrecen (Hungary); Nemet, Katalin; Uher, Ferenc; Sarkadi, Balazs [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary); Apati, Agota, E-mail: apati@kkk.org.hu [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary)

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.

  7. Design and development of TT30, a novel C3d-targeted C3/C5 convertase inhibitor for treatment of human complement alternative pathway-mediated diseases.

    Science.gov (United States)

    Fridkis-Hareli, Masha; Storek, Michael; Mazsaroff, Istvan; Risitano, Antonio M; Lundberg, Ante S; Horvath, Christopher J; Holers, V Michael

    2011-10-27

    To selectively modulate human complement alternative pathway (CAP) activity implicated in a wide range of acute and chronic inflammatory conditions and to provide local cell surface and tissue-based inhibition of complement-induced damage, we developed TT30, a novel therapeutic fusion protein linking the human complement receptor type 2 (CR2/CD21) C3 fragment (C3frag = iC3b, C3dg, C3d)-binding domain with the CAP inhibitory domain of human factor H (fH). TT30 efficiently blocks ex vivo CAP-dependent C3frag accumulation on activated surfaces, membrane attack complex (MAC) formation and hemolysis of RBCs in a CR2-dependent manner, and with a ∼ 150-fold potency gain over fH, without interference of C3 activation or MAC formation through the classic and lectin pathways. TT30 protects RBCs from hemolysis and remains bound and detectable for at least 24 hours. TT30 selectively inhibits CAP in cynomolgus monkeys and is bioavailable after subcutaneous injection. Using a unique combination of targeting and effector domains, TT30 controls cell surface CAP activation and has substantial potential utility for the treatment of human CAP-mediated diseases.

  8. Are there alternative adaptive strategies to human pro-sociality? The role of collaborative morality in the emergence of personality variation and autistic traits

    OpenAIRE

    Spikins, P.; Wright, Barry; Hodgson, Derek

    2016-01-01

    Selection pressures to better understand other’s thoughts and feelings are seen as a primary driving force in human cognitive evolution. Yet might the evolution of social cognition be more complex than we assume, with more than one strategy towards social understanding and developing a positive pro-social reputation? Here we argue that social buffering of vulnerabilities through the emergence of collaborative morality will have opened new niches for adaptive cognitive strategies and widened p...

  9. Reconstituted human corneal epithelium: a new alternative to the Draize eye test for the assessment of the eye irritation potential of chemicals and cosmetic products.

    Science.gov (United States)

    Doucet, O; Lanvin, M; Thillou, C; Linossier, C; Pupat, C; Merlin, B; Zastrow, L

    2006-06-01

    The aim of this study was to evaluate the interest of a new three-dimensional epithelial model cultivated from human corneal cells to replace animal testing in the assessment of eye tolerance. To this end, 65 formulated cosmetic products and 36 chemicals were tested by means of this in vitro model using a simplified toxicokinetic approach. The chemicals were selected from the ECETOC data bank and the EC/HO International validation study list. Very satisfactory results were obtained in terms of concordance with the Draize test data for the formulated cosmetic products. Moreover, the response of the corneal model appeared predictive of human ocular response clinically observed by ophthalmologists. The in vitro scores for the chemicals tested strongly correlated with their respective scores in vivo. For all the compounds tested, the response of the corneal model to irritants was similar regardless of their chemical structure, suggesting a good robustness of the prediction model proposed. We concluded that this new three-dimensional epithelial model, developed from human corneal cells, could be promising for the prediction of eye irritation induced by chemicals and complex formulated products, and that these two types of materials should be tested using a similar protocol. A simple shortening of the exposure period was required for the chemicals assumed to be more aggressively irritant to the epithelial tissues than the cosmetic formulae.

  10. Intronic Alus influence alternative splicing.

    Directory of Open Access Journals (Sweden)

    Galit Lev-Maor

    Full Text Available Examination of the human transcriptome reveals higher levels of RNA editing than in any other organism tested to date. This is indicative of extensive double-stranded RNA (dsRNA formation within the human transcriptome. Most of the editing sites are located in the primate-specific retrotransposed element called Alu. A large fraction of Alus are found in intronic sequences, implying extensive Alu-Alu dsRNA formation in mRNA precursors. Yet, the effect of these intronic Alus on splicing of the flanking exons is largely unknown. Here, we show that more Alus flank alternatively spliced exons than constitutively spliced ones; this is especially notable for those exons that have changed their mode of splicing from constitutive to alternative during human evolution. This implies that Alu insertions may change the mode of splicing of the flanking exons. Indeed, we demonstrate experimentally that two Alu elements that were inserted into an intron in opposite orientation undergo base-pairing, as evident by RNA editing, and affect the splicing patterns of a downstream exon, shifting it from constitutive to alternative. Our results indicate the importance of intronic Alus in influencing the splicing of flanking exons, further emphasizing the role of Alus in shaping of the human transcriptome.

  11. Implication of Akt, ERK1/2 and alternative p38MAPK signalling pathways in human colon cancer cell apoptosis induced by green tea EGCG.

    Science.gov (United States)

    Cerezo-Guisado, María Isabel; Zur, Rafal; Lorenzo, María Jesús; Risco, Ana; Martín-Serrano, Miguel A; Alvarez-Barrientos, Alberto; Cuenda, Ana; Centeno, Francisco

    2015-10-01

    We investigated apoptosis induced by the green tea component the epigallocatechin-3-gallate (EGCG) and the pathways underlying its activity in a colon cancer cell line. A complete understanding of the mechanism(s) and molecules targeted by green tea polyphenols could be useful in developing novel therapeutic approaches for cancer treatment. EGCG, which is the major polyphenol in green tea, has cytotoxic effects and induced cell death in HT-29 cell death. In this study, we evaluated the effect EGCG on mitogen-activated protein kinase (MAPK) and Akt pathways. EGCG treatment increased phospho-ERK1/2, -JNK1/2 and -p38α, -p38γ and -p38δ, as well as phospho-Akt levels. Using a combination of kinase inhibitors, we found that EGCG-induced cell death is partially blocked by inhibiting Akt, ERK1/2 or alternative p38MAPK activity. Our data suggest that these kinase pathways are involved in the anti-cancer effects of EGCG and indicate potential use of this compound as chemotherapeutic agent for colon cancer treatment.

  12. PCBP-1 regulates alternative splicing of the CD44 gene and inhibits invasion in human hepatoma cell line HepG2 cells

    Directory of Open Access Journals (Sweden)

    Ge Changhui

    2010-04-01

    Full Text Available Abstract Background PCBP1 (or alpha CP1 or hnRNP E1, a member of the PCBP family, is widely expressed in many human tissues and involved in regulation of transcription, transportation process, and function of RNA molecules. However, the role of PCBP1 in CD44 variants splicing still remains elusive. Results We found that enforced PCBP1 expression inhibited CD44 variants expression including v3, v5, v6, v8, and v10 in HepG2 cells, and knockdown of endogenous PCBP1 induced these variants splicing. Invasion assay suggested that PCBP1 played a negative role in tumor invasion and re-expression of v6 partly reversed the inhibition effect by PCBP1. A correlation of PCBP1 down-regulation and v6 up-regulation was detected in primary HCC tissues. Conclusions We first characterized PCBP1 as a negative regulator of CD44 variants splicing in HepG2 cells, and loss of PCBP1 in human hepatic tumor contributes to the formation of a metastatic phenotype.

  13. Age-related variations of protein carbonyls in human saliva and plasma: is saliva protein carbonyls an alternative biomarker of aging?

    Science.gov (United States)

    Wang, Zhihui; Wang, Yanyi; Liu, Hongchen; Che, Yuwei; Xu, Yingying; E, Lingling

    2015-06-01

    Free radical hypothesis which is one of the most acknowledged aging theories was developed into oxidative stress hypothesis. Protein carbonylation is by far one of the most widely used markers of protein oxidation. We studied the role of age and gender in protein carbonyl content of saliva and plasma among 273 Chinese healthy subjects (137 females and 136 males aged between 20 and 79) and discussed the correlation between protein carbonyl content of saliva and plasma. Protein carbonyl content of saliva and plasma were, respectively, 2.391 ± 0.639 and 0.838 ± 0.274 nmol/mg. Variations of saliva and plasma different age groups all reached significant differences in both male and female (all p saliva and plasma protein carbonyls were found to be significantly correlated with age (r = 0.6582 and r = 0.5176, all p saliva and plasma protein carbonyl levels (all p > 0.05). Saliva and plasma protein carbonyls were positively related (r = 0.4405, p saliva and plasma protein carbonyls/ferric reducing ability of plasma (FRAP) ratios were proved to be significantly correlated with age (r = 0.7796 and r = 0.6938, all p saliva protein carbonyls/FRAP ratio and plasma protein carbonyls/FRAP ratio were also correlated (r = 0.5573, p saliva protein carbonyls seem to be an alternative biomarker of aging while the mechanisms of protein carbonylation and oxidative stress and the relationship between saliva protein carbonyls and diseases need to be further investigated.

  14. Doxycycline Induces Apoptosis and Inhibits Proliferation and Invasion of Human Cervical Carcinoma Stem Cells.

    Directory of Open Access Journals (Sweden)

    Binlie Yang

    Full Text Available Cancer stem cells (CSCs are proposed to be responsible for high recurrence rate in cervical carcinoma. Reagents that can suppress the proliferation and differentiation of CSCs would provide new opportunities to fight against tumor recurrence. Doxycycline has been reported as a potential anti-cancer compound. However, few studies investigated its inhibitory effect against cervical cancer stem cells.HeLa cells were cultured in cancer stem cell conditional media in a poly-hema-treated dish. In this non-adhesive culture system, HeLa cells were treated with cisplatin until some cells survived and formed spheroids, which were then collected and injected into the immunodeficient mice. Cisplatin was administered every three days for five times. The tumor xenografts with CSC enrichment were cultured in cancer stem cell specific medium again to form tumorsphere, which we called HeLa-CSCs. Expression of cancer stem cell markers in HeLa-CSCs was measured by flow cytometry and qPCR. HeLa-CSCs were then treated with doxycycline. Proliferation and differentiation rates were determined by the size of spheres formed in vitro and tumor formed in vivo.We developed a new strategy to selectively enrich CSCs from human cervical carcinoma cell line HeLa, and these HeLa-CSCs are CD133+/CD49f+ cell populations with significantly enhanced expression of stem cell markers. When these HeLa-CSCs were treated with doxycycline, the colony formation, proliferation, migration and invasion, and differentiation were all suppressed. Meanwhile, stem cell markers SOX-2, OCT-4, NANOG, NOTCH and BMI-1 decreased in doxycycline treated cells, so as the surface markers CD133 and CD49f. Furthermore, proliferation markers Ki67 and PCNA were also decreased by doxycycline treatment in the in vivo xenograft mouse model.Cancer stem cells are enriched from sphere-forming and chemoresistant HeLa-derived tumor xenografts in immunodeficient mice. Doxycycline inhibits proliferation, invasion, and

  15. Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells.

    Science.gov (United States)

    Katz, Adam J; Tholpady, Ashok; Tholpady, Sunil S; Shang, Hulan; Ogle, Roy C

    2005-03-01

    Adult human subcutaneous adipose tissue contains cells with intriguing multilineage developmental plasticity, much like marrow-derived mesenchymal stem cells. Putative stem or progenitor cells from fat have been given many different names in the literature, reflecting an early and evolving consensus regarding their phenotypic characterization. The study reported here used microarrays to evaluate over 170 genes relating to angiogenesis and extracellular matrix in undifferentiated, early-passage human adipose-derived adherent stromal (hADAS) cells isolated from three separate donors. The hADAS populations unanimously transcribed 66% of the screened genes, and 83% were transcribed by at least two of the three populations. The most highly transcribed genes relate to functional groupings such as cell adhesion, matrix proteins, growth factors and receptors, and proteases. The transcriptome of hADAS cells demonstrated by this work reveals many similarities to published profiles of bone marrow mesenchymal stem cells (MSCs). In addition, flow analysis of over 24 hADAS cell surface proteins (n = 7 donors) both confirms and expands on the existing literature and reveals strong intergroup correlation, despite an inconsistent nomenclature and the lack of standardized protocols for cell isolation and culture. Finally, based on flow analysis and reverse transcription polymerase chain reaction studies, our results suggest that hADAS cells do not express several proteins that are implicated as markers of "stemness" in other stem cell populations, including telomerase, CD133, and the membrane transporter ABCG2.

  16. Alternative splicing interference by xenobiotics.

    Science.gov (United States)

    Zaharieva, Emanuela; Chipman, J Kevin; Soller, Matthias

    2012-06-14

    The protein coding sequence of most eukaryotic genes (exons) is interrupted by non-coding parts (introns), which are excised in a process termed splicing. To generate a mature messenger RNA (mRNA) hundreds of combinatorial protein-protein and RNA-protein interactions are required to splice out often very large introns with high fidelity and accuracy. Inherent to splicing is the use of alternative splice sites generating immense proteomic diversity from a limited number of genes. In humans, alternative splicing is a major mode of regulating gene expression, occurs in over 90% of genes and is particularly abundant in the brain. Only recently, it has been recognized that the complexity of the splicing process makes it susceptible to interference by various xenobiotics. These compounds include antineoplastic substances, commonly used drugs and food supplements and cause a spectrum of effects ranging from deleterious inhibition of general splicing to highly specific modifications of alternative splicing affecting only certain genes. Alterations in splicing have been implicated in numerous diseases such as cancer and neurodegeneration. Splicing regulation plays an important role in the execution of programmed cell death. The switch between anti- and pro-apoptotic isoforms by alternative splice site selection and misregulation of a number of splicing factors impacts on cell survival and disease. Here, our current knowledge is summarized on compounds interfering with general and alternative splicing and of the current methodology to study changes in these processes relevant to the field of toxicology and future risk assessments.

  17. Consumer Health: Alternative Therapy

    Science.gov (United States)

    Healthy Lifestyle Consumer health What's considered an alternative therapy is a moving target. Get the facts about what CAM means and ... Original article: http://www.mayoclinic.org/healthy-lifestyle/consumer-health/in-depth/alternative-medicine/art-20045267 . Mayo ...

  18. Quantification of caffeine in human saliva by Nuclear Magnetic Resonance as an alternative method for cytochrome CYP1A2 phenotyping.

    Science.gov (United States)

    Schievano, Elisabetta; Finotello, Claudia; Navarini, Luciano; Mammi, Stefano

    2015-08-01

    The first step in caffeine metabolism is mediated for over 95% by the CYP1A2 isoform of cytochrome P450. Therefore, CYP1A2 activity is most conveniently measured through the determination of caffeine clearance. The HPLC quantification of caffeine is fully validated and is the most widely used method. It can be performed on saliva, which is gaining importance as a diagnostic biofluid and permits easy and low invasive sampling. Here, we present a quantitative (1)H nuclear magnetic resonance (NMR) method to determine caffeine in human saliva. The procedure is simple because it involves only an ultra-filtration step and a direct extraction in a deuterated solvent, yielding a matrix that is then analyzed. The reliability of this NMR method was demonstrated in terms of linearity, accuracy, recovery, and limits of detection (LoD). Good precision (relative standard deviation, RSD 95% and LoD of 6.8·10(-7) mol L(-1) were obtained. The method was applied to samples collected from different volunteers over 24h following a single oral dose of about 100mg of caffeine administered with either coffee beverage or a capsule.

  19. Measurement of electric fields induced in a human subject due to natural movements in static magnetic fields or exposure to alternating magnetic field gradients.

    Science.gov (United States)

    Glover, P M; Bowtell, R

    2008-01-21

    A dual dipole electric field probe has been used to measure surface electric fields in vivo on a human subject over a frequency range of 0.1-800 Hz. The low-frequency electric fields were induced by natural body movements such as walking and turning in the fringe magnetic fields of a 3 T magnetic resonance whole-body scanner. The rate-of-change of magnetic field (dB/dt) was also recorded simultaneously by using three orthogonal search coils positioned near to the location of the electric field probe. Rates-of-change of magnetic field for natural body rotations were found to exceed 1 T s(-1) near the end of the magnet bore. Typical electric fields measured on the upper abdomen, head and across the tongue for 1 T s(-1) rate of change of magnetic field were 0.15+/-0.02, 0.077+/-0.003 and 0.015+/-0.002 V m(-1) respectively. Electric fields on the abdomen and chest were measured during an echo-planar sequence with the subject positioned within the scanner. With the scanner rate-of-change of gradient set to 10 T m(-1) s(-1) the measured rate-of-change of magnetic field was 2.2+/-0.1 T s(-1) and the peak electric field was 0.30+/-0.01 V m(-1) on the chest. The values of induced electric field can be related to dB/dt by a 'geometry factor' for a given subject and sensor position. Typical values of this factor for the abdomen or chest (for measured surface electric fields) lie in the range of 0.10-0.18 m. The measured values of electric field are consistent with currently available numerical modelling results for movement in static magnetic fields and exposure to switched magnetic field gradients.

  20. Measurement of electric fields induced in a human subject due to natural movements in static magnetic fields or exposure to alternating magnetic field gradients

    Science.gov (United States)

    Glover, P. M.; Bowtell, R.

    2008-01-01

    A dual dipole electric field probe has been used to measure surface electric fields in vivo on a human subject over a frequency range of 0.1-800 Hz. The low-frequency electric fields were induced by natural body movements such as walking and turning in the fringe magnetic fields of a 3 T magnetic resonance whole-body scanner. The rate-of-change of magnetic field (dB/dt) was also recorded simultaneously by using three orthogonal search coils positioned near to the location of the electric field probe. Rates-of-change of magnetic field for natural body rotations were found to exceed 1 T s-1 near the end of the magnet bore. Typical electric fields measured on the upper abdomen, head and across the tongue for 1 T s-1 rate of change of magnetic field were 0.15 ± 0.02, 0.077 ± 0.003 and 0.015 ± 0.002 V m-1 respectively. Electric fields on the abdomen and chest were measured during an echo-planar sequence with the subject positioned within the scanner. With the scanner rate-of-change of gradient set to 10 T m-1 s-1 the measured rate-of-change of magnetic field was 2.2 ± 0.1 T s-1 and the peak electric field was 0.30 ± 0.01 V m-1 on the chest. The values of induced electric field can be related to dB/dt by a 'geometry factor' for a given subject and sensor position. Typical values of this factor for the abdomen or chest (for measured surface electric fields) lie in the range of 0.10-0.18 m. The measured values of electric field are consistent with currently available numerical modelling results for movement in static magnetic fields and exposure to switched magnetic field gradients.

  1. Enhanced Design Alternative IV

    Energy Technology Data Exchange (ETDEWEB)

    N. E. Kramer

    1999-05-18

    This report evaluates Enhanced Design Alternative (EDA) IV as part of the second phase of the License Application Design Selection (LADS) effort. The EDA IV concept was compared to the VA reference design using criteria from the ''Design Input Request for LADS Phase II EDA Evaluations'' (CRWMS M&O 1999b) and (CRWMS M&O 1999f). Briefly, the EDA IV concept arranges the waste packages close together in an emplacement configuration known as ''line load''. Continuous pre-closure ventilation keeps the waste packages from exceeding the 350 C cladding and 200 C (4.3.13) drift wall temperature limits. This EDA concept keeps relatively high, uniform emplacement drift temperatures (post-closure) to drive water away from the repository and thus dry out the pillars between emplacement drifts. The waste package is shielded to permit human access to emplacement drifts and includes an integral filler inside the package to reduce the amount of water that can contact the waste form. Closure of the repository is desired 50 years after first waste is emplaced. Both backfill and a drip shields will be emplaced at closure to improve post-closure performance.

  2. Corrections to the human development index and alternative indicators of sustainability%人类发展指数的修正与可持续发展的替代指标

    Institute of Scientific and Technical Information of China (English)

    王玉振(翻译); Alexander Gorobets

    2012-01-01

    本文确认了全球社会-生态问题(过度消费,道德危机)的主要根源并提出了新的可持续发展的愿景,重点要关注精神、道德、心理、生理和人的智力开发(而代替以消费为导主的理念)。讨论了人类发展指标不足所涉及到的因素(寿命预期、人均国内生产总值),提出了测量人类可持续发展的替代指标,即:健康人口的比例。生态效率也被建议作为转化可持续发展的必要条件,人的精神,根据生态中心立场(社会-生态幸福)。通过适当的教育和体制政策而实现环境和人类健康、公平和整个人类的发展。%In this paper, the major roots of the global socio-ecological problems are identified(over consumption, moral crisis)and a new vision of sustainable development, focused on holistic spiritual, moral, psychological, physical and intellectual human development(instead of dominating consumerism)is proposed. The shortcoming of the human development index related to its components (life expectancy at birth and gross domestic product per capita) are discussed and some alternative indicators for measuring sustainable human development are proposed, e.g. proportion of population in full health. While eco-efficiency is suggested as the necessary condition of transition to sustainability, human mentality, based on the eco-centric grounds (socio-ecological wellbeing), environmental and human health, justice and holistic human development is considered as its key condition that can be achieved through an appropriate educational and institutional policy.

  3. 76 FR 30735 - National Center for Complementary & Alternative Medicine; Notice of Closed Meeting

    Science.gov (United States)

    2011-05-26

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary & Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine ] Special... Center for Complementary and Alternative Medicine, National Institutes of Health, 6707...

  4. 75 FR 57970 - National Center for Complementary & Alternative Medicine; Notice of Closed Meeting

    Science.gov (United States)

    2010-09-23

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary & Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special... Officer, Office of Scientific Review, National Center for Complementary, & Alternative Medicine, NIH,...

  5. 76 FR 59707 - National Center for Complementary & Alternative Medicine; Notice of Closed Meetings

    Science.gov (United States)

    2011-09-27

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary & Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special... of Committee: National Center for Complementary and Alternative Medicine Special Emphasis...

  6. 76 FR 6806 - National Center for Complementary & Alternative Medicine; Notice of Closed Meeting

    Science.gov (United States)

    2011-02-08

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary & Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special..., National Center for Complementary and Alternative Medicine, National Institutes of Health, 6707...

  7. 76 FR 35227 - National Center for Complementary & Alternative Medicine; Notice of Closed Meeting

    Science.gov (United States)

    2011-06-16

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary & Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special... Review, National Center for Complementary, and Alternative Medicine, NIH, 6707 Democracy Blvd., Suite...

  8. 75 FR 35075 - National Center for Complementary & Alternative Medicine; Notice of Closed Meeting

    Science.gov (United States)

    2010-06-21

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary & Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special... Review, National Center for Complementary, & Alternative Medicine, NIH, 6707 Democracy Blvd., Suite...

  9. 78 FR 21381 - National Center for Complementary & Alternative Medicine; Notice of Closed Meeting

    Science.gov (United States)

    2013-04-10

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary & Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special... Domestic Assistance Program Nos. 93.213, Research and Training in Complementary and Alternative...

  10. 77 FR 28396 - National Center for Complementary & Alternative Medicine Notice of Closed Meeting

    Science.gov (United States)

    2012-05-14

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary & Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special... Training in Complementary and Alternative Medicine, National Institutes of Health, HHS) Dated: May 8,...

  11. 75 FR 26260 - National Center for Complementary & Alternative Medicine; Notice of Closed Meeting

    Science.gov (United States)

    2010-05-11

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary & Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special... Complementary and Alternative Medicine, National Institutes of Health, HHS) ] Dated: May 4, 2010....

  12. 78 FR 42528 - National Center for Complementary & Alternative Medicine; Notice of Closed Meeting

    Science.gov (United States)

    2013-07-16

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary & Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special... & Alternative Medicine, NIH, 6707 Democracy Blvd., Suite 401, Bethesda, MD 20892, 301-594-3456,...

  13. 76 FR 12744 - National Center for Complementary & Alternative Medicine; Notice of Closed Meeting

    Science.gov (United States)

    2011-03-08

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary & Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special... Assistance Program Nos. 93.213, Research and Training in Complementary and Alternative Medicine,...

  14. 77 FR 52751 - National Center for Complementary & Alternative Medicine; Notice of Closed Meeting

    Science.gov (United States)

    2012-08-30

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary & Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special... Complementary and Alternative Medicine, National Institutes of Health, HHS) Dated: August 23, 2012. Jennifer...

  15. 77 FR 31862 - National Center for Complementary & Alternative Medicine; Notice of Closed Meetings

    Science.gov (United States)

    2012-05-30

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary & Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special..., National Center for Complementary and Alternative Medicine, NIH, 6707 Democracy Blvd., Suite 401,...

  16. 75 FR 63498 - National Center for Complementary and Alternative Medicine; Notice of Closed Meeting

    Science.gov (United States)

    2010-10-15

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary and Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special... Review Officer, National Center for Complementary and Alternative Medicine, National Institutes of...

  17. 76 FR 16433 - National Center for Complementary & Alternative Medicine; Notice of Closed Meeting

    Science.gov (United States)

    2011-03-23

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary & Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special... Complementary and Alternative Medicine, National Institutes of Health, HHS) Dated: March 17, 2011. Jennifer...

  18. 75 FR 6039 - National Center for Complementary & Alternative Medicine; Notice of Closed Meeting

    Science.gov (United States)

    2010-02-05

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary & Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special.... 93.213, Research and Training in Complementary and Alternative Medicine, National Institutes...

  19. 76 FR 79201 - National Center for Complementary & Alternative Medicine; Notice of Closed Meetings

    Science.gov (United States)

    2011-12-21

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary & Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special... Officer, Office of Scientific Review, National Center for Complementary & Alternative Medicine, NIH,...

  20. 78 FR 10184 - National Center For Complementary & Alternative Medicine; Notice of Closed Meetings

    Science.gov (United States)

    2013-02-13

    ... HUMAN SERVICES National Institutes of Health National Center For Complementary & Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special Emphasis Panel; Clinical Studies of Complementary and Alternative Medicine. Date: March 14, 2013. Time:...

  1. 76 FR 29773 - National Center for Complementary & Alternative Medicine; Notice of Closed Meeting

    Science.gov (United States)

    2011-05-23

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary & Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special... Alternative Medicine, National Institutes of Health, HHS) Dated: May 17, 2011. Jennifer S. Spaeth,...

  2. 78 FR 47328 - National Center for Complementary & Alternative Medicine; Notice of Closed Meeting

    Science.gov (United States)

    2013-08-05

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary & Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special..., National Center For Complementary and Alternative Medicine, National Institutes of Health, 6707...

  3. 76 FR 10913 - National Center for Complementary & Alternative Medicine; Notice of Closed Meeting

    Science.gov (United States)

    2011-02-28

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary & Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special... of Scientific Review, National Center for Complementary & Alternative Medicine, NIH, 6707...

  4. 78 FR 66755 - National Center for Complementary & Alternative Medicine; Notice of Closed Meeting

    Science.gov (United States)

    2013-11-06

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary & Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special..., National Center for Complementary, & Alternative Medicine, NIH, 6707 Democracy Blvd., Suite 401,...

  5. 77 FR 1940 - National Center for Complementary and Alternative Medicine; Notice of Closed Meetings

    Science.gov (United States)

    2012-01-12

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary and Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special..., Ph.D., Scientific Review Officer, National Center for Complementary and Alternative...

  6. 75 FR 1796 - National Center for Complementary and Alternative Medicine; Notice of Closed Meetings

    Science.gov (United States)

    2010-01-13

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary and Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special... Complementary, and Alternative Medicine, NIH, 6707 Democracy Blvd., Suite 401, Bethesda, MD 20892,...

  7. 77 FR 10540 - National Center for Complementary and Alternative Medicine Notice of Closed Meeting

    Science.gov (United States)

    2012-02-22

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary and Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special... Shau, Ph.D., Scientific Review Officer, National Center for Complementary and Alternative...

  8. 76 FR 17140 - National Center for Complementary & Alternative Medicine; Notice of Closed Meeting

    Science.gov (United States)

    2011-03-28

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary & Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special... and Training in Complementary and Alternative Medicine, National Institutes of Health, HHS)...

  9. 76 FR 27651 - National Center for Complementary & Alternative Medicine; Notice of Closed Meeting

    Science.gov (United States)

    2011-05-12

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary & Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special... Review, National Center for Complementary & Alternative Medicine, NIH, 6707 Democracy Blvd., Suite...

  10. 75 FR 43994 - National Center for Complementary and Alternative Medicine; Notice of Meeting

    Science.gov (United States)

    2010-07-27

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary and Alternative Medicine... Committee: National Advisory Council for Complementary and Alternative Medicine. Date: September 3, 2010... remarks by the Director of the National Center for Complementary and Alternative Medicine, presentation...

  11. 78 FR 37836 - National Center for Complementary & Alternative Medicine; Notice of Closed Meeting

    Science.gov (United States)

    2013-06-24

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary & Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special... Complementary and Alternative Medicine, National Institutes of Health, HHS) Dated: June 18, 2013. Michelle...

  12. 78 FR 34664 - National Center for Complementary and Alternative Medicine; Notice of Closed Meeting

    Science.gov (United States)

    2013-06-10

    ... HUMAN SERVICES National Institutes of Health National Center for Complementary and Alternative Medicine... personal privacy. Name of Committee: National Center for Complementary and Alternative Medicine Special... Center for Complementary and Alternative Medicine, National Institutes of Health, 6707...