26 results on '"Ruiz-Martinez M"'
Search Results
2. Development and Characterization of Magnetite/Poly(butylcyanoacrylate) Nanoparticles for Magnetic Targeted Delivery of Cancer Drugs
- Author
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López-Viota, Margarita, El-Hammadi, Mazen M., Cabeza, Laura, Prados, José, Melguizo, Consolación, Ruiz Martinez, M. Adolfina, Arias, José L., and Delgado, Ángel V.
- Published
- 2017
- Full Text
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3. La presión arterial domiciliaria se asocia con la hipertrofia concéntrica del ventrículo izquierdo
- Author
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Lozano Olóriz, E., Tomás Rubio, L., Díaz Dorronsoro, A., Ruiz Martínez, M., Buil Cosiales, P., Estremera Urabayen, V., Gasco García, P., Barba Cosials, J., Díez Martínez, J., and Serrano Martínez, M.
- Published
- 2005
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4. Influence of the concentration of a gelling agent and the type of surfactant on the rheological characteristics of oleogels
- Author
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Ruı́z Martı́nez, M <ce:sup loc='post">a</ce:sup>.A, Muñoz de Benavides, M, Morales Hernández, M <ce:sup loc='post">a</ce:sup>.E, and Gallardo Lara, V
- Published
- 2003
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5. Rheological behavior of gels and meloxicam release
- Author
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Ruiz Martinez, M. Adolfina, primary, López-Viota Gallardo, Julián, additional, de Benavides, María Muñoz, additional, de Dios García López-Duran, Juan, additional, and Gallardo Lara, Visitación, additional
- Published
- 2007
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6. Attention deficit and hiperactivity disorder in cocaine addiction
- Author
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Barrigon Estevez, M.L., primary, Fontalba-Navas, A., additional, Ruiz Martinez, M., additional, Joya Redondo, J., additional, Andres Ricoy, O., additional, Sanchez Viñas, A., additional, and Jurado de Flores Yepes, G., additional
- Published
- 2007
- Full Text
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7. DNA sequencing by capillary electrophoresis: use of a two-laser-two-window intensified diode array detection system
- Author
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Carson, S., primary, Cohen, A. S., additional, Belenkii, A., additional, Ruiz-Martinez, M. C., additional, Berka, J., additional, and Karger, B. L., additional
- Published
- 1993
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8. Corticoides tópicos e insuficiencia suprarrenal
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López Ávila, Ángel, Dolores Ruiz Martínez, M., and Ángel Cámara López, Miguel
- Published
- 2001
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9. Opposing regulation of the STING pathway in hepatic stellate cells by NBR1 and p62 determines the progression of hepatocellular carcinoma.
- Author
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Nishimura S, Linares JF, L'Hermitte A, Duran A, Cid-Diaz T, Martinez-Ordoñez A, Ruiz-Martinez M, Kudo Y, Marzio A, Heikenwalder M, Roberts LR, Diaz-Meco MT, and Moscat J
- Subjects
- Animals, Humans, Mice, Ubiquitination, Disease Progression, CD8-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes metabolism, Mice, Inbred C57BL, Mice, Knockout, Ubiquitin-Protein Ligases genetics, Ubiquitin-Protein Ligases metabolism, Cell Line, Tumor, Tumor Microenvironment, Interferons metabolism, Interferons genetics, Autophagy, Endosomes metabolism, Carcinoma, Hepatocellular genetics, Carcinoma, Hepatocellular pathology, Carcinoma, Hepatocellular metabolism, Liver Neoplasms pathology, Liver Neoplasms genetics, Liver Neoplasms metabolism, Hepatic Stellate Cells metabolism, Hepatic Stellate Cells pathology, Membrane Proteins metabolism, Membrane Proteins genetics, Signal Transduction, Sequestosome-1 Protein metabolism, Sequestosome-1 Protein genetics, Intracellular Signaling Peptides and Proteins genetics, Intracellular Signaling Peptides and Proteins metabolism
- Abstract
Hepatocellular carcinoma (HCC) emerges from chronic inflammation, to which activation of hepatic stellate cells (HSCs) contributes by shaping a pro-tumorigenic microenvironment. Key to this process is p62, whose inactivation leads to enhanced hepatocarcinogenesis. Here, we show that p62 activates the interferon (IFN) cascade by promoting STING ubiquitination by tripartite motif protein 32 (TRIM32) in HSCs. p62, binding neighbor of BRCA1 gene 1 (NBR1) and STING, triggers the IFN cascade by displacing NBR1, which normally prevents the interaction of TRIM32 with STING and its subsequent activation. Furthermore, NBR1 also antagonizes STING by promoting its trafficking to the endosome-lysosomal compartment for degradation independent of autophagy. Of functional relevance, NBR1 deletion completely reverts the tumor-promoting function of p62-deficient HSCs by rescuing the inhibited STING-IFN pathway, thus enhancing anti-tumor responses mediated by CD8
+ T cells. Therefore, NBR1 emerges as a synthetic vulnerability of p62 deficiency in HSCs by promoting the STING/IFN pathway, which boosts anti-tumor CD8+ T cell responses to restrain HCC progression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)- Published
- 2024
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10. The hepatokine FGL1 regulates hepcidin and iron metabolism during anemia in mice by antagonizing BMP signaling.
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Sardo U, Perrier P, Cormier K, Sotin M, Personnaz J, Medjbeur T, Desquesnes A, Cannizzo L, Ruiz-Martinez M, Thevenin J, Billoré B, Jung G, Abboud E, Peyssonnaux C, Nemeth E, Ginzburg YZ, Ganz T, and Kautz L
- Subjects
- Mice, Animals, Iron metabolism, Liver metabolism, Bone Morphogenetic Protein 6 genetics, Bone Morphogenetic Protein 6 metabolism, Homeostasis, Hepcidins genetics, Hepcidins metabolism, Anemia genetics, Anemia metabolism
- Abstract
Abstract: As a functional component of erythrocyte hemoglobin, iron is essential for oxygen delivery to all tissues in the body. The liver-derived peptide hepcidin is the master regulator of iron homeostasis. During anemia, the erythroid hormone erythroferrone regulates hepcidin synthesis to ensure the adequate supply of iron to the bone marrow for red blood cell production. However, mounting evidence suggested that another factor may exert a similar function. We identified the hepatokine fibrinogen-like 1 (FGL1) as a previously undescribed suppressor of hepcidin that is induced in the liver in response to hypoxia during the recovery from anemia, and in thalassemic mice. We demonstrated that FGL1 is a potent suppressor of hepcidin in vitro and in vivo. Deletion of Fgl1 in mice results in higher hepcidin levels at baseline and after bleeding. FGL1 exerts its activity by directly binding to bone morphogenetic protein 6 (BMP6), thereby inhibiting the canonical BMP-SMAD signaling cascade that controls hepcidin transcription., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)
- Published
- 2024
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11. Iron chelation improves ineffective erythropoiesis and iron overload in myelodysplastic syndrome mice.
- Author
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An W, Feola M, Levy M, Aluri S, Ruiz-Martinez M, Sridharan A, Fibach E, Zhu X, Verma A, and Ginzburg Y
- Subjects
- Humans, Mice, Animals, Erythropoiesis, Deferiprone, Iron, Mice, Transgenic, Ferritins, Iron Chelating Agents pharmacology, Anemia, Iron Overload complications
- Abstract
Myelodysplastic syndrome (MDS) is a heterogeneous group of bone marrow stem cell disorders characterized by ineffective hematopoiesis and cytopenias, most commonly anemia. Red cell transfusion therapy for anemia in MDS results in iron overload, correlating with reduced overall survival. Whether the treatment of iron overload benefits MDS patients remains controversial. We evaluate underlying iron-related pathophysiology and the effect of iron chelation using deferiprone on erythropoiesis in NUP98-HOXD13 transgenic mice, a highly penetrant well-established MDS mouse model. Our results characterize an iron overload phenotype with aberrant erythropoiesis in these mice which was reversed by deferiprone-treatment. Serum erythropoietin levels decreased while erythroblast erythropoietin receptor expression increased in deferiprone-treated MDS mice. We demonstrate, for the first time, normalized expression of the iron chaperones Pcbp1 and Ncoa4 and increased ferritin stores in late-stage erythroblasts from deferiprone-treated MDS mice, evidence of aberrant iron trafficking in MDS erythroblasts. Importantly, erythroblast ferritin is increased in response to deferiprone, correlating with decreased erythroblast ROS. Finally, we confirmed increased expression of genes involved in iron uptake, sensing, and trafficking in stem and progenitor cells from MDS patients. Taken together, our findings provide evidence that erythroblast-specific iron metabolism is a novel potential therapeutic target to reverse ineffective erythropoiesis in MDS., Competing Interests: WA, MF, ML, SA, MR, AS, EF, XZ, AV No competing interests declared, YG Reviewing editor, eLife, (© 2023, An et al.)
- Published
- 2023
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12. Enhanced SREBP2-driven cholesterol biosynthesis by PKCλ/ι deficiency in intestinal epithelial cells promotes aggressive serrated tumorigenesis.
- Author
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Muta Y, Linares JF, Martinez-Ordoñez A, Duran A, Cid-Diaz T, Kinoshita H, Zhang X, Han Q, Nakanishi Y, Nakanishi N, Cordes T, Arora GK, Ruiz-Martinez M, Reina-Campos M, Kasashima H, Yashiro M, Maeda K, Albaladejo-Gonzalez A, Torres-Moreno D, García-Solano J, Conesa-Zamora P, Inghirami G, Metallo CM, Osborne TF, Diaz-Meco MT, and Moscat J
- Subjects
- Animals, Humans, Mice, Cell Transformation, Neoplastic genetics, Cholesterol, Epithelial Cells metabolism, Protein Kinase C genetics, Protein Kinase C metabolism, Signal Transduction
- Abstract
The metabolic and signaling pathways regulating aggressive mesenchymal colorectal cancer (CRC) initiation and progression through the serrated route are largely unknown. Although relatively well characterized as BRAF mutant cancers, their poor response to current targeted therapy, difficult preneoplastic detection, and challenging endoscopic resection make the identification of their metabolic requirements a priority. Here, we demonstrate that the phosphorylation of SCAP by the atypical PKC (aPKC), PKCλ/ι promotes its degradation and inhibits the processing and activation of SREBP2, the master regulator of cholesterol biosynthesis. We show that the upregulation of SREBP2 and cholesterol by reduced aPKC levels is essential for controlling metaplasia and generating the most aggressive cell subpopulation in serrated tumors in mice and humans. Since these alterations are also detected prior to neoplastic transformation, together with the sensitivity of these tumors to cholesterol metabolism inhibitors, our data indicate that targeting cholesterol biosynthesis is a potential mechanism for serrated chemoprevention., (© 2023. The Author(s).)
- Published
- 2023
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13. The hepatokine FGL1 regulates hepcidin and iron metabolism during the recovery from hemorrhage-induced anemia in mice.
- Author
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Sardo U, Perrier P, Cormier K, Sotin M, Desquesnes A, Cannizzo L, Ruiz-Martinez M, Thevenin J, Billoré B, Jung G, Abboud E, Peyssonnaux C, Nemeth E, Ginzburg YZ, Ganz T, and Kautz L
- Abstract
As a functional component of erythrocyte hemoglobin, iron is essential for oxygen delivery to all tissues in the body. The liver-derived peptide hepcidin is the master regulator of iron homeostasis. During anemia, the erythroid hormone erythroferrone regulates hepcidin synthesis to ensure adequate supply of iron to the bone marrow for red blood cells production. However, mounting evidence suggested that another factor may exert a similar function. We identified the hepatokine FGL1 as a previously undescribed suppressor of hepcidin that is induced in the liver in response to hypoxia during the recovery from anemia and in thalassemic mice. We demonstrated that FGL1 is a potent suppressor of hepcidin in vitro and in vivo . Deletion of Fgl1 in mice results in a blunted repression of hepcidin after bleeding. FGL1 exerts its activity by direct binding to BMP6, thereby inhibiting the canonical BMP-SMAD signaling cascade that controls hepcidin transcription., Key Points: 1/ FGL1 regulates iron metabolism during the recovery from anemia. 2/ FGL1 is an antagonist of the BMP/SMAD signaling pathway.
- Published
- 2023
- Full Text
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14. Hyaluronan driven by epithelial aPKC deficiency remodels the microenvironment and creates a vulnerability in mesenchymal colorectal cancer.
- Author
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Martinez-Ordoñez A, Duran A, Ruiz-Martinez M, Cid-Diaz T, Zhang X, Han Q, Kinoshita H, Muta Y, Linares JF, Kasashima H, Nakanishi Y, Omar M, Nishimura S, Avila L, Yashiro M, Maeda K, Pannellini T, Pigazzi A, Inghirami G, Marchionni L, Sigal D, Diaz-Meco MT, and Moscat J
- Subjects
- Humans, CD8-Positive T-Lymphocytes pathology, Immunotherapy, Sarcoma pathology, Colorectal Neoplasms pathology, Hyaluronic Acid metabolism, Tumor Microenvironment physiology
- Abstract
Mesenchymal colorectal cancer (mCRC) is microsatellite stable (MSS), highly desmoplastic, with CD8
+ T cells excluded to the stromal periphery, resistant to immunotherapy, and driven by low levels of the atypical protein kinase Cs (aPKCs) in the intestinal epithelium. We show here that a salient feature of these tumors is the accumulation of hyaluronan (HA) which, along with reduced aPKC levels, predicts poor survival. HA promotes epithelial heterogeneity and the emergence of a tumor fetal metaplastic cell (TFMC) population endowed with invasive cancer features through a network of interactions with activated fibroblasts. TFMCs are sensitive to HA deposition, and their metaplastic markers have prognostic value. We demonstrate that in vivo HA degradation with a clinical dose of hyaluronidase impairs mCRC tumorigenesis and liver metastasis and enables immune checkpoint blockade therapy by promoting the recruitment of B and CD8+ T cells, including a proportion with resident memory features, and by blocking immunosuppression., Competing Interests: Declaration of interests A.M.-O., A.D., Y.N., J.M., and M.T.D.-M. are co-inventors of Weill Cornell Medicine patent applications covering methods for treating mesenchymal colorectal cancer. J.M., M.T.D.-M., and D.S. are co-founders of Zelambio., (Copyright © 2022 Elsevier Inc. All rights reserved.)- Published
- 2023
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15. The hepcidin regulator erythroferrone is a new member of the erythropoiesis-iron-bone circuitry.
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Castro-Mollo M, Gera S, Ruiz-Martinez M, Feola M, Gumerova A, Planoutene M, Clementelli C, Sangkhae V, Casu C, Kim SM, Ostland V, Han H, Nemeth E, Fleming R, Rivella S, Lizneva D, Yuen T, Zaidi M, and Ginzburg Y
- Subjects
- Animals, Bone Development genetics, Bone Morphogenetic Proteins metabolism, Cells, Cultured, Cytokines genetics, Disease Models, Animal, Erythroblasts, Erythropoiesis, Hepcidins, Male, Mice, Inbred C57BL, Muscle Proteins genetics, beta-Thalassemia genetics, beta-Thalassemia metabolism, Mice, Bone and Bones metabolism, Cytokines metabolism, Muscle Proteins metabolism, Osteoblasts metabolism
- Abstract
Background: Erythroblast erythroferrone (ERFE) secretion inhibits hepcidin expression by sequestering several bone morphogenetic protein (BMP) family members to increase iron availability for erythropoiesis., Methods: To address whether ERFE functions also in bone and whether the mechanism of ERFE action in bone involves BMPs, we utilize the Erfe
-/- mouse model as well as β-thalassemic ( Hbbth3/+ ) mice with systemic loss of ERFE expression. In additional, we employ comprehensive skeletal phenotyping analyses as well as functional assays in vitro to address mechanistically the function of ERFE in bone., Results: We report that ERFE expression in osteoblasts is higher compared with erythroblasts, is independent of erythropoietin, and functional in suppressing hepatocyte hepcidin expression. Erfe-/- mice display low-bone-mass arising from increased bone resorption despite a concomitant increase in bone formation. Consistently, Erfe-/- osteoblasts exhibit enhanced mineralization, Sost and Rankl expression, and BMP-mediated signaling ex vivo. The ERFE effect on osteoclasts is mediated through increased osteoblastic RANKL and sclerostin expression, increasing osteoclastogenesis in Erfe-/- mice. Importantly, Erfe loss in Hbbth3/+ mice, a disease model with increased ERFE expression, triggers profound osteoclastic bone resorption and bone loss., Conclusions: Together, ERFE exerts an osteoprotective effect by modulating BMP signaling in osteoblasts, decreasing RANKL production to limit osteoclastogenesis, and prevents excessive bone loss during expanded erythropoiesis in β-thalassemia., Funding: YZG acknowledges the support of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (R01 DK107670 to YZG and DK095112 to RF, SR, and YZG). MZ acknowledges the support of the National Institute on Aging (U19 AG60917) and NIDDK (R01 DK113627). TY acknowledges the support of the National Institute on Aging (R01 AG71870). SR acknowledges the support of NIDDK (R01 DK090554) and Commonwealth Universal Research Enhancement (CURE) Program Pennsylvania., Competing Interests: MC, SG, MR, MF, AG, MP, CC, VS, CC, SK, EN, RF, SR, DL, TY, YG No competing interests declared, VO, HH is affiliated with Intrinsic Lifesciences, LLC. The author has no other competing interests to declare. MZ Deputy editor, eLife, (© 2021, Castro-Mollo et al.)- Published
- 2021
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16. Identifying High-Risk Stage II Colon Cancer Patients: A Three-MicroRNA-Based Score as a Prognostic Biomarker.
- Author
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Caritg O, Navarro A, Moreno I, Martínez-Rodenas F, Cordeiro A, Muñoz C, Ruiz-Martinez M, Santasusagna S, Castellano JJ, and Monzó M
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- Adult, Aged, Area Under Curve, Colonic Neoplasms genetics, Colonic Neoplasms mortality, Disease-Free Survival, Female, Gene Expression Profiling, History, 16th Century, History, 17th Century, Humans, Kaplan-Meier Estimate, Male, MicroRNAs analysis, Middle Aged, Prognosis, ROC Curve, Risk Factors, Sensitivity and Specificity, Biomarkers, Tumor genetics, Colonic Neoplasms pathology, MicroRNAs biosynthesis
- Abstract
Background: The potential benefit of adjuvant chemotherapy in surgically resected patients with stage II colorectal cancer is controversial. The current guidelines, which are based solely on clinical factors, have limited usefulness, and a clear need exists for biomarkers to supplement the clinical information. MicroRNAs (miRNAs) have previously been shown to be useful cancer biomarkers. In the present study, we assessed the usefulness of a miRNA score to help identify the subset of high-risk patients likely to benefit from adjuvant chemotherapy., Patients and Methods: Six miRNAs previously identified as prognostic markers in Asian patients (miR-21-5p, miR-20a-5p, miR-103a-3p, miR-106b-5p, miR-143-5p, and miR-215) were studied in tumor samples from 71 white patients with stage II colon cancer., Results: Three miRNAs (miR-103a-3p, miR-143-5p, and miR-215) emerged as independent prognostic markers on multivariate analysis and were used to construct a miRNA-based score that classified patients into high- and low-risk groups. The patients in the high-risk group had significantly shorter disease-free survival compared with their low-risk counterparts (P = .003). The time-dependent receiver operating characteristic curve analysis showed that our 3-miRNA score improved the prediction of outcome when added to the clinical features (P = .023)., Conclusion: Our 3-miRNA score added valuable prognostic information to the clinical features in stage II colon cancer. Further research in this field could provide useful tools to determine whether adjuvant chemotherapy would benefit patients with stage II colon cancer after surgery., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
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17. YKT6 expression, exosome release, and survival in non-small cell lung cancer.
- Author
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Ruiz-Martinez M, Navarro A, Marrades RM, Viñolas N, Santasusagna S, Muñoz C, Ramírez J, Molins L, and Monzo M
- Subjects
- A549 Cells, Adult, Aged, Aged, 80 and over, Carcinoma, Non-Small-Cell Lung diagnosis, Carcinoma, Non-Small-Cell Lung pathology, Cell Proliferation genetics, Cell Survival genetics, Female, Gene Expression Regulation, Neoplastic, Humans, Lung Neoplasms diagnosis, Lung Neoplasms pathology, Male, MicroRNAs genetics, Middle Aged, Prognosis, Carcinoma, Non-Small-Cell Lung genetics, Carcinoma, Non-Small-Cell Lung metabolism, Exosomes metabolism, Lung Neoplasms genetics, Lung Neoplasms metabolism, R-SNARE Proteins genetics
- Abstract
Background: Cancer-derived exosomes are involved in metastasis. YKT6 is a SNARE protein that participates in the regulation of exosome production and release, but its role in non-small cell lung cancer (NSCLC) has not been examined., Materials and Methods: Ultracentrifugation-purified exosomes from the A549 cell line were studied by CRYO-TEM, nanoparticle tracking analysis and western blot (TSG101 marker). YKT6 was inhibited using a DsiRNA and selected pre-microRNAs. MicroRNAs targeting YKT6 were validated by Renilla/Luciferase assay and western blot. YKT6 expression and its prognostic impact were analyzed in 98 tissue specimens from resected NSCLC patients., Results: Membranous nanosized vesicles (mode size: 128nm) with TSG101 protein were purified from A549 cells. YKT6 inhibition reduced exosome release by 80.9%. We validated miR-134 and miR-135b as miRNAs targeting YKT6, and transfection with the pre-miRNAs also produced a significant reduction in exosome release. The analysis of YKT6 in tumor samples showed that patients with high levels had shorter disease-free and overall survival., Conclusions: YKT6 is a key molecule in the regulation of exosome release in lung cancer cells and is in turn precisely regulated by miR-134 and miR-135b. Moreover, YKT6 levels impact prognosis of resected NSCLC patients., Competing Interests: All authors declare no conflicts of interest.
- Published
- 2016
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18. Industrial yeast strain engineered to ferment ethanol from lignocellulosic biomass.
- Author
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Khramtsov N, McDade L, Amerik A, Yu E, Divatia K, Tikhonov A, Minto M, Kabongo-Mubalamate G, Markovic Z, Ruiz-Martinez M, and Henck S
- Subjects
- Base Sequence, DNA Primers, Polymerase Chain Reaction, Biomass, Ethanol metabolism, Fermentation, Lignin metabolism, Yeasts metabolism
- Abstract
In this study an industrial Saccharomyces cerevisiae yeast strain capable of fermenting ethanol from pretreated lignocellulosic material was engineered. Genes encoding cellulases (endoglucanase, exoglucanase and β-glucosidase) were integrated into the chromosomal ribosomal DNA and delta regions of a derivative of the K1-V1116 wine yeast strain. The engineered cellulolytic yeast produces ethanol in one step through simultaneous saccharification and fermentation of pretreated biomass without the addition of exogenously produced enzymes. When ethanol fermentation was performed with 10% dry weight of pretreated corn stover, the recombinant strain fermented 63% of the cellulose in 96 h and the ethanol titer reached 2.6% v/v. These results demonstrate that cellulolytic S. cerevisiae strains can be used as a platform for developing an economical advanced biofuel process., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
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19. A transmission imaging spectrograph and microfabricated channel system for DNA analysis.
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Simpson JW, Ruiz-Martinez MC, Mulhern GT, Berka J, Latimer DR, Ball JA, Rothberg JM, and Went GT
- Subjects
- Animals, Base Sequence, Humans, Image Processing, Computer-Assisted, Molecular Sequence Data, DNA analysis, DNA genetics, Electrophoresis methods, Sequence Analysis, DNA methods
- Abstract
In this paper we present the development of a DNA analysis system using a microfabricated channel device and a novel transmission imaging spectrograph which can be efficiently incorporated into a high throughput genomics facility for both sizing and sequencing of DNA fragments. The device contains 48 channels etched on a glass substrate. The channels are sealed with a flat glass plate which also provides a series of apertures for sample loading and contact with buffer reservoirs. Samples can be easily loaded in volumes up to 640 nL without band broadening because of an efficient electrokinetic stacking at the electrophoresis channel entrance. The system uses a dual laser excitation source and a highly sensitive charge-coupled device (CCD) detector allowing for simultaneous detection of many fluorescent dyes. The sieving matrices for the separation of single-stranded DNA fragments are polymerized in situ in denaturing buffer systems. Examples of separation of single-stranded DNA fragments up to 500 bases in length are shown, including accurate sizing of GeneCalling fragments, and sequencing samples prepared with a reduced amount of dye terminators. An increase in sample throughput has been achieved by color multiplexing.
- Published
- 2000
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20. A sample purification method for rugged and high-performance DNA sequencing by capillary electrophoresis using replaceable polymer solutions. A. Development of the cleanup protocol.
- Author
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Ruiz-Martinez MC, Salas-Solano O, Carrilho E, Kotler L, and Karger BL
- Subjects
- Base Sequence, Chlorides, Dideoxynucleosides, Molecular Sequence Data, Polymers, Solutions, Templates, Genetic, DNA isolation & purification, Electrophoresis, Capillary methods, Sequence Analysis, DNA methods
- Abstract
A method for the cleanup of Sanger DNA sequencing reaction products for capillary electrophoresis analysis with replaceable polymer solutions has been developed. A poly(ether sulfone) ultrafiltration membrane pretreated with linear polyacrylamide was first used to remove template DNA from the sequencing samples. Then, gel filtration in a spin column format (two columns per sample) was employed to decrease the concentration of salts below 10 microM in the sample solution. The method was very reproducible and increased the injected amount of the sequencing fragments 10-50-fold compared to traditional cleanup protocols. Using M13mp18 as template, the resulting cleaned-up single DNA sequencing fragments could routinely be separated to more than 1000 bases with a base-calling accuracy of at least 99% for 800 bases. The method is simple and universal and can be easily automated. In the following paper, a systematic study to determine quantitatively the effects of the sample solution components such as high-mobility ions (e.g., chloride and dideoxynucleotides) and template DNA on the injected amount and separation efficiency of the sequencing fragments is presented.
- Published
- 1998
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21. A sample purification method for rugged and high-performance DNA sequencing by capillary electrophoresis using replaceable polymer solutions. B. Quantitative determination of the role of sample matrix components on sequencing analysis.
- Author
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Salas-Solano O, Ruiz-Martinez MC, Carrilho E, Kotler L, and Karger BL
- Subjects
- Chlorides chemistry, Dideoxynucleosides chemistry, Electromagnetic Fields, Osmolar Concentration, Polymers, Solutions, Templates, Genetic, DNA isolation & purification, Electrophoresis, Capillary methods, Sequence Analysis, DNA methods
- Abstract
In the previous paper, a sample cleanup procedure for DNA sequencing reaction products was developed, in which template DNA was removed by ultrafiltration and the total concentration of salts (chloride and di- and deoxynucleotides) was decreased below 10 microM using gel filtration. In this paper, a quantitative study of the effects of these sample solution components on the injected amount and separation efficiency of the sequencing fragments in capillary electrophoresis is presented. The presence of chloride and deoxynucleotides in a total concentration above 10 microM in the sample solution significantly decreased the amount of DNA sequencing fragments injected into the capillary column. However, the separation efficiency was not affected upon increasing the amount of salt. On the other hand, in the presence of only 0.1 microgram of template in the sample (one-third of the lowest quantity recommended in cycle sequencing) and at very low chloride concentration (approximately 5 microM), the separation efficiency decreased by 70%, and the injected amount of DNA sequencing fragments was 40% lower compared to the sample cleaned by the new purification method. The deleterious effect of template DNA on the separation of sequencing fragments was suppressed in the presence of salt in a concentration above 100 microM in the sample solution. Separately, it was found that both the electric field strength and duration of injection affected the resolution of DNA sequencing fragments when the cleaned up sample solution was used. Separation efficiencies of 15 x 10(6) theoretical plates/m were achieved when the sample was loaded at low electric field, e.g., 25 V/cm for 80 s or less. The results demonstrate that the sample solution components (chloride, deoxynucleotides, template DNA) and injection conditions must be controlled to achieve high performance and rugged DNA sequencing analysis.
- Published
- 1998
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22. Characterization of high molecular mass linear polyacrylamide powder prepared by emulsion polymerization as a replaceable polymer matrix for DNA sequencing by capillary electrophoresis.
- Author
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Goetzinger W, Kotler L, Carrilho E, Ruiz-Martinez MC, Salas-Solano O, and Karger BL
- Subjects
- Emulsions, Molecular Weight, Reproducibility of Results, Acrylic Resins, Electrophoresis, Capillary methods, Polymers, Sequence Analysis, DNA methods
- Abstract
In a previous paper, a 2% w/w replaceable high molecular mass linear polyacrylamide solution (high molecular mass LPA) was used to achieve long read-lengths for DNA sequencing by capillary electrophoresis (E. Carrilho et al., Anal. Chem. 1996, 68, 3305-3313). In that work, the polymer was prepared by polymerization in water at 6% w/w, followed by dilution to 2% w/w. In this study, an improved method for preparation of high molecular mass LPA was developed, based on inverse emulsion polymerization. With this polymerization procedure, the LPA results in a molecular mass of approximately 9 MDa, with characteristics of a fine powder of high purity and practically unlimited shelf life. Using size exclusion chromatography (SEC) and viscosity measurements to characterize the polymer, good batch-to-batch reproducibility was found. It was observed that the viscous polymer solutions made from these high molecular mass polymers require careful preparation and handling because the method of dissolution could affect the molecular mass distribution and the resultant separation of DNA components. Solutions containing 2% w/w of LPA made by emulsion polymerization were simple to prepare, resulting in excellent performance as a replaceable matrix for DNA sequencing by capillary electrophoresis. The viscosity of the polymer decreased exponentially when pressure was applied, allowing easy replacement from a capillary using a syringe. With a properly prepared matrix, a read-length of more than 1000 bases in 80 min with an accuracy better than 97%, and better than 99% for the first 800 bases, could be achieved.
- Published
- 1998
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23. Rapid DNA sequencing of more than 1000 bases per run by capillary electrophoresis using replaceable linear polyacrylamide solutions.
- Author
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Carrilho E, Ruiz-Martinez MC, Berka J, Smirnov I, Goetzinger W, Miller AW, Brady D, and Karger BL
- Subjects
- Acrylic Resins, Base Composition, Base Sequence, DNA, Molecular Sequence Data, Molecular Weight, Electrophoresis, Capillary methods, Sequence Analysis, DNA methods
- Abstract
The read length for DNA sequencing using capillary electrophoresis and replaceable linear polyacrylamide (LPA) solutions has been extended to more than 1000 bases with a run time of 80 min. This result was successfully achieved through the combined use of cycle sequencing with dye-labeled primers, improved matrix and separation conditions, and enhanced base-calling software. The influences of LPA molecular weight and concentration on separation were investigated. Additionally, the separation buffer, column temperature, and electric field were adjusted to increase the number of resolvable DNA fragments per run while maintaining an enhanced separation speed. Using low concentrations [2% (w/v)] of high molecular weight LPA polymers (> 5.5 x 10(6) Da), elevated column temperature (50 degrees C) and moderately high field (150 V/cm), rapid sequencing analysis for more than 1000 bases on a model ssM13mp18 template was obtained with 96.8% accuracy.
- Published
- 1996
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24. DNA sequencing by capillary electrophoresis using short oligonucleotide primer libraries.
- Author
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Ruiz-Martinez MC, Carrilho E, Berka J, Kieleczawa J, Miller AW, Foret F, Carson S, and Karger BL
- Subjects
- Base Sequence, DNA Primers genetics, Molecular Sequence Data, Templates, Genetic, Electrophoresis, Capillary methods, Sequence Analysis, DNA methods
- Abstract
Two strategies for DNA sequencing by primer walking using short oligonucleotide primer libraries have been successfully employed along with capillary electrophoresis using replaceable polymer solutions of linear polyacrylamide and fluorescence detection. A 3.5-kb stretch of the single-stranded M13mp18 template was sequenced with T7 PRISM dye-terminator/Sequenase chemistry. An in-house base-calling program offered read lengths of roughly 450 bases with an average of 97.8% accuracy.
- Published
- 1996
- Full Text
- View/download PDF
25. DNA sequencing by capillary electrophoresis with replaceable linear polyacrylamide and laser-induced fluorescence detection.
- Author
-
Ruiz-Martinez MC, Berka J, Belenkii A, Foret F, Miller AW, and Karger BL
- Subjects
- Acrylic Resins chemistry, Base Sequence, DNA, Lasers, Molecular Sequence Data, Software, Electrophoresis, Polyacrylamide Gel methods, Sequence Analysis, DNA, Spectrometry, Fluorescence methods
- Abstract
Replaceable linear polyacrylamide (LPA) has been utilized as a sieving matrix for DNA sequencing by capillary electrophoresis (CE). Difficulties associated with cross-linked polyacrylamide gel stability have been overcome for the routine application of CE to DNA sequencing. A simple laser-induced fluorescence (LIF) detection system based on a single laser and two photomultipliers (PMT) has been adopted for this work. Sequencing information for four bases has been obtained from two fluorescent dyes and two peak height ratios, detected in two optical channels. FAM- and JOE-labeled M13 (-21) primers have been chosen because both dyes are efficiently excited with a low-power argon ion laser, can be optically separated, and exhibit minimal dye-based shifts in DNA fragment mobilities. Addition of denaturants to the electrophoresis running buffer (1 x TBE, 3.5 M urea, 30% formamide) and column operation at 32 degrees C permitted the resolution of difficult compressed sites in the sequence of phage M13mp18. Careful examination of the polymerization reaction of LPA has led to methodology that has proven to be reproducible for obtaining DNA sequencing information of M13mp18 phage for 350 nucleotides in close to 30 min.
- Published
- 1993
- Full Text
- View/download PDF
26. Zinc and liver cirrhosis.
- Author
-
Gil Extremera B, Maldonado Martin A, Ruiz Martinez M, Cantero Hinojosa J, Diez Ruiz A, and Rodrigo Moreno MD
- Subjects
- Adult, Aged, Ascitic Fluid chemistry, Female, Humans, Liver Neoplasms metabolism, Male, Middle Aged, Sulfates administration & dosage, Zinc administration & dosage, Zinc blood, Zinc urine, Zinc Sulfate, Liver Cirrhosis metabolism, Zinc analysis
- Abstract
We have measured zinc levels in serum and urine of healthy controls, patients with liver cirrhosis and patients with cirrhosis and hepatic cancer. In patients with ascitic fluid, we also measured zinc, total protein, albumin and alpha 2-globulin. Basal measurements were performed in blood drawn at 8.00, before the intravenous administration of 8 mg zinc in the form of zinc sulphate. Serum levels were measured at various intervals to a total time of 24 h after overload, and total urine was collected for zinc determinations 24 h before and 24 h after overload. Under basal conditions, cirrhotic patients had lower serum levels and higher rates of urinary excretion of zinc than controls. After overload, blood levels of zinc fell more rapidly in cirrhotic patients than in controls, the former group showing no concomitant rise in urinary zinc excretion, thus suggesting an organic deficit in this trace element. In ascitic fluid, the concentration of zinc was positively correlated with protein content.
- Published
- 1990
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