Untersuchungen zur Substratspezifität von Squalen-Hopen Zyklasen (SHCs)

Cyclic terpenoids form a large group of natural products with various biological functions. About 60,000 different cyclic terpenoids have been identified by now, containing scaffolds of ten to more than 30 carbon atoms. Among this huge amount of different cyclic compounds, there are many well-known flavors and fragrances, such as menthol or limonene, compounds which are widely used for pharmaceutical purposes like the antitumor compound Taxol and the anti-malaria agent artemisinin, or common membrane constituents and hormones such as the sterols. All of these natural products are derived from cyclization reactions of few linear precursor molecules catalyzed by terpenoid cyclases. The main focus of the present work rests on one very interesting family of the terpenoid cyclases, the squalene-hopene cyclases (SHCs). Among the over 300 annotated SHCs, most extensive studies had been carried out characterizing the SHC from the thermophilic bacterium Alicyclobacillus acidocaldarius (AacSHC), solving the crystal structure and the complex cyclization mechanism of the C30 precursor squalene into the pentacyclic products hopene and hopanol. This reaction constitutes one of the most complex reaction mechanisms found in nature, including the stereospecific formation of nine stereocenters and 13 covalent bonds.13 Besides AacSHC, several SHCs were partially characterized in previous works. Our interest was triggered by the ethanol and sugar tolerant strain Zymomonas mobilis which is known as one of the most potent hopanoid producers. Zymomonas mobilis contains two genes encoding for SHCs: ZmoSHC1 and the formerly partially characterized ZmoSHC2. It could be shown that the SHCs are also capable of cyclizing other linear terpenoids. For example, it was found that truncated squalene analogs were accepted as substrates by AacSHC and also the alcohol homofarnesol could be converted into the corresponding cyclic ether ambroxan.16–19 These results indicate that SHCs represent a promising family for catalysis of very different and complex cyclization reactions. Thus, we decided to investigate the SHCs’ potentials regarding their substrate specificities. In order to characterize the squalene-hopene cyclases ZmoSHC1 and ZmoSHC2 and compare them with AacSHC especially regarding their biocatalytic activities towards unnatural substrates, the SHCs were cloned and heterologously expressed in Escherichia coli. Functional expression was confirmed by conversion of the natural substrate squalene. For the direct comparison, a protocol for partial purification of the membrane-anchored SHCs was elaborated and optimized. For this partial purification as well as for the conversion of the hydrophobic substrates in aqueous milieu a suitable detergent had to be selected. ZmoSHC1 was characterized in more detail, retrieving information about pH- and temperature-dependence of the activity and the biocatalytic stability over a long period of time as well as inhibitory effects. All of the three enzymes were tested with unnatural substrates of C10-C18 carbon chain lengths. A special focus was laid on substrates containing functional groups such as hydroxyl , carboxy- or keto-groups expected to participate in the cyclization reaction, as shown for the hydroxyl-group of homofarnesol. Several of the substrates were accepted and cyclic products were generated. Interestingly, the functional groups were integrated in the final ring closure and to products with new properties were obtained. Homofarnesol conversion yielding the cyclic ether ambroxan, which is known as a expensive and rare flavor compound, was observed as reported in the literature. Also the corresponding carboxylic acid, homofarnesoic acid, could be converted into the cyclic lactone sclareolide. The C15 tertiary alcohol nerolidol was accepted as substrate and the bicyclic ether caparrapioxide was formed. Lastly, two ketones were accepted as substrates leading to cyclic enol ether products. Within the present work, all of these new products were characterized after preparative biotransformation and product isolation. Not only the facts that these substrates are much shorter than the natural substrate squalene and possess different functional groups which take part in the cyclization reaction and that useful products containing new properties are formed, but also the different activities of the SHCs towards these substrates are remarkable. Thus, it could be shown that ZmoSHC1 exhibits special biocatalytic properties, as the substrate activity pattern was unexpected. While squalene was converted very poorly, good activity was found towards the reaction of homofarnesol to ambroxan. All of the other substrates were converted in low but significant rates into the corresponding cyclic products. A completely different substrate activity pattern was observed using AacSHC as biocatalyst. Besides very good squalene conversion, much lower activities towards all of the other substrates were found. Using ZmoSHC2, only very low conversion rates were found for squalene and farnesylacetone and no conversion of any of the other substrates. Based on these observations, it can be concluded that ZmoSHC1 represents a versatile biocatalyst for complex cyclization reactions, as it shows unexpected substrate activity towards other substrates than squalene. In the present work, these and further detailed results are described. Besides the examination of the SHCs’ activities towards different substrates there were also several mutants created in order to find explanations for the differences between the SHCs regarding their substrate activities. This characterization of the triterpenoid cyclase ZmoSHC1 and discussion of their special properties leads to new conclusions about the potential of SHCs to serve as potent biocatalysts for new reactions.Die zyklischen Terpenoide stellen eine große Gruppe von Naturstoffen mit verschiedensten biologischen Funktionen dar. Bis heute konnten etwa 60.000 verschiedene zyklische Terpenoide, die aus Gerüsten von zehn bis über 30 Kohlenstoffatomen aufgebaut sind, identifiziert werden. Unter dieser großen Anzahl von zyklischen Stoffen finden sich viele bekannte Duft- und Aromastoffe, wie zum Beispiel Menthol oder Limonen, Verbindungen, die als pharmazeutisch wirksame Inhaltsstoffe in Medikamenten Anwendung finden, wie etwa der gegen Tumor wirksame Stoff Taxol oder das gegen Malaria angewendete Artemisinin oder auch die als Membranbestandteile und Hormone bekannten Steroide. All diese interessanten Naturstoffe werden durch Zyklisierung von wenigen linearen Vorläufermolekülen gebildet. Diese Zyklisierungsreaktionen werden von Terpenoid Zyklasen katalysiert. Der Schwerpunkt der vorliegenden Arbeit liegt auf einer Unterfamilie dieser Enzymgruppe der Terpenoid Zyklasen, den Squalen-Hopen Zyklasen (SHCs). Unter den über 300 annotierten SHCs wurde die SHC vom thermophilen Bakterium Alicyclobacillus acidocaldarius (AacSHC) am besten untersucht. Neben der Kristallstruktur wurde auch der komplexe Mechanismus aufgeklärt, nach dem das lineare C30 Substrat Squalen zu den pentazyklischen Produkten Hopen und Hopanol zyklisiert wird. Der Mechanismus dieser Reaktion, bei der neun Stereozentren und 13 kovalente C-C Bindungen spezifisch entstehen, gilt als einer der komplexesten, die man in der Chemie der Naturstoffe bislang entdecken konnte. Neben AacSHC wurden in vorangehenden Arbeiten auch einige andere SHCs teilweise charakterisiert. Von besonderem Interesse ist hierbei der gegen hohe Alkohol- und Zuckerkonzentrationen tolerante Stamm Zymomonas mobilis, der als einer der besten Hopanoid-produzierenden Bakterienstämme bekannt ist und zwei Gene enthält, die für SHCs codieren: ZmoSHC1 und die in vorherigen Arbeiten partiell charakterisierte ZmoSHC2. Es war gezeigt worden, dass SHCs neben dem natürlichen Substrat Squalen auch einige andere lineare Terpenoide als Substrate akzeptieren und diese zyklisieren. Zum Beispiel konnten verkürzte Squalen-Analoga von AacSHC zyklisiert werden und auch der C16-Alkohol Homofarnesol wurde in den entsprechenden zyklischen Ether Ambroxan umgesetzt. Diese Ergebnisse ließen darauf schließen, dass die SHCs eine vielversprechende Enzymfamilie zur Katalyse von sehr verschiedenen, komplexen Zyklisierungsreaktionen darstellen könnten und deswegen entschieden wir uns dazu, die Substratbreite der SHCs näher zu untersuchen. Um die Squalen-Hopen Zyklasen ZmoSHC1 und ZmoSHC2 zu charakterisieren und ihre biokatalytischen Aktivitäten mit der von AacSHC vergleichen zu können, wurden die für diese Enzyme codierenden Gene kloniert und heterolog in Escherichia coli exprimiert. Die Expression funktioneller Enzyme wurde durch Umsetzung des natürlichen Substrates Squalen bestätigt. Um die Enzyme direkt miteinander vergleichen zu können, wurde ein Protokoll für die partielle Aufreinigung der membrangebundenen SHCs ausgearbeitet und optimiert. Für diese Aufreinigung sowie für die Umsetzung der hydrophoben Substrate in wässrigem Milieu musste ein geeignetes Detergenz verwendet werden. ZmoSHC1 wurde des Weiteren näher charakterisiert, wobei die pH- und Temperaturabhängigkeit der katalytischen Aktivität, die biokatalytische Stabilität des Enzyms über eine längere Zeitdauer sowie Inhibierungseffekte untersucht wurden. Die drei Enzyme wurden auf Aktivität gegenüber unnatürlichen Substraten mit C-Kettenlängen von C10-C18 getestet. Ein besonderer Fokus wurde hierbei auf Substrate gelegt, die funktionelle Gruppen enthalten, wie zum Beispiel Hydroxyl-, Carboxy- oder Ketogruppen, die, wie für die Hydroxylgruppe von Homofarnesol gezeigt, an der Zyklisierungsreaktion teilnehmen könnten. Interessanterweise wurden diese funktionellen Gruppen in den finalen Ring der polyzyklischen Produkte integriert, wodurch Produkte mit neuen, attraktiven Eigenschaften entstanden. Homofarnesol konnte in den zyklischen Ether und bekannten Duftstoff Ambroxan umgesetzt werden. Die entsprechende Carbonsäure Homofarnesolsäure wurde ebenfalls als Substrat akzeptiert und es wurde das zyklische Lakton Sclareolid erhalten. Der tertiäre C15 Alkohol Nerolidol wurde zum bizyklischen Caparrapioxid umgesetzt. Des Weiteren wurden auch zwei Ketone als Substrate akzeptiert und in zyklische Enolether umgesetzt. Im Rahmen der vorliegenden Arbeit wurden die neuen Produkte nach präparativer Biotransformation und Aufreinigung charakterisiert. Nicht nur die Tatsache, dass diese Substrate sehr viel kürzere Kohlenstoff-Ketten als das „natürliche“ Substrat Squalen besitzen und über verschiedene funktionelle Gruppen verfügen, die an den Zyklisierungsreaktionen teilhaben und zu interessanten Produkten umgesetzt werden, sondern auch die unterschiedlichen Aktivitäten der SHCs gegenüber dieser Substrate sind bemerkenswert. Es konnte gezeigt werden, dass ZmoSHC1 über besondere Eigenschaften verfügt, da unerwartete Umsetzungsraten bei der Katalyse mit diesem Enzym bestimmt wurden. Während die Zyklisierung von Squalen von ZmoSHC1 nur sehr gering katalysiert wurde, wurde eine gute Aktivität gegenüber der Reaktion von Homofarnesol zu Ambroxan ermittelt. Alle anderen beschriebenen Substrate wurden in geringen, aber signifikanten Raten umgesetzt. Ein vollkommen anderes Aktivitäts-Muster wurde bei Umsetzungen mit AacSHC erhalten. Hier wurde neben sehr guter Umsetzung von Squalen eine viel geringere Aktivität gegenüber allen anderen Substraten bestimmt. Vom Enzym ZmoSHC2 wurden nur Squalen und Farnesylaceton mit sehr geringer Aktivität als Substrate akzeptiert, alle anderen Substrate wurden nicht umgesetzt. Anhand dieser Ergebnisse kann gefolgert werden, dass SHCs als vielseitige Biokatalysatoren für komplexe Zyklisierungsreaktionen verwendet werden können, da diese Enzyme eine unerwartete Substrataktivität mit anderen Substraten als Squalen zeigen. In der vorliegenden Arbeit werden diese und weitere Ergebnisse im Detail beschrieben. Neben der erwähnten Untersuchung der Aktivität der verschiedenen SHCs gegenüber unterschiedlichen Substraten wurden auch Mutanten hergestellt und untersucht, die zu einer Erklärung der Aktivitätsunterschiede zwischen den verschiedenen Squalen-Hopen Zyklasen verhelfen sollten. Diese Charakterisierung der Triterpen Zyklasen und die Diskussion ihrer besonderen Eigenschaften führen zu neuen Schlussfolgerungen über das Potenzial von SHCs, als fähige Biokatalysatoren für noch nie gezeigte Reaktionen eingesetzt werden zu können

Calcium Electroporation Reduces Viability and Proliferation Capacity of Four Uveal Melanoma Cell Lines in 2D and 3D Cultures

Electrochemotherapy (ECT) is the combination of transient pore formation following electric pulse application with the administration of cytotoxic drugs, which enhances the cytotoxic effect of the applied agent due to membrane changes and permeabilization. Although EP represents an established therapeutic option for solid malignancies, recent advances shift to the investigation of non cytotoxic agents, such as calcium, which can also induce cell death. The present study aims to evaluate the cytotoxic effect, the morphological changes in tumor spheroids, the effect on the cell viability, and the cell-specific growth rate following calcium electroporation (CaEP) in uveal melanoma (UM) 2D monolayer cell cultures as well as in 3D tumor spheroid models. The experiments were conducted in four cell lines, UM92.1, Mel270, and two primary UM cell lines, UPMD2 and UPMM3 (UPM). The 2D and 3D UM cell cultures were electroporated with eight rectangular pulses (100 µs pulse duration, 5 Hz repetition frequency) of a 1000 V/cm pulse strength alone or in combination with 0.11 mg/mL, 0.28 mg/mL, 0.55 mg/mL or 1.11 mg/mL calcium chloride or 1.0 µg/mL or 2.5 µg/mL bleomycin. The application of calcium chloride alone induced an ATP reduction only in the UM92.1 2D cell cultures. Calcium alone had no significant effect on ATP levels in all four UM spheroids. A significant decrease in the intracellular adenosine triphosphate (ATP) level was documented in all four 2D and 3D cell cultures for both CaEP as well as ECT with bleomycin. The results suggest a dose-dependent ATP depletion with a wide range of sensitivity among the tested UM cell lines, control groups, and the applied settings in both 2D monolayer cell cultures and 3D tumor spheroid models. The colony formation capacity of the cell lines after two weeks reduced significantly after CaEP only with 0.5 mg/mL and 1.1 mg/mL, whereas the same effect could be achieved with both applied bleomycin concentrations, 1.0 µg/mL and 2.5 µg/mL, for the ECT group. The specific growth rate on day 7 following CaEP was significantly reduced in UM92.1 cell lines with 0.5 and 1.1 mg/mL calcium chloride, while Mel270 showed a similar effect only after administration of 1.1 mg/mL. UM92.1 and Mel270 spheroids exhibited lower adhesion and density after CaEP on day three in comparison to UPM spheroids showing detachment after day 7 following treatment. CaEP and bleomycin electroporation significantly reduce cell viability at similar applied voltage settings. CaEP may be a feasible and inexpensive therapeutic option for the local tumor control with fewer side effects, in comparison to other chemotherapeutic agents, for the treatment of uveal melanoma. The limited effect on normal cells and the surrounding tissue has already been investigated, but further research is necessary to clarify the effect on the surrounding tissue and to facilitate its application in a clinical setting for the eye

Chick Chorioallantoic Membrane as a Patient-Derived Xenograft Model for Uveal Melanoma : Imaging Modalities for Growth and Vascular Evaluation

Background: Patient-derived tumor xenografts (PDXs) have emerged as valuable preclinical in vivo models in oncology as they largely retain the polygenomic architecture of the human tumors from which they originate. Although animal models are accompanied by cost and time constraints and a low engraftment rate, PDXs have primarily been established in immunodeficient rodent models for the in vivo assessment of tumor characteristics and of novel therapeutic cancer targets. The chick chorioallantoic membrane (CAM) assay represents an attractive alternative in vivo model that has long been used in the research of tumor biology and angiogenesis, and can overcome some of these limitations. Methods: In this study, we reviewed different technical approaches for the establishment and monitoring of a CAM-based uveal melanoma PDX model. Forty-six fresh tumor grafts were acquired after enucleation from six uveal melanoma patients and were implanted onto the CAM on ED7 with Matrigel and a ring (group 1), with Matrigel (group 2), or natively without Matrigel or a ring (group 3). Real-time imaging techniques, such as various ultrasound modalities, optical coherence tomography, infrared imaging, and imaging analyses with Image J for tumor growth and extension, as well as color doppler, optical coherence angiography, and fluorescein angiography for angiogenesis, were performed on ED18 as alternative monitoring instruments. The tumor samples were excised on ED18 for histological assessment. Results: There were no significant differences between the three tested experimental groups regarding the length and width of the grafts during the development period. A statistically significant increase in volume (p = 0.0007) and weight (p = 0.0216) between ED7 and ED18 was only documented for tumor specimens of group 2. A significant correlation of the results for the cross-sectional area, largest basal diameter, and volume was documented between the different imaging and measurement techniques and the excised grafts. The formation of a vascular star around the tumor and of a vascular ring on the base of the tumor was observed for the majority of the viable developing grafts as a sign of successful engraftment. Conclusion: The establishment of a CAM-PDX uveal melanoma model could elucidate the biological growth patterns and the efficacy of new therapeutic options in vivo. The methodological novelty of this study, investigating different implanting techniques and exploiting advances in real-time imaging with multiple modalities, allows precise, quantitative assessment in the field of tumor experimentation, underlying the feasibility of CAM as an in vivo PDX model

Optimisation of the Chicken Chorioallantoic Membrane Assay in Uveal Melanoma Research

The treatment of uveal melanoma and its metastases has not evolved sufficiently over the last decades in comparison to other tumour entities, posing a great challenge in the field of ocular oncology. Despite improvements in the conventional treatment regime and new discoveries about the genetic and molecular background of the primary tumour, effective treatment strategies to either prevent tumours or treat patients with advanced or metastatic disease are still lacking. New therapeutic options are necessary in order to achieve satisfactory local tumour control, reduce the risk of metastasis development, and preserve the eyeball and possibly the visual function of the eye. The development of in vivo model systems remains crucial for the identification and investigation of potential novel treatment modalities. The aim of this study was the optimisation of the chorioallantoic membrane (CAM) model for uveal melanoma research. We analysed the established CAM assay and its modification after the implantation of three-dimensional spheroids. The chorioallantoic membrane of a chick embryo was used to implant uveal melanoma-cell-line-derived spheroids in order to study their growth rate, angiogenic potential, and metastatic capability. Using the UM 92.1, UPMD2, UPMM3, and Mel270 cell lines, we were able to improve the viability of the embryos from 20% to >80% and to achieve up to a fourfold volume increase of the transplanted spheroid masses. The results point to the value of an optimised chicken embryo assay as an in vivo model for testing novel therapies for uveal melanoma by simplifying the research conditions and by contributing to a considerable reduction in animal experiments

Creation of multiple nanodots by single ions

In the challenging search for tools that are able to modify surfaces on the nanometer scale, heavy ions with energies of several 10 MeV are becoming more and more attractive. In contrast to slow ions where nuclear stopping is important and the energy is dissipated into a large volume in the crystal, in the high energy regime the stopping is due to electronic excitations only. Because of the extremely local (< 1 nm) energy deposition with densities of up to 10E19 W/cm^2, nanoscaled hillocks can be created under normal incidence. Usually, each nanodot is due to the impact of a single ion and the dots are randomly distributed. We demonstrate that multiple periodically spaced dots separated by a few 10 nanometers can be created by a single ion if the sample is irradiated under grazing angles of incidence. By varying this angle the number of dots can be controlled.Comment: 12 pages, 6 figure

E 4 Transfer (E=P, As) to Ni Complexes

The use of [Cp′′2Zr(η1:1-E4)] (E=P (1 a), As (1 b), Cp′′=1,3-di-tert-butyl-cyclopentadienyl) as phosphorus or arsenic source, respectively, gives access to novel stable polypnictogen transition metal complexes at ambient temperatures. The reaction of 1 a/1 b with [CpRNiBr]2 (CpR=CpBn (1,2,3,4,5-pentabenzyl-cyclopentadienyl), Cp′′′ (1,2,4-tri-tert-butyl-cyclopentadienyl)) was studied, to yield novel complexes depending on steric effects and stoichiometric ratios. Besides the transfer of the complete En unit, a degradation as well as aggregation can be observed. Thus, the prismane derivatives [(Cp′′′Ni)2(μ,η3:3-E4)] (2 a (E=P); 2 b (E=As)) or the arsenic containing cubane [(Cp′′′Ni)3(μ3-As)(As4)] (5) are formed. Furthermore, the bromine bridged cubanes of the type [(CpRNi)3{Ni(μ-Br)}(μ3-E)4]2 (CpR=Cp′′′: 6 a (E=P), 6 b (E=As), CpR=CpBn: 8 a (E=P), 8 b (E=As)) can be isolated. Here, a stepwise transfer of En units is possible, with a cyclo-E42− ligand being introduced and unprecedented triple-decker compounds of the type [{(CpRNi)3Ni(μ3-E)4}2(μ,η4:4-E′4)] (CpR=CpBn, Cp′′′; E/E′=P, As) are obtained

Does timing of neoadjuvant chemotherapy influence the prognosis in patients with early triple negative breast cancer?

Purpose For patients with triple negative breast cancer (TNBC), the optimal time to initiate neoadjuvant chemotherapy (TTNC) is unknown. This study evaluates the association between TTNC and survival in patients with early TNBC. Methods A retrospective study using data from of a cohort of TNBC patients diagnosed between January 1, 2010 to December 31, 2018 registered in the Tumor Centre Regensburg was performed. Data included demographics, pathology, treatment, recurrence, and survival. Interval to treatment was defined as days from pathology diagnosis of TNBC to first dose of neoadjuvant chemotherapy (NACT). The Kaplan–Meier and Cox regression methods were used to evaluate the impact of TTNC on overall survival (OS) and 5 year OS. Results A total of 270 patients were included. Median follow up was 3.5 years. The 5-year OS estimates according to TTNC were 77.4%, 66.9%, 82.3%, 80.6%, 88.3%, 58.3%, 71.1% and 66.7% in patients who received NACT within 0–14, 15–21, 22–28, 29–35, 36–42, 43–49, 50–56 and > 56 days after diagnosis. Patients who received systemic therapy early had the highest estimated mean OS of 8.4 years, while patients who received systemic therapy after more than 56 days survived an estimated 3.3 years. Conclusion The optimal time interval between diagnosis and NACT remains to be determined. However, starting NACT more than 42 days after diagnosis of TNBC seems to reduce survival. Therefore, it is strongly recommended to carry out the treatment in a certified breast center with appropriate structures, in order to enable an adequate and timely care

Human Biogeography and Demographic Trend in Northpatagonic Monte: An Archaeological Approach from “El Corcovo” (SE of Mendoza)

El presente trabajo aporta al conocimiento de la biogeografía humana en ambientes áridos y las fases de poblamiento en relación a las trayectorias demográficas humanas. En ese marco, se presenta nueva información arqueológica del área El Corcovo, emplazada en el Monte surmendocino/nordpatagónico. Se detallan los trabajos de campo que contemplaron transectas, excavación arqueológica de un sitio y el relevamiento de arte rupestre de otros tres. Los resultados señalan el inicio de las ocupaciones en torno a 1900 años 14C AP y un cambio en el patrón de movilidad en torno a 600 años 14C AP. Ambas situaciones son consistentes con las estimaciones cronológicas relativas existentes para el arte rupestre del área. En base a un modelo demográfico regional del Monte, los resultados encuentran dos pulsos de incremento en la señal humana que se corresponden cronológicamente con la fecha inicial de ocupación estimada para el área y con la fecha obtenida para el cambio propuesto. Al asociarse estos pulsos con disminuciones significativas en el sector fitogeográfico de Patagonia se postulan explicaciones alternativas sobre la dinámica poblacional.The present paper attempt to improve the knowledge of human biogeography in arid environments and, understand the phases of settlement in relation to human demographic trajectories. In this framework, new archaeological information of the El Corcovo area is presented, located in Monte Surmendocino / Nordpatagónico. The field-works that contemplated transects, archaeological excavation of a site and the surveying of rock art of three others are detailed. The results indicate the beginning of occupations around 1900 years 14C AP and a change in the pattern of mobility around 600 years 14C AP. Both situations are consistent with the existing relative chronological estimates for the rock art of the area. Based on a regional demographic model for Monte area, the results show two pulses of increase in the human signal that correspond chronologically with the initial date of occupation estimated for the area and with the date obtained for the proposed change. By associating these pulses with significant falls in the phyto-geographic sector of Patagonia, alternative explanations on population dynamics are postulated.Fil: Gil, Adolfo Fabian. Universidad Tecnologica Nacional. Facultad Reg.san Rafael. Instituto de Evolucion, Ecologia Historica y Ambiente. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mendoza. Instituto de Evolucion, Ecologia Historica y Ambiente.; ArgentinaFil: Sugrañes, Nuria Andrea. Universidad Tecnologica Nacional. Facultad Reg.san Rafael. Instituto de Evolucion, Ecologia Historica y Ambiente. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mendoza. Instituto de Evolucion, Ecologia Historica y Ambiente.; ArgentinaFil: Acevedo, Agustín. Asociación de Investigaciones Antropológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Neme, Gustavo Adolfo. Universidad Tecnologica Nacional. Facultad Reg.san Rafael. Instituto de Evolucion, Ecologia Historica y Ambiente. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mendoza. Instituto de Evolucion, Ecologia Historica y Ambiente.; ArgentinaFil: Salgán, María Laura. Universidad Tecnologica Nacional. Facultad Reg.san Rafael. Instituto de Evolucion, Ecologia Historica y Ambiente. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mendoza. Instituto de Evolucion, Ecologia Historica y Ambiente.; Argentina. Comisión Nacional de Energía Atómica; ArgentinaFil: Giardina, Miguel Angel. Universidad Tecnologica Nacional. Facultad Reg.san Rafael. Instituto de Evolucion, Ecologia Historica y Ambiente. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mendoza. Instituto de Evolucion, Ecologia Historica y Ambiente.; ArgentinaFil: Tucker, Hugo. Centro Regional de Investigación y Desarrollo Cultural. Museo Regional Malargue; ArgentinaFil: Fiore, Danae. Asociación de Investigaciones Antropológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Seitz, Viviana Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Argentino de Investigaciones de las Zonas Áridas. Provincia de Mendoza. Instituto Argentino de Investigaciones de las Zonas Áridas. Universidad Nacional de Cuyo. Instituto Argentino de Investigaciones de las Zonas Áridas; ArgentinaFil: Pompei, María de la Paz. Universidad Tecnologica Nacional. Facultad Reg.san Rafael. Instituto de Evolucion, Ecologia Historica y Ambiente. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mendoza. Instituto de Evolucion, Ecologia Historica y Ambiente.; ArgentinaFil: Ayala, Miriam I.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales. Museo de Historia Natural de San Rafael - Ianigla | Provincia de Mendoza. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales. Museo de Historia Natural de San Rafael - Ianigla | Universidad Nacional de Cuyo. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales. Museo de Historia Natural de San Rafael - Ianigla; Argentin

Discovery of microRNAs and other small RNAs in solid tumors

MicroRNAs (miRNAs) are ∼22-nt long, non-coding RNAs that regulate gene silencing. It is known that many human miRNAs are deregulated in numerous types of tumors. Here we report the sequencing of small RNAs (17–25 nt) from 23 breast, bladder, colon and lung tumor samples using high throughput sequencing. We identified 49 novel miRNA and miR-sized small RNAs. We further validated the expression of 10 novel small RNAs in 31 different types of blood, normal and tumor tissue samples using two independent platforms, namely microarray and RT–PCR. Some of the novel sequences show a large difference in expression between tumor and tumor-adjacent tissues, between different tumor stages, or between different tumor types. We also report the identification of novel small RNA classes in human: highly expressed small RNA derived from Y-RNA and endogenous siRNA. Finally, we identified dozens of new miRNA sequence variants that demonstrate the existence of miRNA-related SNP or post-transcriptional modifications. Our work extends the current knowledge of the tumor small RNA transcriptome and provides novel candidates for molecular biomarkers and drug targets