The C Terminus of Ku80 activates the DNA-dependent protein kinase catalytic subunit

Ku is a heterodimeric protein with double-stranded DNA end-binding activity that operates in the process of nonhomologous end joining. Ku is thought to target the DNA-dependent protein kinase (DNA-PK) complex to the DNA and, when DNA bound, can interact and activate the DNA-PK catalytic subunit (DNA-PKcs). We have carried out a 3′ deletion analysis of Ku80, the larger subunit of Ku, and shown that the C-terminal 178 amino acid residues are dispensable for DNA end-binding activity but are required for efficient interaction of Ku with DNA-PKcs. Cells expressing Ku80 proteins that lack the terminal 178 residues have low DNA-PK activity, are radiation sensitive, and can recombine the signal junctions but not the coding junctions during V(D)J recombination. These cells have therefore acquired the phenotype of mouse SCID cells despite expressing DNA-PKcs protein, suggesting that an interaction between DNA-PKcs and Ku, involving the C-terminal region of Ku80, is required for DNA double-strand break rejoining and coding but not signal joint formation. To gain further insight into important domains in Ku80, we report a point mutational change in Ku80 in the defective xrs-2 cell line. This residue is conserved among species and lies outside of the previously reported Ku70-Ku80 interaction domain. The mutational change nonetheless abrogates the Ku70-Ku80 interaction and DNA end-binding activity

MicroRNA dysregulation and esophageal cancer development depend on the extent of zinc dietary deficiency

open9siopenFong, Louise Y.; Taccioli, Cristian; Jing, Ruiyan; Smalley, Karl J.; Alder, Hansjuerg; Jiang, Yubao; Fadda, Paolo; Farber, John L.; Croce, Carlo M.Fong, Louise Y.; Taccioli, Cristian; Jing, Ruiyan; Smalley, Karl J.; Alder, Hansjuerg; Jiang, Yubao; Fadda, Paolo; Farber, John L.; Croce, Carlo M

Repression of Esophageal Neoplasia and Inflammatory Signaling by Anti-miR-31 Delivery In Vivo.

BACKGROUND: Overexpression of microRNA-31 (miR-31) is implicated in the pathogenesis of esophageal squamous cell carcinoma (ESCC), a deadly disease associated with dietary zinc deficiency. Using a rat model that recapitulates features of human ESCC, the mechanism whereby Zn regulates miR-31 expression to promote ESCC is examined. METHODS: To inhibit in vivo esophageal miR-31 overexpression in Zn-deficient rats (n = 12-20 per group), locked nucleic acid-modified anti-miR-31 oligonucleotides were administered over five weeks. miR-31 expression was determined by northern blotting, quantitative polymerase chain reaction, and in situ hybridization. Physiological miR-31 targets were identified by microarray analysis and verified by luciferase reporter assay. Cellular proliferation, apoptosis, and expression of inflammation genes were determined by immunoblotting, caspase assays, and immunohistochemistry. The miR-31 promoter in Zn-deficient esophagus was identified by ChIP-seq using an antibody for histone mark H3K4me3. Data were analyzed with t test and analysis of variance. All statistical tests were two-sided. RESULTS: In vivo, anti-miR-31 reduced miR-31 overexpression (P = .002) and suppressed the esophageal preneoplasia in Zn-deficient rats. At the same time, the miR-31 target Stk40 was derepressed, thereby inhibiting the STK40-NF-κΒ-controlled inflammatory pathway, with resultant decreased cellular proliferation and activated apoptosis (caspase 3/7 activities, fold change = 10.7, P = .005). This same connection between miR-31 overexpression and STK40/NF-κΒ expression was also documented in human ESCC cell lines. In Zn-deficient esophagus, the miR-31 promoter region and NF-κΒ binding site were activated. Zn replenishment restored the regulation of this genomic region and a normal esophageal phenotype. CONCLUSIONS: The data define the in vivo signaling pathway underlying interaction of Zn deficiency and miR-31 overexpression in esophageal neoplasia and provide a mechanistic rationale for miR-31 as a therapeutic target for ESCC

MDP, a database linking drug response data to genomic information, identifies dasatinib and statins as a combinatorial strategy to inhibit YAP/TAZ in cancer cells

Targeted anticancer therapies represent the most effective pharmacological strategies in terms of clinical responses. In this context, genetic alteration of several oncogenes represents an optimal predictor of response to targeted therapy. Integration of large-scale molecular and pharmacological data from cancer cell lines promises to be effective in the discovery of new genetic markers of drug sensitivity and of clinically relevant anticancer compounds. To define novel pharmacogenomic dependencies in cancer, we created the Mutations and Drugs Portal (MDP, http://mdp.unimore.it), a web accessible database that combines the cell-based NCI60 screening of more than 50,000 compounds with genomic data extracted from the Cancer Cell Line Encyclopedia and the NCI60 DTP projects. MDP can be queried for drugs active in cancer cell lines carrying mutations in specific cancer genes or for genetic markers associated to sensitivity or resistance to a given compound. As proof of performance, we interrogated MDP to identify both known and novel pharmacogenomics associations and unveiled an unpredicted combination of two FDA-approved compounds, namely statins and Dasatinib, as an effective strategy to potently inhibit YAP/TAZ in cancer cells

Definition of miRNAs expression profile in glioblastoma samples: the relevance of non-neoplastic brain reference.

Glioblastoma is the most aggressive brain tumor that may occur in adults. Regardless of the huge improvements in surgery and molecular therapy, the outcome of neoplasia remains poor. MicroRNAs are small molecules involved in several cellular processes, and their expression is altered in the vast majority of tumors. Several studies reported the expression of different miRNAs in glioblastoma, but one of the most critical point in understanding glioblastoma miRNAs profile is the comparison of these studies. In this paper, we focused our attention on the non-neoplastic references used for determining miRNAs expression. The aim of this study was to investigate if using three different non-neoplastic brain references (normal adjacent the tumor, commercial total RNA, and epileptic specimens) could provide discrepant results. The analysis of 19 miRNAs was performed using Real-Time PCR, starting from the set of samples described above and the expression values compared. Moreover, the three different normal RNAs were used to determine the miRNAs profile in 30 glioblastomas. The data showed that different non-neoplastic controls could lead to different results and emphasize the importance of comparing miRNAs profiles obtained using the same experimental condition

Clonado y expresión de ubicuitina en Neurospora crassa

La ubicuitina es una proteína de 76 residuos aminoacídicosaltamente conservada en la escala evolutiva, que se encuentra entodos los organismos eucarióticos, libre o conjugada covalentementea proteínas. La función fisiológica de ubicuitina está relacionada conel camino de degradación proteica dependiente de ATP por vía nolisosomal, con la regulación del ciclo celular y con la respuestaa diferentes condiciones de estrés. A pesar de que la ubicuitina está codificada por una familiamultigenética, ninguno de ellos lo hace para la proteína madura,sino para un precursor en el que la ubicuitina se encuentrafusionada por su extremo C-terminal a si misma (arreglo en tándemcabeza-cola en poliubicuitina), o bien, a una secuencia aminoacídicano relacionada con motivos de unión a ácidos nucleicos. En Neurospora crassa, la inhibición parcial de la sintesisproteica por tratamiento con cicloheximida, pero no con puromicina,conducen a un aumento de la población de transcriptos de poliubicuitina. Otras situaciones de estrés como ayuno, crecimiento en presenciade análogos de aminoácidos, estrés térmico, tratamientocon arsenito de sodio, que son inductores en otros organismos dela transcripción de este gen, no tienen efecto en Neurospora crassa. En nuestro laboratorio se aislaron y caracterizaron dos genesde ubicuitina de Neurospora crassa que corresponden, i) algen de poliubicuitina y ii) a un gen de fusión. Estos genes clonados reconocen especies de ARN de 1,3 y 0,7 Kb, respectivamente en Northern blots. Los análisis de Southern blots parecerían indicar la existenciade un locus único para el gen de poliubicuitina que contiene 4 repeticiones en tándem cabeza-cola con un aminoácido extrafusionado al C-terminal del último monómero. El gen de fusión de Neurospora crassa corresponde a unaunidad que codifica para ubicuitina ligada a una extensión de 78aminoácidos, y su expresión se encuentra particularmente aumentadaen conidias germinadas y en fase logarítmica. Las extensiones de fusión de ubicuitina pertenecen a unafamilia de proteínas ribosomales que además, están involucradasen el procesamiento del rARN de ambas subunidades. En condiciones de mayor replicación celular, los mensajeroscorrespondientes a estos genes de fusión se expresan activamente,como se encontró en Neurospora crassa. Se comprobó que el transcripto de 0,7 Kb no sufre variaciónen respuesta a los estrés estudiados, y que su expresión se reducepor ayuno. La comparación de la secuencia aminoacídica codificada porestos genes con la encontrada en otros organismos, contribuye aconfirmar la alta conservación evolutiva de esta proteína. Sinembargo, el comportamiento particular de la respuesta al estrésen este organismo, plantea algunos nuevos interrogantes respectoa la regulación de la expresión del gen de poliubicuitina.Fil: Taccioli, Guillermo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Comparative analysis of bats and rodents’ genomes suggests a relation between non-LTR retrotransposons, cancer incidence, and ageing

The presence in nature of species showing drastic differences in lifespan and cancer incidence has recently increased the interest of the scientific community. In particular, the adaptations and the genomic features underlying the evolution of cancer-resistant and long-lived organisms have recently focused on transposable elements (TEs). In this study, we compared the content and dynamics of TE activity in the genomes of four rodent and six bat species exhibiting different lifespans and cancer susceptibility. Mouse, rat, and guinea pig genomes (short-lived and cancer-prone organisms) were compared with that of naked mole rat (Heterocephalus glaber) which is a cancer-resistant organism and the rodent with the longest lifespan. The long-lived bats of the genera Myotis, Rhinolophus, Pteropus and Rousettus were instead compared with Molossus molossus, which is one of the organisms with the shortest lifespan among the order Chiroptera. Despite previous hypotheses stating a substantial tolerance of TEs in bats, we found that long-lived bats and the naked mole rat share a marked decrease of non-LTR retrotransposons (LINEs and SINEs) accumulation in recent evolutionary times

Control-Oriented Engine Thermal Model

Abstract The optimization of modern internal combustion engines and vehicles led several researchers to investigate the effects of the coolant system on overall efficiency losses. Electric water pumps have been proposed as a solution to decrease the high power consumption that typically affects mechanically-driven water pumps at high engine speed. Furthermore, decoupling the coolant flow from engine speed allows achieving a better warm-up behavior. The coolant system components, however, also impact vehicle efficiency: the radiator area affects the overall aerodynamic drag coefficient, especially for race vehicles and motorcycles. A thermal model can be used to assess the effects of the components characteristics (pump size, efficiency, speed; radiator surface, fan size, etc.) both on the coolant system capability to reach and maintain the target temperature, and the power it requires. The same model-based approach can be used for optimal thermal management, to control the coolant system actuators (electric pump and valves, fan). The paper shows how the thermal behavior of the engine can be represented by means of a concentrated parameters model, taking into account the main coolant system components features. The model has been calibrated on a set of data referring to a high-performance motorcycle engine, including both idling and high vehicle speed conditions. The good agreement of the model output with experimental data both in static and dynamic conditions confirms that the model is able to catch a large part of the phenomena influencing the coolant temperature

model based control of intake air temperature and humidity on the test bench

Abstract Engine test benches are crucial instruments to perform tests on internal combustion engines. Possible purposes of these tests are to detect the engine performance, check the reliability of the components or make a proper calibration of engine control systems managing the actuations. Since many factors affect tests results in terms of performance, emissions and components durability, an engine test bench is equipped with several conditioning systems (oil, water and air temperature, air humidity, etc.). One of the most important systems is the HVAC (Heating, Ventilating and Air Conditioning), that is essential to control the conditions of the intake air. Intake air temperature, pressure and humidity should be controllable test parameters, because they play a key role on the combustion development. In fact, they can heavily affect the performance detected, such as power and specific consumption, and, in some cases, they may promote knock occurrence. This work presents an HVAC model-based control methodology, where each component of the air treatment system (humidifier, pre-heating and post-heating resistors, chiller and fan) is managed coupling open-loop and closed-loop controls. Each branch of the control model is composed of two parts, the first one to evaluate the target for the given HVAC component, based on the system physical model, the second one is a PID controller based on the difference between the set-point and the feedback values. The control methodology has been validated on an engine test bench where the automation system has been developed on an open software Real-Time compatible platform, allowing the integration of the HVAC control with all other functionalities concerning the test management. The paper shows the plant layout, details the control strategy and finally analyzes experimental results obtained on the test bench, highlighting the benefits of the proposed HVAC management approach