Computer simulation of pulsed field gel runs allows the quantitation of radiation-induced double-strand breaks in yeast

A procedure for the quantification of double-strand breaks in yeast is presented that utilizes pulsed field gel electrophoresis (PFGE) and a comparison of the observed DNA mass distribution in the gel lanes with calculated distributions. Calculation of profiles is performed as follows. If double-strand breaks are produced by sparsely ionizing radiation, one can assume that they are distributed randomly in the genome, and the resulting DNA mass distribution in molecular length can be predicted by means of a random breakage model. The input data for the computation of molecular length profiles are the breakage frequency per unit length, , as adjustable parameter, and the molecular lengths of the intact chromosomes. The obtained DNA mass distributions in molecular length must then be transformed into distributions of DNA mass in migration distance. This requires a calibration of molecular length vs. migration distance that is specific for the gel lane in question. The computed profiles are then folded with a Lorentz distribution with adjusted spread parameter to account for and broadening. The DNA profiles are calculated for different breakage frequencies and for different values of , and the parameters resulting in the best fit of the calculated to the observed profile are determined

Application of Pulsed Field Gel Electrophoresis to Determine γ-ray-induced Double-strand Breaks in Yeast Chromosomal Molecules

The frequency of DNA double-strand breaks (dsb) was determined in yeast cells exposed to γ-rays under anoxic conditions. Genomic DNA of treated cells was separated by pulsed field gel electrophoresis, and two different approaches for the evaluation of the gels were employed: (1) The DNA mass distribution profile obtained by electrophoresis was compared to computed profiles, and the number of DSB per unit length was then derived in terms of a fitting procedure; (2) hybridization of selected chromosomes was performed, and a comparison of the hybridization signals in treated and untreated samples was then used to derive the frequency of dsb

Cellular Radiosensitivity: How much better do we understand it?

Purpose: Ionizing radiation exposure gives rise to a variety of lesions in DNA that result in genetic instability and potentially tumorigenesis or cell death. Radiation extends its effects on DNA by direct interaction or by radiolysis of H2O that generates free radicals or aqueous electrons capable of interacting with and causing indirect damage to DNA. While the various lesions arising in DNA after radiation exposure can contribute to the mutagenising effects of this agent, the potentially most damaging lesion is the DNA double strand break (DSB) that contributes to genome instability and/or cell death. Thus in many cases failure to recognise and/or repair this lesion determines the radiosensitivity status of the cell. DNA repair mechanisms including homologous recombination (HR) and non-homologous end-joining (NHEJ) have evolved to protect cells against DNA DSB. Mutations in proteins that constitute these repair pathways are characterised by radiosensitivity and genome instability. Defects in a number of these proteins also give rise to genetic disorders that feature not only genetic instability but also immunodeficiency, cancer predisposition, neurodegeneration and other pathologies. Conclusions: In the past fifty years our understanding of the cellular response to radiation damage has advanced enormously with insight being gained from a wide range of approaches extending from more basic early studies to the sophisticated approaches used today. In this review we discuss our current understanding of the impact of radiation on the cell and the organism gained from the array of past and present studies and attempt to provide an explanation for what it is that determines the response to radiation

Influence of Gender on Arrhythmia Characteristics and Outcome in the Multicenter UnSustained Tachycardia Trial

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73630/1/j.1540-8167.2004.04050.x.pd

O diálogo necessário entre extensão rural e Agroecologia

The article assumes that rural holds specifics with which it can work other possibilities besides oversizing technology as the only basis for the environmental, social, political and economic development. There is not only one rural society, but rural societies, with their productive specificities linked to the forces of nature and socio-cultural. The approachis focused on the characterization of a new rural as it presupposes the emergence of specific and endogenous forces leading to a global insertion adapted to the local soil and climate needs and socio-economics. The rural extension needs to shape up and get flexibility in the construction process of sustainable development. However, the understanding of sustainability expressed possibilities of retaining aspects geared only to environmental preservation. When considering a new rural with endogenous forces that play key roles in technological advancement and extension intervention, agroecological approach is fundamental. In several plans interact and shape a framework in which it is bio and social diversity, including technological, cultural, political, economic and social aspects. Sustainability, then, implies a breaking of dependence on social actors towards a truly effective participation able to deepen the understanding of reality to a new articulation between research and extension. From this design agroecologicalextension takes on a character of social inclusion and adapted technologies, which affects decisions on public policies in agricultural sciences beyond the production and reproduction needs of the dominant sectors.O artigo parte do pressuposto que o rural detém especificidades com as quais se podem trabalhar outras possibilidades além do superdimensionamento da tecnologia enquanto único embasamento para o desenvolvimento ambiental, social, político e econômico. Não há uma sociedade rural, mas sim sociedades rurais, com suas especificidades produtivas, ligadas às forças da natureza e sócio-culturais. O enfoque está voltado para a caracterização de um novo rural em que se pressupõe a emergência de forças específicas e endógenas que conduza a uma inserção global adaptada às necessidades edafoclimáticas e sócioeconômicas locais. A extensão em ciências agrárias necessita moldar-se e adquirir flexibilidade no processo de construção de projetos de desenvolvimento sustentável. No entanto, o entendimento atual da sustentabilidade expressa possibilidades de reter aspectos voltados apenas à preservação ambiental. Ao se pensar um novo rural com forças endógenas que desempenham papéis fundamentais no avanço tecnológico e na intervenção extensionista, é fundamental uma abordagem agroecológica. Nesta, vários planos interagem e conformam um quadro em que cabe a bio e a sociodiversidade, entre eles aspectos tecnológicos, culturais, políticos, econômicos e sociais. A sustentabilidade, então, implica num rompimento da dependência dos atores sociais na direção de uma participação realmente eficaz, capaz de aprofundar o entendimento da realidade a uma nova articulação entre investigação e extensão. A partir desse delineamento, a extensão agroecológica adquire um caráter de inclusão social e de tecnologias adaptadas, repercutindo nas decisões relativas às políticas públicas em ciências agrárias que ultrapassem as necessidades de produção e reprodução dos setores dominantes

Therapeutic vulnerability of multiple myeloma to MIR17PTi, a first-in-class inhibitor of pri-mir-17-92

The microRNA cluster miR-17-92 is oncogenic and represents a valuable therapeutic target in c-MYC (MYC)-driven malignancies. Here, we developed novel LNA gapmeR antisense oligonucleotides (ASOs) to induce RNase H-mediated degradation of MIR17HG primary transcripts and, consequently, to prevent biogenesis of miR-17-92 microRNAs (miR-17-92s). The leading LNA-ASO, named MIR17PTi, impaired proliferation of several cancer cell lines (n=48) established from both solid and hematologic tumors by on-target antisense activity, and more effectively as compared to miR-17-92s inhibitors. By focusing on multiple myeloma (MM), we found that MIR17PTi triggers apoptosis via impairment of homeostatic MYC/miR-17-92 feed-forward loops (FFLs) in patient-derived MM cells; and induced MYC-dependent synthetic lethality. We show that alteration of a BIM-centered FFL is instrumental for MIR17PTi to induce cytotoxicity in MM cells. MIR17PTi exerts strong in vivo anti-tumor activity in NOD-SCID mice bearing clinically relevant models of MM, with advantageous safety and pharmacokinetics profiles in non-human primates. Altogether, MIR17PTi is a novel pharmacological tool to be tested in early-phase clinical trials against MM and other MYC-driven malignancies

Drugging the lncRNA MALAT1 via LNA gapmeR ASO inhibits gene expression of proteasome subunits and triggers anti-multiple myeloma activity

The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in multiple myeloma (MM) are still to be investigated. Here, we studied the functional significance and the druggability of the oncogenic lncRNA MALAT1 in MM. Targeting MALAT1 by novel LNA-gapmeR antisense oligonucleotide antagonized MM cell proliferation and triggered apoptosis both in vitro and in vivo in a murine xenograft model of human MM. Of note, antagonism of MALAT1 downmodulated the two major transcriptional activators of proteasome subunit genes, namely NRF1 and NRF2, and resulted in reduced trypsin, chymotrypsin and caspase-like proteasome activities and in accumulation of polyubiquitinated proteins. NRF1 and NRF2 decrease upon MALAT1 targeting was due to transcriptional activation of their negative regulator KEAP1, and resulted in reduced expression of anti-oxidant genes and increased ROS levels. In turn, NRF1 promoted MALAT1 expression thus establishing a positive feedback loop. Our findings demonstrate a crucial role of MALAT1 in the regulation of the proteasome machinery, and provide proof-of-concept that its targeting is a novel powerful option for the treatment of MM

Endonuclease-independent LINE-1 retrotransposition at mammalian telomeres

Long interspersed element-1 (LINE-1 or L1) elements are abundant, non-long-terminal-repeat (non-LTR) retrotransposons that comprise 17% of human DNA(1). The average human genome contains similar to 80-100 retrotransposition- competent L1s (ref. 2), and they mobilize by a process that uses both the L1 endonuclease and reverse transcriptase, termed target-site primed reverse transcription(3-5). We have previously reported an efficient, endonuclease-independent L1 retrotransposition pathway (ENi) in certain Chinese hamster ovary (CHO) cell lines that are defective in the non-homologous end-joining (NHEJ) pathway of DNA double-strand-break repair(6). Here we have characterized ENi retrotransposition events generated in V3 CHO cells, which are deficient in DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity and have both dysfunctional telomeres and an NHEJ defect. Notably, similar to 30% of ENi retrotransposition events insert in an orientation-specific manner adjacent to a perfect telomere repeat (5'-TTAGGG-3'). Similar insertions were not detected among ENi retrotransposition events generated in controls or in XR-1 CHO cells deficient for XRCC4, an NHEJ factor that is required for DNA ligation but has no known function in telomere maintenance. Furthermore, transient expression of a dominant-negative allele of human TRF2 ( also called TERF2) in XRCC4-deficient XR-1 cells, which disrupts telomere capping, enables telomere-associated ENi retrotransposition events. These data indicate that L1s containing a disabled endonuclease can use dysfunctional telomeres as an integration substrate. The findings highlight similarities between the mechanism of ENi retrotransposition and the action of telomerase, because both processes can use a 3' OH for priming reverse transcription at either internal DNA lesions or chromosome ends(7,8). Thus, we propose that ENi retrotransposition is an ancestral mechanism of RNA-mediated DNA repair associated with non-LTR retrotransposons that may have been used before the acquisition of an endonuclease domain.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62964/1/nature05560.pd