Voice, body and the transmission of the real in documentary theatre

This article analyses how listening is used to develop performances in Alecky Blythe’s verbatim theatre. Listening includes Blythe’s use of recorded oral interviews for devising performances, and also the actors’ creation of performance by precisely imitating an interviewee’s voice. The article focuses on listening, speaking and embodiment in London Road, Blythe’s recent musical play at London’s National Theatre, which adopted and modified theatre strategies used in her other plays, especially The Girlfriend Experience and Do We look Like Refugees. The article draws on interviews with performers and with Blythe herself, in its critical analysis of how voice legitimates claims to authenticity in performance. The work on Blythe is contextualised by brief comparative analyses. One is Clio Barnard’s film The Arbor, a ‘quasi-documentary’ on the playwright, Andrea Dunbar which makes use of an oral script to which the actors lip-sync. The other comparator is the Wooster Group’s Poor Theater, which attempts to recreate Grotowski's Akropolis via vocal impersonation. The article argues that voice in London Road both claims and defers authenticity and authority, inasmuch as voice signifies presence and embodied identity but the reworking of speech into song signals the absence of the real. The translation of voice into written surtitles works similarly in Do We Look Like Refugees. Blythe’s theatre, Barnard’s film and The Wooster Group’s performances are a useful framework for addressing questions of voice and identity, and authenticity and replication in documentary theatre. The article concludes by placing Blythe’s oral texts amid current debates around theatre’s textual practices.

Mitochondrial genotype segregation and effects during mammalian development: Applications to biotechnology

Mitochondria are endosymbiotic organelles responsible for energy production in practically every eukaryotic cell. Their uniparental fashion of inheritance, maternally inherited in mammals, and the homogeneity of mitochondrial DNA (mtDNA) within individuals and matrilineages, are biological phenomena that remain unexplained. This paper reviews some of the recent findings on mitochondrial influences on the manner in which embryos develop and how their genotypes are inherited in mammals, with particular emphasis on the genetic bottleneck effect. Animal models carrying a mix of mtDNAs (heteroplasmic) have been produced by karyoplast and cytoplast transplantation to analyze the segregation patterns at different stages during embryogenesis, in fetuses and offspring. Comparisons performed between murine and bovine reveal interesting changes in segregation and replication of transplanted mtDNAs. We have recently obtained Bos indicus and Bos taurus fetuses and calves from embryos reconstructed using enucleated polymorphic oocytes of Bos taurus origin. These and other findings on mitochondrial biology will have important implications in determining the cytoplasmic genotype of clones and in the preservation of endangered breeds and species. (C) 1999 by Elsevier B.V.

In vitro and in vivo studies of the Interferon-alpha action on distinct Orthobunyavirus

Oropouche, Caraparu, Guama, Guaroa and Tacaiuma viruses (Orthobunyavirus genus) cause human febrile illnesses and/or encephalitis. To achieve a therapeutical agent to prevent and/or treat these diseases we evaluated the antiviral action of Interferon-alpha (IFN-alpha) on these orthobunyaviruses. In vitro results showed that all the studied orthobunyaviruses are susceptible to antiviral action of IFN-alpha, but this susceptibility is limited and dependent on both concentration of drug and treatment period. In vivo results demonstrated that IFN-alpha present antiviral action on Oropouche and Guaroa viruses when used as a prophylactic treatment. Moreover, a treatment initiated 3 It after infection prevented the death of Guaroa virus infected-mice. Additionally, mortality of mice was related to the migration and replication of viruses in their brains. Our results suggest that IFN-alpha could be potentially useful in the prevention of diseases caused by Oropouche virus and in the prevention and/or treatment of diseases caused by Guaroa virus. (C) 2007 Elsevier B.V. All rights reserved.

Unravelling the Neospora caninum secretome through the secreted fraction (ESA) and quantification of the discharged tachyzoite using high-resolution mass spectrometry-based proteomics

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Protease Inhibitor and Metabolic Alteration

Protease inhibitors (PIs), part of HAART (Highly Active Antiretroviral Therap) are selective, competitive inhibitors of protease, a crucial enzyme to viral maturation, infection and replication. A lipodystrophic syndrome has been reported in individuals treated with HAART, and associated to hyperglycemia, hypercholesterolemia, hypertrigliceridemia, hyperlipidemia, hypertension and hypreinsulinemia. The HAART-associated metabolic abnormalities were first associated with protease inhibitors, Ritonavir mostly, but the mechamisns that underlie these metabolic alterations are to date, not completely understood. Since Pis are candidate to be the drug of choice for other diseases treatment, such as the Hepatitis C, malaria and some types of cancer, it seems to be important to clarify the metabolic alterations associated to PIs. Wistar rats were treated twice a week with 30mg/kg Ritonavir for 4 and 8 weeks. Total cholesterol, HDL, LDL, VLDL, triglycerides and glycemic levels were measured by the end of each period of time selected. To avoid confunding effects of food intake, the animals were fasted 16 hours before. Our results showed rapid increase in serum triglycerides, total cholesterol, LDL-C and glycemic levels. No significant differences were observed for HDL-C or VLDL serum levels. Our study addresses the importance to observe the possible family history of dyslipidemia or diabetes, and control any other cardiovascular and diabetes risk factors when using protease inhibitors

Chromatin Remodeling by BRG1 and SNF2H : Biochemistry and Function

Chromatin is a highly dynamic, regulatory component in the process of transcription, repair, recombination and replication. The BRG1 and SNF2H proteins are ATP-dependent chromatin remodeling proteins that modulate chromatin structure to regulate DNA accessibility for DNA-binding proteins involved in these processes. The BRG1 protein is a central ATPase of the SWI/SNF complexes involved in chromatin remodeling associated with regulation of transcription. SWI/SNF complexes are biochemically hetero-geneous but little is known about the unique functional characteristics of the various forms. We have shown that SWI/SNF activity in SW13 cells affects actin filament organization dependent on the RhoA signaling pathway. We have further shown that the biochemical composition of SWI/SNF complexes qualitatively affects the remodeling activity and that the composition of biochemically purified SWI/SNF complexes does not reflect the patterns of chromatin binding of individual subunits. Chromatin binding assays (ChIP) reveal variations among subunits believed to be constitutive, suggesting that the plasticity in SWI/SNF complex composition is greater than suspected. We have also discovered an interaction between BRG1 and the splicing factor Prp8, linking SWI/SNF activity to mRNA processing. We propose a model whereby parts of the biochemical heterogeneity is a result of function and that the local chromatin environment to which the complex is recruited affect SWI/SNF composition. We have also isolated the novel B-WICH complex that contains WSTF, SNF2H, the splicing factor SAP155, the RNA helicase II/Guα, the transcription factor Myb-binding protein 1a, the transcription factor/DNA repair protein CSB and the RNA processing factor DEK. The formation of this complex is dependent on active transcription and links chromatin remodeling by SNF2H to RNA processing. By linking chromatin remodeling complexes with RNA processing proteins our work has begun to build a bridge between chromatin and RNA, suggesting that factors in chromatin associated assemblies translocate onto the growing nascent RNA.

An integrated system for building and running reproducible research

Our generation of computational scientists is living in an exciting time: not only do we get to pioneer important algorithms and computations, we also get to set standards on how computational research should be conducted and published. From Euclid’s reasoning and Galileo’s experiments, it took hundreds of years for the theoretical and experimental branches of science to develop standards for publication and peer review. Computational science, rightly regarded as the third branch, can walk the same road much faster. The success and credibility of science are anchored in the willingness of scientists to expose their ideas and results to independent testing and replication by other scientists. This requires the complete and open exchange of data, procedures and materials. The idea of a “replication by other scientists” in reference to computations is more commonly known as “reproducible research”. In this context the journal “EAI Endorsed Transactions on Performance & Modeling, Simulation, Experimentation and Complex Systems” had the exciting and original idea to make the scientist able to submit simultaneously the article and the computation materials (software, data, etc..) which has been used to produce the contents of the article. The goal of this procedure is to allow the scientific community to verify the content of the paper, reproducing it in the platform independently from the OS chosen, confirm or invalidate it and especially allow its reuse to reproduce new results. This procedure is therefore not helpful if there is no minimum methodological support. In fact, the raw data sets and the software are difficult to exploit without the logic that guided their use or their production. This led us to think that in addition to the data sets and the software, an additional element must be provided: the workflow that relies all of them.

Hepatitis C virus drug resistance and immune-driven adaptations: relevance to new antiviral therapy

The efficacy of specifically targeted anti-viral therapy for hepatitis C virus (HCV) (STAT-C), including HCV protease and polymerase inhibitors, is limited by the presence of drug-specific viral resistance mutations within the targeted proteins. Genetic diversity within these viral proteins also evolves under selective pressures provided by host human leukocyte antigen (HLA)-restricted immune responses, which may therefore influence STAT-C treatment response. Here, the prevalence of drug resistance mutations relevant to 27 developmental STAT-C drugs, and the potential for drug and immune selective pressures to intersect at sites along the HCV genome, is explored. HCV nonstructural (NS) 3 protease or NS5B polymerase sequences and HLA assignment were obtained from study populations from Australia, Switzerland, and the United Kingdom. Four hundred five treatment-naïve individuals with chronic HCV infection were considered (259 genotype 1, 146 genotype 3), of which 38.5% were coinfected with human immunodeficiency virus (HIV). We identified preexisting STAT-C drug resistance mutations in sequences from this large cohort. The frequency of the variations varied according to individual STAT-C drug and HCV genotype/subtype. Of individuals infected with subtype 1a, 21.5% exhibited genetic variation at a known drug resistance site. Furthermore, we identified areas in HCV protease and polymerase that are under both potential HLA-driven pressure and therapy selection and identified six HLA-associated polymorphisms (P and host immune responses are likely to provide powerful selection forces that shape HCV genetic diversity and replication dynamics. Consideration of HCV viral adaptation in terms of drug resistance as well as host "immune resistance" in the STAT-C treatment era could provide important information toward an optimized and individualized therapy for chronic hepatitis C.

Neuropathology and pathogenesis of HIV encephalopathies

The nervous system is frequently affected in patients with the acquired immune deficiency syndrome (AIDS). In addition to opportunistic CNS infections and cerebral lymphomas, approx. 20% of the patients develop HIV-associated encephalopathies. Two major histopathological manifestations are observed. HIV leukoencephalopathy (progressive diffuse leukoencephalopathy) is characterized by a diffuse loss of myelin in the deep white matter of the cerebral and cerebellar hemispheres, with scattered multinucleated giant cells and microglia but scarce or absent inflammatory reaction. HIV encephalitis (multinucleated giant cell encephalitis) is associated with accumulations of multinucleated giant cells, inflammatory reaction and often focal necroses. In some patients, both patterns may overlap. In order to identify the HIV genome in the CNS, brain tissue from 27 patients was analyzed for the presence of HIV gag sequences using the polymerase chain reaction (PCR) and primers encoding a 109 base pair segment of the gag gene. Amplification of HIV gag succeeded in all 5 patients with clinical and histopathological evidence for HIV encephalopathy but was negative in the 20 AIDS patients with opportunistic bacterial, parasitic and/or viral infections or with cerebral lymphomas. These results strongly suggest that the evolution of histopathologically recognizable HIV-encephalopathies closely correlates with the presence and/or tissue concentration of HIV. Since there were no cases with amplified HIV DNA in the absence of HIV-associated tissue lesions, we conclude that harboring and replication of HIV in the CNS rapidly causes corresponding clinical and morphological changes of HIV-associated encephalopathies. In two children with severe HIV encephalomyelitis, large amounts of HIV gag and env transcripts were detected in affected areas of the brain and spinal cord by in situ hybridization.(ABSTRACT TRUNCATED AT 250 WORDS)

Generation of an hTERT-restricted, CMV-augmented oncolytic adenovirus capable of tumor-specific transgene expression: Development, characterization, and optimization

Current shortcomings in cancer therapy require the generation of new, broadly applicable, potent, targeted treatments. Here, an adenovirus is engineered to replicate specifically in cells with active human telomerase promotion using a modified hTERT promoter, fused to a CMV promoter element. The virus was also modified to contain a visible reporter transgene, GFP. The virus, Ad/hTC-GFP-E1 was characterized in vitro and demonstrated tumor specific activity both by dose and over time course experiments in a variety of cell lines. In vivo, Ad/hTC-GFP-E1 was affected at suppressing tumor growth and providing a survival benefit without causing any measurable toxicity. To increase the host range of the vector, the fiber region was modified to contain an RGD-motif. The vector, AdRGD/hTC-GFP-E1, was recharacterized in vitro, revealing heightened levels of infectivity and toxicity however maintaining a therapeutic window between cancer and normal cell toxicity. AdRGD/hTC-GFP-E1 was administered in vivo by limb perfusion and was observed to be tumor specific both in expression and replication. To further enhance the efficacy of viral vectors in lung delivery, asthma medications were investigated for their abilities to enhance transgene delivery and expression. A combination of bronchodilators, mast cell inhibitors, and mucolytic agents was devised which demonstrated fold increases in expression in immunocompetent mouse lungs as single agents and more homogenous, intense levels of expression when done in combination of all agents. To characterize the methods in which some cancers are resistant or may become resistant to oncolytic treatments, several small molecule inhibitors of metabolic pathways were applied in combination with oncolytic infection in vitro. SP600125 and PD 98059, respective JNK and ERK inhibitors, successfully suppressed oncolytic toxicity, however did not affect infectivity or transgene expression of Ad/hTC-GFP-E1. JNK and ERK inhibition did significantly suppress viral replication, however, as analyzed by lysate transfer and titration assays. In contrast, SB 203580, an inhibitor for p38, did not demonstrate any protective effects with infected cells. Flow cytometric analysis indicated a possible correlation with G1 arrest and suppressed viral production, however more compounds must be investigated to clarify this observation. ^

Digital libraries for the preservation of research methods and associated artifacts

New digital artifacts are emerging in data-intensive science. For example, scientific workflows are executable descriptions of scientific procedures that define the sequence of computational steps in an automated data analysis, supporting reproducible research and the sharing and replication of best-practice and know-how through reuse. Workflows are specified at design time and interpreted through their execution in a variety of situations, environments, and domains. Hence it is essential to preserve both their static and dynamic aspects, along with the research context in which they are used. To achieve this, we propose the use of multidimensional digital objects (Research Objects) that aggregate the resources used and/or produced in scientific investigations, including workflow models, provenance of their executions, and links to the relevant associated resources, along with the provision of technological support for their preservation and efficient retrieval and reuse. In this direction, we specified a software architecture for the design and implementation of a Research Object preservation system, and realized this architecture with a set of services and clients, drawing together practices in digital libraries, preservation systems, workflow management, social networking and Semantic Web technologies. In this paper, we describe the backbone system of this realization, a digital library system built on top of dLibra.

Segregation of viral plasmids depends on tethering to chromosomes and is regulated by phosphorylation

Eukaryotic viruses can maintain latency in dividing cells as extrachromosomal nuclear plasmids. Segregation and nuclear retention of DNA is, therefore, a key issue in retaining copy number. The E2 enhancer protein of the papillomaviruses is required for viral DNA replication and transcription. Viral mutants that prevent phosphorylation of the bovine papillomavirus type 1 (BPV) E2 protein are transformation-defective, despite normal viral gene expression and replication function. Cell colonies harboring such mutants show sectoring of viral DNA and are unable to maintain the episome. We find that transforming viral DNA attaches to mitotic chromosomes, in contrast to the mutant genome encoding the E2 phosphorylation mutant. Second-site suppressor mutations were uncovered in both E1 and E2 genes that allow for transformation, maintenance, and chromosomal attachment. E2 protein was also found to colocalize to mitotic chromosomes, whereas the mutant did not, suggesting a direct role for E2 in viral attachment to chromosomes. Such viral hitch-hiking onto cellular chromosomes is likely to provide a general mechanism for maintaining nuclear plasmids.

Mycobacterium avium complex augments macrophage HIV-1 production and increases CCR5 expression

Infection with HIV-1 results in pronounced immune suppression and susceptibility to opportunistic infections (OI). Reciprocally, OI augment HIV-1 replication. As we have shown for Mycobacterium avium complex (MAC) and Pneumocystis carinii, macrophages infected with opportunistic pathogens and within lymphoid tissues containing OI, exhibit striking levels of viral replication. To explore potential underlying mechanisms for increased HIV-1 replication associated with coinfection, blood monocytes were exposed to MAC antigens (MAg) or viable MAC and their levels of tumor necrosis factor α (TNFα) and HIV-1 coreceptors monitored. MAC enhanced TNFα production in vitro, consistent with its expression in coinfected lymph nodes. Using a polyclonal antibody to the CCR5 coreceptor that mediates viral entry of macrophage tropic HIV-1, a subset of unstimulated monocytes was shown to be CCR5-positive by fluorescence-activated cell sorter analysis. After stimulation with MAg or infection with MAC, CCR5 expression was increased at both the mRNA level and on the cell surface. Up-regulation of CCR5 by MAC was not paralleled by an increase in the T cell tropic coreceptor, CXCR4. Increases in NF-κB, TNFα, and CCR5 were consistent with the enhanced production of HIV-1 in MAg-treated adherent macrophage cultures as measured by HIV-1 p24 levels. Increased CCR5 was also detected in coinfected lymph nodes as compared with tissues with only HIV-1. The increased production of TNFα, together with elevated expression of CCR5, provide potential mechanisms for enhanced infection and replication of HIV-1 by macrophages in OI-infected cells and tissues. Consequently, treating OI may inhibit not only the OI-induced pathology, but also limit the viral burden.

The C-terminal Domain of p21 Inhibits Nucleotide Excision Repair In Vitro and In Vivo

The protein p21Cip1, Waf1, Sdi1 is a potent inhibitor of cyclin-dependent kinases (CDKs). p21 can also block DNA replication through its interaction with the proliferating cell nuclear antigen (PCNA), which is an auxiliary factor for polymerase δ. PCNA is also implicated in the repair resynthesis step of nucleotide excision repair (NER). Previous studies have yielded contradictory results on whether p21 regulates NER through its interaction with PCNA. Resolution of this controversy is of interest because it would help understand how DNA repair and replication are regulated. Hence, we have investigated the effect of p21 on NER both in vitro and in vivo using purified fragments of p21 containing either the CDK-binding domain (N terminus) or the PCNA binding domain (C terminus) of the protein. In the in vitro studies, DNA repair synthesis was measured in extracts from normal human fibroblasts using plasmids damaged by UV irradiation. In the in vivo studies, we used intact and permeabilized cells. The results show that the C terminus of the p21 protein inhibits NER both in vitro and in vivo. These are the first in vivo studies in which this question has been examined, and we demonstrate that inhibition of NER by p21 is not merely an artificial in vitro effect. A 50% inhibition of in vitro NER occurred at a 50:1 molar ratio of p21 C-terminus fragment to PCNA monomer. p21 differentially regulates DNA repair and replication, with repair being much less sensitive to inhibition than replication. Our in vivo results suggest that the inhibition occurs at the resynthesis step of the repair process. It also appears that preassembly of PCNA at repair sites mitigates the inhibitory effect of p21. We further demonstrate that the inhibition of DNA repair is mediated via binding of p21 to PCNA. The N terminus of p21 had no effect on DNA repair, and the inhibition of DNA repair by the C terminus of p21 was relieved by the addition of purified PCNA protein.

Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro

The protein kinase Chk2, the mammalian homolog of the budding yeast Rad53 and fission yeast Cds1 checkpoint kinases, is phosphorylated and activated in response to DNA damage by ionizing radiation (IR), UV irradiation, and replication blocks by hydroxyurea (HU). Phosphorylation and activation of Chk2 are ataxia telangiectasia-mutated (ATM) dependent in response to IR, whereas Chk2 phosphorylation is ATM-independent when cells are exposed to UV or HU. Here we show that in vitro, ATM phosphorylates the Ser-Gln/Thr-Gln (SQ/TQ) cluster domain (SCD) on Chk2, which contains seven SQ/TQ motifs, and Thr68 is the major in vitro phosphorylation site by ATM. ATM- and Rad3-related also phosphorylates Thr68 in addition to Thr26 and Ser50, which are not phosphorylated to a significant extent by ATM in vitro. In vivo, Thr68 is phosphorylated in an ATM-dependent manner in response to IR, but not in response to UV or HU. Substitution of Thr68 with Ala reduced the extent of phosphorylation and activation of Chk2 in response to IR, and mutation of all seven SQ/TQ motifs blocked all phosphorylation and activation of Chk2 after IR. These results suggest that in vivo, Chk2 is directly phosphorylated by ATM in response to IR and that Chk2 is regulated by phosphorylation of the SCD.