Functional characterization of Bartonella effector protein C (BepC) in the context of infection

A wide variety of bacterial pathogens evolved a panel of virulence factors in order to subvert cellular processes and achieve a successful infection. Bacteria of the genus Bartonella translocate a cocktail of effector proteins (Beps) via a type IV secretion system (T4SS) into mammalian cells. BepC, one of the most conserved effectors in the Bartonella species of the lineage 4, has been previously shown to be involved in the internalization of bacterial aggregates and migration defect in vitro. In this work, we show that the effector BepC localizes at cell-to-cell contact and triggers strong actin rearrangements as well as the formation of bacterial aggregates during infection of human cells. The actin phenotype is induced by BepC from different Bartonella species, indicating an important role of this effector during pathogenesis. BepC pull-down from infected cells led to the identification of two interacting partners, GEF-H1 and MRCKα, which are two host proteins involved in the RhoA and Cdc42 pathways, respectively. We demonstrate that the ability of BepC to bind GEF-H1 and MRCKα highly correlates with its ability to trigger actin rearrangements. Accordingly, infected cells show an increase of GTP-bound RhoA and phosphorylated myosin light chain while both RhoA and its downstream effector ROCK are required for actin rearrangements mediated by BepC. Thus, our results indicate that BepC activates the RhoA pathway by interacting with GEF-H1 and thereby inducing actin rearrangements although MRCKα might also be involved. The majority of Beps, including BepC, carries an enzymatic FIC domain that is usually involved in posttranslational modifications. Most Fic proteins carry a canonical FIC motif that is essential for ATP binding and the transfer of AMP onto the target protein (AMPylation). By contrast, BepC is characterized by a non-canonical FIC motif and only displays a weak AMPylation and phosphorylation activity, independently from its conserved motif. Nevertheless, structural analysis and binding assays demonstrate that ATP binds to the FIC domain of BepC and is critical for its thermal stability. In absence of FIC domain, BepC loses its ability to localize at cell junctions, to interact with GEF-H1 and MRCKα, and to trigger actin rearrangement, suggesting a central role for this domain in the effector function. However, a conserved FIC motif is not necessary to trigger actin rearrangements, which indicates that BepC acts by protein-protein interaction rather than by posttranslational modification. Thus, we propose that BepC is recruited to cell contacts where it triggers the activation of the RhoA pathway by interacting with GEF-H1 and eventually leads to actin rearrangements, possibly with the help of MRCKα. Ultimately, the subversion of RhoA signaling by BepC could help Bartonella to interfere with the immune response by preventing phagocytosis or impair cell migration. Furthermore, it could play an important role in the disruption of the endothelial barrier in order to reach the blood and establish a long-lasting bacteremia inside the host

Women as a resource for the flexibility required for high technology innovation

What do women scientists need to know for career advancement into senior level positions? Our declining economic conditions have been the cause for major political and technological changes. The U.S. Congress is turning toward technology to increase our competitive edge in the world. Allowing women scientists, and women engineers in particular, more voice in the decision making process may be an innovative alternative for the diversity and flexibility needed for the unknown technological problems of the future. But first women scientists need to know how the system measures scientific achievement and how to identify the processes needed to increase our technological capability in order for them to formidably compete and win higher ranking positions

Boas razões para um desempenho ruim em testes: o substrato interacional dos exames educacionais

This paper was originally published in 1992. It arose out of a project to study how clinicians tell parents a diagnosis of a developmental disability. That specific project was part of a larger one conducted in 1985-1986 to study the delivery of bad and good news in both ordinary and medical settings (Maynard, 2003). As part of the developmental disabilities study, we also examined how testing was done as a precedent to deciding on diagnosis (Marlaire and Maynard, 1989), and the paper here about the “interactional substrate” was meant to show the orderliness of testing interactions – the basic structures that made it possible to generate valid and reliable examination scores that could lend to the official assessment. More recently (2013-2015), a research team and I returned to the same clinic with a grant from the U.S. National Science Foundation, to study more intensively the testing and diagnosis of Autism Spectrum Disorders (ASD). In the time between the 1985 study and the recent one, the prevalence of ASD had skyrocketed in the U.S. from 1 in 5000 children to the current rate of 1 in 68. A study of the micro-interactions surrounding testing and diagnosis does not explain the increase in prevalence but it does say just how testing is done and how clinicians use results and other information to diagnose children. With regard to testing, in particular, we have come back to the paper on the interactional substrate again and again because probing this substrate and the practices by which it is constituted remains as an avenue in to understanding ASD as not just a condition of the child but as something that is manifested as a feature of social interaction. There are ways in which ASD as a child’s condition is co-produced by way of (i) the orderliness of interactions between clinicians and children, and (ii) how tests constrain both the clinician and the child in terms of what is visible as “competence.” Thus, in current work, we distinguish between what we call first-order, or concrete competence, which, by way of the interactional substrate, allows testing to be done, regardless of what the official results may be, and second-order displays of abstract competence (Maynard and Turowetz, 2016). Abstract competence involves the ability to produce general answers or ones that are shorn of embodied or other contextual orientations to questions or that involve what Donaldson (1978) has called “disembedded knowledge.” The emphasis of clinical tests on measuring second-order, abstract competence may obscure various kinds of first-order, concrete competence and “autistic intelligence” a child displays (Maynard, 2005). By doing so, testing can potentially make the child seem more impaired than he or she is, or at least suppress information that could improve performance and/or be informative for how to design home and schooling environments that enhance a child’s skills and integration into these social units.Keywords: psychological testing, conversation analysis, disability.Este artigo foi originalmente publicado em 1992. Ele surgiu de um projeto para estudar como os médicos apresentam aos pais um diagnóstico de deficiência no desenvolvimento infantil. Tal projeto específico fazia parte de outro mais amplo, conduzido entre 1985-1986, para estudar a comunicação de boas ou más notícias tanto em cenários cotidianos quanto em cenários de tratamento de saúde (Maynard, 2003). No estudo das deficiências de desenvolvimento, também examinamos o modo como eram realizados os testes que precediam a decisão sobre o diagnóstico (Marlaire e Maynard, 1989), sendo que o artigo aqui apresentado, sobre o “substrato interacional”, visava a mostrar o caráter ordenado das interações durante os testes – as estruturas básicas que possibilitavam gerar escores de exames válidos e confiáveis que pudessem levar à avaliação oficial. Mais recentemente (2013-2015), retornei à mesma clínica com um grupo de pesquisa, com financiamento da Fundação Nacional de Pesquisa dos EUA (U.S. National Science Foundation), para estudar mais intensivamente a testagem e o diagnóstico dos Transtornos do Espectro Autista, TEA, (Autism Spectrum Disorders, ASD). Entre o estudo de 1985 e o mais recentemente realizado, a prevalência de TEA nos EUA disparou de 1 em 5000 crianças para a taxa atual de 1 em 68. Um estudo das microinterações envolvidas nos testes e diagnósticos não explica o aumento na prevalência, mas revela, sim, como os testes são realizados e como os médicos usam os seus resultados e outras informações para diagnosticar as crianças. Em relação à testagem, em particular, retomamos inúmeras vezes o artigo de 1992 sobre o substrato interacional, pois o exame desse substrato e das práticas que o constituem continua sendo um caminho para que se compreendam os TEA não só como condição da criança, mas também como algo que se manifesta como traço da interação social. Há, pois, maneiras de se coproduzir os Transtornos do Espectro Autista como uma condição da criança, as quais se prendem a: (i) o caráter ordenado das interações entre médicos e crianças, e (ii) o modo como os testes cerceiam tanto o médico como a criança em termos do que é visível como “competência”. Assim, no trabalho atual, distinguimos entre, primeiro, o que chamamos de competência concreta ou de primeira-ordem, que permite, por meio do substrato interacional, que a testagem seja realizada, independentemente de quais possam ser os resultados oficiais; e, segundo, o que chamamos de demonstrações de competência abstrata, ou de segunda-ordem (Maynard e Turowetz, 2016). A competência abstrata envolve a habilidade de produzir respostas gerais ou que sejam despidas de orientações para a pergunta com base em elementos corporais ou contextuais de outra natureza, ou que envolvam o que Donaldson (1978) chamou de “conhecimento desencaixado” (“disembedded knowledge”). A ênfase dos testes clínicos em medir a competência abstrata, de segunda-ordem, pode tornar obscuros vários tipos de competência concreta, de primeira-ordem, e de “inteligência autista” que uma criança demonstre (Maynard, 2005). Realizada assim, a testagem pode, potencialmente, fazer uma criança parecer mais incapacitada do que realmente é, ou pode, no mínimo, suprimir informação que pudesse melhorar o desempenho e/ou contribuir para projetar ambientes domésticos e escolares que ampliem as habilidades da criança e a sua integração nessas unidades sociais.Palavras-chave: teste psicológico, análise de conversa, deficiência

Good reasons for bad testing performance: The interactional substrate of educational exams

This paper was originally published in 1992. It arose out of a project to study how clinicians tell parents a diagnosis of a developmental disability. That specific project was part of a larger one conducted in 1985-1986 to study the delivery of bad and good news in both ordinary and medical settings (Maynard, 2003). As part of the developmental disabilities study, we also examined how testing was done as a precedent to deciding on diagnosis (Marlaire and Maynard, 1989), and the paper here about the “interactional substrate” was meant to show the orderliness of testing interactions – the basic structures that made it possible to generate valid and reliable examination scores that could lend to the official assessment. More recently (2013-2015), a research team and I returned to the same clinic with a grant from the U.S. National Science Foundation, to study more intensively the testing and diagnosis of Autism Spectrum Disorders (ASD). In the time between the 1985 study and the recent one, the prevalence of ASD had skyrocketed in the U.S. from 1 in 5000 children to the current rate of 1 in 68. A study of the micro-interactions surrounding testing and diagnosis does not explain the increase in prevalence but it does say just how testing is done and how clinicians use results and other information to diagnose children. With regard to testing, in particular, we have come back to the paper on the interactional substrate again and again because probing this substrate and the practices by which it is constituted remains as an avenue in to understanding ASD as not just a condition of the child but as something that is manifested as a feature of social interaction. There are ways in which ASD as a child’s condition is co-produced by way of (i) the orderliness of interactions between clinicians and children, and (ii) how tests constrain both the clinician and the child in terms of what is visible as “competence.” Thus, in current work, we distinguish between what we call first-order, or concrete competence, which, by way of the interactional substrate, allows testing to be done, regardless of what the official results may be, and second-order displays of abstract competence (Maynard and Turowetz, 2016). Abstract competence involves the ability to produce general answers or ones that are shorn of embodied or other contextual orientations to questions or that involve what Donaldson (1978) has called “disembedded knowledge.” The emphasis of clinical tests on measuring second-order, abstract competence may obscure various kinds of first-order, concrete competence and “autistic intelligence” a child displays (Maynard, 2005). By doing so, testing can potentially make the child seem more impaired than he or she is, or at least suppress information that could improve performance and/or be informative for how to design home and schooling environments that enhance a child’s skills and integration into these social units.Keywords: psychological testing, conversation analysis, disability.</p

Boas razões para um desempenho ruim em testes: o substrato interacional dos exames educacionais

This paper was originally published in 1992. It arose out of a project to study how clinicians tell parents a diagnosis of a developmental disability. That specific project was part of a larger one conducted in 1985-1986 to study the delivery of bad and good news in both ordinary and medical settings (Maynard, 2003). As part of the developmental disabilities study, we also examined how testing was done as a precedent to deciding on diagnosis (Marlaire and Maynard, 1989), and the paper here about the “interactional substrate” was meant to show the orderliness of testing interactions – the basic structures that made it possible to generate valid and reliable examination scores that could lend to the official assessment. More recently (2013-2015), a research team and I returned to the same clinic with a grant from the U.S. National Science Foundation, to study more intensively the testing and diagnosis of Autism Spectrum Disorders (ASD). In the time between the 1985 study and the recent one, the prevalence of ASD had skyrocketed in the U.S. from 1 in 5000 children to the current rate of 1 in 68. A study of the micro-interactions surrounding testing and diagnosis does not explain the increase in prevalence but it does say just how testing is done and how clinicians use results and other information to diagnose children. With regard to testing, in particular, we have come back to the paper on the interactional substrate again and again because probing this substrate and the practices by which it is constituted remains as an avenue in to understanding ASD as not just a condition of the child but as something that is manifested as a feature of social interaction. There are ways in which ASD as a child’s condition is co-produced by way of (i) the orderliness of interactions between clinicians and children, and (ii) how tests constrain both the clinician and the child in terms of what is visible as “competence.” Thus, in current work, we distinguish between what we call first-order, or concrete competence, which, by way of the interactional substrate, allows testing to be done, regardless of what the official results may be, and second-order displays of abstract competence (Maynard and Turowetz, 2016). Abstract competence involves the ability to produce general answers or ones that are shorn of embodied or other contextual orientations to questions or that involve what Donaldson (1978) has called “disembedded knowledge.” The emphasis of clinical tests on measuring second-order, abstract competence may obscure various kinds of first-order, concrete competence and “autistic intelligence” a child displays (Maynard, 2005). By doing so, testing can potentially make the child seem more impaired than he or she is, or at least suppress information that could improve performance and/or be informative for how to design home and schooling environments that enhance a child’s skills and integration into these social units.Keywords: psychological testing, conversation analysis, disability.Este artigo foi originalmente publicado em 1992. Ele surgiu de um projeto para estudar como os médicos apresentam aos pais um diagnóstico de deficiência no desenvolvimento infantil. Tal projeto específico fazia parte de outro mais amplo, conduzido entre 1985-1986, para estudar a comunicação de boas ou más notícias tanto em cenários cotidianos quanto em cenários de tratamento de saúde (Maynard, 2003). No estudo das deficiências de desenvolvimento, também examinamos o modo como eram realizados os testes que precediam a decisão sobre o diagnóstico (Marlaire e Maynard, 1989), sendo que o artigo aqui apresentado, sobre o “substrato interacional”, visava a mostrar o caráter ordenado das interações durante os testes – as estruturas básicas que possibilitavam gerar escores de exames válidos e confiáveis que pudessem levar à avaliação oficial. Mais recentemente (2013-2015), retornei à mesma clínica com um grupo de pesquisa, com financiamento da Fundação Nacional de Pesquisa dos EUA (U.S. National Science Foundation), para estudar mais intensivamente a testagem e o diagnóstico dos Transtornos do Espectro Autista, TEA, (Autism Spectrum Disorders, ASD). Entre o estudo de 1985 e o mais recentemente realizado, a prevalência de TEA nos EUA disparou de 1 em 5000 crianças para a taxa atual de 1 em 68. Um estudo das microinterações envolvidas nos testes e diagnósticos não explica o aumento na prevalência, mas revela, sim, como os testes são realizados e como os médicos usam os seus resultados e outras informações para diagnosticar as crianças. Em relação à testagem, em particular, retomamos inúmeras vezes o artigo de 1992 sobre o substrato interacional, pois o exame desse substrato e das práticas que o constituem continua sendo um caminho para que se compreendam os TEA não só como condição da criança, mas também como algo que se manifesta como traço da interação social. Há, pois, maneiras de se coproduzir os Transtornos do Espectro Autista como uma condição da criança, as quais se prendem a: (i) o caráter ordenado das interações entre médicos e crianças, e (ii) o modo como os testes cerceiam tanto o médico como a criança em termos do que é visível como “competência”. Assim, no trabalho atual, distinguimos entre, primeiro, o que chamamos de competência concreta ou de primeira-ordem, que permite, por meio do substrato interacional, que a testagem seja realizada, independentemente de quais possam ser os resultados oficiais; e, segundo, o que chamamos de demonstrações de competência abstrata, ou de segunda-ordem (Maynard e Turowetz, 2016). A competência abstrata envolve a habilidade de produzir respostas gerais ou que sejam despidas de orientações para a pergunta com base em elementos corporais ou contextuais de outra natureza, ou que envolvam o que Donaldson (1978) chamou de “conhecimento desencaixado” (“disembedded knowledge”). A ênfase dos testes clínicos em medir a competência abstrata, de segunda-ordem, pode tornar obscuros vários tipos de competência concreta, de primeira-ordem, e de “inteligência autista” que uma criança demonstre (Maynard, 2005). Realizada assim, a testagem pode, potencialmente, fazer uma criança parecer mais incapacitada do que realmente é, ou pode, no mínimo, suprimir informação que pudesse melhorar o desempenho e/ou contribuir para projetar ambientes domésticos e escolares que ampliem as habilidades da criança e a sua integração nessas unidades sociais.Palavras-chave: teste psicológico, análise de conversa, deficiência

Questioning the answer: questioning style, choice and self-determination in interactions with young people with intellectual disabilities*

For young people with intellectual disabilities (ID), the transition from children's to adult services has long been recognised as a challenging move. One of the aims of the White Paper Valuing People (2001) was to address some of the problems associated with this transition. This paper reports on data from a project which examines the impact of these service changes, and the ways in which transition is negotiated by carers, professionals and users. It presents a conversation analysis of eight tape-recorded formal review meetings at which transition to adult services is discussed. It takes as its starting point the existing interactional work on ID and the way in which this demonstrates the effects of the local and contextual specifics of particular kinds of interaction on the eventual outcomes (e.g. Rapley 2004, Antaki 2001, Maynard and Marlaire 1992). We show that an attempt to allow self-determination in the context of transitions can paradoxically result in undermining user choice and control. We also argue that, while a rule-based approach to practice may offer moral clarity for professionals, it can result in interactional and practical difficulties which cannot be easily reconciled

Nit1 is a metabolite repair enzyme that hydrolyzes deaminated glutathione

The mammalian gene Nit1 (nitrilase-like protein 1) encodes a protein that is highly conserved in eukaryotes and is thought to act as a tumor suppressor. Despite being ∼35% sequence identical to ω-amidase (Nit2), the Nit1 protein does not hydrolyze efficiently α-ketoglutaramate (a known physiological substrate of Nit2), and its actual enzymatic function has so far remained a puzzle. In the present study, we demonstrate that both the mammalian Nit1 and its yeast ortholog are amidases highly active toward deaminated glutathione (dGSH; i.e., a form of glutathione in which the free amino group has been replaced by a carbonyl group). We further show that Nit1-KO mutants of both human and yeast cells accumulate dGSH and the same compound is excreted in large amounts in the urine of Nit1-KO mice. Finally, we show that several mammalian aminotransferases (transaminases), both cytosolic and mitochondrial, can form dGSH via a common (if slow) side-reaction and provide indirect evidence that transaminases are mainly responsible for dGSH formation in cultured mammalian cells. Altogether, these findings delineate a typical instance of metabolite repair, whereby the promiscuous activity of some abundant enzymes of primary metabolism leads to the formation of a useless and potentially harmful compound, which needs a suitable “repair enzyme” to be destroyed or reconverted into a useful metabolite. The need for a dGSH repair reaction does not appear to be limited to eukaryotes: We demonstrate that Nit1 homologs acting as excellent dGSH amidases also occur in Escherichia coli and other glutathione-producing bacteria

This place is not a place: The constructed scene in the works of Sir Walter Scott

This work examines Sir Walter Scott\u27s use of perspective and landscape, focusing mostly on two of his works, The Bride of Lammermoor and Redgauntlet . It is concerned with the influence of societal factors of production on both author, and through author, text, and the interaction of both as they exist within and without a dominant hegemony. There are, in Scott\u27s works, emergent voices coming from the borders of dominant society, as well as once-dominants struggling against the inevitability of their own dissolution. For a variety of reasons, much of the conflict between these voices occurs in the novel\u27s setting. This study discusses the way in which Scott\u27s constructed scene and perspective make past dignities, present actions, and future dooms a predetermined fact of the constructed scene. It addresses formulations of landscape that point to specific ideological implications, sometimes condemning, sometimes privileging segments of a culture as they are connected to and disconnected from specific locales on the textual stage. Scott\u27s use of the scene has several predecessors: it arises from the selective processes involved in picturesque painting, from the landed ontology prevalent in European cultures at this time in social history, from the novelist\u27s growing consciousness of the way in which the world-constructed-in-text influences the way a reading audience sees the world-outside-text. Scott\u27s refraction of the world, his attempts to adjust the conveyed reality against the ambiguating receiver and against the demands of those exterior (and interior) forces that place restrictions on the limits and forms of ideological meaning, leads to a potent fission of meaning within the novel\u27s landscape as an increasingly larger exterior reality is desperately pressed into an increasingly restrictive textual reality. The potency of the medium and its tendency to extend beyond the control of the author lead to more violent attempts to maintain cohesiveness, which in turn powers a novelistic creation that strays further from his control. This study is constructed to identify the gravitational pressures exerted both within the author and without that bend creative intentions and vastly complicate the impact of the created text