Caractérisation structurale et thermodynamique de la reconnaissance du substrat par le ribozyme VS de Neurospora

Les interactions ARN/ARN de type kissing-loop sont des éléments de structure tertiaire qui jouent souvent des rôles clés chez les ARN, tant au niveau fonctionnel que structural. En effet, ce type d’interaction est crucial pour plusieurs processus dépendant des ARN, notamment pour l’initiation de la traduction, la reconnaissance des ARN antisens et la dimérisation de génome rétroviral. Les interactions kissing-loop sont également importantes pour le repliement des ARN, puisqu’elles permettent d’établir des contacts à longue distance entre différents ARN ou encore entre les domaines éloignés d’un même ARN. Ce type d’interaction stabilise aussi les structures complexes des ARN fonctionnels tels que les ARNt, les riborégulateurs et les ribozymes. Comme d’autres ARN fonctionnels, le ribozyme VS de Neurospora contient une interaction kissing-loop importante. Celle-ci est impliquée dans la reconnaissance du substrat et se forme entre la tige-boucle I (stem-loop I, SLI) du substrat et la tige-boucle V (stem-loop V, SLV) du domaine catalytique. Des études biochimiques ont démontré que l’interaction kissing-loop I/V, dépendante du magnésium, implique trois paires de bases Watson-Crick (W-C). De plus, cette interaction est associée à un réarrangement de la structure du substrat, le faisant passer d’une conformation inactive dite unshifted à une conformation active dite shifted. Les travaux présentés dans cette thèse consistent en une caractérisation structurale et thermodynamique de l’interaction kissing-loop I/V du ribozyme VS, laquelle est formée de fragments d’ARN représentant les tige-boucles I et V dérivées du ribozyme VS (SLI et SLV). Cette caractérisation a été réalisée principalement par spectroscopie de résonance magnétique nucléaire (RMN) et par titrage calorimétrique isotherme (isothermal titration calorimetry, ITC) en utilisant différents complexes SLI/SLV dans lesquels l’ARN SLV est commun à tous les complexes, alors que différentes variations de l’ARN SLI ont été utilisées, soit en conformation shiftable ou preshifted. Les données d’ITC ont permis de démontrer qu’en présence d’une concentration saturante de magnésium, l’affinité d’un substrat SLI preshifted pour SLV est extrêmement élevée, rendant cette interaction plus stable que ce qui est prédit pour un duplexe d’ARN équivalent. De plus, l’étude effectuée par ITC montre que des ARN SLI preshifted présentent une meilleure affinité pour SLV que des ARN SLI shiftable, ce qui a permis de calculer le coût énergétique associé au réarrangement de structure du substrat. En plus de confirmer la formation des trois paires de bases W-C prédites à la jonction I/V, les études de RMN ont permis d’obtenir une preuve structurale directe du réarrangement structural des substrats SLI shiftable en présence de magnésium et de l’ARN SLV. La structure RMN d’un complexe SLI/SLV de grande affinité démontre que les boucles terminales de SLI et SLV forment chacune un motif U-turn, ce qui facilite l’appariement W-C intermoléculaire. Plusieurs autres interactions ont été définies à l’interface I/V, notamment des triplets de bases, ainsi que des empilements de bases. Ces interactions contribuent d’ailleurs à la création d’une structure présentant un empilement continu, c’est-à-dire qui se propage du centre de l’interaction jusqu’aux bouts des tiges de SLI et SLV. Ces études de RMN permettent donc de mieux comprendre la stabilité exceptionnelle de l’interaction kissing-loop I/V au niveau structural et mènent à l’élaboration d’un modèle cinétique de l’activation du substrat par le ribozyme VS. En considérant l’ensemble des données d’ITC et de RMN, l’étonnante stabilité de l’interaction I/V s’explique probablement par une combinaison de facteurs, dont les motifs U-turn, la présence d’un nucléotide exclu de la boucle de SLV (U700), la liaison de cations magnésium et l’empilement de bases continu à la jonction I/V.

Identification de caractéristiques communes et rares dans les ARN structurés dans la base de données Rfam

Les ARN non codants (ARNnc) sont des transcrits d'ARN qui ne sont pas traduits en protéines et qui pourtant ont des fonctions clés et variées dans la cellule telles que la régulation des gènes, la transcription et la traduction. Parmi les nombreuses catégories d'ARNnc qui ont été découvertes, on trouve des ARN bien connus tels que les ARN ribosomiques (ARNr), les ARN de transfert (ARNt), les snoARN et les microARN (miARN). Les fonctions des ARNnc sont étroitement liées à leurs structures d’où l’importance de développer des outils de prédiction de structure et des méthodes de recherche de nouveaux ARNnc. Les progrès technologiques ont mis à la disposition des chercheurs des informations abondantes sur les séquences d'ARN. Ces informations sont accessibles dans des bases de données telles que Rfam, qui fournit des alignements et des informations structurelles sur de nombreuses familles d'ARNnc. Dans ce travail, nous avons récupéré toutes les séquences des structures secondaires annotées dans Rfam, telles que les boucles en épingle à cheveux, les boucles internes, les renflements « bulge », etc. dans toutes les familles d'ARNnc. Une base de données locale, RNAstem, a été créée pour faciliter la manipulation et la compilation des données sur les motifs de structure secondaire. Nous avons analysé toutes les boucles terminales et internes ainsi que les « bulges » et nous avons calculé un score d’abondance qui nous a permis d’étudier la fréquence de ces motifs. Tout en minimisant le biais de la surreprésentation de certaines classes d’ARN telles que l’ARN ribosomal, l’analyse des scores a permis de caractériser les motifs rares pour chacune des catégories d’ARN en plus de confirmer des motifs communs comme les boucles de type GNRA ou UNCG. Nous avons identifié des motifs abondants qui n’ont pas été étudiés auparavant tels que la « tetraloop » UUUU. En analysant le contenu de ces motifs en nucléotides, nous avons remarqué que ces régions simples brins contiennent beaucoup plus de nucléotides A et U. Enfin, nous avons exploré la possibilité d’utiliser ces scores pour la conception d’un filtre qui permettrait d’accélérer la recherche de nouveaux ARN non-codants. Nous avons développé un système de scores, RNAscore, qui permet d’évaluer un ARN en se basant sur son contenu en motifs et nous avons testé son applicabilité avec différents types de contrôles.

Variação dos perfis metabólico, hematológico e lácteo em ovinos leiteiros na serra gaúcha

A ovinocultura no Rio Grande do Sul (RS), tem mostrado mudanças no tipo de exploração nos últimos 20 anos. A produção de lã, que por muito tempo movimentou a economia do setor primário, cedeu lugar para a produção de cordeiros. Recentemente, a ovinocultura leiteira começa a dar seus primeiros passos dentro do sistema de produção ovina. Esta dissertação tem por objetivo apresentar a variação dos perfis metabólico, hematológico e lácteo em ovinos leiteiros na Serra Gaúcha e discutir seus resultados. O trabalho foi realizado em ovelhas da raça Lacaune, criadas em regime de confinamento no município de Bento Gonçalves (RS). As observações se estenderam pelo período de dois anos consecutivos. Para determinação do perfil bioquímico foram coletadas amostras de sangue por venipunção jugular sem anticoagulante. O mesmo procedimento foi empregado na coleta de amostras para o hemograma, utilizando-se, nesse caso, tubos contendo EDTA, como anticoagulante. As amostras foram coletadas de sete animais ao acaso nos seguintes períodos fisiológico: (a) ovelhas vazias; (b) início, (c) meio e (d) final da gestação e (e) aos 7, (f) 30, (g) 60 e (h) 140 dias de lactação. Durante os mesmos períodos da lactação foram coletadas amostras para a determinação dos componentes físico-químicos do leite. Os valores dos parâmetros físico-químicos do leite estudados discordam com os resultados citados pela literatura em outros países. A acidez em °Dornic (°D) foi maior que a relatada em outros estudos. Os valores de proteína e gordura foram inferiores aos citados na literatura. Os parâmetros com variação estatística (P<0,05) no período da lactação foram: pH, acidez em °D, densidade, proteína, lactose, gordura, ESTe ESD. O perfil metabólico apresentou as maiores variações com relação à média no final da gestação e início da lactação. Não foi observada diferença significativa nos parâmetros analisados entre o grupo de ovelhas vazias e prenhes, com exceção do cálcio que foi maior nas ovelhas vazias (P<0,05). Dentre os parâmetros do metabolismo nitrogenado, apenas a uréia mostrou variação durante a gestação e a lactação (P<0,05). Os valores de glicose e fructosamina diminuíram e os de BHB aumentaram significativamente (P<0,05) no final da gestação. Durante a lactação a glicose diminuiu e o colesterol aumentou significativamente (P<0,05) com o avanço do período. O nível de magnésio aumentou no final da lactação e o fósforo apresentou um diminuição nos valores aos 30 dias após o parto (P<0,05). Os parâmetros hematológicos mostraram não haver diferença significativa nos diferentes períodos fisiológicos estudados, com exceção dos neutrófilos segmentados que aumentaram com o avanço da gestação. Os dados encontrados servem como referência para estudos sobre nutrição, metabolismo e qualidade do leite em ovelhas leiteiras.

De muros, tempos, artes e pingue-pongue aos genes, anfioxos, mórulas e trissomias : falando do corpo nas práticas escolares

As problematizações que me levaram a realização desse estudo vinculam-se a minha formação como bióloga e professora de Biologia. A partir de aproximações com leituras do campo dos Estudos Culturais, dos Estudos Culturais da Ciência e de estudos com inspiração foucaultiana, passei a questionar tanto a formação acadêmica que me constituiu, quanto o lugar que os objetos, as classificações e as explicações ligados ao corpo no campo da Biologia adquiriam nas práticas escolares. Nesse estudo, tomo o corpo como produção de práticas sociais; inscrito por discursos e práticas de diferentes instâncias culturais que se articulam e se confrontam, constituindo corpos múltiplos, sujeitos particulares. Com esses entendimentos e questionamentos, busquei, nessa dissertação, conhecer e problematizar o corpo nas práticas escolares, ou melhor, como a escola lida com os corpos nas suas práticas cotidianas e naquelas relacionadas ao campo da Biologia, assim como alguns efeitos nos corpos dos estudantes. Realizei esta pesquisa numa escola da rede pública estadual de Porto Alegre. Para tanto, freqüentei o espaço escolar e as aulas de duas turmas do segundo ano do Ensino Médio, por aproximadamente dois meses, e também uma atividade “extra-muros”, um passeio à 4ª Bienal de Arte do Mercosul, com a turma da manhã. Para a realização da pesquisa na escola, utilizei ferramentas de cunho etnográfico e realizei entrevistas com alguns estudantes. Nas análises, fui fazendo relações com autores dos estudos anteriormente citados, conforme as questões que emergiam nessa trajetória. Ao integrar as atividades escolares cotidianas, passei a observar e analisar questões relativas aos efeitos de estratégias disciplinares, direcionadas à fabricação de corpos escolares; ao mesmo tempo, busquei apontar alguns movimentos de resistência e diferentes formas, que estudantes e professores, encontraram para lidar com tais estratégias Procurei mostrar como, nessas relações configuram-se uma pluralidade de sujeitos e práticas que significam o espaço escolar. Da imersão que empreendi na sala de aula, foram criadas questões relativas ao corpo no campo de saberes. Nessa discussão, o corpo associado aos discursos da disciplina biológica foi trazido para a sala de aula vinculado a explicações da área científica, tais como a Embriologia e a Genética, sendo que esse modo de tratar o corpo não articulou-se, muitas vezes, às experiências e problematizações dos estudantes. Por último, analisei uma aula específica, que tratou da temática do aborto numa gestação de um feto com uma patologia grave, sem perspectivas de vida. Essa aula foi uma encenação dos estudantes de um julgamento, em que a mãe pediu autorização na justiça para a interrupção da gravidez. Nesse momento, as problematizações que fiz disseram respeito ao posicionamento de sujeitos — mulher, homem, mãe, pai, filhos, experts, monstros e outros — em nossa sociedade, por diferentes discursos — médicos, biológicos, políticos, religiosos, morais, éticos, etc.

Dançando com gatos e passáros: O movimento ecossistêmico da ludopoiese na educação infantil

This study shows the movement of educators ludopoiese ecosystem of the Center for Early Childhood Education Marise Paiva-CEIMAP. We used the metaphorof dance as an opportunity to stimulate scientific creativity. Ecosystem Thought, complex thinking, the theory of autopoiesis and Flow Theory, constituted the main theoretical steps to understand the phenomenon of ludopoiese, from the look of your totalidadem with the following objectives: 1 - Identify and interpret the process of self ludopoiética CEIMAP of Early Childhood Educators in the actions of the play, care for and educate in school life; 2 - Analyze Ecosystem Thought from ludopoiéticos how these processes affect and / or possible changes and transformations in practice humanescenteseducational CEIMAP. The theoretical metodógicos steps to address the proposed objectives are grounded in existential action research part of the appreciation of the complexity of the real, considering the human being a whole dynamic. In this sense the game of sand, recreational experiences, the systematic studies and video training were explored with a view to the transdisciplinary relevance of everyday phenomena. New knowledge acquired in accordance with the directions given indicating the movement of the ecosystem studied ludopoiese educators, involved in four main streams: love, play, care for and raise it from love streamline interdependently. The ludopoiese eachteacher would then be fed by this web generated by love that permeates all other educational activities, nurturing and maintaining a constant creative self-organization of knowledge and know-how to be teachers. Thus, every network that generates andstream lines the system emerges ludopoiético biology of love, the open dialogue and playing in the wishing well to the student, the aesthetic beauty of caring and educating, as a human conditionand relevant as possible to live / live not only in teaching children, but in other educational contexts of teaching and teacher education

Genomic Signatures Predict Poor Outcome in Undifferentiated Pleomorphic Sarcomas and Leiomyosarcomas

Undifferentiated high-grade pleomorphic sarcomas (UPSs) display aggressive clinical behavior and frequently develop local recurrence and distant metastasis. Because these sarcomas often share similar morphological patterns with other tumors, particularly leiomyosarcomas (LMSs), classification by exclusion is frequently used. In this study, array-based comparative genomic hybridization (array CGH) was used to analyze 20 UPS and 17 LMS samples from untreated patients. The LMS samples presented a lower frequency of genomic alterations compared with the UPS samples. The most frequently altered UPS regions involved gains at 20q13.33 and 7q22.1 and losses at 3p26.3. Gains at 8q24.3 and 19q13.12 and losses at 9p21.3 were frequently detected in the LMS samples. Of these regions, gains at 1q21.3, 11q12.2-q12.3, 16p11.2, and 19q13.12 were significantly associated with reduced overall survival times in LMS patients. A multivariate analysis revealed that gains at 1q21.3 were an independent prognostic marker of shorter survival times in LMS patients (HR = 13.76; P = 0.019). Although the copy number profiles of the UPS and LMS samples could not be distinguished using unsupervised hierarchical clustering analysis, one of the three clusters presented cases associated with poor prognostic outcome (P = 0.022). A relative copy number analysis for the ARNT, SLC27A3, and PBXIP1 genes was performed using quantitative real-time PCR in 11 LMS and 16 UPS samples. Gains at 1q21-q22 were observed in both tumor types, particularly in the UPS samples. These findings provide strong evidence for the existence of a genomic signature to predict poor outcome in a subset of UPS and LMS patients. © 2013 Silveira et al.

The use of AARS activity as a proxy for zooplankton and ichthyoplankton growth rates

Programa de doctorado en Oceanografía

Differential cell type-specific transcriptional regulation of the CYP1A1 gene

Cytochrome P450 1A1 (CYP1A1) monooxygenase plays an important role in the metabolism of environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) and halogenated polycyclic aromatic hydrocarbons (HAHs). Oxidation of these compounds converts them to the metabolites that subsequently can be conjugated to hydrophilic endogenous entities e.g. glutathione. Derivates generated in this way are water soluble and can be excreted in bile or urine, which is a defense mechanism. Besides detoxification, metabolism by CYP1A1 may lead to deleterious effects since the highly reactive intermediate metabolites are able to react with DNA and thus cause mutagenic effects, as it is in the case of benzo(a) pyrene (B[a]P). CYP1A1 is normally not expressed or expressed at a very low level in the cells but it is inducible by many PAHs and HAHs e.g. by B[a]P or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Transcriptional activation of the CYP1A1 gene is mediated by aryl hydrocarbon receptor (AHR), a basic-helix-loop-helix (bHLH) transcription factor. In the absence of a ligand AHR stays predominantly in the cytoplasm. Ligand binding causes translocation of AHR to the nuclear compartment, its heterodimerization with another bHLH protein, the aryl hydrocarbon nuclear translocator (ARNT) and binding of the AHR/ARNT heterodimer to a DNA motif designated dioxin responsive element (DRE). This process leads to the transcriptional activation of the responsive genes containing DREs in their regulatory regions, e.g. that coding for CYP1A1. TCDD is the most potent known agonist of AHR. Since it is not metabolized by the activated enzymes, exposure to this compound leads to a persisting activation of AHR resulting in diverse toxic effects in the organism. To enlighten the molecular mechanisms that mediate the toxicity of xenobiotics like TCDD and related compounds, the AHR-dependent regulation of the CYP1A1 gene was investigated in two cell lines: human cervix carcinoma (HeLa) and mouse hepatoma (Hepa). Study of AHR activation and its consequence concerning expression of the CYP1A1 enzyme confirmed the TCDD-dependent formation of the AHR/ARNT complex on DRE leading to an increase of the CYP1A1 transcription in Hepa cells. In contrast, in HeLa cells formation of the AHR/ARNT heterodimer and binding of a protein complex containing AHR and ARNT to DRE occurred naturally in the absence of TCDD. Moreover, treatment with TCDD did not affect the AHR/ARNT dimer formation and binding of these proteins to DRE in these cells. Even though the constitutive complex on DRE exists in HeLa, transcription of the CYP1A1 gene was not increased. Furthermore, the CYP1A1 level in HeLa cells remained unchanged in the presence of TCDD suggesting repressional mechanism of the AHR complex function which may hinder the TCDD-dependent mechanisms in these cells. Similar to the native, the mouse CYP1A1-driven reporter constructs containing different regulatory elements were not inducible by TCDD in HeLa cells, which supported a presence of cell type specific trans-acting factor in HeLa cells able to repress both the native CYP1A1 and CYP1A1-driven reporter genes rather than species specific differences between CYP1A1 genes of human and rodent origin. The different regulation of the AHR-mediated transcription of CYP1A1 gene in Hepa and HeLa cells was further explored in order to elucidate two aspects of the AHR function: (I) mechanism involved in the activation of AHR in the absence of exogenous ligand and (II) factor that repress function of the exogenous ligand-independent AHR/ARNT complex. Since preliminary studies revealed that the activation of PKA causes an activation of AHR in Hepa cells in the absence of TCDD, the PKA-dependent signalling pathway was the proposed endogenous mechanism leading to the TCDD-independent activation of AHR in HeLa cells. Activation of PKA by forskolin or db-cAMP as well as inhibition of the kinase by H89 in both HeLa and Hepa cells did not lead to alterations in the AHR interaction with ARNT in the absence of TCDD and had no effect on binding of these proteins to DRE. Moreover, the modulators of PKA did not influence the CYP1A1 activity in these cells in the presence and in the absence of TCDD. Thus, an involvement of PKA in the regulation of the CYP1A1 Gen in HeLa cells was not evaluated in the course of this study. Repression of genes by transcription factors bound to their responsive elements in the absence of ligands has been described for nuclear receptors. These receptors interact with protein complex containing histone deacetylase (HDAC), enzyme responsible for the repressional effect. Thus, a participation of histone deacetylase in the transcriptional modulation of CYP1A1 gene by the constitutively DNA-bound AHR/ARNT complex was supposed. Inhibition of the HDAC activity by trichostatin A (TSA) or sodium butyrate (NaBu) led to an increase of the CYP1A1 transcription in the presence but not in the absence of TCDD in Hepa and HeLa cells. Since amount of the AHR and ARNT proteins remained unchanged upon treatment of the cells with TSA or NaBu, the transcriptional upregulation of CYP1A1 gene was not due to an increased expression of the regulatory proteins. These findings strongly suggest an involvement of HDAC in the repression of the CYP1A1 gene. Similar to the native human CYP1A1 also the mouse CYP1A1-driven reporter gene transfected into HeLa cells was repressed by histone deacetylase since the presence of TSA or NaBu led to an increase in the reporter activity. Induction of reporter gene did not require a presence of the promoter or negative regulatory regions of the CYP1A1 gene. A promoter-distal fragment containing three DREs together with surrounding sequences was sufficient to mediate the effects of the HDAC inhibitors suggesting that the AHR/ARNT binding to its specific DNA recognition site may be important for the CYP1A1 repression. Histone deacetylase is recruited to the specific genes by corepressors, proteins that bind to the transcription factors and interact with other members of the HDAC complex. Western blot analyses revealed a presence of HDAC1 and the corepressors mSin3A (mammalian homolog of yeast Sin3) and SMRT (silencing mediator for retinoid and thyroid hormone receptor) in both cell types, while the corepressor NCoR (nuclear receptor corepressor) was expressed exclusively in HeLa cells. Thus the high inducibility of CYP1A1 in Hepa cells may be due to the absence of NCoR in these cells in contrast to the non-responsive HeLa cells, where the presence of NCoR would support repression of the gene by histone deacetylase. This hypothesis was verified in reporter gene experiments where expression constructs coding for the particular members of the HDAC complex were cotransfected in Hepa cells together with the TCDD-inducible reporter constructs containing the CYP1A1 regulatory sequences. An overexpression of NCoR however did not decrease but instead led to a slight increase of the reporter gene activity in the cells. The expected inhibition was observed solely in the case of SMRT that slightly reduced constitutive and TCDD-induced reporter gene activity. A simultaneous expression of NCoR and SMRT shown no further effects and coexpression of HDAC1 with the two corepressors did not alter this situation. Thus, additional factors that are likely involved in the repression of CYP1A1 gene by HDAC complex remained to be identified. Taking together, characterisation of an exogenous ligand independent AHR/ARNT complex on DRE in HeLa cells that repress transcription of the CYP1A1 gene creates a model system enabling investigation of endogenous processes involved in the regulation of AHR function. This study implicates HDAC-mediated repression of CYP1A1 gene that contributes to the xenobiotic-induced expression in a tissue specific manner. Elucidation of these processes gains an insight into mechanisms leading to deleterious effects of TCDD and related compounds.

PAS c als signaltransduzierende Domäne des Sensorproteins DcuS von Escherichia coli

Bakterien besitzen membranintegrierte Sensoren für die Reaktion auf verändernde Umweltbedingungen.rnViele der Sensoren sind Zweikomponenten-Systeme bestehend aus einer Sensorhistidinkinase und einem Responseregulator der die zellulare Antwort auslöst. DcuS, der C4-Dicarboxylat-Sensor von DcuS ist eine membranintegrierte Histidin-Kinase. DcuS ist ein Multidomänen-Protein mit einer sensorischen periplasmatischen PASP (Per-Arnt-Sim) Domäne, zwei Transmembranhelices, eine cytoplasmatische PASC-Domäne und eine C-terminale Kinase-Domäne. PAS-Domänen sind ubiquitäre Signalmodule die in allen Reichen des Lebens zu finden sind. PAS-Domänen detektieren eine Vielfalt von Reizen wie Licht, Sauerstoff, Redoxpotential und verschiedene kleine Moleküle so wie die Modulation von Protein-Protein Interaktionen. PAS-Domänen sind strukturell homolog und besitzen eine charakteristische α/β-Faltung. Eine große Anzahl der sensorischen PAS-Domänen wurden identifiziert, aber viele der PAS-Domänen besitzen keinen apparenten Cofaktor und die Funktion ist unbekannt.rnEine Kombination aus gerichteter und ungerichteter Mutagenese, Protein-Protein-Interaktionsstudien und Festkörper-NMR (ssNMR) Experimente mit strukturellem Modelling wurde zur Untersuchung der Struktur und Funktion der cytoplasmatischen PAS-Domäne des membranintegrierten Sensors DcuS verwendet. Die Experimente zeigen, dass PASC eine wichtige Rolle in die Signaltransduktion von PASP zur C-terminalen Histidin-Kinase von DcuS spielt.rn

Transforming growth factor-beta inhibits the expression of clock genes

Disturbances of sleep-wake rhythms are an important problem in Alzheimer's disease (AD). Circadian rhythms are regulated by clock genes. Transforming growth factor-beta (TGF-β) is overexpressed in neurons in AD and is the only cytokine that is increased in cerebrospinal fluid (CSF). Our data show that TGF-β2 inhibits the expression of the clock genes Period (Per)1, Per2, and Rev-erbα, and of the clock-controlled genes D-site albumin promoter binding protein (Dbp) and thyrotroph embryonic factor (Tef). However, our results showed that TGF-β2 did not alter the expression of brain and muscle Arnt-like protein-1 (Bmal1). The concentrations of TGF-β2 in the CSF of 2 of 16 AD patients and of 1 of 7 patients with mild cognitive impairment were in the dose range required to suppress the expression of clock genes. TGF-β2-induced dysregulation of clock genes may alter neuronal pathways, which may be causally related to abnormal sleep-wake rhythms in AD patients.

5-year results of accelerated partial breast irradiation using sole interstitial multicatheter brachytherapy versus whole-breast irradiation with boost after breast-conserving surgery for low-risk invasive and in-situ carcinoma of the female breast: a randomised, phase 3, non-inferiority trial.

BACKGROUND In a phase 3, randomised, non-inferiority trial, accelerated partial breast irradiation (APBI) for patients with stage 0, I, and IIA breast cancer who underwent breast-conserving treatment was compared with whole-breast irradiation. Here, we present 5-year follow-up results. METHODS We did a phase 3, randomised, non-inferiority trial at 16 hospitals and medical centres in seven European countries. 1184 patients with low-risk invasive and ductal carcinoma in situ treated with breast-conserving surgery were centrally randomised to either whole-breast irradiation or APBI using multicatheter brachytherapy. The primary endpoint was local recurrence. Analysis was done according to treatment received. This trial is registered with ClinicalTrials.gov, number NCT00402519. FINDINGS Between April 20, 2004, and July 30, 2009, 551 patients had whole-breast irradiation with tumour-bed boost and 633 patients received APBI using interstitial multicatheter brachytherapy. At 5-year follow-up, nine patients treated with APBI and five patients receiving whole-breast irradiation had a local recurrence; the cumulative incidence of local recurrence was 1·44% (95% CI 0·51-2·38) with APBI and 0·92% (0·12-1·73) with whole-breast irradiation (difference 0·52%, 95% CI -0·72 to 1·75; p=0·42). No grade 4 late side-effects were reported. The 5-year risk of grade 2-3 late side-effects to the skin was 3·2% with APBI versus 5·7% with whole-breast irradiation (p=0·08), and 5-year risk of grade 2-3 subcutaneous tissue late side-effects was 7·6% versus 6·3% (p=0·53). The risk of severe (grade 3) fibrosis at 5 years was 0·2% with whole-breast irradiation and 0% with APBI (p=0·46). INTERPRETATION The difference between treatments was below the relevance margin of 3 percentage points. Therefore, adjuvant APBI using multicatheter brachytherapy after breast-conserving surgery in patients with early breast cancer is not inferior to adjuvant whole-breast irradiation with respect to 5-year local control, disease-free survival, and overall survival. FUNDING German Cancer Aid.

A genomewide survey of basic helix–loop–helix factors in Drosophila

The basic helix–loop–helix (bHLH) transcription factors play important roles in the specification of tissue type during the development of animals. We have used the information contained in the recently published genomic sequence of Drosophila melanogaster to identify 12 additional bHLH proteins. By sequence analysis we have assigned these proteins to families defined by Atonal, Hairy-Enhancer of Split, Hand, p48, Mesp, MYC/USF, and the bHLH-Per, Arnt, Sim (PAS) domain. In addition, one single protein represents a unique family of bHLH proteins. mRNA in situ analysis demonstrates that the genes encoding these proteins are expressed in several tissue types but are particularly concentrated in the developing nervous system and mesoderm.