Genetic determinants of micronucleus formation in vivo.

Genomic instability arising from defective responses to DNA damage1 or mitotic chromosomal imbalances2 can lead to the sequestration of DNA in aberrant extranuclear structures called micronuclei (MN). Although MN are a hallmark of ageing and diseases associated with genomic instability, the catalogue of genetic players that regulate the generation of MN remains to be determined. Here we analyse 997 mouse mutant lines, revealing 145 genes whose loss significantly increases (n = 71) or decreases (n = 74) MN formation, including many genes whose orthologues are linked to human disease. We found that mice null for Dscc1, which showed the most significant increase in MN, also displayed a range of phenotypes characteristic of patients with cohesinopathy disorders. After validating the DSCC1-associated MN instability phenotype in human cells, we used genome-wide CRISPR-Cas9 screening to define synthetic lethal and synthetic rescue interactors. We found that the loss of SIRT1 can rescue phenotypes associated with DSCC1 loss in a manner paralleling restoration of protein acetylation of SMC3. Our study reveals factors involved in maintaining genomic stability and shows how this information can be used to identify mechanisms that are relevant to human disease biology1.

An integrated characterisation of incineration bottom ashes towards sustainable application: Physicochemical, ecotoxicological, and mechanical properties.

Environmental protection is a central concern regarding municipal solid waste incineration bottom ash (IBA) management, but the assessment of waste Hazardous Property HP14 (ecotoxicity) is still under debate. Civil engineering applications may be a suitable management strategy. This work aimed at evaluating IBA regarding mechanical behaviour and environmental hazardous potential, including a biotest battery for ecotoxicity assessment (comprising miniaturised tests), to explore its potential for safe utilization. Physical, chemical, ecotoxicological (Aliivibrio fischeri, Raphidocelis subcapitata, Lemna minor, Daphnia magna, Lepidium sativum), and mechanical (one-dimensional compressibility, shear strength) analyses were performed. The low leaching for potentially toxic metals and ions complied with European Union (EU) limit values for non-hazardous waste landfills. No relevant ecotoxicological effects were found. The biotest battery seems suitable for ecotoxicological assessment in the aquatic ecosystem, providing wide information on waste impact on different trophic/functional levels and chemical uptake routes, simultaneously involving short-duration tests and reduced amounts of waste. IBA presented more compressibility than sand, but its mixture with sand (30%:70%) was closer to sand compressibility. IBA (lower stresses) and the mixture (higher stresses) showed slightly higher shear strength than sand. Overall, IBA presented the potential for valorisation as loose aggregates from an environmental and mechanical viewpoint in a circular economy framework.

Atuação do psicólogo na equipe multiprofissional da pediatria ­ relato de caso / Psychologist's role in the multi-professional team of pediatrics - case report

INTRODUÇÃO: Durante a gestação, a mãe vivencia diversos sonhos e esperanças ao idealizar o filho perfeito. Ao dar à luz e ser informada que este deve permanecer no hospital por complicações relacionadas à saúde, constata que o filho que nasceu é diferente do que imaginou. Assim, ocorre a ruptura da imagem do filho ideal para o filho real, iniciando um processo de luto. OBJETIVO: Trabalhar a relação mãe-bebê e a comunicação entre a mãe e a equipe médica. MÉTODOS: entrevistas estruturadas e semiestruturadas e atendimentos a beira do leito e Unidade de Tratamento Intensivo (UTI). RELATO DE CASO: P.L ao nascer foi diagnosticado com Síndrome de Down e após uma semana a mãe foi comunicada sobre a cardiopatia congênita severa, o que a deixou revoltada, gerando desconfiança e constantes desentendimentos com a equipe médica. Ao ser internada nesse serviço não suportava permanecer no quarto deixando o bebê aos cuidados da equipe. Referia que se sentia sufocada e não tinha rede de apoio. Inicialmente apresentava dificuldade de interação com o bebê, realizando os cuidados essenciais, porém sem contato visual. Dessa forma, passei a me comunicar com o bebê intermediando a interação entre ambos. O vínculo mãe-terapeuta evoluiu aos poucos e, assim, compreendeu-se a dinâmica familiar e a relação com a equipe. Após um desmaio do bebê a mãe relatou que não suportaria perdê-lo, o que exigiu uma continência efetiva. Contou que devido à progressão da doença, o bebê já não interagia e se desesperou. Frente ao esgotamento físico e emocional, expressou o seu sofrimento deixando o filho sob os cuidados de uma vizinha. P.L foi encaminhado à UTI vindo a óbito poucas horas depois. Ao saber da morte do filho ficou desalentada. No leito, ao se despedir, segurou-o no colo com a ajuda da enfermeira; o abraçou, o beijou e disse o que o amava e pediu para que Deus o protegesse. Ela segurou e apoiou em mim; o que só foi possível pelo vinculo estabelecido. Assim, pude acolher o seu sofrimento proporcionando sustentação para que ela vivenciasse a perda do filho. RESULTADO: Com os atendimentos, observou-se que através do suporte psicológico, a mãe começou a manifestar seus desejos, sonhos e expressar emoções e sentimentos. Notou-se uma melhor interação no vinculo mãe e bebê. Foi possível trabalhar o enfrentamento da morte do filho. Também, mediar o conflito e a comunicação entre equipe e família. CONCLUSÃO: A atuação do psicólogo é essencial no período de internação, intervindo não apenas com o paciente e familiar, mas também com a equipe multidisciplinar. (AU)

From Meiosis to Mitosis: The Astonishing Flexibility of Cell Division Mechanisms in Early Mammalian Development.

The execution of female meiosis and the establishment of the zygote is arguably the most critical stage of mammalian development. The egg can be arrested in the prophase of meiosis I for decades, and when it is activated, the spindle is assembled de novo. This spindle must function with the highest of fidelity and yet its assembly is unusually achieved in the absence of conventional centrosomes and with minimal influence of chromatin. Moreover, its dramatic asymmetric positioning is achieved through remarkable properties of the actin cytoskeleton to ensure elimination of the polar bodies. The second meiotic arrest marks a uniquely prolonged metaphase eventually interrupted by egg activation at fertilization to complete meiosis and mark a period of preparation of the male and female pronuclear genomes not only for their entry into the mitotic cleavage divisions but also for the imminent prospect of their zygotic expression.

Chromatin remodeling and neuronal function: exciting links.

Regulation of gene expression occurs at different levels, from DNA to protein, and through various mechanisms. One of them is modification of the chromatin structure, which is involved in the definition of transcriptional active and inactive regions of the chromosomes. These phenomena are associated with reversible chemical modifications of the genetic material rather than with variability within the DNA sequences inherited by the individual and are therefore called 'epigenetic' modifications. Ablation of the molecular players responsible for epigenetic modifications often gives rise to neurological and behavioral phenotypes in humans and in mouse models, suggesting a relevant function for chromatin remodeling in central nervous system function, particularly in the adaptive response of the brain to stimuli. We will discuss several human disorders that are due to altered epigenetic mechanisms, with special focus on Rett syndrome.

A role for Drosophila SMC4 in the resolution of sister chromatids in mitosis.

BACKGROUND: Faithful segregation of the genome during mitosis requires interphase chromatin to be condensed into well-defined chromosomes. Chromosome condensation involves a multiprotein complex known as condensin that associates with chromatin early in prophase. Until now, genetic analysis of SMC subunits of the condensin complex in higher eukaryotic cells has not been performed, and consequently the detailed contribution of different subunits to the formation of mitotic chromosome morphology is poorly understood. RESULTS: We show that the SMC4 subunit of condensin is encoded by the essential gluon locus in Drosophila. DmSMC4 contains all the conserved domains present in other members of the structural-maintenance-of-chromosomes protein family. DmSMC4 is both nuclear and cytoplasmic during interphase, concentrates on chromatin during prophase, and localizes to the axial chromosome core at metaphase and anaphase. During decondensation in telophase, most of the DmSMC4 leaves the chromosomes. An examination of gluon mutations indicates that SMC4 is required for chromosome condensation and segregation during different developmental stages. A detailed analysis of mitotic chromosome structure in mutant cells indicates that although the longitudinal axis can be shortened normally, sister chromatid resolution is strikingly disrupted. This phenotype then leads to severe chromosome segregation defects, chromosome breakage, and apoptosis. CONCLUSIONS: Our results demonstrate that SMC4 is critically important for the resolution of sister chromatids during mitosis prior to anaphase onset.

Pattern of chromosomal localization of the Hoppel transposable element family in the Drosophila melanogaster subgroup.

We have isolated a Hoppel-like transposon from heterochromatin of the second chromosome of Drosophila melanogaster and used a conserved DNA sequence between the different elements of this family to determine their distribution in both mitotic and polytene chromosomes. The hybridization pattern of polytene chromosomes extends throughout the entire chromocentre, as well as a substantial portion of the fourth chromosome. Analysis of different wild-type strains of D. melanogaster shows variation in euchromatic insertion sites, although most insertions are found near the chromocentre. The positions and the number of heterochromatic clusters of Hoppel on mitotic chromosomes are conserved among the several strains analysed. Accurate mapping of this element was achieved by in situ hybridization on D. melanogaster mitotic chromosomes that had previously been banded with Hoechst 33258. To evaluate the evolutionary stability of this pattern, different species were analysed by in situ hybridization and Southern blotting. We conclude that Hoppel has a conserved distribution in mitotic heterochromatin within the D. melanogaster subgroup, established around 5 million years ago. The overall conservation of heterochormatic organization supports the notion that heterochormatin does perform important structural and functional roles.

Identification of Porto-1, a new repeated sequence that localises close to the centromere of chromosome 2 of Drosophila melanogaster.

We have used the polymerase chain reaction (PCR) technique to search the Drosophila melanogaster genome for the presence of sequences with homology to mammalian and yeast centromeric DNA. Using primers based on the human CENP-B box present in alpha-satellite DNA and part of the Saccharomyces cerevisiae CDEIII centromeric sequence, a number of specific DNA fragments were amplified from total genomic DNA. In situ hybridization to polytene and mitotic chromosomes showed these fragments to localise to centromeric and pericentromeric regions. Direct cloning of the amplified fragments into conventional plasmids proved unsuccessful. However, a recombinant P1 clone containing D. melanogaster genomic DNA that supports PCR amplification by the primers was identified. Molecular characterisation of this clone revealed a DNA fragment that localises primarily to the centromere of chromosome 2. Sequence analysis indicated that this fragment contains at least four different repeats, including Rsp, transposable elements, Bari-1 and a new AT-rich repeated sequence that we have designated Porto-1. Detailed fluorescence in situ hybridization analysis shows that Porto-1 is localised very close to the primary constriction of chromosome 2. Sequence analysis suggests that this repeat was specifically amplified by our primers, although limited homology to the CENP-B box or CDEIII elements was found. In situ hybridization to a number of Drosophila species shows Porto-1 to be present only in D. melanogaster.

The elusive centromere: sequence divergence and functional conservation.

The centromere is an essential cis-acting structure present in the chromosomes of all eukaryotes, central to the mechanism that ensures proper segregation during meiosis and mitosis. Molecular characterization of centromeres in the budding and fission yeasts has advanced significantly over the last few years due to their relatively small size and the availability of functional assays. However, identification and characterization of centromeric sequences from multicellular organisms has proven to be slow and difficult in the absence of direct functional tests. Molecular data have recently become available on the centromere of Drosophila, making it possible to bridge a long-standing gap in our knowledge on the general structure of centromeres. An evaluation of the available data from yeast to man suggests that centromere sequence and centromere sequence organization have diverged significantly, even amongst different chromosomes of a single organism; however, overall centromere organization and kinetochore components might be significantly more conserved than thought previously.