Micronuclei from misaligned chromosomes that satisfy the spindle assembly checkpoint in cancer cells.

Chromosome alignment to the spindle equator is a hallmark of mitosis thought to promote chromosome segregation fidelity in metazoans. Yet chromosome alignment is only indirectly supervised by the spindle assembly checkpoint (SAC) as a byproduct of chromosome bi-orientation, and the consequences of defective chromosome alignment remain unclear. Here, we investigated how human cells respond to chromosome alignment defects of distinct molecular nature by following the fate of live HeLa cells after RNAi-mediated depletion of 125 proteins previously implicated in chromosome alignment. We confirmed chromosome alignment defects upon depletion of 108/125 proteins. Surprisingly, in all confirmed cases, depleted cells frequently entered anaphase after a delay with misaligned chromosomes. Using depletion of prototype proteins resulting in defective chromosome alignment, we show that misaligned chromosomes often satisfy the SAC and directly missegregate without lagging behind in anaphase. In-depth analysis of specific molecular perturbations that prevent proper kinetochore-microtubule attachments revealed that misaligned chromosomes that missegregate frequently result in micronuclei. Higher-resolution live-cell imaging indicated that, contrary to most anaphase lagging chromosomes that correct and reintegrate the main nuclei, misaligned chromosomes are a strong predictor of micronuclei formation in a cancer cell model of chromosomal instability, but not in non-transformed near-diploid cells. We provide evidence supporting that intrinsic differences in kinetochore-microtubule attachment stability on misaligned chromosomes account for this distinct outcome. Thus, misaligned chromosomes that satisfy the SAC may represent a previously overlooked mechanism driving chromosomal/genomic instability during cancer cell division, and we unveil genetic conditions predisposing for these events.

An anaphase surveillance mechanism prevents micronuclei formation from frequent chromosome segregation errors.

Micronuclei are a hallmark of cancer and several other human disorders. Recently, micronuclei were implicated in chromothripsis, a series of massive genomic rearrangements that may drive tumor evolution and progression. Here, we show that Aurora B kinase mediates a surveillance mechanism that integrates error correction during anaphase with spatial control of nuclear envelope reassembly to prevent micronuclei formation. Using high-resolution live-cell imaging of human cancer and non-cancer cells, we uncover that anaphase lagging chromosomes are more frequent than previously anticipated, yet they rarely form micronuclei. Micronuclei formation from anaphase lagging chromosomes is prevented by a midzone-based Aurora B phosphorylation gradient that stabilizes kinetochore-microtubule attachments and assists spindle forces required for anaphase error correction while delaying nuclear envelope reassembly on lagging chromosomes, independently of microtubule density. We propose that a midzone-based Aurora B phosphorylation gradient actively monitors and corrects frequent chromosome segregation errors to prevent micronuclei formation during human cell division.

Paragem cardiorrespiratória em contexto de Unidade de Cuidados Continuados: vivências dos enfermeiro / Cardiorespiratory arrest in a Long Term Care Unit context: nurses' experiences

A Paragem Cardiorrespiratória é uma situação que exige uma resposta imediata e eficiente por parte dos profissionais de saúde. Por este facto, os enfermeiros têm de estar preparados para atuar perante estas situações. Quando confrontados com estes eventos, os enfermeiros sentem muitas dificuldades e preocupações, uma vez que, nas Unidades de Cuidados Continuados, a paragem cardiorrespiratória ocorre com pouca frequência, gerando sentimentos de insegurança, stress e dilemas éticos. Desta forma, torna-se pertinente perceber quais as vivências dos enfermeiros perante este evento, para que as práticas clínicas no atendimento a estes utentes, possam levar a uma reflexão e consequente melhoria na qualidade de cuidados. Estruturamos este estudo com o objetivo de compreender as vivências dos enfermeiros na intervenção face à pessoa em Paragem Cardiorrespiratória, numa Unidade de Cuidados Continuados. A análise seguiu uma abordagem qualitativa, de cariz exploratório-descritivo. Como instrumento de recolha de dados recorreu-se à entrevista semiestruturada dirigida a enfermeiros que trabalham numa unidade de Cuidados Continuados da zona norte de Portugal. Os procedimentos ético-moral foram respeitados. Deste estudo resultaram seis áreas temáticas aglutinando várias categorias e sub categorias. Através da análise de dados foi possível verificar que perante uma situação de Paragem Cardiorrespiratória, os enfermeiros experienciam sentimentos e emoções como o stress, a ansiedade, o medo, a tristeza, o confronto com a morte, a dúvida face à qualidade da sua intervenção e a impotência, perante o evento critico. No que respeita à perceção dos enfermeiros em relação à sua prática durante a Paragem Cardiorrespiratória, estes veem a mesma como uma situação de aprendizagem e como um fator de ajuda na gestão de emoções. Como fatores dificultadores foram enumerados pelos participantes, os seguintes: inexperiência profissional; escassez de ocorrências de Paragem Cardiorrespiratória; infuncionalidade ou escassez equipamento/material; recursos humanos insuficientes; cansaço físico; dificuldades na gestão de emoções; qualidade de atuação das equipas de emergência pré-hospitalar deficitária. Já os fatores potencializadores da qualidade de atuação partilhados pelos entrevistados foram o trabalho em equipa; formação pessoal e da equipa; experiência profissional; exposição a situações de Paragem Cardiorrespiratória e gestão de emoções adequada. Como estratégias de melhoria de intervenção os enfermeiros referiram o debriefing, a formação continua e propostas à instituição, como a manutenção de equipamento e a aquisição de material. Referem como principal implicação da atuação em Paragem Cardiorrespiratória, o desenvolvimento de competências, nesta área específica. Estes resultados sugerem que é importante implementar estratégias que favoreçam a formação continua e a partilha de experiências entre as equipas, que resultarão numa melhor intervenção na Paragem Cardiorrespiratória, das equipas das Unidades de Cuidados Continuados.

Cardiopulmonary arrest is a situation that requires immediate and efficient response from health professionals. For this reason, nurses have to be prepared to act in these situations. When confronted with these events, nurses feel many difficulties and worry, since in the Continuous Care Units, a CRP occurs infrequently, generating feelings of insecurity, stress and ethical problems. Thus, it is pertinent to understand what are the experiences of nurses considered this event, so that clinical practices in the care of patients in CRP can lead to reflection and consequent improvement in the quality of care. We performed this study to understand the feelings of the nurses when they face a cardiopulmonary arrest in a Continuing Care Unit. The analysis followed a qualitative, exploratory-descriptive approach. Regards data collection, we used a semi-structured interview directed to nurses in a continuous care unit in the north of Portugal. All ethicalmoral procedures were respected. This study resulted in six thematic areas bringing together several categories and sub categories. Through data analysis, it was possible to verify that, in a cardiopulmonary arrest situation, nurses experience feelings and emotions such as stress, anxiety, fear, sadness, confrontation with death, doubt regarding the quality of their intervention and impotence in the face of the critical event. Concerning nurses' perception of their practice during the Cardiopulmonary Resuscitation, they see it as a learning situation and as a help factor in managing emotions. The following factors were difficult factors listed by the participants: professional inexperience; scarcity of occurrences of cardiac arrest; nonfunctionality or scarcity equipment / material; insufficient human resources; physical tiredness; difficulties in managing emotions; quality of action of the prehospital emergency teams with a deficit. On the other hand, the factors that potentiate the quality of the actions shared by the interviewees were teamwork; personal and team training; professional experience; exposure to cardiopulmonary arrest situations and proper emotion management. As intervention improvement strategies, nurses referred to debriefing, continuing education and proposals to the institution, such as equipment maintenance and material acquisition. Refer as the main implication of acting in Cardiorespiratory arrest, the development of skills in this specific area. These results suggest that it is important to implement strategies that favor continuous training and the sharing of experiences between the teams, which will result in a better intervention in the cardiopulmonary arrest of the teams of the Continuous Care Units.

Mechanisms of Chromosome Congression during Mitosis.

Chromosome congression during prometaphase culminates with the establishment of a metaphase plate, a hallmark of mitosis in metazoans. Classical views resulting from more than 100 years of research on this topic have attempted to explain chromosome congression based on the balance between opposing pulling and/or pushing forces that reach an equilibrium near the spindle equator. However, in mammalian cells, chromosome bi-orientation and force balance at kinetochores are not required for chromosome congression, whereas the mechanisms of chromosome congression are not necessarily involved in the maintenance of chromosome alignment after congression. Thus, chromosome congression and maintenance of alignment are determined by different principles. Moreover, it is now clear that not all chromosomes use the same mechanism for congressing to the spindle equator. Those chromosomes that are favorably positioned between both poles when the nuclear envelope breaks down use the so-called "direct congression" pathway in which chromosomes align after bi-orientation and the establishment of end-on kinetochore-microtubule attachments. This favors the balanced action of kinetochore pulling forces and polar ejection forces along chromosome arms that drive chromosome oscillatory movements during and after congression. The other pathway, which we call "peripheral congression", is independent of end-on kinetochore microtubule-attachments and relies on the dominant and coordinated action of the kinetochore motors Dynein and Centromere Protein E (CENP-E) that mediate the lateral transport of peripheral chromosomes along microtubules, first towards the poles and subsequently towards the equator. How the opposite polarities of kinetochore motors are regulated in space and time to drive congression of peripheral chromosomes only now starts to be understood. This appears to be regulated by position-dependent phosphorylation of both Dynein and CENP-E and by spindle microtubule diversity by means of tubulin post-translational modifications. This so-called "tubulin code" might work as a navigation system that selectively guides kinetochore motors with opposite polarities along specific spindle microtubule populations, ultimately leading to the congression of peripheral chromosomes. We propose an integrated model of chromosome congression in mammalian cells that depends essentially on the following parameters: (1) chromosome position relative to the spindle poles after nuclear envelope breakdown; (2) establishment of stable end-on kinetochore-microtubule attachments and bi-orientation; (3) coordination between kinetochore- and arm-associated motors; and (4) spatial signatures associated with post-translational modifications of specific spindle microtubule populations. The physiological consequences of abnormal chromosome congression, as well as the therapeutic potential of inhibiting chromosome congression are also discussed.

Determination of ABCB1 polymorphisms and haplotypes frequencies in a French population.

The ATP-binding cassette (ABC) transporter ABCB1, or P-glycoprotein, is a transmembrane efflux pump well known for its implication in drug transport and chemoresistance. ABCB1 substrates include either drugs, such as antiretrovirals and immunomodulators, or physiological molecules like phospholipids. Pharmacogenetic analysis of ABCB1 polymorphisms, in addition to other xenobiotic metabolizing enzymes, might help to personalize and optimize drug therapy. Indeed, some polymorphisms of ABCB1 have been implicated in susceptibility to diseases, changes in drug pharmacokinetics, and in variation of the biological response to drug treatment. In addition, variant and haplotype distributions differ depending on ethnicity. Thus, some ethnies may be at higher risk for adverse events, inefficacy of treatment or prevalence of pathologies. This study aimed to determine frequencies of ABCB1 polymorphisms and haplotypes in a sample of French healthy individuals. DNA was isolated from blood-EDTA. Polymerase chain reaction-restriction fragment length polymorphism and TaqMan single nucleotide polymorphism genotyping assays were used to genotype 227 individuals for T-129C, G-1A, A61G, G1199A, C1236T, T-76A, G2677T/A and C3435T polymorphisms. The observed frequencies of the variant allele for these eight polymorphisms are 0.04, 0.08, 0.09, 0.06, 0.42, 0.46, 0.45 and 0.46 respectively. These polymorphisms are in linkage disequilibrium and haplotype frequencies were determined, the most frequent haplotype being the one with variants at position 1236, 2677 and 3435 and wild-type alleles at the other positions. Finally, the frequencies of these eight ABCB1 polymorphisms in our French individuals supposed to be healthy population are quite similar to those described in other Caucasian populations except for the C3435T polymorphism.