Twenty years of merotelic kinetochore attachments: a historical perspective.

Micronuclei, small DNA-containing structures separate from the main nucleus, were used for decades as an indicator of genotoxic damage. Micronuclei containing whole chromosomes were considered a biomarker of aneuploidy and were believed to form, upon mitotic exit, from chromosomes that lagged behind in anaphase as all other chromosomes segregated to the poles of the mitotic spindle. However, the mechanism responsible for inducing anaphase lagging chromosomes remained unknown until just over twenty years ago. Here, I summarize what preceded and what followed this discovery, highlighting some of the open questions and opportunities for future investigation.

A guide to classifying mitotic stages and mitotic defects in fixed cells.

Cell division is fundamental to life and its perturbation can disrupt organismal development, alter tissue homeostasis, and cause disease. Analysis of mitotic abnormalities provides insight into how certain perturbations affect the fidelity of cell division and how specific cellular structures, molecules, and enzymatic activities contribute to the accuracy of this process. However, accurate classification of mitotic defects is instrumental for correct interpretation of data and formulation of new hypotheses. In this article, we provide guidelines for identifying specific mitotic stages and for classifying normal and deviant mitotic phenotypes. We hope this will clarify confusion about how certain defects are classified and help investigators avoid misnomers, misclassification, and/or misinterpretation, thus leading to a unified and standardized system to classify mitotic defects.

Exogenous glucocorticoids amplify the costs of infection by reducing resistance and tolerance, but effects are mitigated by co-infection.

Individual variation in parasite defences, such as resistance and tolerance, can underlie heterogeneity in fitness and could influence disease transmission dynamics. Glucocorticoid hormone concentrations often change in response to fluctuating environmental conditions and mediate changes in immune function, resource allocation and tissue repair. Thus, changes in glucocorticoid hormone concentrations might mediate individual variation in investment in resistance versus tolerance. In this study, we experimentally increased glucocorticoid concentrations in red-winged blackbirds ( Agelaius phoeniceus) that were naturally infected with haemosporidian parasites, and assessed changes in resistance and tolerance of infection. Glucocorticoid treatment increased burdens of Plasmodium, the parasite causing avian malaria, but only in the absence of co-infection with another Haemosporidian, Haemoproteus. Thus, glucocorticoids might reduce resistance to infection, but co-infection can mitigate the negative consequences of increased hormone concentrations. Glucocorticoid treatment also decreased tolerance of infection. We found no evidence that the inflammatory immune response or rate of red blood cell production underlie the effects of glucocorticoids on resistance and tolerance. Our findings suggest that exogenous glucocorticoids can increase the costs of haemosporidian infections by both increasing parasite numbers and reducing an individual's ability to cope with infection. These effects could scale up to impact populations of both host and parasite.

Near-tetraploid cancer cells show chromosome instability triggered by replication stress and exhibit enhanced invasiveness.

A considerable proportion of tumors exhibit aneuploid karyotypes, likely resulting from the progressive loss of chromosomes after whole-genome duplication. Here, by using isogenic diploid and near-tetraploid (4N) single-cell-derived clones from the same parental cell lines, we aimed at exploring how polyploidization affects cellular functions and how tetraploidy generates chromosome instability. Gene expression profiling in 4N clones revealed a significant enrichment of transcripts involved in cell cycle and DNA replication. Increased levels of replication stress in 4N cells resulted in DNA damage, impaired proliferation caused by a cell cycle delay during S phase, and higher sensitivity to S phase checkpoint inhibitors. In fact, increased levels of replication stress were also observed in nontransformed, proliferative posttetraploid RPE1 cells. Additionally, replication stress promoted higher levels of intercellular genomic heterogeneity and ongoing genomic instability, which could be explained by high rates of mitotic defects, and was alleviated by the supplementation of exogenous nucleosides. Finally, our data found that 4N cancer cells displayed increased migratory and invasive capacity, both in vitro and in primary colorectal tumors, indicating that tetraploidy can promote aggressive cancer cell behavior.-Wangsa, D., Quintanilla, I., Torabi, K., Vila-Casadesús, M., Ercilla, A., Klus, G., Yuce, Z., Galofré, C., Cuatrecasas, M., Lozano, J. J., Agell, N., Cimini, D., Castells, A., Ried, T., Camps, J. Near-tetraploid cancer cells show chromosome instability triggered by replication stress and exhibit enhanced invasiveness.

[Vaccination among healthcare workers in Italy: a narrative review]. / La vaccinazione negli operatori sanitari in Italia: una revisione narrativa di letteratura.

Vaccination of healthcare workers (HCWs) is a public health tool of the utmost importance and the Italian National Vaccine Prevention Plan (PNPV) 2017-2019 recommends several vaccinations in this population group. Nevertheless, vaccine hesitancy is influencing HCWs' attitude towards vaccination. Moreover, a large number of measles cases have been reported in Italy among HCWs in 2017 and 2018. In Italy there is no national registry for vaccinations, so data on vaccine coverage among HCWs are not readily accessible. The aim of this literature review is to describe the most recent data about vaccination coverage among HCWs in Italy. We also report studies that evaluated the effectiveness of strategies to increase influenza vaccine uptake. We included all studies conducted in Italy and published between 2008 and 2018, regarding vaccines recommended by the PNPV 2017-2019 (hepatitis B, influenza, pertussis, measles, mumps, rubella, varicella, and tuberculosis). Our findings confirm that low vaccination coverage levels among HCWs exist in several Italian regions and cities, highlighting a relevant gap towards targets set by the PNPV. Studies that evaluated the effectiveness of multicomponent interventions to increase vaccination coverage found only minimal to moderate increases in uptake levels. It is therefore crucial to tackle vaccine hesitancy in HCWs, by identifying effective strategies able to significantly increase vaccine coverage, in order to decrease the risk of nosocomial infections, prevent transmission of preventable diseases to patients, and reduce indirect costs related to HCW absenteeism due to illness.

Cyclophilin 20-3 relays a 12-oxo-phytodienoic acid signal during stress responsive regulation of cellular redox homeostasis.

The jasmonate family of phytohormones plays central roles in plant development and stress acclimation. However, the architecture of their signaling circuits remains largely unknown. Here we describe a jasmonate family binding protein, cyclophilin 20-3 (CYP20-3), which regulates stress-responsive cellular redox homeostasis. (+)-12-Oxo-phytodienoic acid (OPDA) binding promotes CYP20-3 to form a complex with serine acetyltransferase 1, which triggers the formation of a hetero-oligomeric cysteine synthase complex with O-acetylserine(thiol)lyase B in chloroplasts. The cysteine synthase complex formation then activates sulfur assimilation that leads to increased levels of thiol metabolites and the buildup of cellular reduction potential. The enhanced redox capacity in turn coordinates the expression of a subset of OPDA-responsive genes. Thus, we conclude that CYP20-3 is a key effector protein that links OPDA signaling to amino acid biosynthesis and cellular redox homeostasis in stress responses.

Enhancing routine immunization efforts for older adults and frail individuals: Good practices during the SARS-CoV-2 pandemic in Italy.

Infectious diseases pose a significant burden on the general population, particularly older adults who are more susceptible to severe complications. Immunization plays a crucial role in preventing infections and securing a healthier aging, but actual vaccination rates among older adults and frail individuals (OAFs) remains far from recommended targets. This study aims to collect and share good practices implemented in several Italian local health districts during the SARS-CoV-2 pandemic to ease routine immunization for OAFs. A 28-items questionnaire has been developed to collect information on organization aspect of immunization services and local good practices implemented before and during the SARS-CoV-2 pandemic. Twelve Public Health managers representative of 9 Italian Regions were further interviewed between January and March 2021. Despite literature suggests several effective interventions to increase vaccine demand, improve vaccine access, and enhance healthcare providers' performance, our survey highlighted substantial heterogeneity in their implementation at local level. Seven good local practices have been identified and described: mass vaccination centers; vaccination mobile units; drive-through vaccination; co-administration; tailored pathways; cooperation among providers involved in vaccination; digitization. Our survey pointed out valuable strategies for enhancing routine immunization for OAFs. Providers should combine effective interventions adequate to their specific context and share good practices.

The fate of extra centrosomes in newly formed tetraploid cells: should I stay, or should I go?

An increase in centrosome number is commonly observed in cancer cells, but the role centrosome amplification plays along with how and when it occurs during cancer development is unclear. One mechanism for generating cancer cells with extra centrosomes is whole genome doubling (WGD), an event that occurs in over 30% of human cancers and is associated with poor survival. Newly formed tetraploid cells can acquire extra centrosomes during WGD, and a generally accepted model proposes that centrosome amplification in tetraploid cells promotes cancer progression by generating aneuploidy and chromosomal instability. Recent findings, however, indicate that newly formed tetraploid cells in vitro lose their extra centrosomes to prevent multipolar cell divisions. Rather than persistent centrosome amplification, this evidence raises the possibility that it may be advantageous for tetraploid cells to initially restore centrosome number homeostasis and for a fraction of the population to reacquire additional centrosomes in the later stages of cancer evolution. In this review, we explore the different evolutionary paths available to newly formed tetraploid cells, their effects on centrosome and chromosome number distribution in daughter cells, and their probabilities of long-term survival. We then discuss the mechanisms that may alter centrosome and chromosome numbers in tetraploid cells and their relevance to cancer progression following WGD.

A fine balance among key biophysical factors is required for recovery of bipolar mitotic spindle from monopolar and multipolar abnormalities.

During mitosis, equal partitioning of chromosomes into two daughter cells requires assembly of a bipolar mitotic spindle. Because the spindle poles are each organized by a centrosome in animal cells, centrosome defects can lead to monopolar or multipolar spindles. However, the cell can effectively recover the bipolar spindle by separating the centrosomes in monopolar spindles and clustering them in multipolar spindles. To interrogate how a cell can separate and cluster centrosomes as needed to form a bipolar spindle, we developed a biophysical model, based on experimental data, which uses effective potential energies to describe key mechanical forces driving centrosome movements during spindle assembly. Our model identified general biophysical factors crucial for robust bipolarization of spindles that start as monopolar or multipolar. These factors include appropriate force fluctuation between centrosomes, balance between repulsive and attractive forces between centrosomes, exclusion of the centrosomes from the cell center, proper cell size and geometry, and a limited centrosome number. Consistently, we found experimentally that bipolar centrosome clustering is promoted as mitotic cell aspect ratio and volume decrease in tetraploid cancer cells. Our model provides mechanistic explanations for many more experimental phenomena and a useful theoretical framework for future studies of spindle assembly.

High-Frequency Dielectrophoresis Reveals That Distinct Bio-Electric Signatures of Colorectal Cancer Cells Depend on Ploidy and Nuclear Volume.

Aneuploidy, or an incorrect chromosome number, is ubiquitous among cancers. Whole-genome duplication, resulting in tetraploidy, often occurs during the evolution of aneuploid tumors. Cancers that evolve through a tetraploid intermediate tend to be highly aneuploid and are associated with poor patient prognosis. The identification and enrichment of tetraploid cells from mixed populations is necessary to understand the role these cells play in cancer progression. Dielectrophoresis (DEP), a label-free electrokinetic technique, can distinguish cells based on their intracellular properties when stimulated above 10 MHz, but DEP has not been shown to distinguish tetraploid and/or aneuploid cancer cells from mixed tumor cell populations. Here, we used high-frequency DEP to distinguish cell subpopulations that differ in ploidy and nuclear size under flow conditions. We used impedance analysis to quantify the level of voltage decay at high frequencies and its impact on the DEP force acting on the cell. High-frequency DEP distinguished diploid cells from tetraploid clones due to their size and intracellular composition at frequencies above 40 MHz. Our findings demonstrate that high-frequency DEP can be a useful tool for identifying and distinguishing subpopulations with nuclear differences to determine their roles in disease progression.

Computer simulations predict that chromosome movements and rotations accelerate mitotic spindle assembly without compromising accuracy.

The mitotic spindle self-assembles in prometaphase by a combination of centrosomal pathway, in which dynamically unstable microtubules search in space until chromosomes are captured, and a chromosomal pathway, in which microtubules grow from chromosomes and focus to the spindle poles. Quantitative mechanistic understanding of how spindle assembly can be both fast and accurate is lacking. Specifically, it is unclear how, if at all, chromosome movements and combining the centrosomal and chromosomal pathways affect the assembly speed and accuracy. We used computer simulations and high-resolution microscopy to test plausible pathways of spindle assembly in realistic geometry. Our results suggest that an optimal combination of centrosomal and chromosomal pathways, spatially biased microtubule growth, and chromosome movements and rotations is needed to complete prometaphase in 10-20 min while keeping erroneous merotelic attachments down to a few percent. The simulations also provide kinetic constraints for alternative error correction mechanisms, shed light on the dual role of chromosome arm volume, and compare well with experimental data for bipolar and multipolar HT-29 colorectal cancer cells.

Whole-Genome Duplication Shapes the Aneuploidy Landscape of Human Cancers.

Aneuploidy is a hallmark of cancer with tissue-specific prevalence patterns that suggest it plays a driving role in cancer initiation and progression. However, the contribution of aneuploidy to tumorigenesis depends on both cellular and genomic contexts. Whole-genome duplication (WGD) is a common macroevolutionary event that occurs in more than 30% of human tumors early in tumorigenesis. Although tumors that have undergone WGD are reported to be more permissive to aneuploidy, it remains unknown whether WGD also affects aneuploidy prevalence patterns. Here we analyzed clinical tumor samples from 5,586 WGD- tumors and 3,435 WGD+ tumors across 22 tumor types and found distinct patterns of aneuploidy in WGD- and WGD+ tumors. WGD+ tumors were characterized by more promiscuous aneuploidy patterns, in line with increased aneuploidy tolerance. Moreover, the genetic interactions between chromosome arms differed between WGD- and WGD+ tumors, giving rise to distinct cooccurrence and mutual exclusivity aneuploidy patterns. The proportion of whole-chromosome aneuploidy compared with arm-level aneuploidy was significantly higher in WGD+ tumors, indicating distinct dominant mechanisms for aneuploidy formation. Human cancer cell lines successfully reproduced these WGD/aneuploidy interactions, confirming the relevance of studying this phenomenon in culture. Finally, induction of WGD and assessment of aneuploidy in isogenic WGD-/WGD+ human colon cancer cell lines under standard or selective conditions validated key findings from the clinical tumor analysis, supporting a causal link between WGD and altered aneuploidy landscapes. We conclude that WGD shapes the aneuploidy landscape of human tumors and propose that this interaction contributes to tumor evolution. SIGNIFICANCE: These findings suggest that the interactions between whole-genome duplication and aneuploidy are important for tumor evolution, highlighting the need to consider genome status in the analysis and modeling of cancer aneuploidy.

Kinesin 5-independent poleward flux of kinetochore microtubules in PtK1 cells.

Forces in the spindle that align and segregate chromosomes produce a steady poleward flux of kinetochore microtubules (MTs [kMTs]) in higher eukaryotes. In several nonmammalian systems, flux is driven by the tetrameric kinesin Eg5 (kinesin 5), which slides antiparallel MTs toward their minus ends. However, we find that the inhibition of kinesin 5 in mammalian cultured cells (PtK1) results in only minor reduction in the rate of kMT flux from approximately 0.7 to approximately 0.5 microm/min, the same rate measured in monopolar spindles that lack antiparallel MTs. These data reveal that the majority of poleward flux of kMTs in these cells is not driven by Eg5. Instead, we favor a polar "pulling-in" mechanism in which a depolymerase localized at kinetochore fiber minus ends makes a major contribution to poleward flux. One candidate, Kif2a (kinesin 13), was detected at minus ends of fluxing kinetochore fibers. Kif2a remains associated with the ends of K fibers upon disruption of the spindle by dynein/dynactin inhibition, and these K fibers flux.

Survey of diagnostic and typing capacity for enterovirus infection in Italy and identification of two echovirus 30 outbreaks.

BACKGROUND: Enterovirus infections can cause a variety of illnesses, ranging from asymptomatic infections to severe illness and death. AIM: To support polio eradication activities, in February 2019, the WHO Regional Reference Laboratory for polio in Italy, at the National Institute of Public Health (Istituto Superiore di Sanità), promoted an investigation on non-polio enterovirus laboratory capacity, with the support of the Italian Ministry of Health. The aim was to collect data on the assays used routinely for diagnostic purposes and to characterize enterovirus outbreaks strains by sequence analysis of the Viral Protein 1 region. METHODS: A questionnaire was administered to public health laboratories through all Italian Regions for 2018 and subsequently, an electronic form for lab-confirmed enterovirus infection reported from February 2019 to January 2020, including patients clinical characteristics, and laboratory data was distributed through 25 laboratories participating the survey. RESULTS: Overall, a homogenous laboratory capacity for enterovirus infection diagnosis was found and 21,000 diagnostic tests were retrospectively reported in 2018. Then, in 2019, two outbreaks of Echovirus 30 were identified and confirmed by molecular analyses. CONCLUSION: These results underline the need monitor the circulation of non-polio enterovirus to ascertain the real burden of the disease in the country.

Aneuploidy: a matter of bad connections.

Proper chromosome segregation is required to maintain the appropriate number of chromosomes from one cell generation to the next and to prevent aneuploidy, the condition in which a cell has gained or lost one or several chromosomes during cell division. Aneuploidy is a hallmark associated with birth defects and cancer, and is observed at relatively high frequencies in human somatic cells. Recent studies in mammalian tissue culture cells suggest that the persistence of kinetochore-microtubule misattachments through mitosis is a major cause of chromosome mis-segregation and aneuploidy. Furthermore, studies in mice and humans suggest that small changes in the expression, rather than complete inactivation, of genes encoding specific proteins might be associated with aneuploidy in living organisms. In this article (which is part of the Chromosome Segregation and Aneuploidy series), we survey the outcome of these studies, focusing on the importance of kinetochore misattachments in producing aneuploid cells.

Spindle Architectural Features Must Be Considered Along With Cell Size to Explain the Timing of Mitotic Checkpoint Silencing.

Mitosis proceeds through a defined series of events that is largely conserved, but the amount of time needed for their completion can vary in different cells and organisms. In many systems, mitotic duration depends on the time required to satisfy and silence the spindle assembly checkpoint (SAC), also known as the mitotic checkpoint. Because SAC silencing involves trafficking SAC molecules among kinetochores, spindle, and cytoplasm, the size and geometry of the spindle relative to cell volume are expected to affect mitotic duration by influencing the timing of SAC silencing. However, the relationship between SAC silencing, cell size, and spindle dimensions is unclear. To investigate this issue, we used four DLD-1 tetraploid (4N) clones characterized by small or large nuclear and cell size. We found that the small 4N clones had longer mitotic durations than the parental DLD-1 cells and that this delay was due to differences in their metaphase duration. Leveraging a previous mathematical model for spatiotemporal regulation of SAC silencing, we show that the difference in metaphase duration, i.e., SAC silencing time, can be explained by the distinct spindle microtubule densities and sizes of the cell, spindle, and spindle poles in the 4N clones. Lastly, we demonstrate that manipulating spindle geometry can alter mitotic and metaphase duration, consistent with a model prediction. Our results suggest that spindle size does not always scale with cell size in mammalian cells and cell size is not sufficient to explain the differences in metaphase duration. Only when a number of spindle architectural features are considered along with cell size can the kinetics of SAC silencing, and hence mitotic duration, in the different clones be explained.

Asymmetric clustering of centrosomes defines the early evolution of tetraploid cells.

Tetraploidy has long been of interest to both cell and cancer biologists, partly because of its documented role in tumorigenesis. A common model proposes that the extra centrosomes that are typically acquired during tetraploidization are responsible for driving tumorigenesis. However, tetraploid cells evolved in culture have been shown to lack extra centrosomes. This observation raises questions about how tetraploid cells evolve and more specifically about the mechanisms(s) underlying centrosome loss. Here, using a combination of fixed cell analysis, live cell imaging, and mathematical modeling, we show that populations of newly formed tetraploid cells rapidly evolve in vitro to retain a near-tetraploid chromosome number while losing the extra centrosomes gained at the time of tetraploidization. This appears to happen through a process of natural selection in which tetraploid cells that inherit a single centrosome during a bipolar division with asymmetric centrosome clustering are favored for long-term survival.

Merotelic kinetochore orientation, aneuploidy, and cancer.

Accurate chromosome segregation in mitosis is crucial to maintain a diploid chromosome number. A majority of cancer cells are aneuploid and chromosomally unstable, i.e. they tend to gain and lose chromosomes at each mitotic division. Chromosome mis-segregation can arise when cells progress through mitosis with mis-attached kinetochores. Merotelic kinetochore orientation, a type of mis-attachment in which a single kinetochore binds microtubules from two spindle poles rather than just one, can represent a particular threat for dividing cells, as: (i) it occurs frequently in early mitosis; (ii) it is not detected by the spindle assembly checkpoint (unlike other types of mis-attachments); (iii) it can lead to chromosome mis-segregation, and, hence, aneuploidy. A number of studies have recently started to unveil the cellular and molecular mechanisms involved in merotelic kinetochore formation and correction. Here, I review these studies and discuss the relevance of merotelic kinetochore orientation in cancer cell biology.

Aurora kinase promotes turnover of kinetochore microtubules to reduce chromosome segregation errors.

Merotelic kinetochore orientation is a misattachment in which a single kinetochore binds microtubules from both spindle poles rather than just one and can produce anaphase lagging chromosomes, a major source of aneuploidy. Merotelic kinetochore orientation occurs frequently in early mitosis, does not block chromosome alignment at the metaphase plate, and is not detected by the spindle checkpoint. However, microtubules to the incorrect pole are usually significantly reduced or eliminated before anaphase. We discovered that the frequency of lagging chromosomes in anaphase is very sensitive to partial inhibition of Aurora kinase activity by ZM447439 at a dose, 3 microM, that has little effect on histone phosphorylation, metaphase chromosome alignment, and cytokinesis in PtK1 cells. Partial Aurora kinase inhibition increased the frequency of merotelic kinetochores in late metaphase, and the fraction of microtubules to the incorrect pole. Measurements of fluorescence dissipation after photoactivation showed that kinetochore-microtubule turnover in prometaphase is substantially suppressed by partial Aurora kinase inhibition. Our results support a preanaphase correction mechanism for merotelic attachments in which correct plus-end attachments are pulled away from high concentrations of Aurora B at the inner centromere, and incorrect merotelic attachments are destabilized by being pulled toward the inner centromere.