Nutritional Status and Daytime Pattern of Protein Intake on Match, Post-Match, Rest and Training Days in Senior Professional and Youth Elite Soccer Players

The nutritional status of elite soccer players across match, post-match, training and rest days has not been defined. Recent evidence suggests the pattern of dietary protein intake impacts the daytime turnover of muscle proteins and, as such, influences muscle recovery. We assessed the nutritional status and daytime pattern of protein intake in senior professional and elite youth soccer players and compared findings against published recommendations. Fourteen senior professional (SP) and fifteen youth elite (YP)soccer players fromtheDutch premier division completed nutritional assessments using a 24-h web-based recall method. Recall days consisted of a match, post-match, rest and training day. Daily energy intake over the 4-day period was similar between SP (2988±583 kcal/day) and YP (2938±465 kcal/day;p=0.800). Carbohydrate intake over the combined 4-day period was lower in SP (4.7±0.7 g·kg-1BM·day-1)vs.YP(6.0±1.5 g·kg-1BM·day-1,p=0.006) and SP failed to meet recommended carbohydrate intakes on match and training days. Conversely, recommended protein intakes were met for SP (1.9±0.3 g·kg-1BM·day-1) and YP (1.7±0.4 g·kg-1BM·day-1), with no differences between groups (p=0.286). Accordingly, both groups met or exceeded recommended daily protein intakes on individual match, post-match, rest and training days. A similar ‘balanced’ daytime pattern of protein intake was observed in SP and YP. To conclude, SP increased protein intake on match and training days to a greater extent than YP, however at the expense of carbohydrate intake. The daytime distribution of protein intake for YP and SP aligned with current recommendations of a balanced protein meal pattern

A kinesin-based approach for inducing chromosome-specific mis-segregation in human cells

Various cancer types exhibit characteristic and recurrent aneuploidy patterns. The origins of these cancer type-specific karyotypes are still unknown, partly because introducing or eliminating specific chromosomes in human cells still poses a challenge. Here, we describe a novel strategy to induce mis-segregation of specific chromosomes in different human cell types. We employed Tet repressor or nuclease-dead Cas9 to link a microtubule minus-end-directed kinesin (Kinesin14VIb) from Physcomitrella patens to integrated Tet operon repeats and chromosome-specific endogenous repeats, respectively. By live- and fixed-cell imaging, we observed poleward movement of the targeted loci during (pro)metaphase. Kinesin14VIb-mediated pulling forces on the targeted chromosome were counteracted by forces from kinetochore-attached microtubules. This tug-of-war resulted in chromosome-specific segregation errors during anaphase and revealed that spindle forces can heavily stretch chromosomal arms. By single-cell whole-genome sequencing, we established that kinesin-induced targeted mis-segregations predominantly result in chromosomal arm aneuploidies after a single cell division. Our kinesin-based strategy opens the possibility to investigate the immediate cellular responses to specific aneuploidies in different cell types; an important step toward understanding how tissue-specific aneuploidy patterns evolve.</p

Gene copy-number changes and chromosomal instability induced by aneuploidy confer resistance to chemotherapy

Mitotic errors lead to aneuploidy, a condition of karyotype imbalance, frequently found in cancer cells. Alterations in chromosome copy number induce a wide variety of cellular stresses, including genome instability. Here, we show that cancer cells might exploit aneuploidy-induced genome instability and the resulting gene copy-number changes to survive under conditions of selective pressure, such as chemotherapy. Resistance to chemotherapeutic drugs was dictated by the acquisition of recurrent karyotypes, indicating that gene dosage might play a role in driving chemoresistance. Thus, our study establishes a causal link between aneuploidy-driven changes in gene copy number and chemoresistance and might explain why some chemotherapies fail to succeed

A kinesin-based approach for inducing chromosome-specific mis-segregation in human cells

Various cancer types exhibit characteristic and recurrent aneuploidy patterns. The origins of these cancer type-specific karyotypes are still unknown, partly because introducing or eliminating specific chromosomes in human cells still poses a challenge. Here, we describe a novel strategy to induce mis-segregation of specific chromosomes in different human cell types. We employed Tet repressor or nuclease-dead Cas9 to link a microtubule minus-end-directed kinesin (Kinesin14VIb) from Physcomitrella patens to integrated Tet operon repeats and chromosome-specific endogenous repeats, respectively. By live- and fixed-cell imaging, we observed poleward movement of the targeted loci during (pro)metaphase. Kinesin14VIb-mediated pulling forces on the targeted chromosome were counteracted by forces from kinetochore-attached microtubules. This tug-of-war resulted in chromosome-specific segregation errors during anaphase and revealed that spindle forces can heavily stretch chromosomal arms. By single-cell whole-genome sequencing, we established that kinesin-induced targeted mis-segregations predominantly result in chromosomal arm aneuploidies after a single cell division. Our kinesin-based strategy opens the possibility to investigate the immediate cellular responses to specific aneuploidies in different cell types; an important step toward understanding how tissue-specific aneuploidy patterns evolve

A kinesin-based approach for inducing chromosome-specific mis-segregation in human cells

Various cancer types exhibit characteristic and recurrent aneuploidy patterns. The origins of these cancer type-specific karyotypes are still unknown, partly because introducing or eliminating specific chromosomes in human cells still poses a challenge. Here, we describe a novel strategy to induce mis-segregation of specific chromosomes in different human cell types. We employed Tet repressor or nuclease-dead Cas9 to link a microtubule minus-end-directed kinesin (Kinesin14VIb) from Physcomitrella patens to integrated Tet operon repeats and chromosome-specific endogenous repeats, respectively. By live- and fixed-cell imaging, we observed poleward movement of the targeted loci during (pro)metaphase. Kinesin14VIb-mediated pulling forces on the targeted chromosome were counteracted by forces from kinetochore-attached microtubules. This tug-of-war resulted in chromosome-specific segregation errors during anaphase and revealed that spindle forces can heavily stretch chromosomal arms. By single-cell whole-genome sequencing, we established that kinesin-induced targeted mis-segregations predominantly result in chromosomal arm aneuploidies after a single cell division. Our kinesin-based strategy opens the possibility to investigate the immediate cellular responses to specific aneuploidies in different cell types; an important step toward understanding how tissue-specific aneuploidy patterns evolve

Genetic instability from a single S phase after whole-genome duplication

Diploid and stable karyotypes are associated with health and fitness in animals. By contrast, whole-genome duplications—doublings of the entire complement of chromosomes—are linked to genetic instability and frequently found in human cancers(1–3). It has been established that whole-genome duplications fuel chromosome instability through abnormal mitosis(4–8); however, the immediate consequences of tetraploidy in the first interphase are not known. This is a key question because single whole-genome duplication events such as cytokinesis failure can promote tumorigenesis(9). Here we find that human cells undergo high rates of DNA damage during DNA replication in the first S phase following induction of tetraploidy. Using DNA combing and single-cell sequencing, we show that DNA replication dynamics is perturbed, generating under- and over-replicated regions. Mechanistically, we find that these defects result from a shortage of proteins during the G1/S transition, which impairs the fidelity of DNA replication. This work shows that within a single interphase, unscheduled tetraploid cells can acquire highly abnormal karyotypes. These findings provide an explanation for the genetic instability landscape that favours tumorigenesis after tetraploidization

Chromosomal Instability Characterizes Pediatric Medulloblastoma but Is Not Tolerated in the Developing Cerebellum

Medulloblastoma is a pediatric brain malignancy that consists of four transcriptional subgroups. Structural and numerical aneuploidy are common in all subgroups, although they are particularly profound in Group 3 and Group 4 medulloblastoma and in a subtype of SHH medulloblastoma termed SHH alpha. This suggests that chromosomal instability (CIN), the process leading to aneuploidy, is an important player in medulloblastoma pathophysiology. However, it is not known if there is ongoing CIN in medulloblastoma or if CIN affects the developing cerebellum and promotes tumor formation. To investigate this, we performed karyotyping of single medulloblastoma cells and demonstrated the presence of distinct tumor cell clones harboring unique copy number alterations, which is suggestive of ongoing CIN. We also found enrichment for processes related to DNA replication, repair, and mitosis in both SHH medulloblastoma and in the highly proliferative compartment of the presumed tumor cell lineage-of-origin, the latter also being sensitive to genotoxic stress. However, when challenging these tumor cells-of-origin with genetic lesions inducing CIN using transgenic mouse modeling, we found no evidence for large chromosomal aberrations in the cerebellum or for medulloblastoma formation. We therefore conclude that without a background of specific genetic mutations, CIN is not tolerated in the developing cerebellum in vivo and, thus, by itself is not sufficient to initiate medulloblastoma

A living biobank of ovarian cancer ex vivo models reveals profound mitotic heterogeneity

High-grade serous ovarian carcinoma is characterised by TP53 mutation and extensive chromosome instability (CIN). Because our understanding of CIN mechanisms is based largely on analysing established cell lines, we developed a workflow for generating ex vivo cultures from patient biopsies to provide models that support interrogation of CIN mechanisms in cells not extensively cultured in vitro. Here, we describe a “living biobank” of ovarian cancer models with extensive replicative capacity, derived from both ascites and solid biopsies. Fifteen models are characterised by p53 profiling, exome sequencing and transcriptomics, and karyotyped using single-cell whole-genome sequencing. Time-lapse microscopy reveals catastrophic and highly heterogeneous mitoses, suggesting that analysis of established cell lines probably underestimates mitotic dysfunction in advanced human cancers. Drug profiling reveals cisplatin sensitivities consistent with patient responses, demonstrating that this workflow has potential to generate personalized avatars with advantages over current pre-clinical models and the potential to guide clinical decision making

The H3.3K27M oncohistone affects replication stress outcome and provokes genomic instability in pediatric glioma

While comprehensive molecular profiling of histone H3.3 mutant pediatric high-grade glioma has revealed extensive dysregulation of the chromatin landscape, the exact mechanisms driving tumor formation remain poorly understood. Since H3.3 mutant gliomas also exhibit high levels of copy number alterations, we set out to address if the H3.3K27M oncohistone leads to destabilization of the genome. Hereto, we established a cell culture model allowing inducible H3.3K27M expression and observed an increase in mitotic abnormalities. We also found enhanced interaction of DNA replication factors with H3.3K27M during mitosis, indicating replication defects. Further functional analyses revealed increased genomic instability upon replication stress, as represented by mitotic bulky and ultrafine DNA bridges. This co-occurred with suboptimal 53BP1 nuclear body formation after mitosis in vitro, and in human glioma. Finally, we observed a decrease in ultrafine DNA bridges following deletion of the K27M mutant H3F3A allele in primary high-grade glioma cells. Together, our data uncover a role for H3.3 in DNA replication under stress conditions that is altered by the K27M mutation, promoting genomic instability and potentially glioma development

Aneuploidy renders cancer cells vulnerable to mitotic checkpoint inhibition

Selective targeting of aneuploid cells is an attractive strategy for cancer treatment(1). Here, we mapped the aneuploidy landscapes of ~1,000 human cancer cell lines, and analyzed genetic and chemical perturbation screens(2–9) to reveal aneuploidy-associated cellular vulnerabilities. We identified and validated an increased sensitivity of aneuploid cancer cells to genetic perturbation of core components of the spindle assembly checkpoint (SAC), which ensures the proper segregation of chromosomes during mitosis(10). Surprisingly, we also found aneuploid cancer cells to be less sensitive to short-term exposures to multiple SAC inhibitors. Indeed, aneuploid cancer cells became increasingly more sensitive to SAC inhibition (SACi) over time. Aneuploid cells exhibited aberrant spindle geometry and dynamics, and kept dividing in the presence of SACi, resulting in accumulating mitotic defects, and in unstable and less fit karyotypes. Therefore, although aneuploid cancer cells could overcome SACi more readily than diploid cells, their long-term proliferation was jeopardized. We identified a specific mitotic kinesin, KIF18A, whose activity was perturbed in aneuploid cancer cells. Aneuploid cancer cells were particularly vulnerable to KIF18A depletion, and KIF18A overexpression restored their response to SACi. Our study reveals a novel, therapeutically-relevant, synthetic lethal interaction between aneuploidy and the SAC