CENP-C unwraps the human CENP-A nucleosome through the H2A C-terminal tail

Centromeres are defined epigenetically by nucleosomes containing the histone H3 variant CENP-A, upon which the constitutive centromere-associated network of proteins (CCAN) is built. CENP-C is considered to be a central organizer of the CCAN. We provide new molecular insights into the structure of human CENP-A nucleosomes, in isolation and in complex with the CENP-C central region (CENP-C-CR), the main CENP-A binding module of human CENP-C. We establish that the short alpha N helix of CENP-A promotes DNA flexibility at the nucleosome ends, independently of the sequence it wraps. Furthermore, we show that, in vitro, two regions of human CENP-C (CENP-C-CR and CENP-C-motif) both bind exclusively to the CENP-A nucleosome. We find CENP-C-CR to bind with high affinity due to an extended hydrophobic area made up of CENP-A(V)(532) and CENP-A(V)(533). Importantly, we identify two key conformational changes within the CENP-A nucleosome upon CENP-C binding. First, the loose DNA wrapping of CENP-A nucleosomes is further exacerbated, through destabilization of the H2A C-terminal tail. Second, CENP-C-CR rigidifies the N-terminal tail of H4 in the conformation favoring H4(K20) monomethylation, essential for a functional centromere

Toward prevention of doping in youth sport: Cross-sectional analysis of correlates of doping tendency in swimming

Doping is recognized as one of the most important problems in sports, but a limited number of studies have investigated doping problems in youth athletes. This study aimed to evaluate doping tendency (potential doping behavior (PDB)) and correlates of PDB in youth age swimmers. The participants were 241 competitive swimmers (131 females; 15.3 ± 1.1 years of age, all under 18 years old). Variables included predictors and PDB (criterion). Predictors consisted of sociodemographic factors (gender and age), sport-related variables (i.e., experience in swimming and sport achievement), variables explaining coaching strategy and training methodology, consumption of dietary supplements (DS), knowledge about doping, and knowledge about sports nutrition and DS (KSN). In addition to the descriptive statistics and differences between genders, a multinomial regression using PDB as the criterion (negative-, neutral-, or positive-PDB, with a negative-PDB as the reference value) was calculated to define associations between predictors and criterion. With only 71% of swimmers who declared negative-PDB results indicated an alarming figure. Boys with better KSN were more negatively oriented toward positive-PDB (OR: 0.77, 95%CI: 0.60–0.95). In girls, lower competitive achievement was evidenced as a risk factor for neutral-PDB (OR: 0.39, 95%CI: 0.24–0.63). Also, higher neutral-PDB (OR: 0.88, 95%CI: 0.81–0.96) and positivePDB (OR: 0.90, 95%CI: 0.83–0.99) were identified in girls who began with intensive training in younger age. Because of the alarming figures of PDB, there is an evident need for the development of systematic antidoping educational programs in youth swimming. In doing so, focus should be placed on girls who began intensive training at an earlier age and those who did not achieve high competitive results

Identification of factors associated with potential doping behavior in sports: A cross-sectional analysis in high-level competitive swimmers

Background: Doping behavior, including the misuse of performance-enhancing drugs, is currently a serious problem in sports, and the efficacy of preventive efforts directly depends on information regarding the associations among different precipitating factors (PF) and doping behavior. This study aimed to establish the PF of potential doping behavior (PDB) in competitive swimmers. Methods: The study included 301 swimmers from Slovenia (153 females, 16.4 \ub1 2.4 years), tested during the 2017 National Championship. Variables were collected by previously validated questionnaires, which included questions on sociodemographics, sports-related factors, consumption of dietary supplements, knowledge of doping, factors of hesitation, and doping-related factors (i.e., number of doping tests, opinion about the presence of doping in sport). The PDB (positive, neutral, or negative intention toward doping) was observed as a criterion, while other variables were included as predictors in multinomial regression analyses (with \u201cnegative\u201d as reference value), which additionally controlled for gender and age category (cadet-, junior-, and senior-level) as confounders. Results: The results confirmed higher susceptibility to doping in males (positive: odds ratio (OR): 2.77, 95% confidence interval (CI): 1.27\u20136.04), those swimmers who reported higher alcohol consumption (neutral: OR: 2.18, 95%CI: 1.06\u20134.16, positive: OR: 2.14, 95%CI: 1.05\u20134.37), and those regularly used dietary supplements (positive: OR: 3.62, 95%CI: 1.25\u201310.52). Competitive achievement in Olympic-(neutral: OR: 0.57, 95%CI: 0.41\u20130.81, positive: OR: 0.59, 95%CI: 0.39\u20130.88), and non-Olympic disciplines (positive: OR: 0.54, 95%CI: 0.35\u20130.83) was protective against PDB. Swimmers who were more concerned about the negative social consequences of doping behavior (i.e., condemnation by family and friends) were less likely to declare a positive intention toward the PDB (family condemnation: OR: 0.69, 95%CI: 0.56\u20130.86, friends\u2019 condemnation: OR: 0.65, 95%CI: 0.52\u20130.80). Conclusions: The results of the study provide more precise insight into the specific factors associated with PDB in swimming. The established precipitating factors should be incorporated into targeted anti-doping campaigns in this sport

Evaluation of pre-analytical factors affecting plasma DNA analysis.

Pre-analytical factors can significantly affect circulating cell-free DNA (cfDNA) analysis. However, there are few robust methods to rapidly assess sample quality and the impact of pre-analytical processing. To address this gap and to evaluate effects of DNA extraction methods and blood collection tubes on cfDNA yield and fragment size, we developed a multiplexed droplet digital PCR (ddPCR) assay with 5 short and 4 long amplicons targeting single copy genomic loci. Using this assay, we compared 7 cfDNA extraction kits and found cfDNA yield and fragment size vary significantly. We also compared 3 blood collection protocols using plasma samples from 23 healthy volunteers (EDTA tubes processed within 1 hour and Cell-free DNA Blood Collection Tubes processed within 24 and 72 hours) and found no significant differences in cfDNA yield, fragment size and background noise between these protocols. In 219 clinical samples, cfDNA fragments were shorter in plasma samples processed immediately after venipuncture compared to archived samples, suggesting contribution of background DNA by lysed peripheral blood cells. In summary, we have described a multiplexed ddPCR assay to assess quality of cfDNA samples prior to downstream molecular analyses and we have evaluated potential sources of pre-analytical variation in cfDNA studies

Parental and familial factors influencing physical activity levels in early adolescence: a prospective study

Parental/familial factors are important determinants of the physical activity level (PAL) in children and adolescents, but studies rarely prospectively evaluate their relationships. This study aimed to evaluate the changes in physical activity levels among adolescents from Bosnia and Herzegovina over a two-year period and to determine parental/familial predictors of PAL in early adolescence. A total of 651 participants (50.3% females) were tested at baseline (beginning of high school education; 14 years old on average) and at follow-up (approximately 20 months later). The predictors included sociodemographic characteristics (age, gender) and parental/familial factors (socioeconomic status of the family, maternal and paternal education, conflict with parents, parental absence from home, parental questioning, and parental monitoring). Physical activity levels were evidenced by the Physical Activity Questionnaire for Adolescents (PAQ-A; criterion). Boys were more active than girls, both at baseline (t-test = 3.09, p < 0.001) and at follow-up (t-test = 3.4, p < 0.001). Physical activity level decreased over the observed two-year period (t-test = 16.89, p < 0.001), especially in boys, which is probably a consequence of drop-out from the sport in this period. Logistic regression evidenced parental education as a positive predictor of physical activity level at baseline (OR [95% CI]; 1.38 [1.15–170], 1.35 [1.10–1.65]), and at follow-up (1.35 [1.11–1.69], 1.29 [1.09–1.59], for maternal and paternal education, respectively). Parents with a higher level of education are probably more informed about the importance of physical activity on health status, and thus transfer this information to their children as well. The age from 14 to 16 years is likely a critical period for maintaining physical activity levels in boys, while further studies of a younger age are necessary to evaluate the dynamics of changes in physical activity levels for girls. For maintaining physical activity levels in adolescence, special attention should be paid to children whose parents are less educated, and to inform them of the benefits of an appropriate physical activity level and its necessity for maintaining proper health and growth

Prospective analysis of the influence of sport and educational factors on the prevalence and initiation of smoking in older adolescents from Croatia

The prevalence of smoking among Croatian adolescents is alarmingly high, but no previous study has prospectively examined the sport- and academic-factors associated with smoking and smoking initiation. This study aimed to prospectively examine the associations between scholastic (educational) achievement and sport factors and smoking in 16- to 18-year-old adolescents. This twoyear prospective cohort study included 644 adolescents who were 16 years of age at baseline (46% females). Baseline testing was implemented at the beginning of the 3rd year of high school (September 2014) when participants were 16 years old. Follow-up testing was completed at the end of the fourth year of high school, which occurred 20 months later. The evaluated predictor variables were educational-achievement- and sport-related-factors. The outcome variables were (i) smoking at baseline; (ii) smoking at follow-up; and (iii) smoking initiation over the course of the study. We assessed the associations between predictors and outcomes using logistic regression models adjusted for age, gender, socioeconomic status, and conflict with parents. The educational variables were consistently associated with smoking, with lower grade-point-average (Baseline: odd ratio (OR): 2.01, 95% confidence interval (CI): 1.61\u20132.55; Follow-up: 1.59, 1.31\u20131.94), more frequent absence from school (Baseline: OR: 1.40, 95% CI: 1.19\u20131.69; Follow-up: 1.30, 1.08\u20131.58), and lower behavioral grades (Baseline: OR: 1.80, 95% CI: 1.10\u20132.89; Follow-up: 1.57, 1.03\u20132.41) in children who smoke. Adolescents who reported quitting sports were at greater odds of being smokers (Baseline: 2.07, 1.31\u20133.32; Followup: 1.66, 1.09\u20132.56). Sport competitive achievement at baseline was protective against smoking initiation during following two-year period (0.45, 0.21\u20130.91). While the influence of the educational variables on smoking initiation has been found to be established earlier; sport achievement was identified as a significant protective factor against initiating smoking in older adolescents. Results should be used in development of an anti-smoking preventive campaign in older adolescents

Chromosomes. CENP-C reshapes and stabilizes CENP-A nucleosomes at the centromere

Inheritance of each chromosome depends upon its centromere. A histone H3 variant, centromere protein A (CENP-A), is essential for epigenetically marking centromere location. We find that CENP-A is quantitatively retained at the centromere upon which it is initially assembled. CENP-C binds to CENP-A nucleosomes and is a prime candidate to stabilize centromeric chromatin. Using purified components, we find that CENP-C reshapes the octameric histone core of CENP-A nucleosomes, rigidifies both surface and internal nucleosome structure, and modulates terminal DNA to match the loose wrap that is found on native CENP-A nucleosomes at functional human centromeres. Thus, CENP-C affects nucleosome shape and dynamics in a manner analogous to allosteric regulation of enzymes. CENP-C depletion leads to rapid removal of CENP-A from centromeres, indicating their collaboration in maintaining centromere identity.NIH grants: (GM082989, CA186430, GM008275, GM008216, GM007229); American Heart Association predoctoral fellowship; American Cancer Society postdoctoral fellowship; NSF grant: (agreement DMR-0944772)

Holocentric Chromosomes of Luzula elegans Are Characterized by a Longitudinal Centromere Groove, Chromosome Bending, and a Terminal Nucleolus Organizer Region

The structure of holocentric chromosomes was analyzed in mitotic cells of Luzula elegans. Light and scanning electron microscopy observations provided evidence for the existence of a longitudinal groove along each sister chromatid. The centromere-specific histone H3 variant, CENH3, colocalized with this groove and with microtubule attachment sites. The terminal chromosomal regions were CENH3-negative. During metaphase to anaphase transition, L. elegans chromosomes typically curved to a sickle-like shape, a process that is likely to be influenced by the pulling forces of microtubules along the holocentric axis towards the corresponding microtubule organizing regions. A single pair of 45S rDNA sites, situated distal to Arabidopsis-telomere repeats, was observed at the terminal region of one chromosome pair. We suggest that the 45S rDNA position in distal centromere-free regions could be required to ensure chromosome stability. Copyright (C) 2011 S. Karger AG, Base

Hedgehog pathway mutations drive oncogenic transformation in high-risk T-cell acute lymphoblastic leukemia.

The role of Hedgehog signaling in normal and malignant T-cell development is controversial. Recently, Hedgehog pathway mutations have been described in T-ALL, but whether mutational activation of Hedgehog signaling drives T-cell transformation is unknown, hindering the rationale for therapeutic intervention. Here, we show that Hedgehog pathway mutations predict chemotherapy resistance in human T-ALL, and drive oncogenic transformation in a zebrafish model of the disease. We found Hedgehog pathway mutations in 16% of 109 childhood T-ALL cases, most commonly affecting its negative regulator PTCH1. Hedgehog mutations were associated with resistance to induction chemotherapy (P = 0.009). Transduction of wild-type PTCH1 into PTCH1-mutant T-ALL cells induced apoptosis (P = 0.005), a phenotype that was reversed by downstream Hedgehog pathway activation (P = 0.007). Transduction of most mutant PTCH1, SUFU, and GLI alleles into mammalian cells induced aberrant regulation of Hedgehog signaling, indicating that these mutations are pathogenic. Using a CRISPR/Cas9 system for lineage-restricted gene disruption in transgenic zebrafish, we found that ptch1 mutations accelerated the onset of notch1-induced T-ALL (P = 0.0001), and pharmacologic Hedgehog pathway inhibition had therapeutic activity. Thus, Hedgehog-activating mutations are driver oncogenic alterations in high-risk T-ALL, providing a molecular rationale for targeted therapy in this disease