Dicentric chromosomes are resolved through breakage and repair at their centromeres.

Chromosomes with two centromeres provide a unique opportunity to study chromosome breakage and DNA repair using completely endogenous cellular machinery. Using a conditional transcriptional promoter to control the second centromere, we are able to activate the dicentric chromosome and follow the appearance of DNA repair products. We find that the rate of appearance of DNA repair products resulting from homology-based mechanisms exceeds the expected rate based on their limited centromere homology (340 bp) and distance from one another (up to 46.3 kb). In order to identify whether DNA breaks originate in the centromere, we introduced 12 single-nucleotide polymorphisms (SNPs) into one of the centromeres. Analysis of the distribution of SNPs in the recombinant centromeres reveals that recombination was initiated with about equal frequency within the conserved centromere DNA elements CDEII and CDEIII of the two centromeres. The conversion tracts range from about 50 bp to the full length of the homology between the two centromeres (340 bp). Breakage and repair events within and between the centromeres can account for the efficiency and distribution of DNA repair products. We propose that in addition to providing a site for kinetochore assembly, the centromere may be a point of stress relief in the face of genomic perturbations.

Polymer models reveal how chromatin modification can modulate force at the kinetochore.

A key feature of chromosome segregation is the ability to sense tension between sister kinetochores. DNA between sister kinetochores must be packaged in a way that sustains tension propagation from one kinetochore to its sister, approximately 1 micron away. A molecular bottlebrush consisting of a primary axis populated with a crowded array of side chains provides a means to build tension over length scales considerably larger than the stiffness of the individual elements, that is, DNA polymer. Evidence for the bottlebrush organization of chromatin between sister kinetochores comes from genetic, cell biological, and polymer modeling of the budding yeast centromere. In this study, we have used polymer dynamic simulations of the bottlebrush to recapitulate experimental observations of kinetochore structure. Several aspects of the spatial distribution of kinetochore proteins and their response to perturbation lack a mechanistic understanding. Changes in physical parameters of bottlebrush, DNA stiffness, and DNA loops directly impact the architecture of the inner kinetochore. This study reveals that the bottlebrush is an active participant in building tension between sister kinetochores and proposes a mechanism for chromatin feedback to the kinetochore.

Behavior of dicentric chromosomes in budding yeast.

DNA double-strand breaks arise in vivo when a dicentric chromosome (two centromeres on one chromosome) goes through mitosis with the two centromeres attached to opposite spindle pole bodies. Repair of the DSBs generates phenotypic diversity due to the range of monocentric derivative chromosomes that arise. To explore whether DSBs may be differentially repaired as a function of their spatial position in the chromosome, we have examined the structure of monocentric derivative chromosomes from cells containing a suite of dicentric chromosomes in which the distance between the two centromeres ranges from 6.5 kb to 57.7 kb. Two major classes of repair products, homology-based (homologous recombination (HR) and single-strand annealing (SSA)) and end-joining (non-homologous (NHEJ) and micro-homology mediated (MMEJ)) were identified. The distribution of repair products varies as a function of distance between the two centromeres. Genetic dependencies on double strand break repair (Rad52), DNA ligase (Lif1), and S phase checkpoint (Mrc1) are indicative of distinct repair pathway choices for DNA breaks in the pericentromeric chromatin versus the arms.

Statistical mechanics of chromosomes: in vivo and in silico approaches reveal high-level organization and structure arise exclusively through mechanical feedback between loop extruders and chromatin substrate properties.

The revolution in understanding higher order chromosome dynamics and organization derives from treating the chromosome as a chain polymer and adapting appropriate polymer-based physical principles. Using basic principles, such as entropic fluctuations and timescales of relaxation of Rouse polymer chains, one can recapitulate the dominant features of chromatin motion observed in vivo. An emerging challenge is to relate the mechanical properties of chromatin to more nuanced organizational principles such as ubiquitous DNA loops. Toward this goal, we introduce a real-time numerical simulation model of a long chain polymer in the presence of histones and condensin, encoding physical principles of chromosome dynamics with coupled histone and condensin sources of transient loop generation. An exact experimental correlate of the model was obtained through analysis of a model-matching fluorescently labeled circular chromosome in live yeast cells. We show that experimentally observed chromosome compaction and variance in compaction are reproduced only with tandem interactions between histone and condensin, not from either individually. The hierarchical loop structures that emerge upon incorporation of histone and condensin activities significantly impact the dynamic and structural properties of chromatin. Moreover, simulations reveal that tandem condensin-histone activity is responsible for higher order chromosomal structures, including recently observed Z-loops.

Attentional Avoidance of Historically Relevant, Task-Irrelevant Facial Affect: Past Depression, Interpersonal Characteristics, and Ethnicity.

Experiential background can influence how individuals respond to affective interpersonal information. For formerly depressed individuals, sad facial expressions are presumably salient. If so, when performing affectively neutral daily tasks, these individuals would find peripheral sad faces particularly distracting, and thus, they might shift their attention from them. The present study examined this hypothesis by comparing how euthymic formerly depressed and never depressed adults attended to sad and happy task-irrelevant emotional facial expression stimuli. The study also measured constructs linked to interpersonal functioning and depression and conducted exploratory analyses to examine whether Hispanic ethnicity status would moderate effects of study outcomes. Results of analyses indicated that formerly depressed individuals directed more attention away from sad faces than never depressed individuals. There were no significant between-group effects for happy faces and no moderation by ethnicity on attention to affective faces. However, irrespective of depression history, Hispanic individuals reported lower fear of negative evaluation compared to non-Hispanic Caucasian individuals. Findings are in line with hypothesized attentional avoidance among formerly depressed individuals and consistent with prior research suggesting that some Hispanic individuals experience protective mental health benefits through engagement with aspects of their culture. Directions for future research are discussed.

Fork pausing allows centromere DNA loop formation and kinetochore assembly.

De novo kinetochore assembly, but not template-directed assembly, is dependent on COMA, the kinetochore complex engaged in cohesin recruitment. The slowing of replication fork progression by treatment with phleomycin (PHL), hydroxyurea, or deletion of the replication fork protection protein Csm3 can activate de novo kinetochore assembly in COMA mutants. Centromere DNA looping at the site of de novo kinetochore assembly can be detected shortly after exposure to PHL. Using simulations to explore the thermodynamics of DNA loops, we propose that loop formation is disfavored during bidirectional replication fork migration. One function of replication fork stalling upon encounters with DNA damage or other blockades may be to allow time for thermal fluctuations of the DNA chain to explore numerous configurations. Biasing thermodynamics provides a mechanism to facilitate macromolecular assembly, DNA repair, and other nucleic acid transactions at the replication fork. These loop configurations are essential for sister centromere separation and kinetochore assembly in the absence of the COMA complex.

Impact of formal training on agreement of videofluoroscopic swallowing study interpretation across and within disciplines.

PURPOSE: Formal agreement studies on interpretation of the videofluoroscopic swallowing study (VFSS) procedure among speech-language pathologists, radiology house officers, and staff radiologists have not been pursued. Each of these professions participates in the procedure, interprets the examination, and writes separate reports on the findings. The aim of this study was to determine reliability of interpretation between and within the disciplines and to determine if structured training improved reliability. METHODS: Thirteen speech-language pathologists (SLPs), ten diagnostic radiologists (RADs) and twenty-one diagnostic radiology house officers (HOs) participated in this study. Each group viewed 24 VFSS samples and rated the presence or absence of seven aberrant swallowing features as well as the presence of dysphagia and identification of oral dysphagia, pharyngeal dysphagia, or both. During part two, the groups were provided with a training session on normal and abnormal swallowing, using different VFSS samples from those in part one, followed by re-rating of the original 24 VFSS samples. A generalized estimating equations (GEE) approach with a binomial link function was used to examine each question separately. For each cluster of tests, as example, all pairwise comparisons between the three groups in the pretraining period, a Hochberg's correction for multiple testing was used to determine significance. A GEE approach with a binomial link function was used to compare the premeasure to postmeasure for each of the three groups of raters stratified by experience. RESULTS: The primary result revealed that the HO group scored significantly lower than the SLP and RAD group on identification of the presence of dysphagia (p = 0.008; p = 0.001, respectively), identification of oral phase dysphagia (p = 0.003; p = 0.001, respectively), and identification of both oral and pharyngeal phase dysphagia, (p = 0.014, p = 0.001, respectively) pretraining. Post training there was no statistically significant difference between the three groups on identification of dysphagia and identification of combined oral and pharyngeal dysphagia. CONCLUSIONS: Formal training to identify oropharyngeal dysphagia characteristics appears to improve accuracy of interpretation of the VFSS procedure for radiology house officers. Consideration to include formal training in this area for radiology residency training programs is recommended.

Stu2 uses a 15-nm parallel coiled coil for kinetochore localization and concomitant regulation of the mitotic spindle.

XMAP215/Dis1 family proteins are potent microtubule polymerases, critical for mitotic spindle structure and dynamics. While microtubule polymerase activity is driven by an N-terminal tumor overexpressed gene (TOG) domain array, proper cellular localization is a requisite for full activity and is mediated by a C-terminal domain. Structural insight into the C-terminal domain's architecture and localization mechanism remain outstanding. We present the crystal structure of the Saccharomyces cerevisiae Stu2 C-terminal domain, revealing a 15-nm parallel homodimeric coiled coil. The parallel architecture of the coiled coil has mechanistic implications for the arrangement of the homodimer's N-terminal TOG domains during microtubule polymerization. The coiled coil has two spatially distinct conserved regions: CRI and CRII. Mutations in CRI and CRII perturb the distribution and localization of Stu2 along the mitotic spindle and yield defects in spindle morphology including increased frequencies of mispositioned and fragmented spindles. Collectively, these data highlight roles for the Stu2 dimerization domain as a scaffold for factor binding that optimally positions Stu2 on the mitotic spindle to promote proper spindle structure and dynamics.

Enrichment of dynamic chromosomal crosslinks drive phase separation of the nucleolus.

Regions of highly repetitive DNA, such as those found in the nucleolus, show a self-organization that is marked by spatial segregation and frequent self-interaction. The mechanisms that underlie the sequestration of these sub-domains are largely unknown. Using a stochastic, bead-spring representation of chromatin in budding yeast, we find enrichment of protein-mediated, dynamic chromosomal cross-links recapitulates the segregation, morphology and self-interaction of the nucleolus. Rates and enrichment of dynamic crosslinking have profound consequences on domain morphology. Our model demonstrates the nucleolus is phase separated from other chromatin in the nucleus and predicts that multiple rDNA loci will form a single nucleolus independent of their location within the genome. Fluorescent labeling of budding yeast nucleoli with CDC14-GFP revealed that a split rDNA locus indeed forms a single nucleolus. We propose that nuclear sub-domains, such as the nucleolus, result from phase separations within the nucleus, which are driven by the enrichment of protein-mediated, dynamic chromosomal crosslinks.

Microtubule dynamics drive enhanced chromatin motion and mobilize telomeres in response to DNA damage.

Chromatin exhibits increased mobility on DNA damage, but the biophysical basis for this behavior remains unknown. To explore the mechanisms that drive DNA damage-induced chromosome mobility, we use single-particle tracking of tagged chromosomal loci during interphase in live yeast cells together with polymer models of chromatin chains. Telomeres become mobilized from sites on the nuclear envelope and the pericentromere expands after exposure to DNA-damaging agents. The magnitude of chromatin mobility induced by a single double-strand break requires active microtubule function. These findings reveal how relaxation of external tethers to the nuclear envelope and internal chromatin-chromatin tethers, together with microtubule dynamics, can mobilize the genome in response to DNA damage.

A cohort study of the morbidity of combined anterior-posterior cervical spinal fusions: incidence and predictors of postoperative dysphagia.

PURPOSE: To identify risk factors that may lead to the development of dysphagia after combined anterior and posterior (360°) cervical fusion surgery. METHODS: A single center, retrospective analysis of patients who had same-day, 360° fusion at Henry Ford Hospital between 2008 and 2012 was performed. Variables analyzed included demographics, medical co-morbidities, levels fused, and degree of dysphagia. RESULTS: The overall dysphagia rate was 37.7 %. Patients with dysphagia had a longer mean length of stay (p < 0.001), longer mean operative time (p < 0.001), greater intraoperative blood loss (p = 0.002), and fusion above the fourth cervical vertebra, C4, (p = 0.007). There were no differences in the rates of dysphagia when comparing patients undergoing primary or revision surgery (p = 0.554). CONCLUSION: Prolonged surgery and fusion above C4 lead to higher rates of dysphagia after 360° fusions. Prior anterior cervical fusion does not increase the risk of dysphagia development.

Parental Emotion Coaching: Associations With Self-Regulation in Aggressive/Rejected and Low Aggressive/Popular Children.

This study investigated associations between maternal and paternal emotion coaching and the self-regulation skills of kindergarten and first-grade children. Participants were 54 children categorized as either aggressive/rejected or low aggressive/popular by peer reports. Findings indicated a statistical trend for fathers of low aggressive/popular children to engage in more emotion coaching than fathers of aggressive/rejected children. Paternal emotion coaching accounted for significant variance in children's regulation of attention. Maternal emotion coaching moderated the relation between children's status and regulation of emotion. Findings suggest that interventions focused on parental emotion coaching may prove beneficial for increasing the self-regulation and attention skills of children with social and conduct problems.

Fathead minnow response to broad-range exposure of ß-sitosterol concentrations during life-cycle testing.

The ß-sitosterol concentration in pulp and paper mill effluents is typically greater than that of other phytosterols and has been shown to cause a variety of effects in fish. The authors exposed fathead minnow (Pimephales promelas) to low (22 ± 0.93 µg/L), medium-low (70 ± 2.1 µg/L), medium-high (237 ± 5.5 µg/L), and high (745 ± 16.2 µg/L) concentrations of ß-sitosterol as well as negative (water), positive (ethynyl estradiol, 16 ± 0.58 ng/L), and carrier (0.6 mL/L acetone) controls. Fish were monitored over a full life cycle for population-level endpoints including growth and survival, reproductive endpoints (e.g. fecundity, sex steroids and vitellogenin, gonado-/hepatosomatic indices, and gonad histology). No significant differences were seen in fish growth, mortality, or reproduction with ß-sitosterol exposure, although a trend for lower egg production in ß-sitosterol exposures relative to the water control may be related to the acetone carrier. All ethynyl estradiol-exposed fish were smaller, showed female characteristics, and did not spawn. Sex steroid and vitellogenin were highly variable with no detectable treatment-related differences. Gonadal tissue showed no ß-sitosterol-related differences in reproductive development and spawning capability, although most ethynyl estradiol-exposed males had ovarian tissue and were not spawning-capable. The results indicate that ß-sitosterol exposure had little apparent impact on fathead minnow survival, growth, and reproduction even at concentrations >10 times that of typical effluents, although small sample size and variability precluded fully evaluating treatment responses on sex steroids and vitellogenin.

Responses of fathead minnows (Pimephales promelas) during life-cycle exposures to pulp mill effluents at four long-term receiving water study sites.

We exposed fathead minnows (Pimephales promelas to 7 concentrations of effluents from pulp mills at 4 Long-Term Receiving Water Study (LTRWS) sites. The primary objective of these investigations was to determine the potential for toxicity, particularly on fish reproduction, of the pulp mill effluents using laboratory tests. These tests were performed as LTRWS fish community assessments were being completed, thus results of the laboratory fish reproduction tests could be compared to in-stream fish community measurements. In general, bioindicators measured during the life-cycle tests, including gonadosomatic index (GSI), hepatosomatic index, condition factor, numbers of tubercles on heads of males and females, and gonadal histology did not show consistent patterns or dose response and did not predict effects on egg production. Gonadosomatic indexes and tubercles also did not indicate estrogenic or androgenic responses to the effluents during the life-cycle tests. The most consistently sensitive test endpoint showing a dose response was the 25% inhibition concentration (IC25) for egg production. Based on this endpoint all 4 effluents had effects on fish reproduction from 8% by volume to 100% effluent. However, in-stream effects on fish reproduction would not be expected based on these 4 life-cycle tests for any of the LTRWS stream sites. The mean effluent concentration in Codorus Creek, Pennsylvania, USA was approximately 32%, and the IC25 for the life-cycle test was 100% effluent, providing a margin of safety of approximately 3 times. The margins of safety at the other sites are much greater: 34 times for Leaf River, Mississippi, USA (IC25 = 69%, 2% mean receiving water concentration), 36 times for the McKenzie River, Oregon, USA (IC25 = 18%, 0.5% mean receiving water concentration), and 40 times for the Willamette River, Oregon, USA (IC25 = 8%, 0.20% mean receiving water concentration). Effects on fish numbers, diversity, and community structure due to the effluent were also not found during the LTRWS, which is consistent with these laboratory results. These findings indicate that in this case, when laboratory results combined with in-stream effluent concentrations suggest in-stream effects on fish population are not expected, the laboratory results are consistent with the in-stream observations. However, inferences about situations where laboratory results predict in-stream effects cannot be made from these data.

A long-term, multitrophic level study to assess pulp and paper mill effluent effects on aquatic communities in four US receiving waters: characteristics of the study streams, sample sites, mills, and mill effluents.

Watershed characteristics, study streams, sample sites, mills, and mill effluents are provided for 4 streams included in a long-term study to assess potential effects of pulp and paper mill effluents on US receiving waters. The study streams are Codorus Creek (Pennsylvania, USA), Leaf River (Mississippi, USA) and McKenzie and Willamette rivers (Oregon, USA) and were chosen to represent a blend of mill process types, effluent concentrations, and coldwater/warmwater stream systems. The described effluent quality, water quality, and habitat data sets encompass the initial 7 to 8 y of a study anticipated to continue >10 y and provide a backdrop to a series of articles describing periphyton, macroinvertebrate, and fish community properties in these same streams. The mean in-stream waste concentration (IWC) for these 4 effluent discharges was 32.4%, 2.0%, 0.5%, and 0.2% v/v for Codorus Creek and Leaf, McKenzie, and Willamette rivers, respectively, as compared with a median of 0.4% for US mills. Effluent quality measurements included Selenastrum capricornutum, Ceriodaphnia dubia, and Pimephales promelas chronic bioassays as sanctioned by the US Environmental Protection Agency for estimating effluent effects on receiving-water aquatic communities. Based on mean bioassay inhibition concentration for a 25% effect and on mean IWC, a margin of safety against adverse biological effects of 2, 25, 137, and 150 times was indicated for Codorus Creek and Leaf, McKenzie, and Willamette rivers, respectively. Habitat and water quality assessment was carried out over a gradient of sample sites above and below the effluent discharge to determine nonmill-related conditions that might interfere with interpretation of effluent effects. Noneffluent related localized differences in conditions for some parameters, including current velocity (McKenzie River), and surface incident photosynthetically active radiation (Codorus Creek and Willamette River) occurred at the sample stations immediately upstream or downstream of the effluent discharge. In addition, broader watershed differences were evident on Codorus Creek, where a relatively rich riparian corridor and stream structure occurred upstream in contrast to areas of canopy and stream-structure loss in the downstream urban area. The mill effluent discharges contributed to increases in receiving-water color and conductivity, although upstream tributaries contributed additional conductivity to Codorus Creek and color to the Leaf River. The McKenzie River provided the only example of a nutrient increase immediately downstream of a mill discharge. This increase in total nitrogen (0.11 vs 0.16 mg/L) could not, however, be differentiated with respect to whether it was of mill effluent or tributary stream origin. Tributary streams were potentially important total nitrogen contributors on Codorus Creek and the Willamette River. As an integrated study, the effluent quality and physical/chemical watershed descriptions provided here represent 1 component of the broader study addressing potential point-source effluent effects within the context of the larger watershed and a multiyear timescale. The absence of effluent-related in-stream chemical/physical responses, other than increases in conductivity and color, and a considerable bioassay-based margin of safety, provides for a working hypothesis that there will be no effluent-related biological population/community responses from these 4 mill discharges. This hypothesis, as it relates to periphyton, macroinvertebrate, and fish communities, will be addressed in other articles in this series.