The p150N domain of chromatin assembly factor-1 regulates Ki-67 accumulation on the mitotic perichromosomal layer

Chromatin assembly factor 1 (CAF-1) deposits histones during DNA synthesis. The p150 subunit of human CAF-1 contains an N-terminal domain (p150N) that is dispensable for histone deposition but promotes the localization of specific loci (nucleolar-associated domains [NADs]) and proteins to the nucleolus during interphase. One of the p150N-regulated proteins is proliferation antigen Ki-67, whose depletion also decreases the nucleolar association of NADs. Ki-67 is also a fundamental component of the perichromosomal layer (PCL), a sheath of proteins surrounding condensed chromosomes during mitosis. We show here that a subset of p150 localizes to the PCL during mitosis and that p150N is required for normal levels of Ki-67 accumulation on the PCL. This activity requires the sumoylation-interacting motif within p150N, which is also required for the nucleolar localization of NADs and Ki-67 during interphase. In this manner, p150N coordinates both interphase and mitotic nuclear structures via Ki67

Biochemical Analysis of Dimethyl Suberimidate-crosslinked Yeast Nucleosomes

Nucleosomes are the fundamental unit of eukaryotic chromosome packaging, comprised of 147 bp of DNA wrapped around two molecules of each of the core histone proteins H2A, H2B, H3, and H4. Nucleosomes are symmetrical, with one axis of symmetry centered on the homodimeric interaction between the C-termini of the H3 molecules. To explore the functional consequences of nucleosome symmetry, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, allowing us to compare cells with single or double H3 alterations. Our biochemical validation of the heterodimeric X-Y interaction included intra-nucleosomal H3 crosslinking using dimethyl suberimidate (DMS). Here, we provide a detailed protocol for the use of DMS to analyze yeast nucleosomes

Reduced-rank spatio-temporal modeling of air pollution concentrations in the Multi-Ethnic Study of Atherosclerosis and Air Pollution

There is growing evidence in the epidemiologic literature of the relationship between air pollution and adverse health outcomes. Prediction of individual air pollution exposure in the Environmental Protection Agency (EPA) funded Multi-Ethnic Study of Atheroscelerosis and Air Pollution (MESA Air) study relies on a flexible spatio-temporal prediction model that integrates land-use regression with kriging to account for spatial dependence in pollutant concentrations. Temporal variability is captured using temporal trends estimated via modified singular value decomposition and temporally varying spatial residuals. This model utilizes monitoring data from existing regulatory networks and supplementary MESA Air monitoring data to predict concentrations for individual cohort members. In general, spatio-temporal models are limited in their efficacy for large data sets due to computational intractability. We develop reduced-rank versions of the MESA Air spatio-temporal model. To do so, we apply low-rank kriging to account for spatial variation in the mean process and discuss the limitations of this approach. As an alternative, we represent spatial variation using thin plate regression splines. We compare the performance of the outlined models using EPA and MESA Air monitoring data for predicting concentrations of oxides of nitrogen (NO$_x$)-a pollutant of primary interest in MESA Air-in the Los Angeles metropolitan area via cross-validated $R^2$. Our findings suggest that use of reduced-rank models can improve computational efficiency in certain cases. Low-rank kriging and thin plate regression splines were competitive across the formulations considered, although TPRS appeared to be more robust in some settings.Comment: Published in at http://dx.doi.org/10.1214/14-AOAS786 the Annals of Applied Statistics (http://www.imstat.org/aoas/) by the Institute of Mathematical Statistics (http://www.imstat.org

An asymmetric centromeric nucleosome

Nucleosomes contain two copies of each core histone, held together by a naturally symmetric, homodimeric histone H3-H3 interface. This symmetry has complicated efforts to determine the regulatory potential of this architecture. Through molecular design and in vivo selection, we recently generated obligately heterodimeric H3s, providing a powerful tool for discovery of the degree to which nucleosome symmetry regulates chromosomal functions in living cells (Ichikawa et al., 2017). We now have extended this tool to the centromeric H3 isoform (Cse4/CENP-A) in budding yeast. These studies indicate that a single Cse4 N- or C-terminal extension per pair of Cse4 molecules is sufficient for kinetochore function, and validate previous experiments indicating that an octameric centromeric nucleosome is required for viability in this organism. These data also support the generality of the H3 asymmetric interface for probing general questions in chromatin biology

Novel paths to antifungal therapeutics

Adhesion to medical device and host cell surfaces are crucial steps during pathogenesis by fungi such as Candida albicans, which is especially dangerous to immunocompromised individuals such as AIDS patients. We have identified a small molecule that inhibits adhesion of C. albicans to polystyrene and to cultured human epithelial cells. Moreover, this compound is able to coat plastic surfaces and make them resistant to colonization by fungal biofilms. Therefore, this compound has the potential to be widely useful as a novel therapeutic and/or as a coating on medical devices. Rationale: C. albicans is the most widespread fungal pathogen of humans and one of the most frequent hospital-acquired infections. The estimated annual cost of treating nosocomial Candida infections exceeds $1 billion per year. As an opportunistic pathogen, it is responsible for common clinical problems including oral thrush and vaginitis, but can also lead to life-threatening systemic infections (candidiasis) in immunocompromised individuals, resulting in 30-50% mortality rates. Contributing to these problems is the ability of C. albicans to develop resistance to antifungal drugs. Moreover, most effective antifungal drugs also cause serious side effects, in many cases because of the significant homology between mammalian and fungal drug targets. Therefore, new antifungal drugs are a high medical priority. Surface adhesion, morphological switching, and biofilm formation are interrelated factors that contribute directly to C. albicans virulence. Therefore, compounds that impair these processes would have promising properties as first step towards new antifungal therapeutics. Preliminary Studies: Efficient adhesion is required for formation of aggressive biofilms, which in turn make Candida a successful pathogen. Therefore, we identified compounds that prevent adhesion of Candida albicans to polystyrene surfaces. Because the assays in this proposal are based on altering the behavior of intact cells, we avoid the complication of compounds unable to cross the cell wall and membranes. Our initial search for adhesion inhibitors was conceptually simple, based on dye binding to monitor yeast adhesion to surfaces. We identified 41 compounds that reduced adhesion to Candidastrain confirmed that most of the reordered compounds indeed inhibit adhesion to polystyrene (Figure 1). Figure 1. Compound #4 inhibits C. albicans adhesion to polystyrene. GFP-expressing wild type or non-adherent edt1 mutant C. albicans cells were plated into 96 well plates with DMSO or 25 mM compound 4 as indicated. Plates were incubated for 4 hours at 37oC. Media was then decanted and plates were washed 3 times prior to fluorescence microscopy. Human cell adhesion: To determine whether any candidate compounds would affect interactions with biological targets as well as inert surfaces, we also tested how the candidate compounds affect C. albicans adherence to human cells, using monolayers of human lung epithelial cells. The GFP-encoding cells allowed us to use both microscopy and fluorescence measurements to detect fungal cells that remained bound after washing. We observed that “compound #4”, but not other candidate compounds, reduced the interaction of C. albicans with the human cells to background levels. We also verified that compound 4 did not affect the viability of this human cell line, even at concentrations much higher (250 mM) than those used in the adhesion assay (data not shown). Plastic coating: Compound 4 also inhibits Candida adhesion to polystyrene when it is incubated with the plastic prior to the addition of the cells. Therefore, compound 4 not only has effects on Candida cell morphology in the absence of surface adhesion, it also renders plastic surfaces resistant to subsequent Candida binding. Together, our data suggest that compound 4 may not only be effective at combating fungal infections, but could also have potential use as a compound to prevent multiple types of unwanted microbial colonization

Pragmatic Estimation of a Spatio-Temporal Air Quality Model With Irregular Monitoring Data

Statistical analyses of the health effects of air pollution have increasingly used GIS-based covariates for prediction of ambient air quality in “land-use” regression models. More recently these regression models have accounted for spatial correlation structure in combining monitoring data with land-use covariates. The current paper builds on these concepts to address spatio-temporal prediction of ambient concentrations of particulate matter with aerodynamic diameter less than 2.5 μm (PM2.5) on the basis of a model representing spatially varying seasonal trends and spatial correlation structures. Our hierarchical methodology provides a pragmatic approach that fully exploits regulatory and other supplemental monitoring data which jointly define a complex spatio-temporal monitoring design. We explain the elements of the computational approach, including estimation of smoothed empirical orthogonal functions (SEOFs) as basis functions for temporal trend, spatial (“land use”) regression by Partial Least Squares (PLS), modeling of spatio-temporal correlation structure, and generalized universal kriging prediction of ambient exposure for subjects in the Multi-Ethnic Study of Atherosclerosis and Air Pollution (MESA Air) project. Analyses are demonstrated in detail for the South California study area of the MESA Air project using AQS monitoring data from 2000 to 2006 and supplemental MESA Air monitoring data beginning in 2005. Results of application of the modeling and estimation methodology are presented also for five other MESA Air metropolitan study areas across the country with comments on current and future research developments

Massachusetts Bar Discipline: History, Practice, and Procedure

While there have been several publications describing the bar discipline process for violations of the Massachusetts Rules of Professional Conduct, no work has described that process in great detail while also analyzing the related sanctions. The treatise seeks to fill this gap and make the practice more accessible to participants and observers by referencing, in a single volume, materials that had not previously been collected and analyzed. We begin with an overview of the history of bar discipline in the Commonwealth, followed by a discussion of the participants in, and structure of, the disciplinary process. Part II takes the reader through the steps of a typical bar discipline case from the initial complaint through the hearing. Part III discusses in detail some typical misconduct, including that related to competence, confidentiality, safekeeping of trust property, and advertising—and the sanctions typically imposed for it. This is followed by a discussion in Part IV of conduct that may aggravate or mitigate the presumptive sanction. Lastly, Part V discusses the post-hearing process as well as reciprocal discipline, resignations, duties after suspension or disbarment, reinstatement, and registration. While this volume is not a dissertation on legal ethics per se, it addresses the Rules of Professional Conduct as they arise in bar discipline

Structure of the yeast histone H3-ASF1 interaction: implications for chaperone mechanism, species-specific interactions, and epigenetics

BACKGROUND: The histone H3/H4 chaperone Asf1 (anti-silencing function 1) is required for the establishment and maintenance of proper chromatin structure, as well as for genome stability in eukaryotes. Asf1 participates in both DNA replication-coupled (RC) and replication-independent (RI) histone deposition reactions in vitro and interacts with complexes responsible for both pathways in vivo. Asf1 is known to directly bind histone H3, however, high-resolution structural information about the geometry of this interaction was previously unknown. RESULTS: Here we report the structure of a histone/histone chaperone interaction. We have solved the 2.2 Å crystal structure of the conserved N-terminal immunoglobulin fold domain of yeast Asf1 (residues 2–155) bound to the C-terminal helix of yeast histone H3 (residues 121–134). The structure defines a histone-binding patch on Asf1 consisting of both conserved and yeast-specific residues; mutation of these residues abrogates H3/H4 binding affinity. The geometry of the interaction indicates that Asf1 binds to histones H3/H4 in a manner that likely blocks sterically the H3/H3 interface of the nucleosomal four-helix bundle. CONCLUSION: These data clarify how Asf1 regulates histone stoichiometry to modulate epigenetic inheritance. The structure further suggests a physical model in which Asf1 contributes to interpretation of a "histone H3 barcode" for sorting H3 isoforms into different deposition pathways