ABSTRACT
Sumoylation is emerging as a posttranslation modification important for regulating chromosome duplication and stability. The origin recognition complex (ORC) that directs DNA replication initiation by loading the MCM replicative helicase onto origins is sumoylated in both yeast and human cells. However, the biological consequences of ORC sumoylation are unclear. Here we report the effects of hypersumoylation and hyposumoylation of yeast ORC on ORC activity and origin function using multiple approaches. ORC hypersumoylation preferentially reduced the function of a subset of early origins, while Orc2 hyposumoylation had an opposing effect. Mechanistically, ORC hypersumoylation reduced MCM loading in vitro and diminished MCM chromatin association in vivo. Either hypersumoylation or hyposumoylation of ORC resulted in genome instability and the dependence of yeast on other genome maintenance factors, providing evidence that appropriate ORC sumoylation levels are important for cell fitness. Thus, yeast ORC sumoylation status must be properly controlled to achieve optimal origin function across the genome and genome stability.
ABSTRACT
In Saccharomyces cerevisiae, the forkhead (Fkh) transcription factor Fkh1 (forkhead homolog) enhances the activity of many DNA replication origins that act in early S-phase (early origins). Current models posit that Fkh1 acts directly to promote these origins' activity by binding to origin-adjacent Fkh1 binding sites (FKH sites). However, the post-DNA binding functions that Fkh1 uses to promote early origin activity are poorly understood. Fkh1 contains a conserved FHA (forkhead associated) domain, a protein-binding module with specificity for phosphothreonine (pT)-containing partner proteins. At a small subset of yeast origins, the Fkh1-FHA domain enhances the ORC (origin recognition complex)-origin binding step, the G1-phase event that initiates the origin cycle. However, the importance of the Fkh1-FHA domain to either chromosomal replication or ORC-origin interactions at genome scale is unclear. Here, S-phase SortSeq experiments were used to compare genome replication in proliferating FKH1 and fkh1-R80A mutant cells. The Fkh1-FHA domain promoted the activity of ≈ 100 origins that act in early to mid- S-phase, including the majority of centromere-associated origins, while simultaneously inhibiting ≈ 100 late origins. Thus, in the absence of a functional Fkh1-FHA domain, the temporal landscape of the yeast genome was flattened. Origins are associated with a positioned nucleosome array that frames a nucleosome depleted region (NDR) over the origin, and ORC-origin binding is necessary but not sufficient for this chromatin organization. To ask whether the Fkh1-FHA domain had an impact on this chromatin architecture at origins, ORC ChIPSeq data generated from proliferating cells and MNaseSeq data generated from G1-arrested and proliferating cell populations were assessed. Origin groups that were differentially regulated by the Fkh1-FHA domain were characterized by distinct effects of this domain on ORC-origin binding and G1-phase chromatin. Thus, the Fkh1-FHA domain controlled the distinct chromatin architecture at early origins in G1-phase and regulated origin activity in S-phase.
Subject(s)
Chromatin , DNA Replication , G1 Phase , Origin Recognition Complex , Replication Origin , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Replication Origin/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , DNA Replication/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Chromatin/genetics , Chromatin/metabolism , Origin Recognition Complex/genetics , Origin Recognition Complex/metabolism , G1 Phase/genetics , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , S Phase/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Protein Domains/genetics , Binding Sites , Protein Binding , Chromosomes, Fungal/genetics , Chromosomes, Fungal/metabolism , Nucleosomes/metabolism , Nucleosomes/geneticsABSTRACT
The pioneer event in eukaryotic DNA replication is binding of chromosomal DNA by the origin recognitioncomplex (ORC). The ORC-DNA complex directs the formation of origins, the specific chromosomal regions where DNA synthesis initiates. In all eukaryotes, incompletely understood features of chromatin promote ORC-DNA binding. Here, we uncover a role for the Fkh1 (Forkhead homolog) protein and its forkhead associated (FHA) domain in promoting ORC-origin binding and origin activity at a subset of origins in Saccharomyces cerevisiae. Several of the FHA-dependent origins examined required a distinct Fkh1 binding site located 5' of and proximal to their ORC sites (5'-FKH-T site). Genetic and molecular experiments provided evidence that the Fkh1-FHA domain promoted origin activity directly through Fkh1 binding to this 5' FKH-T site. Nucleotide substitutions within two relevant origins that enhanced their ORC-DNA affinity bypassed the requirement for their 5' FKH-T sites and for the Fkh1-FHA domain. Significantly, assessment of ORC-origin binding by ChIPSeq provided evidence that this mechanism was relevant at â¼25% of yeast origins. Thus, the FHA domain of the conserved cell-cycle transcription factor Fkh1 enhanced origin selection in yeast at the level of ORC-origin binding.
Subject(s)
Cell Cycle Proteins/metabolism , DNA, Fungal/metabolism , Forkhead Transcription Factors/metabolism , Origin Recognition Complex/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , DNA Replication , Protein Binding , Protein DomainsABSTRACT
A eukaryotic chromosome relies on the function of multiple spatially distributed DNA replication origins for its stable inheritance. The spatial location of an origin is determined by the chromosomal position of an MCM complex, the inactive form of the DNA replicative helicase that is assembled onto DNA in G1-phase (also known as origin licensing). While the biochemistry of origin licensing is understood, the mechanisms that promote an adequate spatial distribution of MCM complexes across chromosomes are not. We have elucidated a role for the Sir2 histone deacetylase in establishing the normal distribution of MCM complexes across Saccharomyces cerevisiae chromosomes. In the absence of Sir2, MCM complexes accumulated within both early-replicating euchromatin and telomeric heterochromatin, and replication activity within these regions was enhanced. Concomitantly, the duplication of several regions of late-replicating euchromatin were delayed. Thus, Sir2-mediated attenuation of origin licensing within both euchromatin and telomeric heterochromatin established the normal spatial distribution of origins across yeast chromosomes important for normal genome duplication.
Subject(s)
Euchromatin/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Silent Information Regulator Proteins, Saccharomyces cerevisiae/metabolism , Sirtuin 2/metabolism , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone , Chromosomes , DNA Helicases , DNA Replication , Heterochromatin , Replication Origin/geneticsABSTRACT
Bicaudal-C (Bicc1) is a conserved RNA-binding protein that represses the translation of selected mRNAs to control development. In Xenopus embryos, Bicc1 binds and represses specific maternal mRNAs to control anterior-posterior cell fates. However, it is not known how Bicc1 binds its RNA targets or how binding affects Bicc1-dependent embryogenesis. Focusing on the KH domains, we analyzed Bicc1 mutants for their ability to bind RNA substrates in vivo and in vitro Analyses of these Bicc1 mutants demonstrated that a single KH domain, KH2, was crucial for RNA binding in vivo and in vitro, while the KH1 and KH3 domains contributed minimally. The Bicc1 mutants were also assayed for their ability to repress translation, and results mirrored the RNA-binding data, with KH2 being the only domain essential for repression. Finally, maternal knockdown and rescue experiments indicated that the KH domains were essential for the regulation of embryogenesis by Bicc1. These data advance our understanding of how Bicc1 selects target mRNAs and provide the first direct evidence that the RNA binding functions of Bicc1 are essential for both Bicc1-dependent translational repression and maternal vertebrate development.
Subject(s)
RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Xenopus Proteins/metabolism , 3' Untranslated Regions/genetics , 3' Untranslated Regions/physiology , Animals , Embryonic Development/genetics , Embryonic Development/physiology , Female , Immunoblotting , Immunoprecipitation , Protein Binding , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , Xenopus Proteins/genetics , Xenopus laevisABSTRACT
Most active DNA replication origins are found within euchromatin, while origins within heterochromatin are often inactive or inhibited. In yeast, origin activity within heterochromatin is negatively controlled by the histone H4K16 deacetylase, Sir2, and at some heterochromatic loci also by the nucleosome binding protein, Sir3. The prevailing view has been that direct functions of Sir2 and Sir3 are confined to heterochromatin. However, growth defects in yeast mutants compromised for loading the MCM helicase, such as cdc6-4, are suppressed by deletion of either SIR2 or SIR3. While these and other observations indicate that SIR2,3 can have a negative impact on at least some euchromatic origins, the genomic scale of this effect was unknown. It was also unknown whether this suppression resulted from direct functions of Sir2,3 within euchromatin, or was an indirect effect of their previously established roles within heterochromatin. Using MCM ChIP-Seq, we show that a SIR2 deletion rescued MCM complex loading at ~80% of euchromatic origins in cdc6-4 cells. Therefore, Sir2 exhibited a pervasive effect at the majority of euchromatic origins. Using MNase-H4K16ac ChIP-Seq, we show that origin-adjacent nucleosomes were depleted for H4K16 acetylation in a SIR2-dependent manner in wild type (i.e. CDC6) cells. In addition, we present evidence that both Sir2 and Sir3 bound to nucleosomes adjacent to euchromatic origins. The relative levels of each of these molecular hallmarks of yeast heterochromatin-SIR2-dependent H4K16 hypoacetylation, Sir2, and Sir3 -correlated with how strongly a SIR2 deletion suppressed the MCM loading defect in cdc6-4 cells. Finally, a screen for histone H3 and H4 mutants that could suppress the cdc6-4 growth defect identified amino acids that map to a surface of the nucleosome important for Sir3 binding. We conclude that heterochromatin proteins directly modify the local chromatin environment of euchromatic DNA replication origins.
Subject(s)
DNA, Fungal/metabolism , Euchromatin/metabolism , Saccharomyces cerevisiae/genetics , Silent Information Regulator Proteins, Saccharomyces cerevisiae/genetics , Sirtuin 2/genetics , Acetylation , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromatin Immunoprecipitation , DNA Copy Number Variations , DNA Replication , DNA, Fungal/genetics , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , F-Box Proteins/genetics , F-Box Proteins/metabolism , Gene Expression Regulation, Fungal , Heterochromatin/metabolism , High-Throughput Nucleotide Sequencing , Histones/genetics , Histones/metabolism , Minichromosome Maintenance Proteins/metabolism , Mutagenesis, Site-Directed , Nucleosomes/genetics , Nucleosomes/metabolism , Replication Origin , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Silent Information Regulator Proteins, Saccharomyces cerevisiae/metabolism , Sirtuin 2/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
Undergraduate nursing education programs can play an integral role in developing the next generation of nurses by incorporating more oncology content to meet the needs of the increasing numbers of patients diagnosed with cancer. While oncology nursing is a specialized area of practice, student nurses and new graduates will come in contact with patients who have been diagnosed with cancer whether they work on a specialized unit or not. Increasing the amount of oncology content provided in undergraduate nursing programs can help to encourage interest in this specialty area and improve the ability of new graduates to care for this patient population.
ABSTRACT
Biochemical, proteomic, and epigenetic studies of chromatin rely on the ability to efficiently isolate native nucleosomes in high yield and purity. However, isolation of native chromatin suitable for many downstream experiments remains a challenging task. This is especially true for the budding yeast Saccharomyces cerevisiae, which continues to serve as an important model organism for the study of chromatin structure and function. Here, we developed a time- and cost-efficient universal protocol for isolation of native chromatin fragments from yeast, insect, and mammalian cells. The resulting protocol preserves histone posttranslational modification in the native chromatin state and is applicable for both parallel multisample spin-column purification and large-scale isolation. This protocol is based on the efficient and stable purification of polynucleosomes and features a combination of optimized cell lysis and purification conditions, three options for chromatin fragmentation, and a novel ion-exchange chromatographic purification strategy. The procedure will aid chromatin researchers interested in isolating native chromatin material for biochemical studies and serve as a mild, acid- and detergent-free sample preparation method for MS analysis.
Subject(s)
Chemistry Techniques, Analytical/methods , Chromatin/isolation & purification , Chromatography, Ion Exchange/methods , Saccharomyces cerevisiae Proteins/isolation & purification , Saccharomyces cerevisiae/chemistry , Cell Nucleus/chemistry , Chromatin/chemistry , Saccharomyces cerevisiae Proteins/chemistryABSTRACT
The Saccharomyces cerevisiae Fkh1 protein has roles in cell-cycle regulated transcription as well as a transcription-independent role in recombination donor preference during mating-type switching. The conserved FHA domain of Fkh1 regulates donor preference by juxtaposing two distant regions on chromosome III to promote their recombination. A model posits that this Fkh1-mediated long-range chromosomal juxtaposition requires an interaction between the FHA domain and a partner protein(s), but to date no relevant partner has been described. In this study, we used structural modeling, 2-hybrid assays, and mutational analyses to show that the predicted phosphothreonine-binding FHA domain of Fkh1 interacted with multiple partner proteins. The Fkh1 FHA domain was important for its role in cell-cycle regulation, but no single interaction partner could account for this role. In contrast, Fkh1's interaction with the Mph1 DNA repair helicase regulated donor preference during mating-type switching. Using 2-hybrid assays, co-immunoprecipitation, and fluorescence anisotropy, we mapped a discrete peptide within the regulatory Mph1 C-terminus required for this interaction and identified two threonines that were particularly important. In vitro binding experiments indicated that at least one of these threonines had to be phosphorylated for efficient Fkh1 binding. Substitution of these two threonines with alanines (mph1-2TA) specifically abolished the Fkh1-Mph1 interaction in vivo and altered donor preference during mating-type switching to the same degree as mph1Δ. Notably, the mph1-2TA allele maintained other functions of Mph1 in genome stability. Deletion of a second Fkh1-interacting protein encoded by YMR144W also resulted in a change in Fkh1-FHA-dependent donor preference. We have named this gene FDO1 for Forkhead one interacting protein involved in donor preference. We conclude that a phosphothreonine-mediated protein-protein interface between Fkh1-FHA and Mph1 contributes to a specific long-range chromosomal interaction required for mating-type switching, but that Fkh1-FHA must also interact with several other proteins to achieve full functionality in this process.
Subject(s)
Cell Cycle Proteins/metabolism , DEAD-box RNA Helicases/metabolism , DNA Helicases/metabolism , Forkhead Transcription Factors/metabolism , Genes, Mating Type, Fungal/genetics , Phosphopeptides/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomycetales/metabolism , Cell Cycle/genetics , DNA Repair/genetics , Gene Expression Regulation, Fungal/genetics , Phosphothreonine/metabolism , Recombination, Genetic/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomycetales/genetics , Transcription Factors/metabolismABSTRACT
The origin recognition complex (ORC) binds to the specific positions on chromosomes that serve as DNA replication origins. Although ORC is conserved from yeast to humans, the DNA sequence elements that specify ORC binding are not. In particular, metazoan ORC shows no obvious DNA sequence specificity, whereas yeast ORC binds to a specific DNA sequence within all yeast origins. Thus, whereas chromatin must play an important role in metazoan ORC's ability to recognize origins, it is unclear whether chromatin plays a role in yeast ORC's recognition of origins. This study focused on the role of the conserved N-terminal bromo-adjacent homology domain of yeast Orc1 (Orc1BAH). Recent studies indicate that BAH domains are chromatin-binding modules. We show that the Orc1BAH domain was necessary for ORC's stable association with yeast chromosomes, and was physiologically relevant to DNA replication in vivo. This replication role was separable from the Orc1BAH domain's previously defined role in transcriptional silencing. Genome-wide analyses of ORC binding in ORC1 and orc1bahDelta cells revealed that the Orc1BAH domain contributed to ORC's association with most yeast origins, including a class of origins highly dependent on the Orc1BAH domain for ORC association (orc1bahDelta-sensitive origins). Orc1bahDelta-sensitive origins required the Orc1BAH domain for normal activity on chromosomes and plasmids, and were associated with a distinct local nucleosome structure. These data provide molecular insights into how the Orc1BAH domain contributes to ORC's selection of replication origins, as well as new tools for examining conserved mechanisms governing ORC's selection of origins within eukaryotic chromosomes.
Subject(s)
Chromatin/genetics , Origin Recognition Complex/genetics , Origin Recognition Complex/metabolism , Replication Origin/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Acetyltransferases/genetics , Acetyltransferases/metabolism , Binding Sites , Conserved Sequence , DNA Replication , Protein Structure, Tertiary , Sequence Deletion/geneticsABSTRACT
The selective translation of maternal mRNAs encoding cell-fate determinants drives the earliest decisions of embryogenesis that establish the vertebrate body plan. This chapter will discuss studies in Xenopus laevis that provide insights into mechanisms underlying this translational control. Xenopus has been a powerful model organism for many discoveries relevant to the translational control of maternal mRNAs because of the large size of its oocytes and eggs that allow for microinjection of molecules and the relative ease of manipulating the oocyte to egg transition (maturation) and fertilization in culture. Consequently, many key studies have focused on the expression of maternal mRNAs during the oocyte to egg transition (the meiotic cell cycle) and the rapid cell divisions immediately following fertilization. This research has made seminal contributions to our understanding of translational regulatory mechanisms, but while some of the mRNAs under consideration at these stages encode cell-fate determinants, many encode cell cycle regulatory proteins that drive these early cell cycles. In contrast, while maternal mRNAs encoding key developmental (i.e., cell-fate) regulators that function after the first cleavage stages may exploit aspects of these foundational mechanisms, studies reveal that these mRNAs must also rely on distinct and, as of yet, incompletely understood mechanisms. These findings are logical because the functions of such developmental regulatory proteins have requirements distinct from cell cycle regulators, including becoming relevant only after fertilization and then only in specific cells of the embryo. Indeed, key maternal cell-fate determinants must be made available in exquisitely precise amounts (usually low), only at specific times and in specific cells during embryogenesis. To provide an appreciation for the regulation of maternal cell-fate determinant expression, an overview of the maternal phase of Xenopus embryogenesis will be presented. This section will be followed by a review of translational mechanisms operating in oocytes, eggs, and early cleavage-stage embryos and conclude with a discussion of how the regulation of key maternal cell-fate determinants at the level of translation functions in Xenopus embryogenesis. A key theme is that the molecular asymmetries critical for forming the body axes are established and further elaborated upon by the selective temporal and spatial regulation of maternal mRNA translation.
Subject(s)
Embryonic Development/genetics , Protein Biosynthesis , RNA, Messenger/biosynthesis , Xenopus laevis/growth & development , Animals , Cell Cycle/genetics , Female , Gene Expression Regulation, Developmental , Oocytes/growth & development , Oocytes/metabolism , RNA, Messenger/genetics , Transcription, Genetic , Xenopus laevis/geneticsABSTRACT
Conditions of chronic stress are associated with genetic instability in many organisms, but the roles of stress responses in mutagenesis have so far been elucidated only in bacteria. Here, we present data demonstrating that the environmental stress response (ESR) in yeast functions in mutagenesis induced by proteotoxic stress. We show that the drug canavanine causes proteotoxic stress, activates the ESR, and induces mutagenesis at several loci in an ESR-dependent manner. Canavanine-induced mutagenesis also involves translesion DNA polymerases Rev1 and Polζ and non-homologous end joining factor Ku. Furthermore, under conditions of chronic sub-lethal canavanine stress, deletions of Rev1, Polζ, and Ku-encoding genes exhibit genetic interactions with ESR mutants indicative of ESR regulating these mutagenic DNA repair processes. Analyses of mutagenesis induced by several different stresses showed that the ESR specifically modulates mutagenesis induced by proteotoxic stress. Together, these results document the first known example of an involvement of a eukaryotic stress response pathway in mutagenesis and have important implications for mechanisms of evolution, carcinogenesis, and emergence of drug-resistant pathogens and chemotherapy-resistant tumors.
Subject(s)
DNA Repair/genetics , DNA-Directed DNA Polymerase/genetics , Nucleotidyltransferases/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/physiology , Stress, Physiological , Canavanine/toxicity , DNA Damage/genetics , Mutagenesis/drug effects , Mutagenesis/genetics , Mutation , Saccharomyces cerevisiae/geneticsABSTRACT
Eukaryotic DNA replication origins are selected in G1-phase when the origin recognition complex (ORC) binds chromosomal positions and triggers molecular events culminating in the initiation of DNA replication (a.k.a. origin firing) during S-phase. Each chromosome uses multiple origins for its duplication, and each origin fires at a characteristic time during S-phase, creating a cell-type specific genome replication pattern relevant to differentiation and genome stability. It is unclear whether ORC-origin interactions are relevant to origin activation time. We applied a novel genome-wide strategy to classify origins in the model eukaryote Saccharomyces cerevisiae based on the types of molecular interactions used for ORC-origin binding. Specifically, origins were classified as DNA-dependent when the strength of ORC-origin binding in vivo could be explained by the affinity of ORC for origin DNA in vitro, and, conversely, as 'chromatin-dependent' when the ORC-DNA interaction in vitro was insufficient to explain the strength of ORC-origin binding in vivo. These two origin classes differed in terms of nucleosome architecture and dependence on origin-flanking sequences in plasmid replication assays, consistent with local features of chromatin promoting ORC binding at 'chromatin-dependent' origins. Finally, the 'chromatin-dependent' class was enriched for origins that fire early in S-phase, while the DNA-dependent class was enriched for later firing origins. Conversely, the latest firing origins showed a positive association with the ORC-origin DNA paradigm for normal levels of ORC binding, whereas the earliest firing origins did not. These data reveal a novel association between ORC-origin binding mechanisms and the regulation of origin activation time.
Subject(s)
Chromatin/genetics , DNA Replication/genetics , Origin Recognition Complex/genetics , Saccharomyces cerevisiae/genetics , Binding Sites , Chromosomes/genetics , DNA/genetics , DNA-Binding Proteins/genetics , G1 Phase/genetics , Nucleosomes/genetics , Protein Binding , Replication Origin/geneticsABSTRACT
The posttranslational addition of palmitate to cysteines occurs ubiquitously in eukaryotic cells, where it functions in anchoring target proteins to membranes and in vesicular trafficking. Here we show that the Saccharomyces cerevisiae palmitoyltransferase Pfa4 enhanced heterochromatin formation at the cryptic mating-type loci HMR and HML via Rif1, a telomere regulatory protein. Acylated Rif1 was detected in extracts from wild-type but not pfa4Δ mutant cells. In a pfa4Δ mutant, Rif1-GFP dispersed away from foci positioned at the nuclear periphery into the nucleoplasm. Sir3-GFP distribution was also perturbed, indicating a change in the nuclear dynamics of heterochromatin proteins. Genetic analyses indicated that PFA4 functioned upstream of RIF1. Surprisingly, the pfa4Δ mutation had only mild effects on telomeric regulation, suggesting Rif1's roles at HM loci and telomeres were more complexly related than previously thought. These data supported a model in which Pfa4-dependent palmitoylation of Rif1 anchored it to the inner nuclear membrane, influencing its role in heterochromatin dynamics.
Subject(s)
Heterochromatin/metabolism , Repressor Proteins/physiology , Saccharomyces cerevisiae Proteins/physiology , Saccharomycetales/genetics , Telomere-Binding Proteins/physiology , Acylation , Acyltransferases/physiology , Lipoylation , Silent Information Regulator Proteins, Saccharomyces cerevisiae/genetics , TelomereABSTRACT
Recovery approaches to health care now feature in the mental health policies of many Western countries. There are, however, continuing challenges to the operationalization of these approaches. This study aimed to identify the nature of these challenges for a public mental health service organization located in a major urban center in southeastern Australia, where Recovery-oriented services have been implemented; and to develop recommendations to address these challenges. These aims were achieved by asking mental health consumers about their experiences of the implementation of Recovery-oriented services. Research participants described an uncertainty in health professionals and consumers alike about how to practice within a Recovery model, with many health professionals taking a "hands off" approach in the name of Recovery, rather than working in partnership with consumers and other stakeholders, including the community managed organizations. Solutions to these challenges included more targeted, practice-focused education for consumers and health professionals, with this education provided by consumer representatives. Insights derived from this research add to the growing body of evidence related to the implementation of Recovery-oriented services in Western countries.
Subject(s)
Mental Disorders/rehabilitation , Mental Health Services/organization & administration , Patient Satisfaction , Patient-Centered Care/organization & administration , Adult , Australia , Female , Focus Groups , Hospitals, Urban , Humans , Male , Middle Aged , Organizational Case Studies , Patient Education as Topic , Recovery of FunctionABSTRACT
In Saccharomyces cerevisiae, the forkhead (Fkh) transcription factor Fkh1 (forkhead homolog) enhances the activity of many DNA replication origins that act in early S-phase (early origins). Current models posit that Fkh1 acts directly to promote these origins' activity by binding to origin-adjacent Fkh1 binding sites (FKH sites). However, the post-DNA binding functions that Fkh1 uses to promote early origin activity are poorly understood. Fkh1 contains a conserved FHA (forkhead associated) domain, a protein-binding module with specificity for phosphothreonine (pT)-containing partner proteins. At a small subset of yeast origins, the Fkh1-FHA domain enhances the ORC (origin recognition complex)-origin binding step, the G1-phase event that initiates the origin cycle. However, the importance of the Fkh1-FHA domain to either chromosomal replication or ORC-origin interactions at genome scale is unclear. Here, S-phase SortSeq experiments were used to compare genome replication in proliferating FKH1 and fkh1-R80A mutant cells. The Fkh1-FHA domain promoted the activity of 100 origins that act in early to mid- S-phase, including the majority of centromere-associated origins, while simultaneously inhibiting 100 late origins. Thus, in the absence of a functional Fkh1-FHA domain, the temporal landscape of the yeast genome was flattened. Origins are associated with a positioned nucleosome array that frames a nucleosome depleted region (NDR) over the origin, and ORC-origin binding is necessary but not sufficient for this chromatin organization. To ask whether the Fkh1-FHA domain had an impact on this chromatin architecture at origins, ORC ChIPSeq data generated from proliferating cells and MNaseSeq data generated from G1-arrested and proliferating cell populations were assessed. Origin groups that were differentially regulated by the Fkh1-FHA domain were characterized by distinct effects of this domain on ORC-origin binding and G1-phase chromatin. Thus, the Fkh1-FHA domain controlled the distinct chromatin architecture at early origins in G1-phase and regulated origin activity in S-phase.
ABSTRACT
OBJECTIVES: The aim of the study was to describe the relation between sensory issues and medical complexity in a series of patients presenting to an outpatient multidisciplinary feeding team for evaluation, by a standardized measure of sensory-processing abilities. METHODS: A retrospective chart review of all of the patients seen from 2004 to 2009 on 2 key variables: medical diagnostic category and short sensory profile (SSP) score. RESULTS: On the SSP, 67.6% of children scored in the clinical ("definite difference") range. The most common diagnostic categories were developmental (n = 23), gastrointestinal (n = 16), and neurological (n = 13). Behavioral and cardiorespiratory medical diagnostic categories were significantly related to SSP total score and SSP definite difference score. CONCLUSIONS: Children who present for feeding evaluation do indeed tend to have clinically elevated scores regarding sensory processing, and these elevated scores are significantly related to certain medical diagnostic categories. Future research is needed to determine why these significant relations exist as well as their implications for treatment of feeding-related issues.
Subject(s)
Developmental Disabilities/complications , Feeding and Eating Disorders of Childhood/etiology , Gastrointestinal Diseases/complications , Nervous System Diseases/complications , Perception , Sensation Disorders/etiology , Sensation , Adolescent , Ambulatory Care Facilities , Child , Child, Preschool , Developmental Disabilities/diagnosis , Eating/psychology , Feeding and Eating Disorders of Childhood/psychology , Female , Gastrointestinal Diseases/diagnosis , Humans , Infant , Male , Nervous System Diseases/diagnosis , Retrospective Studies , Sensation Disorders/psychologyABSTRACT
In budding yeast, the eukaryotic initiator protein ORC (origin recognition complex) binds to a bipartite sequence consisting of an 11 bp ACS element and an adjacent B1 element. However, the genome contains many more matches to this consensus than actually bind ORC or function as origins in vivo. Although ORC-dependent loading of the replicative MCM helicase at origins is enhanced by a distal B2 element, less is known about this element. Here, we analyzed four highly active origins (ARS309, ARS319, ARS606 and ARS607) by linker scanning mutagenesis and found that sequences adjacent to the ACS contributed substantially to origin activity and ORC binding. Using the sequences of four additional B2 elements we generated a B2 multiple sequence alignment and identified a shared, degenerate 8 bp sequence that was enriched within 228 known origins. In addition, our high-resolution analysis revealed that not all origins exist within nucleosome free regions: a class of Sir2-regulated origins has a stably positioned nucleosome overlapping or near B2. This study illustrates the conserved yet flexible nature of yeast origin architecture to promote ORC binding and origin activity, and helps explain why a strong match to the ORC binding site is insufficient to identify origins within the genome.
Subject(s)
Origin Recognition Complex/metabolism , Replication Origin , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Base Sequence , Binding Sites , Consensus Sequence , DNA Helicases/metabolism , Molecular Sequence Data , Nucleosomes/metabolism , Sequence AlignmentABSTRACT
Introduction: The limited data indicate that pediatric medical errors in the outpatient setting, including at home, are common. This study is the first step of our Ambulatory Pediatric Patient Safety Learning Lab to address medication errors and treatment delays among children with T1D in the outpatient setting. We aimed to identify failures and potential solutions associated with medication errors and treatment delays among outpatient children with T1D. Methods: A transdisciplinary team of parents, safety researchers, and clinicians used Systems Engineering Initiative for Patient Safety (SEIPS) based process mapping of data we collected through in-home medication review, observation of administration, chart reviews, parent surveys, and failure modes and effects analysis (FMEA). Results: Eight (57%) of the 14 children who had home visits experienced 18 errors (31 per 100 medications). Four errors in two children resulted in harm, and 13 had the potential for harm. Two injuries occurred when parents failed to treat severe hypoglycemia and lethargy, and two were due to repeated failures to administer insulin at home properly. In SEIPS-based process maps, high-risk errors occurred during communication between the clinic and home or in management at home. Two FMEAs identified interventions to better communicate with families and support home care, especially during evolving illness. Conclusion: Using SEIPS-based process maps informed by multimodal methods to identify medication errors and treatment delays, we found errors were common. Better support for managing acute illness at home and improved communication between the clinic and home are potentially high-yield interventions.