Miracle-dz/UniFAR-FTUs huggingface dataset:
Geminin Inhibits a Late Step in the Formation of Human Pre-replicative Complexes

Background: Initiation of metazoan DNA replication is regulated by Geminin. Results: HsGeminin binds to HsCdt1, HsMcm3, and HsMcm5 and inhibits formation of salt-stable complexes of purified human pre-replication proteins in vitro. Conclusion: Geminin does not prevent association of the pre-replication proteins, but blocks a late step in pre-RC assembly. Significance: This work provides mechanistic insight into the regulation of initiation of human DNA replication. The initial step in initiation of eukaryotic DNA replication involves the assembly of pre-replicative complexes (pre-RCs) at origins of replication during the G1 phase of the cell cycle. In metazoans initiation is inhibited by the regulatory factor Geminin. We have purified the human pre-RC proteins, studied their interactions in vitro with each other and with origin DNA, and analyzed the effects of HsGeminin on formation of DNA-protein complexes. The formation of an initial complex containing the human origin recognition complex (HsORC), HsCdt1, HsCdc6, and origin DNA is cooperative, involving all possible binary interactions among the components. Maximal association of HsMCM2–7, a component of the replicative helicase, requires HsORC, HsCdc6, HsCdt1, and ATP, and is driven by interactions of HsCdt1 and HsCdc6 with multiple HsMCM2–7 subunits. Formation of stable complexes, resistant to high salt, requires ATP hydrolysis. In the absence of HsMCM proteins, HsGeminin inhibits the association of HsCdt1 with DNA or with HsORC-HsCdc6-DNA complexes. However, HsGeminin does not inhibit recruitment of HsMCM2–7 to DNA to form complexes containing all of the pre-RC proteins. In fact, HsGeminin itself is a component of such complexes, and interacts directly with the HsMcm3 and HsMcm5 subunits of HsMCM2–7, as well as with HsCdt1. Although HsGeminin does not prevent the initial formation of DNA-protein complexes containing the pre-RC proteins, it strongly inhibits the formation of stable pre-RCs that are resistant to high salt. We suggest that bound HsGeminin prevents transition of the pre-RC to a state that is competent for initiation of DNA replication.
Crystal Structures of β-Primeverosidase in Complex with Disaccharide Amidine Inhibitors*

Background: β-Primeverosidase hydrolyzes disaccharide conjugates in tea leaves and releases aromatic volatiles. Results: Crystal structures of β-primeverosidase in complex with disaccharide analogues were determined. Conclusion: β-Primeverosidase has a disaccharide-binding site, and the molecular architecture for disaccharide recognition was elucidated. Significance: β-Primeverosidase adapts the active site to the 6-O-β-d-xylopyranosyl modification of β-glucoside and contributes multiple volatile emissions in tea leaves. β-Primeverosidase (PD) is a disaccharide-specific β-glycosidase in tea leaves. This enzyme is involved in aroma formation during the manufacturing process of oolong tea and black tea. PD hydrolyzes β-primeveroside (6-O-β-d-xylopyranosyl-β-d-glucopyranoside) at the β-glycosidic bond of primeverose to aglycone, and releases aromatic alcoholic volatiles of aglycones. PD only accepts primeverose as the glycone substrate, but broadly accepts various aglycones, including 2-phenylethanol, benzyl alcohol, linalool, and geraniol. We determined the crystal structure of PD complexes using highly specific disaccharide amidine inhibitors, N-β-primeverosylamidines, and revealed the architecture of the active site responsible for substrate specificity. We identified three subsites in the active site: subsite −2 specific for 6-O-β-d-xylopyranosyl, subsite −1 well conserved among β-glucosidases and specific for β-d-glucopyranosyl, and wide subsite +1 for hydrophobic aglycone. Glu-470, Ser-473, and Gln-477 act as the specific hydrogen bond donors for 6-O-β-d-xylopyranosyl in subsite −2. On the other hand, subsite +1 was a large hydrophobic cavity that accommodates various aromatic aglycones. Compared with aglycone-specific β-glucosidases of the glycoside hydrolase family 1, PD lacks the Trp crucial for aglycone recognition, and the resultant large cavity accepts aglycone and 6-O-β-d-xylopyranosyl together. PD recognizes the β-primeverosides in subsites −1 and −2 by hydrogen bonds, whereas the large subsite +1 loosely accommodates various aglycones. The glycone-specific activity of PD for broad aglycone substrates results in selective and multiple release of temporally stored alcoholic volatile aglycones of β-primeveroside.
Electrostatic Interactions in the Binding Pathway of a Transient Protein Complex Studied by NMR and Isothermal Titration Calorimetry*

Background: Electrostatic interactions are known accelerate binding in protein complexes with long lifetimes (minutes to hours). Results: NMR and calorimetry were used to characterize binding kinetics in short lived (∼1 ms) protein-peptide complexes. Conclusion: Electrostatic enhancement is much less and basal (electrostatic-free) association rates are greater than previously observed. Significance: Electrostatics may play different roles in short lived and long lived protein complexes. Much of our knowledge of protein binding pathways is derived from extremely stable complexes that interact very tightly, with lifetimes of hours to days. Much less is known about weaker interactions and transient complexes because these are challenging to characterize experimentally. Nevertheless, these types of interactions are ubiquitous in living systems. The combination of NMR relaxation dispersion Carr–Purcell–Meiboom–Gill (CPMG) experiments and isothermal titration calorimetry allows the quantification of rapid binding kinetics for complexes with submillisecond lifetimes that are difficult to study using conventional techniques. We have used this approach to investigate the binding pathway of the Src homology 3 (SH3) domain from the Fyn tyrosine kinase, which forms complexes with peptide targets whose lifetimes are on the order of about a millisecond. Long range electrostatic interactions have been shown to play a critical role in the binding pathways of tightly binding complexes. The role of electrostatics in the binding pathways of transient complexes is less well understood. Similarly to previously studied tight complexes, we find that SH3 domain association rates are enhanced by long range electrostatics, whereas short range interactions are formed late in the docking process. However, the extent of electrostatic association rate enhancement is several orders of magnitudes less, whereas the electrostatic-free basal association rate is significantly greater. Thus, the SH3 domain is far less reliant on electrostatic enhancement to achieve rapid association kinetics than are previously studied systems. This suggests that there may be overall differences in the role played by electrostatics in the binding pathways of extremely stable versus transient complexes.
Directed Expression of a Chimeric Type II Keratin Partially Rescues Keratin 5-null Mice*

Background: The role of self-mediated keratin filament cross-linking is addressed in transgenic mice. Results: A chimeric type II keratin, K8bc, partially rescues Krt5-null mice. Conclusion: Despite similar assembly properties, K8bc is functionally distinct from its natural counterpart K5 in skin tissue in vivo. Significance: Type II keratins possess unique properties beyond their potential for filament formation subject to cell type-intrinsic regulation. The crucial role of structural support fulfilled by keratin intermediate filaments (IFs) in surface epithelia likely requires that they be organized into cross-linked networks. For IFs comprised of keratins 5 and 14 (K5 and K14), which occur in basal keratinocytes of the epidermis, formation of cross-linked bundles is, in part, self-driven through cis-acting determinants. Here, we targeted the expression of a bundling-competent KRT5/KRT8 chimeric cDNA (KRT8bc) or bundling-deficient wild type KRT8 as a control to the epidermal basal layer of Krt5-null mice to assess the functional importance of keratin IF self-organization in vivo. Such targeted expression of K8bc rescued Krt5-null mice with a 47% frequency, whereas K8 completely failed to do so. This outcome correlated with lower than expected levels of K8bc and especially K8 mRNA and protein in the epidermis of E18.5 replacement embryos. Ex vivo culture of embryonic skin keratinocytes confirmed the ability of K8bc to form IFs in the absence of K5. Additionally, electron microscopy analysis of E18.5 embryonic skin revealed that the striking defects observed in keratin IF bundling, cytoarchitecture, and mitochondria are partially restored by K8bc expression. As young adults, viable KRT8bc replacement mice develop alopecia and chronic skin lesions, indicating that the skin epithelia are not completely normal. These findings are consistent with a contribution of self-mediated organization of keratin IFs to structural support and cytoarchitecture in basal layer keratinocytes of the epidermis and underscore the importance of context-dependent regulation for keratin genes and proteins in vivo.
Rheumatoid arthritis and birth outcomes: a Danish and Swedish nationwide prevalence study

Abstract. Nørgaard M, Larsson H, Pedersen L, Granath F, Askling J, Kieler H, Ekbom A, Sørensen HT, Stephansson O (Aarhus University Hospital, Denmark, Karolinska Institutet; Karolinska University Hospital, Solna; and Karolinska Institutet, Solna; Stockholm, Sweden). Rheumatoid arthritis and birth outcomes: a Danish and Swedish nationwide prevalence study. J Intern Med 2010; 268: 329–337.
Interferon γ-inducible Protein (IFI) 16 Transcriptionally Regulates Type I Interferons and Other Interferon-stimulated Genes and Controls the Interferon Response to both DNA and RNA Viruses*

Background: IFI16 binds dsDNA and elicits a type I interferon response. Results: IFI16 knockdown cells show a decrease in interferon production and ISG expression in response to DNA and RNA ligands and cyclic dinucleotides. Conclusion: IFI16 transcriptionally regulates ISGs to enhance IFN responses to multiple IFN-inducing ligands. Significance: IFI16 has a broader role in the regulation of ISG expression. The interferon γ-inducible protein 16 (IFI16) has recently been linked to the detection of nuclear and cytosolic DNA during infection with herpes simplex virus-1 and HIV. IFI16 binds dsDNA via HIN200 domains and activates stimulator of interferon genes (STING), leading to TANK (TRAF family member-associated NF-κB activator)-binding kinase-1 (TBK1)-dependent phosphorylation of interferon regulatory factor (IRF) 3 and transcription of type I interferons (IFNs) and related genes. To better understand the role of IFI16 in coordinating type I IFN gene regulation, we generated cell lines with stable knockdown of IFI16 and examined responses to DNA and RNA viruses as well as cyclic dinucleotides. As expected, stable knockdown of IFI16 led to a severely attenuated type I IFN response to DNA ligands and viruses. In contrast, expression of the NF-κB-regulated cytokines IL-6 and IL-1β was unaffected in IFI16 knockdown cells, suggesting that the role of IFI16 in sensing these triggers was unique to the type I IFN pathway. Surprisingly, we also found that knockdown of IFI16 led to a severe attenuation of IFN-α and the IFN-stimulated gene retinoic acid-inducible gene I (RIG-I) in response to cyclic GMP-AMP, a second messenger produced by cyclic GMP-AMP synthase (cGAS) as well as RNA ligands and viruses. Analysis of IFI16 knockdown cells revealed compromised occupancy of RNA polymerase II on the IFN-α promoter in these cells, suggesting that transcription of IFN-stimulated genes is dependent on IFI16. These results indicate a broader role for IFI16 in the regulation of the type I IFN response to RNA and DNA viruses in antiviral immunity.
Addressing Adherence to Antiretroviral Therapy in Ouagadougou, Burkina Faso: Insights from hospital ethnography

The human immunodeficiency virus (HIV) epidemic is the most pressing public health crisis of modern times. Present international focus targets expanding access to life-saving antiretroviral treatment (ART) for people living with HIV/AIDS – referred to as treatment scale-upin contexts with limited resources. Near perfect adherence to ART regimens is required to maximize individual and public health outcomes. ART is being scaled-up in African countries with under-funded, fragile health systems. These health systems are further plagued by a shortage of health professionals who play a critical role in the implementation and sustainability of ART programmes, including the provision of adherence support. This ethnographic study sought to explore the experience of health providers in addressing adherence to ART in settings of limited resources where ART is being scaled-up. The study took place in two hospital centres in the capital city of Burkina Faso; Ouagadougou. The study led to three broad conclusions. First, good organization is imperative in the delivery of adherence support services, both in terms of logistics and materiality. This infrastructure must extend beyond the level of the hospital ward, to the level of the health sector in order to ensure a sustainable impact. Second, the provision of ART within a comprehensive package of care is an essential component of effective adherence support. This involves equipping providers with tools to assist them in their clinical practice (laboratory monitoring, treatment for opportunistic infections, and additional staff) as well as mechanisms for them to help patients negotiate the trials of daily life (gratuity of care, literacy support, psychosocial support). Third, there is a need for improved coordination of nationally and internationally-led HIV programming for coherent service provision at the local level. Limited and short-term external funding affects the long-term availability of material and human resources, as well as working conditions and service provision on the wards. The results of the study contribute to the existing academic literature on ART adherence, to the design of future research projects, and to the organization of adherence support services.
Taught postgraduate education in tourism and hospitality in an Irish Institute of Technology : student, graduate, educator and employer perspectives

This thesis was guided by the research question ‘what is the perceived value of a taught postgraduate qualification in hospitality and tourism? ’ The topic was justified, given the dearth of previous research in this area, and the researcher’s positionality as an educator on two taught MSc programmes, in the field of tourism and hospitality, in an institute of technology in the Republic of Ireland. In order to address this question, various stakeholder voices, comprising 15 current students on the two MSc programmes, 122 graduates, 11 educators and 2 employers were included in the primary research. This thesis included a review of secondary literature on education in the field of tourism and hospitality, an investigation into why graduates completed a taught MSc programme, and an assessment of their experiences during their programme of study and subsequent to graduation. The views and experiences of educators on the MSc programmes were explored, as were those of employers. A pragmatist theoretical lens and mixed research methods were employed to fulfil the objectives. Generally, student and graduate satisfaction with the programmes was high, though aspects of module delivery attracted suggestions for improvement. Graduates questioned the value attributed to formal education compared to industry experience. However, educators believed that whilst the MSc contributed to the overall professionalism of the tourism and hospitality industries, academic / industry dialogue was limited. Employers were positive about the role of formal education yet, also, emphasised the importance of attitude and interpersonal skills in the people centred industry that is tourism and hospitality. Educators, students and industry can benefit from the findings of this study.
Understanding consumer preferences for care of adolescents with pelvic inflammatory disease.

Objective The objective of this study is to estimate consumers' maximum willingness-to-pay (WTP) for follow-up PID services by physicians and community health nurses (CHNs), differences by consumer type (adolescents versus parents), and the differences in health-provider predicted WTP consumer estimates and actual consumer WTP estimates. Methods In this IRB-approved study, a contingent valuation method was used to collect WTP data regarding co-payments to physicians or nurses for clinical service delivery from the consumers of adolescent PID services (parents and adolescents) and health providers using a national convenience sample. Consumers were recruited from an academic pediatric and adolescent medicine clinic and five health department school-based health clinics in a large urban community with high (sexually transmitted infection) STI prevalence. Participants completed a web-based survey. Data were analyzed using linear regression analyses. Results Adolescents were willing to pay $36 more (95 % Cl : $27.9-44.3) for community health nursing care and parents were willing to pay $48 more dollars (95 % Cl : $40.3-$57.4) than physician's predicted. There were no significant differences in adolescent and parents WTP for physician or nursing services Consumers (adolescents & parents) WTP for physician PID services were on average $18.50 higher than CHN PID services (p = 0.01). Using physician estimates for WTP as the reference group, adolescents were willing to pay $56 more (95 % Cl : $48.6-$63.4) for physician care and parents were willing to pay $66 more (95 % Cl : $59.0-$72.8) than physician's predicted. Conclusion Adolescents and parents are willing to pay more for physician follow-up for PID, but they are open to CHN follow-up visits based on the mean WTP for CHN visits. Since WTP also reflects the value that individuals place on a service, our data demonstrate that providers consistently underestimate the value consumers place on clinical services for x adolescents with PID.
Faecal calprotectin: a biomarker of gastrointestinal disease in systemic sclerosis

Abstract. Andréasson K, Scheja A, Saxne T, Ohlsson B, Hesselstrand R (Section for Rheumatology; Section for Gastroenterology and Hepatology, Lund University, Lund, Sweden). Faecal calprotectin: a biomarker of gastrointestinal disease in systemic sclerosis. J Intern Med 2011; 270: 50–57.
Exploring Cancer Risk Perceptions of Turf and Lawn Pesticide Professionals in New York State

A pilot study was conducted to better understand the knowledge, perceptions, and concerns of turf and lawn care pesticide professionals in New York State (NYS) regarding cancer and other health risks. Data were collected from 31 focus group participants and 104 questionnaire respondents in applicator trainings and other meetings around the state. Three distinct themes emerged from focus group discussions: cultural shift in pesticide management practices, costs and benefits of pesticide regulations, and challenges of understanding and communicating about health risks. Factor analysis yielded three subscales: control disease, reduce chemical risk, and concern. Exploratory regression analysis yielded predictive relationships between work experience, cancer risk knowledge, and attitudes in support of reducing chemical risk, which in turn predicted precautionary pesticide behavior, such as reading the label and using personal protective equipment. Results suggest the need to incorporate cancer risk education into continuing education for turf and lawn care professionals.
The Molecular Chaperone HSP70 Binds to and Stabilizes NOD2, an Important Protein Involved in Crohn Disease*

Background: NOD2, an innate immune receptor, senses bacterial cell wall fragments. NOD2 mutations are linked to Crohn disease. Results: HSP70 enhances NOD2's activity and increases its half-life. Conclusion: NOD2 mutants are less stable than the wild type. HSP70 overexpression stabilizes the NOD2 Crohn mutants and rescues its activity. Significance: Stabilization of the NOD2 mutants may provide an effective therapy for Crohn disease. Microbes are detected by the pathogen-associated molecular patterns through specific host pattern recognition receptors. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an intracellular pattern recognition receptor that recognizes fragments of the bacterial cell wall. NOD2 is important to human biology; when it is mutated it loses the ability to respond properly to bacterial cell wall fragments. To determine the mechanisms of misactivation in the NOD2 Crohn mutants, we developed a cell-based system to screen for protein-protein interactors of NOD2. We identified heat shock protein 70 (HSP70) as a protein interactor of both wild type and Crohn mutant NOD2. HSP70 has previously been linked to inflammation, especially in the regulation of anti-inflammatory molecules. Induced HSP70 expression in cells increased the response of NOD2 to bacterial cell wall fragments. In addition, an HSP70 inhibitor, KNK437, was capable of decreasing NOD2-mediated NF-κB activation in response to bacterial cell wall stimulation. We found HSP70 to regulate the half-life of NOD2, as increasing the HSP70 level in cells increased the half-life of NOD2, and down-regulating HSP70 decreased the half-life of NOD2. The expression levels of the Crohn-associated NOD2 variants were less compared with wild type. The overexpression of HSP70 significantly increased NOD2 levels as well as the signaling capacity of the mutants. Thus, our study shows that restoring the stability of the NOD2 Crohn mutants is sufficient for rescuing the ability of these mutations to signal the presence of a bacterial cell wall ligand.