Characterization Of Drug Interactions With Serum Proteins by Using High-Performance Affinity Chromatography

The binding of drugs with serum proteins can affect the activity, distribution, rate of excretion, and toxicity of pharmaceutical agents in the body. One tool that can be used to quickly analyze and characterize these interactions is high-performance affinity chromatography (HPAC). This review shows how HPAC can be used to study drug-protein binding and describes the various applications of this approach when examining drug interactions with serum proteins. Methods for determining binding constants, characterizing binding sites, examining drug-drug interactions, and studying drug-protein dissociation rates will be discussed. Applications that illustrate the use of HPAC with serum binding agents such as human serum albumin, α1-acid glycoprotein, and lipoproteins will be presented. Recent developments will also be examined, such as new methods for immobilizing serum proteins in HPAC columns, the utilization of HPAC as a tool in personalized medicine, and HPAC methods for the high-throughput screening and characterization of drug-protein binding

Characterization Of Drug Interactions With Serum Proteins by Using High-Performance Affinity Chromatography

The binding of drugs with serum proteins can affect the activity, distribution, rate of excretion, and toxicity of pharmaceutical agents in the body. One tool that can be used to quickly analyze and characterize these interactions is high-performance affinity chromatography (HPAC). This review shows how HPAC can be used to study drug-protein binding and describes the various applications of this approach when examining drug interactions with serum proteins. Methods for determining binding constants, characterizing binding sites, examining drug-drug interactions, and studying drug-protein dissociation rates will be discussed. Applications that illustrate the use of HPAC with serum binding agents such as human serum albumin, α1-acid glycoprotein, and lipoproteins will be presented. Recent developments will also be examined, such as new methods for immobilizing serum proteins in HPAC columns, the utilization of HPAC as a tool in personalized medicine, and HPAC methods for the high-throughput screening and characterization of drug-protein binding

RNA polymerase II stalling promotes nucleosome occlusion and pTEFb recruitment to drive immortalization by Epstein-Barr virus

Epstein-Barr virus (EBV) immortalizes resting B-cells and is a key etiologic agent in the development of numerous cancers. The essential EBV-encoded protein EBNA 2 activates the viral C promoter (Cp) producing a message of ~120 kb that is differentially spliced to encode all EBNAs required for immortalization. We have previously shown that EBNA 2-activated transcription is dependent on the activity of the RNA polymerase II (pol II) C-terminal domain (CTD) kinase pTEFb (CDK9/cyclin T1). We now demonstrate that Cp, in contrast to two shorter EBNA 2-activated viral genes (LMP 1 and 2A), displays high levels of promoter-proximally stalled pol II despite being constitutively active. Consistent with pol II stalling, we detect considerable pausing complex (NELF/DSIF) association with Cp. Significantly, we observe substantial Cp-specific pTEFb recruitment that stimulates high-level pol II CTD serine 2 phosphorylation at distal regions (up to +75 kb), promoting elongation. We reveal that Cp-specific pol II accumulation is directed by DNA sequences unfavourable for nucleosome assembly that increase TBP access and pol II recruitment. Stalled pol II then maintains Cp nucleosome depletion. Our data indicate that pTEFb is recruited to Cp by the bromodomain protein Brd4, with polymerase stalling facilitating stable association of pTEFb. The Brd4 inhibitor JQ1 and the pTEFb inhibitors DRB and Flavopiridol significantly reduce Cp, but not LMP1 transcript production indicating that Brd4 and pTEFb are required for Cp transcription. Taken together our data indicate that pol II stalling at Cp promotes transcription of essential immortalizing genes during EBV infection by (i) preventing promoter-proximal nucleosome assembly and ii) necessitating the recruitment of pTEFb thereby maintaining serine 2 CTD phosphorylation at distal regions

Modulation of enhancer looping and differential gene targeting by Epstein-Barr virus transcription factors directs cellular reprogramming

Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors, we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets (WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular basis for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of enhancer-promoter interactions by viral transcription factors

Effect modification of greenness on the association between heat and mortality: A multi-city multi-country study

Background: Identifying how greenspace impacts the temperature-mortality relationship in urban environments is crucial, especially given climate change and rapid urbanization. However, the effect modification of greenspace on heat-related mortality has been typically focused on a localized area or single country. This study examined the heat-mortality relationship among different greenspace levels in a global setting. Methods: We collected daily ambient temperature and mortality data for 452 locations in 24 countries and used Enhanced Vegetation Index (EVI) as the greenspace measurement. We used distributed lag non-linear model to estimate the heat-mortality relationship in each city and the estimates were pooled adjusting for city-specific average temperature, city-specific temperature range, city-specific population density, and gross domestic product (GDP). The effect modification of greenspace was evaluated by comparing the heat-related mortality risk for different greenspace groups (low, medium, and high), which were divided into terciles among 452 locations. Findings: Cities with high greenspace value had the lowest heat-mortality relative risk of 1·19 (95% CI: 1·13, 1·25), while the heat-related relative risk was 1·46 (95% CI: 1·31, 1·62) for cities with low greenspace when comparing the 99th temperature and the minimum mortality temperature. A 20% increase of greenspace is associated with a 9·02% (95% CI: 8·88, 9·16) decrease in the heat-related attributable fraction, and if this association is causal (which is not within the scope of this study to assess), such a reduction could save approximately 933 excess deaths per year in 24 countries. Interpretation: Our findings can inform communities on the potential health benefits of greenspaces in the urban environment and mitigation measures regarding the impacts of climate change.Research in context - I-Evidence before this study: Urbanization and climate change have resulted in changes to the urban environment, including the urban heat island effect and contributions to other extreme weather events. Recently, as metropolitan areas have become denser due to rapid urbanization, environmental problems such as high temperatures are also worsening. Many studies showed that high temperatures increase health risks, including mortality. Therefore, identifying factors that could mitigate the high-temperature conditions in urban environments are a crucial part of climate change mitigation strategies. Many studies found that urban green spaces may play an important role in mitigating heat. Specifically, large green spaces have shown a significant and positive cooling effect. Vegetation can promote air convection through shading and evapotranspiration, which indicates that dense vegetation can lower air temperature. Therefore, more greenspace could result in lower temperatures during the warm season, which would lower exposure to high temperatures that impact human health. Importantly, while greenspace can lower exposure to heat, this study examined how greenspace modifies the heat-health relationship. Some studies have investigated this issue. For example, studies found that heat-related mortality and ambulance calls are negatively correlated with the amount of greenspace coverage. However, most previous work on how greenspace modifies the heat-health relationship was based on one country or region. Research is needed on a global scale to understand how greenspace in urban areas among different countries, with different populations, levels of urbanization, and types of greenspace, can modify the relationship between extreme temperatures and health. As climate change is anticipated to increase temperatures and the associated health consequences worldwide, greenspace may be a plausible mitigation strategy for cities in order to address heat-related health impacts at present and in the future. II-Added value of this study: In this study, we explored the effect modification of greenspace on the heat-mortality relationship on a global scale. With a dataset of 452 locations from 24 countries located in various climate zones and continents, this study incorporated variability in greenspace, temperature, and population characteristics. We found that, based on 452 locations, the heat-mortality risks differed with greenspace category and the cities with higher greenspace values had lower heat-mortality risk than those with lower greenspace values. III-Implications of all the available evidence: Our findings provide evidence that higher greenspace reduces the heat-related mortality, which is similar to other previous smaller studies, and our study results were consistent in different countries around various climate zones. These findings indicate that disparate greenspace levels, temperature, and environment settings should be considered when developing policies and strategies in climate change mitigation and public health adaptation. This study adds to the existing literature that greenspace can reduce the urban heat island effect, by providing evidence for the theory that greenspace can also lower the heat-mortality association, and documents such impacts on a global scale.This publication was developed under Assistance Agreement No. RD83587101 awarded by the U.S. Environmental Protection Agency to Yale University. Research reported in this publication was also supported by the National Institute on Minority Health and Health Disparities of the National Institutes of Health under Award Number R01MD012769. Also, this work has been supported by the National Research Foundation of Korea (2021R1A6A3A03038675), Medical Research Council-UK (MR/V034162/1 and MR/R013349/1), Natural Environment Research Council UK (Grant ID: NE/R009384/1), Academy of Finland (Grant ID: 310372), European Union's Horizon 2020 Project Exhaustion (Grant ID: 820655 and 874990), Czech Science Foundation (22-24920S), Emory University's NIEHS-funded HERCULES Center (Grant ID: P30ES019776), and Grant CEX2018-000794-S funded by MCIN/AEI/ 10.13039/501100011033.info:eu-repo/semantics/publishedVersio

Projections of Excess Mortality Related to Diurnal Temperature Range Under Climate Change Scenarios: a multi-country modelling study

Various retrospective studies have reported on the increase of mortality risk due to higher diurnal temperature range (DTR). This study projects the effect of DTR on future mortality across 445 communities in 20 countries and regions

The Complete Spectrum of Yeast Chromosome Instability Genes Identifies Candidate CIN Cancer Genes and Functional Roles for ASTRA Complex Components

Chromosome instability (CIN) is observed in most solid tumors and is linked to somatic mutations in genome integrity maintenance genes. The spectrum of mutations that cause CIN is only partly known and it is not possible to predict a priori all pathways whose disruption might lead to CIN. To address this issue, we generated a catalogue of CIN genes and pathways by screening ∼2,000 reduction-of-function alleles for 90% of essential genes in Saccharomyces cerevisiae. Integrating this with published CIN phenotypes for other yeast genes generated a systematic CIN gene dataset comprised of 692 genes. Enriched gene ontology terms defined cellular CIN pathways that, together with sequence orthologs, created a list of human CIN candidate genes, which we cross-referenced to published somatic mutation databases revealing hundreds of mutated CIN candidate genes. Characterization of some poorly characterized CIN genes revealed short telomeres in mutants of the ASTRA/TTT components TTI1 and ASA1. High-throughput phenotypic profiling links ASA1 to TTT (Tel2-Tti1-Tti2) complex function and to TORC1 signaling via Tor1p stability, consistent with the role of TTT in PI3-kinase related kinase biogenesis. The comprehensive CIN gene list presented here in principle comprises all conserved eukaryotic genome integrity pathways. Deriving human CIN candidate genes from the list allows direct cross-referencing with tumor mutational data and thus candidate mutations potentially driving CIN in tumors. Overall, the CIN gene spectrum reveals new chromosome biology and will help us to understand CIN phenotypes in human disease

A Conceptual Framework for Healthy Eating Behavior in Ecuadorian Adolescents: A Qualitative Study

Objective: The objective of this study was to identify factors influencing eating behavior of Ecuadorian adolescents - from the perspective of parents, school staff and adolescents - to develop a conceptual framework for adolescents’ eating behavior. Study design: Twenty focus groups (N = 144 participants) were conducted separately with adolescents aged 11–15 y (n (focus groups) = 12, N (participants) = 80), parents (n = 4, N = 32) and school staff (n = 4, N = 32) in rural and urban Ecuador. A semi-structured questioning route was developed based on the ‘Attitude, Social influences and Self-efficacy’ model and the socio-ecological model to assess the relevance of behavioral and environmental factors in low- and middle-income countries. Two researchers independently analyzed verbatim transcripts for emerging themes, using deductive thematic content analysis. Data were analyzed using NVivo 8. Results: All groups recognized the importance of eating healthily and key individual factors in Ecuadorian adolescents’ food choices were: financial autonomy, food safety perceptions, lack of self-control, habit strength, taste preferences and perceived peer norms. Environmental factors included the poor nutritional quality of food and its easy access at school. In their home and family environment, time and convenience completed the picture as barriers to eating healthily. Participants acknowledged the impact of the changing socio-cultural environment on adolescents’ eating patterns. Availability of healthy food at home and financial constraints differed between settings and socio-economic groups. Conclusion: Our findings endorse the importance of investigating behavioral and environmental factors that influence and mediate healthy dietary behavior prior to intervention development. Several culture-specific factors emerged that were incorporated into a conceptual framework for developing health promotion interventions in Ecuador