Biosensor-based studies on coumarins

Polyclonal antibodies to 7-hydroxycoumarin - the main metabolite of coumarin, a plant constituent with many clinical applications - were produced, purified and characterised. This antibody preparation was used to develop a competitive immunoassay for 7- hydroxycoumarin which was carried out on the BIAcore, an optical biosensor based on the phenomenon of surface plasmon resonance (SPR). The immunoassay was optimised and internally validated. Monoclonal antibodies were generated by somatic cell fusion using the spleens of mice immunised with a 7-hydroxycoumarin-protein conjugate. The screening of cell culture supernatants by BIAcore and ELISA was compared, and all of 13 clones isolated by limiting dilution were found to be reactive to the drug-protein conjugate, but not to free drug. The panning of phage displayed antibodies from a naïve library using the BIAcore was investigated, and, although the amount of bound phage was insufficient to generate an SPR signal, subsequent analysis of eluate demonstrated that enrichment had taken place. BIAcore was also used to affinity rank a panel of 3 genetically-produced single chain Fv antibodies against coumarin-BSA and to examine kinetic and affinity data for the interaction of one of these antibodies with immobilised drug-protein conjugate. The interaction of two antibody preparations against the fungal toxin aflatoxin Bi (AFBj), a member of the coumarin family, was studied using both BIAcore and ELISA methods. High affinity constants rendered the monoclonal and polyclonal antibodies unsuitable for use in regenerable immunosensor formats. A range of competitive and sandwich ELISAs for the detection of AFBi were developed using both types of antibody. In addition, methods to facilitate better design of formats for use with BIAcore were established using ELISA to mimic regeneration conditions on the sensor chip surface, and to calculate equilibrium affinity constants. Supercoiled plasmid DNA was also immobilised on the BIAcore sensor surface, and the inhibition of the enzyme topoisomerase II by the coumarin antibiotic novobiocin, as well as the direct binding of a range of coumarin-protein conjugates to nucleic acids, were investigated

Differences in visceral adipose tissue and biochemical cardiometabolic risk markers in elite rugby union athletes of Caucasian and Polynesian descent

Polynesian individuals are leaner with greater musculature than Caucasians of an equivalent size, and this genetically different morphology provides a physique that is often compatible with success in a number of sports, including rugby union. Evidence indicates that Polynesians have greater stores of absolute and relative abdominal fat mass and this is known to confer cardiometabolic risk. The aims of this study were to (1) explore the relationship between ethnicity, visceral adipose tissue (VAT), and cardiometabolic disease risk markers in elite Caucasian and Polynesian rugby union athletes, and (2) assess the impact of a pre-season training programme on these markers. Twenty-two professional rugby union athletes of Caucasian (n = 11) and Polynesian (n = 11) descent underwent physique assessment via surface anthropometry, dual-energy X-ray absorptiometry, and magnetic resonance imaging before and after an 11-week pre-season. A fasted blood test was undertaken at both time points. Compared to Caucasians, at baseline Polynesians displayed significantly higher VAT (771 ± 609 cm3 vs 424 ± 235 cm3; p = 0.043), triglycerides (1.0 ± 0.9 mmol/L vs 0.6 ± 0.2 mmol/L; p = 0.050), and low-density lipoprotein cholesterol (3.1 ± 0.9 mmol/L vs 2.3 ± 0.7 mmol/L; p = 0.019). Similar changes were observed in both groups over the pre-season period in VAT and blood biochemical markers. Polynesian rugby union athletes were more likely than Caucasians to exhibit risk factors associated with cardiometabolic disease, such as elevated VAT and unfavourable lipid profiles. Further longitudinal research is required to identify and explain the short- and long-term risk of cardiometabolic disease in athletes of Polynesian descent

Longitudinal changes in body composition assessed using DXA and surface anthropometry show good agreement in elite Rugby Union athletes

Rugby union athletes have divergent body composition based on the demands of their on-field playing position and ethnicity. With an established association between physique traits and positional requirements, body composition assessment is routinely undertaken. Surface anthropometry and dual-energy X-ray absorptiometry (DXA) are the most common assessment techniques utilised, often undertaken synchronously. This study aims to investigate the association between DXA and surface anthropometry when assessing longitudinal changes in fat free mass (FFM) and fat mass (FM) in rugby union athletes. Thirty-nine elite male rugby union athletes (age 25.7 ± 3.1 years; stature 187.6 ± 7.7 cm; mass 104.1 ± 12.2 kg) underwent assessment via DXA and surface anthropometry multiple times over three consecutive international seasons. Changes in the lean mass index (LMI), an empirical measure to assess proportional variation in FFM, showed large agreement with changes in DXA FFM (r=0.54, SEE=1.5%,

Investigating the influence of African American and African Caribbean race on primary care doctors' decision making about depression

This paper explores differences in how primary care doctors process the clinical presentation of depression by African American and African-Caribbean patients compared with white patients in the US and the UK. The aim is to gain a better understanding of possible pathways by which racial disparities arise in depression care. One hundred and eight doctors described their thought processes after viewing video recorded simulated patients presenting with identical symptoms strongly suggestive of depression. These descriptions were analysed using the CliniClass system, which captures information about micro-components of clinical decision making and permits a systematic, structured and detailed analysis of how doctors arrive at diagnostic, intervention and management decisions. Video recordings of actors portraying black (both African American and African-Caribbean) and white (both White American and White British) male and female patients (aged 55 years and 75 years) were presented to doctors randomly selected from the Massachusetts Medical Society list and from Surrey/South West London and West Midlands National Health Service lists, stratified by country (US v.UK), gender, and years of clinical experience (less v. very experienced). Findings demonstrated little evidence of bias affecting doctors' decision making processes, with the exception of less attention being paid to the potential outcomes associated with different treatment options for African American compared with White American patients in the US. Instead, findings suggest greater clinical uncertainty in diagnosing depression amongst black compared with white patients, particularly in the UK. This was evident in more potential diagnoses. There was also a tendency for doctors in both countries to focus more on black patients' physical rather than psychological symptoms and to identify endocrine problems, most often diabetes, as a presenting complaint for them. This suggests that doctors in both countries have a less well developed mental model of depression for black compared with white patients

Reducing the health disparities of Indigenous Australians: time to change focus

Background: Indigenous peoples have worse health than non-Indigenous, are over-represented amongst the poor and disadvantaged, have lower life expectancies, and success in improving disparities is limited. To address this, research usually focuses on disadvantaged and marginalised groups, offering only partial understanding of influences underpinning slow progress. Critical analysis is also required of those with the power to perpetuate or improve health inequities. In this paper, using Australia as a case example, we explore the effects of ‘White’, Anglo-Australian cultural dominance in health service delivery to Indigenous Australians. We address the issue using race as an organising principle, underpinned by relations of power.Methods: Interviews with non-Indigenous medical practitioners in Western Australia with extensive experience in Indigenous health encouraged reflection and articulation of their insights into factors promoting or impeding quality health care to Indigenous Australians. Interviews were audio-taped and transcribed. An inductive, exploratory analysis identified key themes that were reviewed and interrogated in light of existing literature on health care to Indigenous people, race and disadvantage. The researchers’ past experience, knowledge and understanding of health care and Indigenous health assisted with data interpretation. Informal discussions were also held with colleagues working professionally in Indigenous policy, practice and community settings.Results: Racism emerged as a key issue, leading us to more deeply interrogate the role ‘Whiteness’ plays in Indigenous health care. While Whiteness can refer to skin colour, it also represents a racialized social structure where Indigenous knowledge, beliefs and values are subjugated to the dominant western biomedical model in policy and practice. Racism towards Indigenous patients in health services was institutional and interpersonal. Internalised racism was manifest when Indigenous patients incorporated racist attitudes and beliefs into their lived experience, lowering expectations and their sense of self-worth.Conclusions: Current health policies and practices favour standardised care where the voice of those who are marginalised is often absent. Examining the effectiveness of such models in reducing health disparities requires health providers to critically reflect on whether policies and practices promote or compromise Indigenous health and wellbeing - an important step in changing the discourse that places Indigenous people at the centre of the problem

Identification, Replication, and Fine-Mapping of Loci Associated with Adult Height in Individuals of African Ancestry

Adult height is a classic polygenic trait of high heritability (h2 ∼0.8). More than 180 single nucleotide polymorphisms (SNPs), identified mostly in populations of European descent, are associated with height. These variants convey modest effects and explain ∼10% of the variance in height. Discovery efforts in other populations, while limited, have revealed loci for height not previously implicated in individuals of European ancestry. Here, we performed a meta-analysis of genome-wide association (GWA) results for adult height in 20,427 individuals of African ancestry with replication in up to 16,436 African Americans. We found two novel height loci (Xp22-rs12393627, P = 3.4×10−12 and 2p14-rs4315565, P = 1.2×10−8). As a group, height associations discovered in European-ancestry samples replicate in individuals of African ancestry (P = 1.7×10−4 for overall replication). Fine-mapping of the European height loci in African-ancestry individuals showed an enrichment of SNPs that are associated with expression of nearby genes when compared to the index European height SNPs (P<0.01). Our results highlight the utility of genetic studies in non-European populations to understand the etiology of complex human diseases and traits

Quantifying neutralising antibody responses against SARS-CoV-2 in dried blood spots (DBS) and paired sera

The ongoing SARS-CoV-2 pandemic was initially managed by non-pharmaceutical interventions such as diagnostic testing, isolation of positive cases, physical distancing and lockdowns. The advent of vaccines has provided crucial protection against SARS-CoV-2. Neutralising antibody (nAb) responses are a key correlate of protection, and therefore measuring nAb responses is essential for monitoring vaccine efficacy. Fingerstick dried blood spots (DBS) are ideal for use in large-scale sero-surveillance because they are inexpensive, offer the option of self-collection and can be transported and stored at ambient temperatures. Such advantages also make DBS appealing to use in resource-limited settings and in potential future pandemics. In this study, nAb responses in sera, venous blood and fingerstick blood stored on filter paper were measured. Samples were collected from SARS-CoV-2 acutely infected individuals, SARS-CoV-2 convalescent individuals and SARS-CoV-2 vaccinated individuals. Good agreement was observed between the nAb responses measured in eluted DBS and paired sera. Stability of nAb responses was also observed in sera stored on filter paper at room temperature for 28 days. Overall, this study provides support for the use of filter paper as a viable sample collection method to study nAb responses.</p

Correction: Identification, Replication, and Fine-Mapping of Loci Associated with Adult Height in Individuals of African Ancestry

Adult height is a classic polygenic trait of high heritability (h 2 ∼0.8). More than 180 single nucleotide polymorphisms (SNPs), identified mostly in populations of European descent, are associated with height. These variants convey modest effects and explain ∼10% of the variance in height. Discovery efforts in other populations, while limited, have revealed loci for height not previously implicated in individuals of European ancestry. Here, we performed a meta-analysis of genome-wide association (GWA) results for adult height in 20,427 individuals of African ancestry with replication in up to 16,436 African Americans. We found two novel height loci (Xp22-rs12393627, P = 3.4×10−12 and 2p14-rs4315565, P = 1.2×10−8). As a group, height associations discovered in European-ancestry samples replicate in individuals of African ancestry (P = 1.7×10−4 for overall replication). Fine-mapping of the European height loci in African-ancestry individuals showed an enrichment of SNPs that are associated with expression of nearby genes when compared to the index European height SNPs (P<0.01). Our results highlight the utility of genetic studies in non-European populations to understand the etiology of complex human diseases and traits

Genetic mechanisms of critical illness in COVID-19.

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice