Monitoring glycation through the intrinsic fluorescence of biological fluorophores

The high blood glucose levels associated with diabetes affect various cells and proteins in the body. In response to high blood glucose the proteins collagen, keratin, and human serum albumin (HSA) experience glycation, and the cofactor reduced nicotinamide adenine dinucleotide (NADH) is also known to be affected in some way. This work aims to establish if the intrinsic fluorescence of these compounds could be used to monitor the impact of glucose, and thus offer an alternative method to monitoring long term glycaemic control. We have studied the evolution of the intrinsic fluorescence of four compounds in response to glucose in vitro using steady state and time-resolved fluorescence spectroscopy techniques. For NADH, although significant changes were seen in the sample when in a phosphate buffered saline (PBS) buffer, no similar changes in fluorescence were observed when a Trizma buffer was used. As such, we conclude that glycation does not have an impact on the intrinsic fluorescence of NADH. Changes in the intrinsic fluorescence of collagen, keratin, and HSA however were observed. For collagen, TRES revealed changes in the intrinsic fluorescence kinetics, caused by both collagen aggregation and glycation. In keratin, the addition of glucose caused an increase in the fluorescence intensity at the characteristic wavelength of 460 nm, due to faster formation of new cross-links, and glucose may also cause the formation of two new fluorescent complexes that emit at longer wavelengths. For HSA, fluorescence intensity decay analysis indicates that glycation can be detected through a decrease in the short lifetime component when decays are fitted to a 2-exponential model, however fitting to a non-Debye model more clearly highlights the impact of glucose. We also studied the intrinsic fluoresce of mouse skin and human fingernails. In an initial pilot study on mouse skin, our results suggested that collagen, keratin, and NADH can be detected from a skin autofluorescence (AF) measurement, and that there may be a relationship between skin AF and blood glucose. Using fingernails, it appeared that although both the peak emission wavelengths, and the fitted exponential parameters indicated that we can detect keratin fluorescence, there was no evidence that glycated keratin can be sensed through a nail clipping. Furthermore, neither our steady state or time resolved analysis exposed a correlation between any of the extracted fluorescence parameters and glycated haemoglobin (HbA1c). In conclusion, monitoring the intrinsic fluorescence of particular biological proteins in vitro suggests that this method could be used as a method to monitor long term glycaemic control in patients with diabetes. Further studies would be required to translate these findings into an in vivo environment.The high blood glucose levels associated with diabetes affect various cells and proteins in the body. In response to high blood glucose the proteins collagen, keratin, and human serum albumin (HSA) experience glycation, and the cofactor reduced nicotinamide adenine dinucleotide (NADH) is also known to be affected in some way. This work aims to establish if the intrinsic fluorescence of these compounds could be used to monitor the impact of glucose, and thus offer an alternative method to monitoring long term glycaemic control. We have studied the evolution of the intrinsic fluorescence of four compounds in response to glucose in vitro using steady state and time-resolved fluorescence spectroscopy techniques. For NADH, although significant changes were seen in the sample when in a phosphate buffered saline (PBS) buffer, no similar changes in fluorescence were observed when a Trizma buffer was used. As such, we conclude that glycation does not have an impact on the intrinsic fluorescence of NADH. Changes in the intrinsic fluorescence of collagen, keratin, and HSA however were observed. For collagen, TRES revealed changes in the intrinsic fluorescence kinetics, caused by both collagen aggregation and glycation. In keratin, the addition of glucose caused an increase in the fluorescence intensity at the characteristic wavelength of 460 nm, due to faster formation of new cross-links, and glucose may also cause the formation of two new fluorescent complexes that emit at longer wavelengths. For HSA, fluorescence intensity decay analysis indicates that glycation can be detected through a decrease in the short lifetime component when decays are fitted to a 2-exponential model, however fitting to a non-Debye model more clearly highlights the impact of glucose. We also studied the intrinsic fluoresce of mouse skin and human fingernails. In an initial pilot study on mouse skin, our results suggested that collagen, keratin, and NADH can be detected from a skin autofluorescence (AF) measurement, and that there may be a relationship between skin AF and blood glucose. Using fingernails, it appeared that although both the peak emission wavelengths, and the fitted exponential parameters indicated that we can detect keratin fluorescence, there was no evidence that glycated keratin can be sensed through a nail clipping. Furthermore, neither our steady state or time resolved analysis exposed a correlation between any of the extracted fluorescence parameters and glycated haemoglobin (HbA1c). In conclusion, monitoring the intrinsic fluorescence of particular biological proteins in vitro suggests that this method could be used as a method to monitor long term glycaemic control in patients with diabetes. Further studies would be required to translate these findings into an in vivo environment

Keratin intrinsic fluorescence as a mechanism for non-invasive monitoring of its glycation

We have studied the evolution of keratin intrinsic fluorescence as an indicator of its glycation. Steadystate and time-resolved fluorescence of free keratin and keratin-glucose samples were detected in PBS solutions in vitro. The changes in the fluorescence response demonstrate that the effect of glucose is manifest in the accelerated formation of fluorescent cross-links with an emission peak at 460 nm and formation of new cross-links with emission peaks at 525 nm and 575 nm. The fluorescence kinetics of these structures is studied and their potential application for the detection of long-term complications of diabetes discussed

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

Dipeptidyl peptidase-1 inhibition in patients hospitalised with COVID-19: a multicentre, double-blind, randomised, parallel-group, placebo-controlled trial

Background Neutrophil serine proteases are involved in the pathogenesis of COVID-19 and increased serine protease activity has been reported in severe and fatal infection. We investigated whether brensocatib, an inhibitor of dipeptidyl peptidase-1 (DPP-1; an enzyme responsible for the activation of neutrophil serine proteases), would improve outcomes in patients hospitalised with COVID-19. Methods In a multicentre, double-blind, randomised, parallel-group, placebo-controlled trial, across 14 hospitals in the UK, patients aged 16 years and older who were hospitalised with COVID-19 and had at least one risk factor for severe disease were randomly assigned 1:1, within 96 h of hospital admission, to once-daily brensocatib 25 mg or placebo orally for 28 days. Patients were randomly assigned via a central web-based randomisation system (TruST). Randomisation was stratified by site and age (65 years or ≥65 years), and within each stratum, blocks were of random sizes of two, four, or six patients. Participants in both groups continued to receive other therapies required to manage their condition. Participants, study staff, and investigators were masked to the study assignment. The primary outcome was the 7-point WHO ordinal scale for clinical status at day 29 after random assignment. The intention-to-treat population included all patients who were randomly assigned and met the enrolment criteria. The safety population included all participants who received at least one dose of study medication. This study was registered with the ISRCTN registry, ISRCTN30564012. Findings Between June 5, 2020, and Jan 25, 2021, 406 patients were randomly assigned to brensocatib or placebo; 192 (47·3%) to the brensocatib group and 214 (52·7%) to the placebo group. Two participants were excluded after being randomly assigned in the brensocatib group (214 patients included in the placebo group and 190 included in the brensocatib group in the intention-to-treat population). Primary outcome data was unavailable for six patients (three in the brensocatib group and three in the placebo group). Patients in the brensocatib group had worse clinical status at day 29 after being randomly assigned than those in the placebo group (adjusted odds ratio 0·72 [95% CI 0·57–0·92]). Prespecified subgroup analyses of the primary outcome supported the primary results. 185 participants reported at least one adverse event; 99 (46%) in the placebo group and 86 (45%) in the brensocatib group. The most common adverse events were gastrointestinal disorders and infections. One death in the placebo group was judged as possibly related to study drug. Interpretation Brensocatib treatment did not improve clinical status at day 29 in patients hospitalised with COVID-19

Collagen glycation detected by its intrinsic fluorescence

Collagen's long half-life (in skin approximately 10 years) makes this protein highly susceptible to glycation and formation of the advanced glycation end products (AGEs). Accumulation of cross-linking AGEs in the skin collagen has several detrimental effects; thus, the opportunity for non-invasive monitoring of skin glycation is essential, especially for diabetic patients. In this paper, we report using the time-resolved intrinsic fluorescence of collagen as a biomarker of its glycation. Contrary to the traditional fluorescence intensity decay measurement at the arbitrarily selected excitation and detection wavelengths, we conducted systematic wavelength- and time-resolved measurements to achieve time-resolved emission spectra. Changes in the intrinsic fluorescence kinetics, caused by both collagen aggregation and glycation, have been detected

Multiple loci on 8q24 associated with prostate cancer susceptibility

Previous studies have identified multiple loci on 8q24 associated with prostate cancer risk. We performed a comprehensive analysis of SNP associations across 8q24 by genotyping tag SNPs in 5,504 prostate cancer cases and 5,834 controls. We confirmed associations at three previously reported loci and identified additional loci in two other linkage disequilibrium blocks (rs1006908: per-allele OR = 0.87, P = 7.9 x 10(-8); rs620861: OR = 0.90, P = 4.8 x 10(-8)). Eight SNPs in five linkage disequilibrium blocks were independently associated with prostate cancer susceptibility

Identification of seven new prostate cancer susceptibility loci through a genome-wide association study

Prostate cancer (PrCa) is the most frequently diagnosed male cancer in developed countries. To identify common PrCa susceptibility alleles, we have previously conducted a genome-wide association study in which 541, 129 SNPs were genotyped in 1,854 PrCa cases with clinically detected disease and 1,894 controls. We have now evaluated promising associations in a second stage, in which we genotyped 43,671 SNPs in 3,650 PrCa cases and 3,940 controls, and a third stage, involving an additional 16,229 cases and 14,821 controls from 21 studies. In addition to previously identified loci, we identified a further seven new prostate cancer susceptibility loci on chromosomes 2, 4, 8, 11, and 22 (P=1.6×10−8 to P=2.7×10−33)

Genomic reconstruction of the SARS-CoV-2 epidemic in England

AbstractThe evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus leads to new variants that warrant timely epidemiological characterization. Here we use the dense genomic surveillance data generated by the COVID-19 Genomics UK Consortium to reconstruct the dynamics of 71 different lineages in each of 315 English local authorities between September 2020 and June 2021. This analysis reveals a series of subepidemics that peaked in early autumn 2020, followed by a jump in transmissibility of the B.1.1.7/Alpha lineage. The Alpha variant grew when other lineages declined during the second national lockdown and regionally tiered restrictions between November and December 2020. A third more stringent national lockdown suppressed the Alpha variant and eliminated nearly all other lineages in early 2021. Yet a series of variants (most of which contained the spike E484K mutation) defied these trends and persisted at moderately increasing proportions. However, by accounting for sustained introductions, we found that the transmissibility of these variants is unlikely to have exceeded the transmissibility of the Alpha variant. Finally, B.1.617.2/Delta was repeatedly introduced in England and grew rapidly in early summer 2021, constituting approximately 98% of sampled SARS-CoV-2 genomes on 26 June 2021.</jats:p