Enzyme Immobilization Strategies and Electropolymerization Conditions to Control Sensitivity and Selectivity Parameters of a Polymer-Enzyme Composite Glucose Biosensor

In an ongoing programme to develop characterization strategies relevant to biosensors for in-vivo monitoring, glucose biosensors were fabricated by immobilizing the enzyme glucose oxidase (GOx) on 125 μm diameter Pt cylinder wire electrodes (PtC), using three different methods: before, after or during the amperometric electrosynthesis of poly(ortho-phenylenediamine), PoPD, which also served as a permselective membrane. These electrodes were calibrated with H2O2 (the biosensor enzyme signal molecule), glucose, and the archetypal interference compound ascorbic acid (AA) to determine the relevant polymer permeabilities and the apparent Michaelis-Menten parameters for glucose. A number of selectivity parameters were used to identify the most successful design in terms of the balance between substrate sensitivity and interference blocking. For biosensors electrosynthesized in neutral buffer under the present conditions, entrapment of the GOx within the PoPD layer produced the design (PtC/PoPD-GOx) with the highest linear sensitivity to glucose (5.0 ± 0.4 μA cm−2 mM−1), good linear range (KM = 16 ± 2 mM) and response time (< 2 s), and the greatest AA blocking (99.8% for 1 mM AA). Further optimization showed that fabrication of PtC/PoPD-GOx in the absence of added background electrolyte (i.e., electropolymerization in unbuffered enzyme-monomer solution) enhanced glucose selectivity 3-fold for this one-pot fabrication protocol which provided AA-rejection levels at least equal to recent multi-step polymer bilayer biosensor designs. Interestingly, the presence of enzyme protein in the polymer layer had opposite effects on permselectivity for low and high concentrations of AA, emphasizing the value of studying the concentration dependence of interference effects which is rarely reported in the literature

Determination of Dosage Compensation of the Mammalian X Chromosome by RNA-seq is Dependent on Analytical Approach

Background An enduring question surrounding sex chromosome evolution is whether effective hemizygosity in the heterogametic sex leads inevitably to dosage compensation of sex-linked genes, and whether this compensation has been observed in a variety of organisms. Incongruence in the conclusions reached in some recent reports has been attributed to different high-throughput approaches to transcriptome analysis. However, recent reports each utilizing RNA-seq to gauge X-linked gene expression relative to autosomal gene expression also arrived at diametrically opposed conclusions regarding X chromosome dosage compensation in mammals. Results Here we analyze RNA-seq data from X-monosomic female human and mouse tissues, which are uncomplicated by genes that escape X-inactivation, as well as published RNA-seq data to describe relative X expression (RXE). We find that the determination of RXE is highly dependent upon a variety of computational, statistical and biological assumptions underlying RNA-seq analysis. Parameters implemented in short-read mapping programs, choice of reference genome annotation, expression data distribution, tissue source for RNA and RNA-seq library construction method have profound effects on comparing expression levels across chromosomes. Conclusions Our analysis shows that the high number of paralogous gene families on the mammalian X chromosome relative to autosomes contributes to the ambiguity in RXE calculations, RNA-seq analysis that takes into account that single- and multi-copy genes are compensated differently supports the conclusion that, in many somatic tissues, the mammalian X is up-regulated compared to the autosomes

RASSF1A–LATS1 signalling stabilizes replication forks by restricting CDK2-mediated phosphorylation of BRCA2

Genomic instability is a key hallmark of cancer leading to tumour heterogeneity and therapeutic resistance. ​BRCA2 has a fundamental role in error-free DNA repair but also sustains genome integrity by promoting ​RAD51 nucleofilament formation at stalled replication forks. ​CDK2 phosphorylates ​BRCA2 (pS3291-​BRCA2) to limit stabilizing contacts with polymerized ​RAD51; however, how replication stress modulates ​CDK2 activity and whether loss of pS3291-​BRCA2 regulation results in genomic instability of tumours are not known. Here we demonstrate that the Hippo pathway kinase ​LATS1 interacts with ​CDK2 in response to genotoxic stress to constrain pS3291-​BRCA2 and support ​RAD51 nucleofilaments, thereby maintaining genomic fidelity during replication stalling. We also show that ​LATS1 forms part of an ​ATR-mediated response to replication stress that requires the tumour suppressor ​RASSF1A. Importantly, perturbation of the ​ATR–​RASSF1A–​LATS1 signalling axis leads to genomic defects associated with loss of ​BRCA2 function and contributes to genomic instability and ‘BRCA-ness’ in lung cancers

Multi-omic analysis of Esophageal Adenocarcinoma uncovers candidate therapeutic targets and cancer-selective post-transcriptional regulation

Background: Efforts to address the poor prognosis associated with esophagealadenocarcinoma (EAC) have been hampered by a lack of biomarkers to identify early disease and therapeutic targets. Despite extensive efforts to understand the somatic mutations associated with EAC over the past decade, a gap remains in understanding how the atlas of genomic aberrations in this cancer impacts the proteome and which somatic variants are of importance for the disease phenotype.Methods: We performed a quantitative proteomic analysis of 23 EACs and matched adjacent normal esophageal and gastric tissues. We explored the correlation of transcript and protein abundance using tissue-matched RNA-seq and proteomic data from 7 patients and further integrated these data with a cohort of EAC RNA-seq data (n=264 patients), EAC whole-genome sequencing (n=454 patients) and external published datasets.Central findings: We quantified protein expression from 5879 genes in EAC and patient-matched normal tissues. Several biomarker candidates with EAC-selective expression were identified including the transmembrane protein GPA33. We further verified the EAC-enriched expression of GPA33 in an external cohort of 115 patients and confirm this as an attractive diagnostic and therapeutic target. To further extend the insights gained from our proteomic data, an integrated analysis of protein and RNA expression in EAC and normal tissues revealed several genes with poorly correlated Protein and RNA abundance, suggesting post-transcriptional regulation of protein expression. These outlier genes including SLC25A30, TAOK2, and AGMAT, only rarely demonstrated somatic mutation suggesting post-transcriptional drivers for this EAC-specific phenotype. AGMAT was demonstrated to be over-expressed at the protein level in EAC compared to adjacent normal tissues with an EAC-selective, post-transcriptional mechanism of regulation of protein abundance proposed.Conclusions: By quantitative proteomic analysis we have identified GPA33 as an EAC-selective biomarker. Integrated analysis of proteome, transcriptome, and genome in EAC hasJournal Pre-proof Running Title: Multi-omic analysis of Esophageal Adenocarcinoma uncovers candidate therapeutic targets and cancer-selective post-transcriptional regulation revealed several genes with tumor-selective, post-transcriptional regulation of protein expression which may be an exploitable vulnerabilit

Insights into toxic prymnesium parvum blooms:The role of sugars and algal viruses

Prymnesium parvum is a toxin-producing microalga that causes harmful algal blooms globally, which often result in large-scale fish kills that have severe ecological and economic implications. Although many toxins have previously been isolated from P. parvum, ambiguity still surrounds the responsible ichthyotoxins in P. parvum blooms and the biotic and abiotic factors that promote bloom toxicity. A major fish kill attributed to P. parvum occurred in Spring 2015 on the Norfolk Broads, a low-lying set of channels and lakes (Broads) found on the East of England. Here, we discuss how water samples taken during this bloom have led to diverse scientific advances ranging from toxin analysis to discovery of a new lytic virus of P. parvum, P. parvum DNA virus (PpDNAV-BW1). Taking recent literature into account, we propose key roles for sialic acids in this type of viral infection. Finally, we discuss recent practical detection and management strategies for controlling these devastating blooms

A national registry study of patient and renal survival in adult nephrotic syndrome

Introduction: We aimed to determine the mortality rate, cause of death, and rate of end-stage kidney disease (ESKD) in adults with nephrotic syndrome (NS). Methods: We conducted a national registry–based study, including all 522 adults who had a kidney biopsy for NS in Scotland in 2014–2017. We linked the Scottish Renal Registry to death certificate data. We performed survival and Cox proportional hazards analyses, accounting for competing risks of death and ESKD. We compared mortality rates with those in the age- and sex-matched general population. Results: A total of 372 patients had primary NS; 150 had secondary NS. Over a median follow-up of 866 days, 110 patients (21%) died. In patients with primary NS, observed versus population 3-year mortality was 2.1% (95% CI 0.0%–4.6%) versus 0.9% (0.8%–1.0%) in patients aged &lt;60 years and 24.9% (18.4%–30.8%) versus 9.4% (8.3%–10.5%) in those aged ≥60 years. In secondary NS, this discrepancy was 17.1% (5.6%–27.2%) versus 1.1% (0.9%–1.2%) in &lt;60-year-olds and 49.4% (36.6%–59.7%) versus 8.1% (6.6%–9.6%) in ≥60-year-olds. In primary NS, cardiovascular causes accounted for 28% of deaths, compared with 18% in the general population. Eighty patients (15%) progressed to ESKD. Incidence of ESKD by 3 years was 8.4% (95% CI 4.9%–11.7%) in primary and 35.1% (24.3%–44.5%) in secondary NS. Early remission of proteinuria and the absence of early acute kidney injury (AKI) were associated with lower rates of death and ESKD. Conclusions: Adults with NS have high rates of death and ESKD. Cardiovascular causes account for excess mortality in primary NS

The androgen receptor gene CAG repeat in relation to 4-year changes in androgen-sensitive endpoints in community-dwelling older European men

Context: The Androgen Receptor (AR) gene exon 1 CAG repeat length has been proposed to be a determinant of between-individual variations in androgen action in target tissues, which might regulate phenotypic differences of human ageing. However, findings on its phenotypic effects are inconclusive.Objective: To assess whether the AR CAG repeat length is associated with longitudinal changes in endpoints which are influenced by testosterone (T) levels in middle-aged and elderly European men.Design: Multinational European observational prospective cohort studyParticipants: 1887 men (mean±sd age: 63±11 years; median follow-up: 4.3 years) from centres of 8 European countries comprised the analysis sample after exclusion of those with diagnosed diseases of the hypothalamic-pituitary-testicular (HPT) axis.Main outcome measures: Longitudinal associations between the AR CAG repeat and changes in androgen-sensitive endpoints (ASEs) and medical conditions were assessed using regression analysis adjusting for age and centre. The AR CAG repeat length was treated both as a continuous and categorical (6-20; 21-23; 24-39 repeats) predictor. Additional analysis investigated whether results were independent of baseline T or oestradiol (E2) levels.Results: The AR CAG repeat, when used as a continuous or categorical predictor, was not associated with longitudinal changes in ASEs or medical conditions after adjustments. These results were independent of T and E2 levels.Conclusion: Within a 4-year timeframe, variations in the AR CAG repeat do not contribute to the rate of phenotypic ageing, over and above, that, which might be associated with the age-related decline in T levels

Assessing the ecological impacts of invasive species based on their functional responses and abundances

Invasive species management requires allocation of limited resources towards the proactive mitigation of those species that could elicit the highest ecological impacts. However, we lack predictive capacity with respect to the identities and degree of ecological impacts of invasive species. Here, we combine the relative per capita effects and relative field abundances of invader as compared to native species into a new metric, “Relative Impact Potential” (RIP), and test whether this metric can reliably predict high impact invaders. This metric tests the impact of invaders relative to the baseline impacts of natives on the broader ecological community. We first derived the functional responses (i.e. per capita effects) of two ecologically damaging invasive fish species in Europe, the Ponto-Caspian round goby (Neogobius melanostomus) and Asian topmouth gudgeon (Pseudorasbora parva), and their native trophic analogues, the bullhead (Cottus gobio; also C. bairdi) and bitterling (Rhodeus amarus), towards several prey species. This establishes the existence and relative strengths of the predator-prey relationships. Then, we derived ecologically comparable field abundance estimates of the invader and native fish from surveys and literature. This establishes the multipliers for the above per capita effects. Despite both predators having known severe detrimental field impacts, their functional responses alone were of modest predictive power in this regard; however, incorporation of their abundances relative to natives into the RIP metric gave high predictive power. We present invader/native RIP biplots that provide an intuitive visualisation of comparisons among the invasive and native species, reflecting the known broad ecological impacts of the invaders. Thus, we provide a mechanistic understanding of invasive species impacts and a predictive tool for use by practitioners, for example, in risk assessments

Modifications of poly(o-phenylenediamine) permselective layer on Pt-Ir for biosensor application in neurochemical monitoring

Reports that globular proteins could enhance the interference blocking ability of the PPD (poly(o-phenylenediamine) layer used as a permselective barrier in biosensor design, prompted this study where a variety of modifying agents were incorporated into PPD during its electrosynthesis on Pt-Ir electrodes. Trapped molecules, including fibrous proteins and β-cyclodextrin, altered the polymer/modifier composite selectivity by affecting the sensitivity to both H2O2 (signal molecule in many enzyme-based biosensors) and the archetypal interference species, ascorbic acid. A comparison of electrochemical properties of Pt and a Pt-Ir alloy suggests that the benefits of the latter, more rigid, metal can be exploited in PPD-based biosensor design without significant loss of backward compatibility with studies involving pure Pt