The Association Between Self-Rated Fitness and Cardiorespiratory Fitness in Adults.

To assess criterion validity of a single item question on self-rated physical fitness against objectively measured cardiorespiratory fitness. From the Health2008 study 749 men and women between 30 and 60 years of age rated their fitness as excellent, very good, good, fair or poor. Cardiorespiratory fitness was estimated with the watt-max test. Agreement between self-rated and objectively measured physical fitness was assessed by Cohen's weighted kappa coefficient. Correlation was determined by Goodman & Kruskal's gamma correlation coefficient. All analyses were stratified according to gender. Data from 323 men and 426 women were analysed. There was a slight agreement between self-rated and objectively measured fitness in men (weighted kappa: 0.18, [95%CI: 0.13;0.23]) and a fair agreement in women (weighted kappa: 0.27, [95%CI: 0.22;0.32]). In both genders, self-rated fitness was positively correlated with objectively measured fitness (moderate correlation; gamma correlation coefficient for men: 0.63 [95%CI: 0.54;0.72] and women: 0.67 [95%CI: 0.59;0.75]). There was a slight to fair agreement and moderate, positive correlations between self-rated physical fitness and watt-max estimated cardiorespiratory fitness. Hence, a single-item question on physical fitness may be a cost-effective method of assessing fitness in large population studies, but is not valid for individual assessments.

The validity of pathology codes for biopsy-confirmed kidney disease in the Danish National Patobank.

Background: This study validates the application of Systematized Nomenclature of Medicine second edition (SNOMED II) codes used to describe medical kidney biopsies in Denmark in encoded form, aiming to support robust epidemiological research on the causes, treatments and prognosis of kidney diseases. Methods: Kidney biopsy reports from 1 January 1998 to 31 December 2018 were randomly extracted from the Danish National Patobank, using SNOMED codes. A 5% sample was selected, and nephrologists assessed the corresponding medical records, assigning each case the applied clinical diagnoses. Sensitivity, specificity, positive predictive values (PPV), negative predictive values and Cohen's kappa coefficient for the retrieved SNOMED codes were calculated. Results: A total of 613 kidney biopsies were included. The primary clinical disease groups were glomerular disease (n = 368), tubulointerstitial disease (n = 67), renal vascular disease (n = 51), diabetic nephropathy (n = 51) and various renal disorders (n = 40). Several SNOMED codes were used to describe each clinical disease group and PPV for the combined SNOMED codes were high for glomerular disease (94%), diabetic nephropathy (85%) and systemic diseases affecting the kidney (96%). Conversely, tubulointerstitial disease (62%), renal vascular disease (60%) and other renal disorders (17%) showed lower PPV. Conclusions: SNOMED codes have a high PPV for glomerular diseases, diabetic nephropathy and systemic diseases affecting the kidney, in which they could be applied for future epidemiological research.

Design and methodology of the PRIMETIME 1 cohort study: PRecIsion MEdicine based on kidney TIssue Molecular interrogation in diabetic nEphropathy.

Background: Clinical features of diabetic kidney disease alone cannot differentiate between the histopathology that defines diabetic nephropathy (DN) and non-diabetic nephropathy (NDN). A kidney biopsy is necessary to make the definitive diagnosis of DN. However, there is no consensus on when to perform a kidney biopsy in individuals with diabetes and kidney disease. Furthermore, the implications of NDN versus DN for management, morbidity and kidney prognosis are unclear. To address the gap in knowledge, we aimed to create a national retrospective cohort of people with diabetes and a performed kidney biopsy. Methods: Adults diagnosed with diabetes in Denmark between 1996 and 2020 who had a kidney biopsy performed were included. The cohort was established by linking a nationwide diabetes registry with the Danish Pathology Registry. Data from 11 national registries and databases were compiled. The type of kidney disease was classified using a three-step analysis of Systematized Nomenclature of Medicine codes reported in relation to the histopathological examinations of kidney tissue. The final cohort and classification of kidney disease was as follows: out of 485 989 individuals with diabetes 2586 were included, 2259 of whom had type 2 diabetes. We were able to classify 599 (26.5%) with DN, 703 (31.1%) with NDN and 165 (7.3%) with mixed disease in individuals with type 2 diabetes. In individuals with type 1 diabetes, 132 (40.4%) had DN, 73 (22.3%) NDN and 39 (11.9%) mixed disease. The remaining could not be classified or had normal histology. The overall median (Q1-Q3) follow-up time was 3.8 (1.6-7.2) years. Conclusions: This cohort is a novel platform based on high-quality registry data for important longitudinal studies of the impact of kidney disease diagnosis on prognosis. With regular updates of data from the Danish registries, the presented follow-up will increase over time and is only limited by emigration or death.

Development, implementation and evaluation of patient decision aids supporting shared decision making in women with recurrent ovarian cancer.

Objective: Shared decision making (SDM) and use of patient decision aids (PtDAs) are key components in patient-centered care in relapsed ovarian cancer. This paper describes the development and implementation process of PtDAs into a clinical routine in three departments. Methods: Two PtDAs were developed in collaboration between patients and clinicians. Acceptability and usability of the PtDAs were tested on clinicians and patients using items from the internationally validated questionnaire "Preparation for Decision Making Scale". Results: Ten patients and 15 clinicians participated in the study. Most patients indicated that PtDAs would be helpful as preparation for the decision-making process with the clinicians. Ten (75%) of the clinicians responded that the PtDAs helped the patients to understand the benefits and disadvantages of each treatment option. Generally, the clinicians indicated that they would use SDM if they had a PtDA tailored to the clinical situation. Conclusions: Two PtDAs were systematically developed, tested, and implemented thereby supporting an SDM intervention. The PtDAs are still in use at the participating departments. Innovation: This study was successful in reusing a generic template for a patient decision aid (PtDA) developed at one institution and implemented in two other institutions. This was guided by a well-described systematic development process for PtDAs.

Rationale and design of a prospective, clinical study of kidney biopsies in people with type 2 diabetes and severely increased albuminuria (the PRIMETIME 2 study).

INTRODUCTION: Diabetic kidney disease is a severe complication of diabetes. The diagnosis is based on clinical characteristics such as persistently elevated albuminuria, hypertension and decline in kidney function, although this definition is not specific to kidney disease caused by diabetes. The only way to establish an accurate diagnosis-diabetic nephropathy-is by performing a kidney biopsy. The histological presentation of diabetic nephropathy can be associated with a heterogeneous range of histological features with many pathophysiological factors involved demonstrating the complexity of the condition. Current treatment strategies aim to slow disease progression and are not specific to the underlying pathological processes.This study will investigate the prevalence of diabetic nephropathy in individuals with type 2 diabetes (T2D) and severely elevated albuminuria. The deep molecular characterisation of the kidney biopsy and biological specimens may pave the way for improved diagnostic accuracy and a better understanding of the pathological processes involved and may also reveal new targets for individualised treatment. METHODS AND ANALYSIS: In the PRecIsion MEdicine based on kidney TIssue Molecular interrogation in diabetic nEphropathy 2 study, research kidney biopsies will be performed in 300 participants with T2D, urine albumin/creatinine ratio ≥700 mg/g and estimated glomerular filtration ratio >30 mL/min/1.73 m2. Cutting-edge molecular technologies will be applied to the kidney, blood, urine, faeces and saliva samples for comprehensive multi-omics profiling. The associated disease course and clinical outcomes will be assessed by annual follow-up for 20 years. ETHICS AND DISSEMINATION: The Danish Regional Committee on Health Research Ethics and the Knowledge Center on Data Protection (in the Capital Region of Denmark) have granted approval for the study. The results will be published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT04916132.

Report of the first nationally implemented clinical routine screening for fetal RHD in D- pregnant women to ascertain the requirement for antenatal RhD prophylaxis.

BACKGROUND: A combination of antenatal and postnatal RhD prophylaxis is more effective in reducing D immunization in pregnancy than postnatal RhD prophylaxis alone. Based on the result from antenatal screening for the fetal RHD gene, antenatal RhD prophylaxis in Denmark is given only to those D- women who carry a D+ fetus. We present an evaluation of the first national clinical application of antenatal RHD screening. STUDY DESIGN AND METHODS: In each of the five Danish health care regions, blood samples were drawn from D- women in Gestational Week 25. DNA was extracted from the maternal plasma and analyzed for the presence of the RHD gene by real-time polymerase chain reaction targeting two RHD exons. Prediction of the fetal RhD type was compared with serologic typing of the newborn in 2312 pregnancies, which represented the first 6 months of routine analysis. RESULTS: For the detection of fetal RHD, the sensitivity was 99.9%. The accuracy was 96.5%. The recommendation for unnecessary antenatal RhD prophylaxis for women carrying a D- fetus was correctly avoided in 862 cases (37.3%), while 39 women (1.7%) were recommended for antenatal RhD prophylaxis unnecessarily. Two RHD+ fetuses (0.087%) were not detected, and antenatal RhIG was not given. CONCLUSION: These data represent the first demonstration of the reliability of routine antenatal fetal RHD screening in D-, pregnant women to ascertain the requirement for antenatal RhD prophylaxis. Our findings should encourage the implementation of such screening programs worldwide, to reduce the unnecessary use of RhIG.

A generalised module for the selective extracellular accumulation of recombinant proteins.

BACKGROUND: It is widely believed that laboratory strains of Escherichia coli, including those used for industrial production of proteins, do not secrete proteins to the extracellular milieu. RESULTS: Here, we report the development of a generalised module, based on an E. coli autotransporter secretion system, for the production of extracellular recombinant proteins. We demonstrate that a wide variety of structurally diverse proteins can be secreted as soluble proteins when linked to the autotransporter module. Yields were comparable to those achieved with other bacterial secretion systems. CONCLUSIONS: The advantage of this module is that it relies on a relatively simple and easily manipulated secretion system, exhibits no apparent limitation to the size of the secreted protein and can deliver proteins to the extracellular environment at levels of purity and yields sufficient for many biotechnological applications.

The porin OmpD from nontyphoidal Salmonella is a key target for a protective B1b cell antibody response.

Invasive nontyphoidal Salmonella (NTS), including Salmonella typhimurium (STm), are major yet poorly-recognized killers of infants in sub-Saharan Africa. Death in these children is usually associated with bacteremia, commonly in the absence of gastrointestinal symptoms. Evidence from humans and animal studies suggest that severe infection and bacteremia occur when specific Ab is lacking. Understanding how Ab responses to Salmonella are regulated will help develop vaccines against these devastating infections. STm induces atypical Ab responses characterized by prominent, accelerated, extrafollicular T-independent (TI) Ab against a range of surface antigens. These responses develop without concomitant germinal centers, which only appear as infection resolves. Here, we show STm rapidly induces a population of TI B220(+)CD5(-) B1b cells during infection and TI Ab from B1b cells targets the outer membrane protein (Omp) porins OmpC, OmpD and OmpF but not flagellin. When porins are used as immunogens they can ablate bacteremia and provide equivalent protection against STm as killed bacterial vaccine and this is wholly B cell-dependent. Furthermore Ab from porin-immunized chimeras, that have B1b cells, is sufficient to impair infection. Infecting with porin-deficient bacteria identifies OmpD, a protein absent from Salmonella Typhi, as a key target of Ab in these infections. This work broadens the recognized repertoire of TI protein antigens and highlights the importance of Ab from different B cell subsets in controlling STm infection. OmpD is a strong candidate vaccine target and may, in part, explain the lack of cross-protection between Salmonella Typhi and STm infections.

Shared decision making in recurrent ovarian cancer: Implementation of patient decision aids across three departments of oncology in Denmark.

Objective: Patients with relapsed ovarian cancer are offered multiple treatment options. To match treatment with the individual patient's life situation and preferences, healthcare professionals can apply shared decision making (SDM) including patient decision aids (PtDAs).This study aimed to evaluate the implementation of two different PtDAs in consultations with patients suffering from relapsed ovarian cancer. Methods: We analyzed the following data before and after implementation of the PtDAs: 1) observed SDM using the OPTION instrument, 2) physician treatment recommendations, and 3) patients' and physicians' evaluations of SDM in consultations using the CollaboRATE, SDM-Q-9, and SDM-Q-Doc. Results: Significant improvement in observed SDM was found after the implementation (p = 0.002). Improvement of SDM was detected in consultations conducted by physicians reporting more than two hours of SDM-training (p < 0.001), but not when physicians reported less than two hours of SDM-training.No before/after differences in treatment recommendations and in patients' and physicians' evaluations were found. Conclusion: Implementation of PtDAs improved the level of observed SDM. Training of physicians in SDM is necessary for improved SDM practice. Innovation: Discussing oncological treatment options with the use of PtDAs is not standard practice in Denmark. The present study is one of the first Danish studies focusing on how to implement SDM and PtDAs in oncological consultations.

The essential ß-barrel assembly machinery complex components BamD and BamA are required for autotransporter biogenesis.

Autotransporter biogenesis is dependent upon BamA, a central component of the ß-barrel assembly machinery (BAM) complex. In this report, we detail the role of the other BAM components (BamB-E). We identify the importance of BamD in autotransporter biogenesis and show that BamB, BamC, and BamE are not required.

SadA, a trimeric autotransporter from Salmonella enterica serovar Typhimurium, can promote biofilm formation and provides limited protection against infection.

Salmonella enterica is a major cause of morbidity worldwide and mortality in children and immunocompromised individuals in sub-Saharan Africa. Outer membrane proteins of Salmonella are of significance because they are at the interface between the pathogen and the host, they can contribute to adherence, colonization, and virulence, and they are frequently targets of antibody-mediated immunity. In this study, the properties of SadA, a purported trimeric autotransporter adhesin of Salmonella enterica serovar Typhimurium, were examined. We demonstrated that SadA is exposed on the Salmonella cell surface in vitro and in vivo during infection of mice. Expression of SadA resulted in cell aggregation, biofilm formation, and increased adhesion to human intestinal Caco-2 epithelial cells. Immunization of mice with folded, full-length, purified SadA elicited an IgG response which provided limited protection against bacterial challenge. When anti-SadA IgG titers were enhanced by administering alum-precipitated protein, a modest additional protection was afforded. Therefore, despite SadA having pleiotropic functions, it is not a dominant, protective antigen for antibody-mediated protection against Salmonella.

The impact of shared decision making on time consumption and clinical decisions. A prospective cohort study.

OBJECTIVE: Concerns of increased time consumption and of the impact on clinical decisions may restrain doctors from shared decision making (SDM). This paper evaluates consultation length and decisions made when using an in-consult patient decision aid (PtDA). METHODS: This prospective cohort study compared an unexposed cohort with a cohort exposed to SDM and a PtDA in two preference-sensitive decision situations: invasive lung cancer diagnostics and adjuvant treatment for early breast cancer. Outcome measures were consultation length and decisions made. RESULTS: The study included 261 consultations, 115 were in the SDM-exposed cohort. Consultations were inconsiderably longer in the SDM cohort; 2 min, 11 s (p = 0.2217) for lung cancer diagnostics and 3 min, 57 s (p = 0.1128) for adjuvant breast cancer treatment. In lung cancer diagnostics, consultation length became more uniform and decisions tended to become conservative after introduction of SDM. For adjuvant breast cancer, slightly more patients in the SDM cohort chose to decline treatment. CONCLUSION: Shared decision making did not take significantly longer time and led to slightly more conservative decisions. PRACTICE IMPLICATIONS: SDM may be implemented without considerable impact on consultation length. The impact on clinical decisions depends mainly on the clinical situation.

Nutrients, Diet, and Other Factors in Prenatal Life and Bone Health in Young Adults: A Systematic Review of Longitudinal Studies.

Optimizing skeletal health in early life has potential effects on bone health later in childhood and in adulthood. We aimed to evaluate the existing evidence that maternal exposures during pregnancy have an impact on the subsequent bone health among offspring in young adults aged between 16 and 30 years. The protocol is registered in the International Prospective Register of Systematic Reviews (PROSPERO) (ID: CRD42019126890). The search was conducted up to 2 April 2019. We included seven observational prospective cohort studies that examined the association between maternal dietary factors, vitamin D concentration, age, preeclampsia, and smoking with any bone indices among offspring. The results indicated that high concentrations of maternal vitamin D; low fat intake; and high intakes of calcium, phosphorus, and magnesium may increase the bone mineral density in offspring at age 16. Evidence also suggests that the offspring of younger mothers may have a higher peak bone mass. It remains inconclusive whether there is an influence of preeclampsia or maternal smoking on bone health among young adults. Our assessment of internal validity warrants a cautious interpretation of these results, as all of the included studies were judged to have serious risks of bias. High-quality studies assessing whether prenatal prognostic factors are associated with bone health in young adults are needed.

A prospective cohort study of shared decision making in lung cancer diagnostics: Impact of using a patient decision aid.

OBJECTIVE: The objective of this study was to describe the impact on patient-reported outcomes of introducing Shared Decision Making (SDM) and a Patient Decision Aid (PtDA) in the initial process of lung cancer diagnostics. METHODS: We conducted a prospective cohort study, where a control cohort was consulted according to usual clinical practice. After introducing SDM through a PtDA and training of the staff, the SDM cohort was enrolled in the study. All patients completed four questionnaires: the Decisional Conflict Scale (DCS) before and after the consultation, the CollaboRATE scale after the consultation, and the Decision Regret Scale (DRS). RESULTS: Patients exposed to SDM and a PtDA had significantly improved DCS scores after the consultation compared to the control group (a difference of 10.26, p = 0.0128) and significantly lower DRS scores (a difference of 8.98, p = 0.0197). Of the 82 control patients and 52 SDM patients 29% and 54%, respectively, gave the maximum score on the CollaboRATE scale (Pearson's chi2 8.0946, p = 0.004). CONCLUSION: The use of SDM and a PtDA had significant positive impact on patient-reported outcomes. PRACTICE IMPLICATIONS: Our results may encourage the increased uptake of SDM in the initial process of lung cancer diagnostics.

Sexual rehabilitation for cardiac patients with erectile dysfunction: a randomised clinical trial.

BACKGROUND: Sexual dysfunction is common in patients with either ischaemic heart disease (IHD) or implantable cardioverter defibrillator (ICD) and has a negative impact on quality of life. Non-pharmacological treatment options are lacking. The purpose of this trial was to assess the effect of sexual rehabilitation versus usual care for males with erectile dysfunction and either IHD and/or ICD. METHODS: Participants with erectile dysfunction and IHD and/or ICD were randomised to 12 weeks of sexual rehabilitation consisting of physical exercise training, pelvic floor exercise and psychoeducation, or usual care. PRIMARY OUTCOME: sexual function by the International Index of Erectile Function (IIEF). Secondary outcome: sexual function by the Psychosocial Adjustment to Illness Scale. Exploratory outcomes: exercise capacity, pelvic floor strength/endurance, self-reported health and mental health. RESULTS: 154 participants were included, mean age 61.6 years (SD 6.1). Sexual rehabilitation compared with usual care improved sexual function with a mean difference IIEF score of 6.7 (95% CI 3.1 to 10.4, p<0.0003) at 4 months between groups (unadjusted IIEF mean scores 36.4 vs 31.3) and a mean difference of 6.7, 95% CI 3.2 to 10.1 (p<0.0002) at 6 months between groups (unadjusted mean scores IIEF 37.1 vs 32.2). No effects were seen on the secondary outcome. Sexual rehabilitation improved exercise capacity on cycle ergometer measured by Watt max with a mean difference of 10.3, 95% CI 3.6 to 16.9 (p<0.003) and pelvic floor strength (p<0.01). No differences were seen on self-reported health and mental health. CONCLUSION: Sexual rehabilitation compared with usual care improves sexual function and exercise capacity. TRIAL REGISTRATION: NCT01796353; Results.

ReactELISA method for quantifying methylglyoxal levels in plasma and cell cultures.

Methylglyoxal (MG) is a toxic glycolytic by-product associated with increased levels of inflammation and oxidative stress and has been linked to ageing-related diseases, such as diabetes and Alzheimer's disease. As MG is a highly reactive dicarbonyl compound, forming both reversible and irreversible adducts with a range of endogenous nucleophiles, measuring endogenous levels of MG are quite troublesome. Furthermore, as MG is a small metabolite it is not very immunogenic, excluding conventional ELISA for detection purposes, thus only more instrumentally demanding LC-MS/MS-based methods have demonstrated convincing quantitative data. In the present work we develop a novel bifunctional MG capture probe as well as a high specificity monoclonal antibody to finally setup a robust reaction-based ELISA (ReactELISA) method for detecting the highly reactive and low-level (nM) metabolite MG in human biological specimens. The assay is tested and validated against the current golden standard LC-MS/MS method in human blood plasma and cell-culture media. Furthermore, we demonstrate the assays ability to measure small perturbations of MG levels in growth media caused by a small molecule drug buthionine sulfoximine (BSO) of current clinical relevance. Finally, the assay is converted into a homogenous (no-wash) AlphaLISA version (ReactAlphaLISA), which offers the potential for operationally simple screening of further small molecules capable of perturbing cellular MG. Such compounds could be of relevance as probes to gain insight into MG metabolism as well as drug-leads to alleviate ageing-related diseases.

Mutations in transhydrogenase change the fluorescence emission state of TRP72 from 1La to 1Lb.

The dI component of Rhodospirillum rubrum transhydrogenase has a single Trp residue (Trp(72)), which has distinctive optical properties, including short-wavelength fluorescence emission with clear vibrational fine structure, and long-lived, well-resolved phosphorescence emission. We have made a set of mutant dI proteins in which residues contacting Trp(72) are conservatively substituted. The room-temperature fluorescence-emission spectra of our three Met(97) mutants are blue shifted by approximately 4 nm, giving them a shorter-wavelength emission than any other protein described in the literature, including azurin from Pseudomonas aeruginosa. Fluorescence spectra in low-temperature glasses show equivalent well-resolved vibrational bands in wild-type and the mutant dI proteins, and in azurin. Substitution of Met(97) in dI changes the relative intensities of some of these vibrational bands. The analysis supports the view that fluorescence from the Met(97) mutants arises predominantly from the (1)L(b) excited singlet state of Trp(72), whereas (1)L(a) is the predominant emitting state in wild-type dI. It is suggested that the sulfur atom of Met(97) promotes greater stabilization of (1)L(a) than either (1)L(b) or the ground state. The phosphorescence spectra of Met(97) mutants are also blue-shifted, indicating that the sulfur atom decreases the transition energy between the (3)L(a) state of the Trp and the ground state.

The interaction of the brominated flame retardant: tetrabromobisphenol A with phospholipid membranes.

Tetrabromobisphenol A (TBBPA) is one of the most widely used members of the family of brominated flame retardants (BFRs). BFRs, including TBBPA have been shown to be widely distributed within the environment and there is growing evidence of their bio-accumulation within animals and man. Toxicological studies have shown that TBBPA can be harmful to cells by modulating a number of cell signalling processes. In this study, we employed fluorescence spectroscopy and differential scanning calorimetry to investigate the interaction of TBBPA with phospholipid membranes, as this is the most likely route for it to influence membrane-associated cellular processes. TBBPA readily and randomly partitions throughout all regions of the phospholipid bilayer with high efficacy [partition coefficient (Log K(p))=5.7+/-0.7]. A decrease in membrane fluidity in both liquid-crystalline and gel-phase membranes was detected at concentrations of TBBPA as low as 2.5 microM. TBBPA also decreases the phase transition temperature of dipalmitoyl phoshatidylcholine (DPPC) membranes and broadened transition peaks, in a fashion similar to that for cholesterol. TBBPA, however, also prefers to partition into membrane regions not too highly enriched with cholesterol. Our findings therefore suggests that, the toxic effects of TBBPA, may at least in part, be due to its lipid membrane binding/perturbing effects, which in turn, could influence biological processes involving cell membranes.

The effect of HOCl-induced modifications on phosphatase and tensin homologue (PTEN) structure and function.

Oxidation by reactive species can cause changes in protein function and affect cell signalling pathways. Phosphatase and tensin homologue (PTEN) is a negative regulator of the PI3K/AKT pathway and is known to be inhibited by oxidation, but its oxidation by the myeloperoxidase-derived oxidant hypochlorous acid (HOCl) has not previously been investigated. PTEN-GST was treated with HOCl:protein ratios from 15:1 to 300:1. Decreases in PTEN phosphatase activity were observed at treatment ratios of 60:1 and higher, which correlated with the loss of the intact protein band and appearance of high molecular weight aggregates in SDS-PAGE. LC-MSMS was used to map oxidative modifications (oxPTMs) in PTEN-GST tryptic peptides and label-free quantitative proteomics used to determine their relative abundance. Twenty different oxPTMs of PTEN were identified, of which 14 were significantly elevated upon HOCl treatment in a dose-dependent manner. Methionine and cysteine residues were the most heavily oxidised; the percentage modification depended on their location in the sequence, reflecting differences in susceptibility. Other modifications included tyrosine chlorination and dichlorination, and hydroxylations of tyrosine, tryptophan, and proline. Much higher levels of oxidation occurred in the protein aggregates compared to the monomeric protein for certain methionine and tyrosine residues located in the C2 and C-terminal domains, suggesting that their oxidation promoted protein destabilisation and aggregation; many of the residues modified were classified as buried according to their solvent accessibility. This study provides novel information on the susceptibility of PTEN to the inflammatory oxidant HOCl and its effects on the structure and activity of the protein.

A high-sensitivity electrochemiluminescence-based ELISA for the measurement of the oxidative stress biomarker, 3-nitrotyrosine, in human blood serum and cells.

The generation of 3-nitrotyrosine, within proteins, is a post-translational modification resulting from oxidative or nitrative stress. It has been suggested that this modification could be used as a biomarker for inflammatory diseases. Despite the superiority of mass spectrometry-based determinations of nitrotyrosine, in a high-throughput clinical setting the measurement of nitrotyrosine by an enzyme-linked immunosorbent assay (ELISA) is likely to be more cost-effective. ELISAs offer an alternative means to detect nitrotyrosine, but many commercially available ELISAs are insufficiently sensitive to detect nitrotyrosine in healthy human serum. Here, we report the development, validation and clinical application of a novel electrochemiluminescence-based ELISA for nitrotyrosine which provides superior sensitivity (e.g. a 50-fold increase in sensitivity compared with one of the tested commercial colorimetric ELISAs). This nitrotyrosine ELISA has the following characteristics: a lower limit of quantitation of 0.04 nM nitrated albumin equivalents; intra- and inter-assay coefficients of variation of 6.5% and 11.3%, respectively; a mean recovery of 106 ±â€¯3% and a mean linearity of 0.998 ±â€¯0.001. Far higher nitration levels were measured in normal human blood cell populations when compared to plasma. Mass spectrometry was used to validate the new ELISA method. The analysis of the same set of chemically modified albumin samples using the ELISA method and mass spectrometry showed good agreement for the relative levels of nitration present in each sample. The assay was applied to serum samples from patients undergoing elective surgery which induces the human inflammatory response. Matched samples were collected before and one day after surgery. An increase in nitration was detected following surgery (median (IQR): 0.59 (0.00-1.34) and 0.97 (0.00-1.70) nitrotyrosine (fmol of nitrated albumin equivalents/mg protein) for pre- and post-surgery respectively. The reported assay is suitable for nitrotyrosine determination in patient serum samples, and may also be applicable as a means to determine oxidative stress in primary and cultured cell populations.