Add-on testing: stability assessment of 63 biochemical analytes in centrifuged and capped samples stored at 16 °C.

OBJECTIVES: Integration of add-on testing in high-scale automated clinical laboratories constitute a valuable instrument not only for the clinicians and the general patient care, but also for the laboratory itself. Knowledge on sample quality and analytical stability upon storage is necessary to be able to offer add-on testing. The objectives of this study were to examine the analytical stability of 63 biochemical analytes in plasma and urine samples stored at 16 °C. METHODS: Samples were collected by professional laboratory technicians, analyzed at automated analyzers and stored in their primary, capped tube without separator for 10, 12, 16, 20 or 24 h at 16 °C. Stability was assessed by inspecting mean concentration of samples at baseline and examining if (A) mean concentration over time violated limits of bias, or if (B) individual sample concentrations violated limits of total error. RESULTS: The majority of the 63 analytes were stable for up to 24 h of storage. Few of the analytes were only suitable for add-on testing for 4, 6, 10, 12, 16 or 20 h of storage. One analyte, P-lactate dehydrogenase, was not found suitable for add-on testing when stored at 16 °C. CONCLUSIONS: Due to the increasing number of intelligent solutions for high-scale clinical laboratories, add-on testing has come to stay. Loss of stability could not be demonstrated for the majority of analytes after 10, 12, 16, 20 or 24 h of storage. This feature of analytical stability suggests that add-on testing is an acceptable tool for these analytes.

Faecal haemoglobin concentration predicts all-cause mortality.

BACKGROUND: Population-based screening for colorectal cancer by a faecal immunochemical test (FIT) is recommended by the European Union. Detectable faecal haemoglobin can indicate colorectal neoplasia as well as other conditions. A positive FIT predicts an increased risk of death from colorectal cancer but might also predict an increased risk of all-cause mortality. METHODS: A cohort of screening participants was followed using the Danish National Register of Causes of Death. Data were retrieved from the Danish Colorectal Cancer Screening Database supplemented with FIT concentrations. Colorectal cancer specific and all-cause mortality were compared between FIT concentration groups using multivariate cox proportional hazards regression models. FINDINGS: In 444,910 Danes invited for the screening program, 25,234 (5·7%) died during a mean follow-up of 56·5 months. Colorectal cancer caused 1120 deaths. The risk of colorectal cancer death increased with the increasing FIT concentration. The hazard ratios ranged from 2·6 to 25·9 compared to individuals with FIT concentrations <4 µg hb/g faeces. Causes other than colorectal cancer caused 24,114 deaths. The risk of all-cause death increased with the increasing FIT concentration, with the hazard ratios ranging from 1·6 to 5·3 compared to individuals with FIT concentrations <4 µg hb/g faeces. INTERPRETATION: The risk of colorectal cancer mortality increased with the increasing FIT concentrations even for FIT concentrations considered negative in all European screening programs. The risk of all-cause mortality was also increased for individuals with detectable faecal blood. For colorectal cancer specific mortality and all-cause mortality, the risk was increased at the FIT concentrations as low as 4-9 µg hb/g faeces. FUNDING: The study was funded by the Odense University Hospital grants A3610 and A2359.

Quantifying SARS-CoV-2 nucleocapsid antigen in oropharyngeal swabs using single molecule array technology.

This study aimed to develop a highly sensitive SARS-CoV-2 nucleocapsid antigen assay using the single molecule array (Simoa) technology and compare it with real time RT-PCR as used in routine clinical practice with the ambition to achieve a comparative technical and clinical sensitivity. Samples were available from 148 SARS-CoV-2 real time RT-PCR positive and 73 SARS-CoV-2 real time RT-PCR negative oropharyngeal swabs. For determination of technical sensitivity SARS-CoV-2 virus culture material was used. The samples were treated with lysis buffer and analyzed using both an in-house and a pre-commercial SARS-CoV-2 nucleocapsid antigen assay on Simoa. Both nucleocapsid antigen assays have a technical sensitivity corresponding to around 100 SARS-CoV-2 RNA molecules/mL. Using a cut-off at 0.1 pg/mL the pre-commercial SARS-CoV-2 nucleocapsid antigen assay had a sensitivity of 96% (95% CI 91.4-98.5%) and specificity of 100% (95% CI 95.1-100%). In comparison the in-house nucleocapsid antigen assay had sensitivity of 95% (95% CI 89.3-98.1%) and a specificity of 100% (95% CI 95.1-100%) using a cut-off at 0.01 pg/mL. The two SARS-CoV-2 nucleocapsid antigen assays correlated with r = 0.91 (P < 0.0001). The in-house and the pre-commercial SARS-CoV-2 nucleocapsid antigen assay demonstrated technical and clinical sensitivity comparable to real-time RT-PCR methods for identifying SARS-CoV-2 infected patients and thus can be used clinically as well as serve as a reference method for antigen Point of Care Testing.

The Soluble Urokinase-Type Plasminogen Activator Receptor as a Biomarker for Survival and Early Treatment Effect in Metastatic Colorectal Cancer.

The soluble urokinase-type plasminogen activator receptor (suPAR) is prognostic for overall survival (OS) in colorectal cancer (CRC). Our study explored the association between baseline suPAR and OS and progression-free survival (PFS) in metastatic CRC (mCRC). It is also the first study to explore the association between the initial change in suPAR level and OS, PFS and the first CT response evaluation. The study included 132 patients with mCRC treated with chemotherapy (FOLFIRI) with or without an EGFR-inhibitor. Blood samples were drawn before the first treatment cycle and in between the first and second treatment cycle. suPAR levels were determined using an ELISA assay. Using the Kaplan-Meyer method, we demonstrated a significantly shorter OS for patients with suPAR levels above the median (HR = 1.79, 95%CI = 1.10-2.92, p = 0.01). We also showed association between plasma suPAR level, gender and performance status (PS). However, we could not show any association with PFS, and analysis on the change in suPAR level provided no significant results. The results showing association between baseline suPAR and OS are in line with previous findings.

Daily monitoring of viral load measured as SARS-CoV-2 antigen and RNA in blood, IL-6, CRP and complement C3d predicts outcome in patients hospitalized with COVID-19.

OBJECTIVES: We hypothesized that the amount of antigen produced in the body during a COVID-19 infection might differ between patients, and that maximum concentrations would predict the degree of both inflammation and outcome for patients. METHODS: Eighty-four hospitalized and SARS-CoV-2 PCR swab-positive patients, were followed with blood sampling every day until discharge or death. A total of 444 serial EDTA plasma samples were analyzed for a range of biomarkers: SARS-CoV-2 nuclear antigen and RNA concentration, complement activation as well as several inflammatory markers, and KL-6 as a lung marker. The patients were divided into outcome groups depending on need of respiratory support and death/survival. RESULTS: Circulating SARS-CoV-2 nuclear antigen levels were above the detection limit in blood in 65 out of 84 COVID-19 PCR swab-positive patients on day one of hospitalization, as was viral RNA in plasma in 30 out of 84. In all patients, complete antigen clearance was observed within 24 days. There were definite statistically significant differences between the groups depending on their biomarkers, showing that the concentrations of virus RNA and antigen were correlated to the inflammatory biomarker levels, respiratory treatment and death. CONCLUSIONS: Viral antigen is cleared in parallel with the virus RNA levels. The levels of antigens and SARS-CoV-2 RNA in the blood correlates with the level of IL-6, inflammation, respiratory failure and death. We propose that the antigens levels together with RNA in blood can be used to predict the severity of disease, outcome, and the clearance of the virus from the body.

Deglycosylation by the Acidic Glycosidase PNGase H+ Enables Analysis of N-Linked Glycoproteins by Hydrogen/Deuterium Exchange Mass Spectrometry.

Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become an important method to study the structural dynamics of proteins. However, glycoproteins represent a challenge to the traditional HDX-MS workflow for determining the deuterium uptake of the protein segments that contain the glycan. We have recently demonstrated the utility of the glycosidase PNGase A to enable HDX-MS analysis of N-glycosylated protein regions. Here, we have investigated the use of the acidic glycosidase PNGase H+, which has a pH optimum at 2.6, to efficiently deglycosylate N-linked glycosylated peptides during HDX-MS analysis of glycoproteins. Our results show that PNGase H+ retains high deglycosylation activity at HDX quench conditions. When used in an HDX-MS workflow, PNGase H+ allowed the extraction of HDX data from all five glycosylated regions of the serpin α1-antichymotrypsin. We demonstrate that PNGase A and PNGase H+ are capable of similar deglycosylation performance during HDX-MS analysis of α1-antichymotrypsin and the IgG1 antibody trastuzumab (TZ). However, PNGase H+ provides broader specificity and greater tolerance to the disulfide-bond reducing agent TCEP, while PNGase A offers advantages in terms of commercial availability and purity. Overall, our findings demonstrate the unique features of PNGase H+ for improving conformational analysis of glycoproteins by HDX-MS, in particular, challenging glycoproteins containing both glycosylations and disulfide bonds.

Biochemical and structural analyses suggest that plasminogen activators coevolved with their cognate protein substrates and inhibitors.

Protein sequences of members of the plasminogen activation system are present throughout the entire vertebrate phylum. This important and well-described proteolytic cascade is governed by numerous protease-substrate and protease-inhibitor interactions whose conservation is crucial to maintaining unchanged protein function throughout evolution. The pressure to preserve protein-protein interactions may lead to either co-conservation or covariation of binding interfaces. Here, we combined covariation analysis and structure-based prediction to analyze the binding interfaces of urokinase (uPA):plasminogen activator inhibitor-1 (PAI-1) and uPA:plasminogen complexes. We detected correlated variation between the S3-pocket-lining residues of uPA and the P3 residue of both PAI-1 and plasminogen. These residues are known to form numerous polar interactions in the human uPA:PAI-1 Michaelis complex. To test the effect of mutations that correlate with each other and have occurred during mammalian diversification on protein-protein interactions, we produced uPA, PAI-1, and plasminogen from human and zebrafish to represent mammalian and nonmammalian orthologs. Using single amino acid point substitutions in these proteins, we found that the binding interfaces of uPA:plasminogen and uPA:PAI-1 may have coevolved to maintain tight interactions. Moreover, we conclude that although the interaction areas between protease-substrate and protease-inhibitor are shared, the two interactions are mechanistically different. Compared with a protease cleaving its natural substrate, the interaction between a protease and its inhibitor is more complex and involves a more fine-tuned mechanism. Understanding the effects of evolution on specific protein interactions may help further pharmacological interventions of the plasminogen activation system and other proteolytic systems.

Conformational preludes to the latency transition in PAI-1 as determined by atomistic computer simulations and hydrogen/deuterium-exchange mass spectrometry.

Both function and dysfunction of serine protease inhibitors (serpins) involve massive conformational change in their tertiary structure but the dynamics facilitating these events remain poorly understood. We have studied the dynamic preludes to conformational change in the serpin plasminogen activator inhibitor 1 (PAI-1). We report the first multi-microsecond atomistic molecular dynamics simulations of PAI-1 and compare the data with experimental hydrogen/deuterium-exchange data (HDXMS). The simulations reveal notable conformational flexibility of helices D, E and F and major fluctuations are observed in the W86-loop which occasionally leads to progressive detachment of ß-strand 2 A from ß-strand 3 A. An interesting correlation between Cα-RMSD values from simulations and experimental HDXMS data is observed. Helices D, E and F are known to be important for the overall stability of active PAI-1 as ligand binding in this region can accelerate or decelerate the conformational inactivation. Plasticity in this region may thus be mechanistically linked to the conformational change, possibly through facilitation of further unfolding of the hydrophobic core, as previously reported. This study provides a promising example of how computer simulations can help tether out mechanisms of serpin function and dysfunction at a spatial and temporal resolution that is far beyond the reach of any experiment.

An RNA Aptamer Inhibits a Mutation-Induced Inactivating Misfolding of a Serpin.

Most serpins are fast and specific inhibitors of extracellular serine proteases controlling biological processes such as blood coagulation, fibrinolysis, tissue remodeling, and inflammation. The inhibitory activity of serpins is based on a conserved metastable structure and their conversion to a more stable state during reaction with the target protease. However, the metastable state also makes serpins vulnerable to mutations, resulting in disease caused by inactive and misfolded monomeric or polymeric forms ("serpinopathy"). Misfolding can occur either intracellularly (type-I serpinopathies) or extracellularly (type-II serpinopathies). We have isolated a 2'-fluoropyrimidine-modified RNA aptamer, which inhibits a mutation-induced inactivating misfolding of the serpin α1-antichymotrypsin. It is the first agent able to stabilize a type-II mutation of a serpin without interfering with the inhibitory mechanism, thereby presenting a solution for the long-standing challenge of preventing pathogenic misfolding without compromising the inhibitory function.

Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin.

Components of the plasminogen activation system including urokinase (uPA), its inhibitor (PAI-1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin. We now report that uPA and plasmin induces a potent negative feedback on cell adhesion through specific cleavage of the RGD motif in vitronectin. Cleavage of vitronectin by uPA displays a remarkable receptor dependence and requires concomitant binding of both uPA and vitronectin to uPAR Moreover, we show that PAI-1 counteracts the negative feedback and behaves as a proteolysis-triggered stabilizer of uPAR-mediated cell adhesion to vitronectin. These findings identify a novel and highly specific function for the plasminogen activation system in the regulation of cell adhesion to vitronectin. The cleavage of vitronectin by uPA and plasmin results in the release of N-terminal vitronectin fragments that can be detected in vivo, underscoring the potential physiological relevance of the process.

Local transient unfolding of native state PAI-1 associated with serpin metastability.

The metastability of the native fold makes serpin (serine protease inhibitor) proteins prone to pathological conformational change, often by insertion of an extra ß-strand into the central ß-sheet A. How this insertion is made possible is a hitherto unresolved question. By the use of advanced hydrogen/deuterium-exchange mass spectrometry (HDX-MS) it is shown that the serpin plasminogen activator inhibitor 1 (PAI-1) transiently unfolds under native condition, on a second-to-minute time scale. The unfolding regions comprise ß-strand 5A as well as the underlying hydrophobic core, including ß-strand 6B and parts of helices A, B, and C. Based thereon, a mechanism is proposed by which PAI-1 makes transitions through progressively more unfolded states along the reaction coordinate to the inactive, so-called latent form. Our results highlight the profound utility of HDX-MS in detecting sparsely populated, transiently unfolded protein states.

Dissecting the effect of RNA aptamer binding on the dynamics of plasminogen activator inhibitor 1 using hydrogen/deuterium exchange mass spectrometry.

RNA aptamers, selected from large synthetic libraries, are attracting increasing interest as protein ligands, with potential uses as prototype pharmaceuticals, conformational probes, and reagents for specific quantification of protein levels in biological samples. Very little is known, however, about their effects on protein conformation and dynamics. We have employed hydrogen/deuterium exchange (HDX) mass spectrometry to study the effect of RNA aptamers on the structural flexibility of the serpin plasminogen activator inhibitor-1 (PAI-1). The aptamers have characteristic effects on the biochemical properties of PAI-1. In particular, they are potent inhibitors of the structural transition of PAI-1 from the active state to the inactive, so-called latent state. This transition is one of the largest conformational changes of a folded protein domain without covalent modification. Binding of the aptamers to PAI-1 is associated with substantial and widespread protection against deuterium uptake in PAI-1. The aptamers induce protection against exchange with the solvent both in the protein-aptamer interface as well as in other specific areas. Interestingly, the aptamers induce substantial protection against exchange in α-helices B, C and I. This observation substantiates the relevance of structural instability in this region for transition to the latent state and argues for involvement of flexibility in regions not commonly associated with regulation of latency transition in serpins.

RNA aptamers as conformational probes and regulatory agents for plasminogen activator inhibitor-1.

The hallmark of serpins is the ability to undergo the so-called "stressed-to-relaxed" switch during which the surface-exposed reactive center loop (RCL) becomes incorporated as strand 4 in central beta-sheet A. RCL insertion drives not only the inhibitory reaction of serpins with their target serine proteases but also the conversion to the inactive latent state. RCL insertion is coupled to conformational changes in the flexible joint region flanking beta-sheet A. One interesting serpin is plasminogen activator inhibitor-1 (PAI-1), a fast and specific inhibitor of the serine proteases tissue-type and urokinase-type plasminogen activator. Via its flexible joints' region, native PAI-1 binds vitronectin and relaxed, protease-complexed PAI-1 certain endocytosis receptors. From a library of 35-nucleotides long 2'-fluoropyrimidine-containing RNA oligonucleotides, we have isolated two aptamers binding PAI-1 by the flexible joint region with low nanomolar K(D) values. One of the aptamers exhibited measurable binding to native PAI-1 only, while the other also bound relaxed PAI-1. While none of the aptamers inhibited the antiproteolytic effect of PAI-1, both aptamers inhibited vitronectin binding and the relaxed PAI-1-binding aptamer also endocytosis receptor binding. The aptamer binding exclusively to native PAI-1 increased the half-life for the latency transition to more than 6 h, manyfold more than vitronectin. Contact with Lys124 in the flexible joint region was critical for strong inhibition of the latency transition and the lack of binding to relaxed PAI-1. We conclude that aptamers yield important information about the serpin conformational switch and, because they can compete with high-affinity protein-protein interactions, may provide leads for pharmacological intervention.

Subtle structural differences between human and mouse PAI-1 reveal the basis for biochemical differences.

Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor (serpin) that plays an important role in cardiovascular disorders and tumor development. The potential role of PAI-1 as a drug target has been evaluated in various animal models (e.g. mouse and rat). Sensitivity to PAI-1 inhibitory agents varied in different species. To date, absence of PAI-1 structures from species other than human hampers efforts to reveal the molecular basis for the observed species differences. Here we describe the structure of latent mouse PAI-1. Comparison with available structures of human PAI-1 reveals (1) a differential positioning of α-helix A; (2) differences in the gate region; and (3) differences in the reactive center loop position. We demonstrate that the optimal binding site of inhibitors may be dependent on the orthologs, and our results affect strategies in the rational design of a pharmacologically active PAI-1 inhibitor.