Micro-bead injection spectroscopy for label-free automated determination of immunoglobulin G in human serum

Immunoglobulin G (IgG) represents the major fraction of antibodies in healthy adult human serum, and deviations from physiological levels are a generic marker of disease corresponding to different pathologies. Therefore, screening methods for IgG evaluation are a valuable aid to diagnostics. The present work proposes a rapid, automatic, and miniaturized method based on UV-vis micro-bead injection spectroscopy (μ-BIS) for the real-time determination of human serum IgG with label-free detection. Relying on attachment of IgG in rec-protein G immobilized in Sepharose 4B, a bioaffinity column is automatically assembled, where IgG is selectively retained and determined by on-column optical density measurement. A "dilution-and-shoot" approach (50 to 200 times) was implemented without further sample treatment because interferences were flushed out of the column upon sample loading, with minimization of carryover and cross-contamination by automatically discarding the sorbent (0.2 mg) after each determination. No interference from human serum albumin at 60 mg mL-1 in undiluted sample was found. The method allowed IgG determination in the range 100-300 μg mL-1 (corresponding to 5.0-60 mg mL-1 in undiluted samples), with a detection limit of 33 μg mL-1 (1.7 mg mL-1 for samples, dilution factor of 50). RSD values were < 9.4 and < 11.7%, for intra and inter-assay precision, respectively, while recovery values for human serum spiked with IgG at high pathological levels were 97.8-101.4%. Comparison to commercial ELISA kit showed no significant difference for tested samples (n = 8). Moreover, time-to-result decreased from several hours to < 5 min and analysis cost decreased 10 times, showing the potential of the proposed approach as a point-of-care method. Graphical abstract Micro-Bead Injection Spectroscopy method for real time, automated and label-free determination of total serum human Immunoglobulin G (IgG). The method was designed for Lab-on-Valve (LOV) platforms using a miniaturised protein G bioaffinity separative approach. IgG are separated from serum matrix components upon quantification with low non-specific binding in less than 5 min.info:eu-repo/semantics/publishedVersio

Screening of fluoroquinolones in environmental waters using disk-based solid-phase extraction combined to microplate fluorimetric determination and LC-MS/MS

Fluoroquinolones are in the order of the day concerning environmental contamination through anthropogenic activities, resulting in increased risk for antibiotic resistance dissemination. In this context, accessible, low-cost analytical methods are required for implementation of comprehensive surveillance and screening schemes. In this work, we propose a down-scaled disk-based solid-phase extraction system from which the eluate can be first screened by miniaturized fluorimetric reading, followed by individual determination of target fluoroquinolones (ciprofloxacin, norfloxacin, and enrofloxacin) by liquid chromatography combined to tandem mass spectrometry. The fluorimetric measurement is based on the intrinsic fluorescence of fluoroquinolones. Disk-based retention was performed after sample acidification (pH 4.0) by mixed-mode cation exchange using polystyrene divinylbenzene sulphonated sorbent. Sample loading was precisely controlled in a dedicated flow system operating at 4.0 mL min−1. Different eluent compositions were tested, with elution performed by 1.00 mL of methanol-ammonium hydroxide (98:2, v/v), with subsequent reading of eluate in both detectors. Quantification was attained for 2–25 µg L−1 range, with LOD values at 1 µg L−1. The proposed approach was successfully applied to estuarine waters from the Douro River, with comparable results to a conventional SPE-LC-MS/MS procedure.info:eu-repo/semantics/publishedVersio

Automatic solid-phase extraction by programmable flow injection coupled to chromatographic fluorimetric determination of fluoroquinolones

Fluoroquinolones are broad-spectrum bactericidal agents applied for the treatment of human and veterinary diseases. Their common use and their incorrect disposal foster environmental contamination, namely in water resources, increasing the risk of antimicrobial resistance. Hence, a method based on automatic solid-phase extraction coupled to liquid chromatography and fluorimetric detection is proposed for the determination of fluoroquinolones in environmental waters. For the solid-phase extraction procedure, a commercially available molecularly imprinted polymer targeting fluoroquinolones was trapped inside a flow-through extraction column, integrated into a programmable flow injection system using multisyringe flow injection analysis, where all steps concerning sorbent conditioning, sample loading, matrix removal, and analyte elution were performed under computer control. The eluate resulting from the sample preparation was collected and transferred at-line to chromatographic analysis using a reversed-phase monolithic column coupled to a fluorimetric detector, and isocratic elution with methanol-phosphoric acid (pH 3.0; 5.0 mM) (17.5:82.5, v/v) at a flow rate of 3.5 mL min-1. Sample treatment and chromatographic analysis were performed in tandem, with sample throughput limited by the sample treatment step. Calibration curves based on fluorescence intensity vs. analyte mass were obtained in the range of 10 to 1000 pg for norfloxacin, ciprofloxacin, and enrofloxacin with LOD values of 6-19 ng L-1 for a sample volume of 100 mL, and RSD < 11% at 0.7 ¿g L-1. The method was successfully applied to estuarine river water analysis.info:eu-repo/semantics/publishedVersio

Intergroup Emotions, Perceived Threats and Hostility against Foreigners: Comparing Brazil, Portugal, and Spain

This study analyzed whether hostility towards foreigners perceived as competitors in the labor market would be related to the perception of threat (symbolic and economic) and negative intergroup emotions. 270 people between 18 and 63 years participated. In Brazil (N = 89), mostly men (59%) and average age 32.5 years; In Portugal (N = 87), mostly men (56.3%) and average age 39.9 years; in Spain (N = 94), mostly women (53.2%) and average age of 32.8 years. The results (t-test, analysis of variance and multiple regressions) indicated that in Spain negative emotions are associated with less hostility towards immigrants; In Brazil, positive emotions are associated with hostile attitudes. In Portugal, the relationship between positive emotions and hostility is positive, but not signifi cant. The symbolic threat was the best predictor of hostility only in the Brazilian sample. Positive emotions predispose to a greater kindness towards foreigners in the three countries. The main conclusion of the study is that the economic crisis does not seem to be associated with the perception of competitiveness of foreigners in the local labor market and hostility towards this social group.O estudo analisou se a hostilidade para com estrangeiros percebidos como concorrentes no mercado de trabalho estaria relacionada com a percepção de ameaça (simbólica e econômica) e as emoções intergrupais negativas. Participaram 270 pessoas entre 18 e 63 anos, assim distribuídas: Brasil (N = 89), idade média de 32.5 anos, maioria de homens (59%); Portugal (N = 87), idade média de 39.9 anos, maioria de homens (56.3%); e Espanha (N=94), idade média de 32.8 anos, maioria de mulheres (53.2%). Os resultados do Teste-t, análise da variância e regressões múltiplas indicaram que enquanto na Espanha emoções negativas se associam a menos hostilidade para com imigrantes, no Brasil é a expressão de emoções positivas que se encontra associada a tais atitudes hostis. Em Portugal a relação entre emoções positivas e hostilidade é positiva, mas não signifi cativa. A ameaça simbólica foi o melhor preditor de hostilidade somente na amostra brasileira. Emoções positivas predizem maior amabilidade para com estrangeiros nas amostras dos três países. A principal conclusão do estudo é que a crise econômica não parece estar associada com a percepção de competitividade do estrangeiro no mercado de trabalho local e a hostilidade para com este grupo social.info:eu-repo/semantics/publishedVersio

Photocatalytic ozonation of urban wastewater and surface water using immobilized TiO2 with LEDs: Micropollutants, antibiotic resistance genes and estrogenic activity

Photocatalytic ozonation was employed for the first time in continuous mode with TiO2-coated glass Raschig rings and light emitting diodes (LEDs) to treat urban wastewater as well as surface water collected from the supply area of a drinking water treatment plant (DWTP). Different levels of contamination and types of contaminants were considered in this work, including chemical priority substances (PSs) and contaminants of emerging concern (CECs), as well as potential human opportunistic antibiotic resistant bacteria and their genes (ARB&ARG). Photocatalytic ozonation was more effective than single ozonation (or even than TiO2 catalytic ozonation) in the degradation of typical reaction by-products (such as oxalic acid), and more effective than photocatalysis to remove the parent micropollutants determined in urban wastewater. In fact, only fluoxetine, clarithromycin, erythromycin and 17-alpha-ethinylestradiol (EE2) were detected after photocatalytic ozonation, by using solid-phase extraction (SPE) pre-concentration and LC-MS/MS analysis. In surface water, this treatment allowed the removal of all determined micropollutants to levels below the limit of detection (0.01-0.20 ng L(-1)). The efficiency of this process was then assessed based on the capacity to remove different groups of cultivable microorganisms and housekeeping (16S rRNA) and antibiotic resistance or related genes (intI1, blaTEM, qnrS, sul1). Photocatalytic ozonation was observed to efficiently remove microorganisms and ARGs. Although after storage total heterotrophic and ARB (to ciprofloxacin, gentamicin, meropenem), fungi, and the genes 16S rRNA and intI1, increased to values close to the pre-treatment levels, the ARGs (blaTEM, qnrS and sul1) were reduced to levels below/close to the quantification limit even after 3-days storage of treated surface water or wastewater. Yeast estrogen screen (YES), thiazolyl blue tetrazolium reduction (MTT) and lactate dehydrogenase (LDH) assays were also performed before and after photocatalytic ozonation to evaluate the potential estrogenic activity, the cellular metabolic activity and the cell viability. Compounds with estrogenic effects and significant differences concerning cell viability were not observed in any case. A slight cytotoxicity was only detected for Caco-2 and hCMEC/D3 cell lines after treatment of the urban wastewater, but not for L929 fibroblasts.info:eu-repo/semantics/acceptedVersio

Autoantibodies neutralizing type I IFNs are present in ~4% of uninfected individuals over 70 years old and account for ~20% of COVID-19 deaths

Publisher Copyright: © 2021 The Authors, some rights reserved.Circulating autoantibodies (auto-Abs) neutralizing high concentrations (10 ng/ml; in plasma diluted 1:10) of IFN-alpha and/or IFN-omega are found in about 10% of patients with critical COVID-19 (coronavirus disease 2019) pneumonia but not in individuals with asymptomatic infections. We detect auto-Abs neutralizing 100-fold lower, more physiological, concentrations of IFN-alpha and/or IFN-omega (100 pg/ml; in 1:10 dilutions of plasma) in 13.6% of 3595 patients with critical COVID-19, including 21% of 374 patients >80 years, and 6.5% of 522 patients with severe COVID-19. These antibodies are also detected in 18% of the 1124 deceased patients (aged 20 days to 99 years; mean: 70 years). Moreover, another 1.3% of patients with critical COVID-19 and 0.9% of the deceased patients have auto-Abs neutralizing high concentrations of IFN-beta. We also show, in a sample of 34,159 uninfected individuals from the general population, that auto-Abs neutralizing high concentrations of IFN-alpha and/or IFN-omega are present in 0.18% of individuals between 18 and 69 years, 1.1% between 70 and 79 years, and 3.4% >80 years. Moreover, the proportion of individuals carrying auto-Abs neutralizing lower concentrations is greater in a subsample of 10,778 uninfected individuals: 1% of individuals 80 years. By contrast, auto-Abs neutralizing IFN-beta do not become more frequent with age. Auto-Abs neutralizing type I IFNs predate SARS-CoV-2 infection and sharply increase in prevalence after the age of 70 years. They account for about 20% of both critical COVID-19 cases in the over 80s and total fatal COVID-19 cases.Peer reviewe

The risk of COVID-19 death is much greater and age dependent with type I IFN autoantibodies

SignificanceThere is growing evidence that preexisting autoantibodies neutralizing type I interferons (IFNs) are strong determinants of life-threatening COVID-19 pneumonia. It is important to estimate their quantitative impact on COVID-19 mortality upon SARS-CoV-2 infection, by age and sex, as both the prevalence of these autoantibodies and the risk of COVID-19 death increase with age and are higher in men. Using an unvaccinated sample of 1,261 deceased patients and 34,159 individuals from the general population, we found that autoantibodies against type I IFNs strongly increased the SARS-CoV-2 infection fatality rate at all ages, in both men and women. Autoantibodies against type I IFNs are strong and common predictors of life-threatening COVID-19. Testing for these autoantibodies should be considered in the general population