RESUMO
The viral genome of the SARS-CoV-2 coronavirus, the aetiologic agent of COVID-19, encodes structural, non-structural, and accessory proteins. Most of these components undergo rapid genetic variations, though to a lesser extent the essential viral proteases. Consequently, the protease and/or deubiquitinase activities of the cysteine proteases Mpro and PLpro became attractive targets for the design of antiviral agents. Here, we develop and evaluate new bis(benzylidene)cyclohexanones (BBC) and identify potential antiviral compounds. Three compounds were found to be effective in reducing the SARS-CoV-2 load, with EC50 values in the low micromolar concentration range. However, these compounds also exhibited inhibitory activity IC50 against PLpro at approximately 10-fold higher micromolar concentrations. Although originally developed as PLpro inhibitors, the comparison between IC50 and EC50 of BBC indicates that the mechanism of their in vitro antiviral activity is probably not directly related to inhibition of viral cysteine proteases. In conclusion, our study has identified new potential noncytotoxic antiviral compounds suitable for in vivo testing and further improvement.
Assuntos
COVID-19 , Cisteína Proteases , Humanos , SARS-CoV-2 , Cisteína Endopeptidases/metabolismo , Proteínas não Estruturais Virais/química , Antivirais/farmacologia , Antivirais/química , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , Simulação de Acoplamento MolecularRESUMO
BACKGROUND: Aberrant glycosylation is a hallmark of cancer and thereby has an excellent potential for the discovery of novel biomarkers. Impairments in the glycan composition of lipoproteins impact their functional properties and can be associated with various diseases, including cancer. This research is still in its infancy; however, it can lead to the development of new diagnostic and disease stratification approaches as well as therapeutic strategies. Therefore, we aimed to evaluate anomalies in O-glycosylation of apolipoprotein C-III (apoC-III) in colorectal carcinoma (CRC) patients' sera, in comparison with sera from healthy individuals, and assess the disparities of O-glycoforms on apoC-III in CRC. METHODS: The choice of patients (n = 42) was based on the same tumor type (adenocarcinoma) and tumor size (T3), without or with inconsiderable lymph node infiltration. Patients with comorbidities were excluded from the study. The control healthy individuals (n = 40) were age- and sex-matched with patients. We used an approach based on the MALDI-TOF MS in linear positive ion mode, allowing simple analysis of O-glycosylation on intact apoC-III molecules in the serum samples directly, without the need for specific protein isolation. This approach enables relatively simple and high-throughput analysis. RESULTS: In CRC patients' sera samples, we observed significantly elevated apoC-III sialylation. Fully sialylated (disialylated) O-glycans had 1.26 times higher relative abundance in CRC samples compared to controls with a p-value of Mann-Whitney U test of 0.0021. CONCLUSIONS: We found altered O-glycosylation of apoC-III in the serum of CRC patients. However, it can be non-specific as it may be associated with another process such as ongoing inflammation. Therefore, to establish it as a potential novel non-invasive biomarker for CRC in suspected patients, further studies interrogating the changes in apoC-III O-glycosylation and the robustness of this biomarker need to be performed and evaluated.
Assuntos
Neoplasias Colorretais , Polissacarídeos , Humanos , Apolipoproteína C-III , Glicosilação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Biomarcadores , Neoplasias Colorretais/diagnósticoRESUMO
Blue C-phycocyanin (C-PC), the major Spirulina protein with innumerable health-promoting benefits, is an attractive colourant and food supplement. A crucial obstacle to its more extensive use is its relatively low stability. This study aimed to screen various food-derived ligands for their ability to bind and stabilise C-PC, utilising spectroscopic techniques and molecular docking. Among twelve examined ligands, the protein fluorescence quenching revealed that only quercetin, coenzyme Q10 and resveratrol had a moderate affinity to C-PC (Ka of 2.2 to 3.7 × 105 M-1). Docking revealed these three ligands bind more strongly to the C-PC hexamer than the trimer, with the binding sites located at the interface of two (αß)3 trimers. UV/VIS absorption spectroscopy demonstrated the changes in the C-PC absorption spectra in a complex with quercetin and resveratrol compared to the spectra of free protein and ligands. Selected ligands did not affect the secondary structure content, but they induced changes in the tertiary protein structure in the CD study. A fluorescence-based thermal stability assay demonstrated quercetin and coenzyme Q10 increased the C-PC melting point by nearly 5 °C. Our study identified food-derived ligands that interact with C-PC and improve its thermal stability, indicating their potential as stabilising agents for C-PC in the food industry.
Assuntos
Proteína C , Spirulina , Animais , Ubiquinona , Antioxidantes/farmacologia , Ficocianina , Simulação de Acoplamento Molecular , Quercetina , Resveratrol/farmacologia , Aditivos Alimentares , Decapodiformes , Suplementos NutricionaisRESUMO
In this study, one hundred serum samples from healthy people and patients with rheumatoid arthritis (RA) were analyzed. Standard immunoassays for detection of 10 different RA markers and analysis of glycan markers on antibodies in 10 different assay formats with several lectins were applied for each serum sample. A dataset containing 2000 data points was data mined using artificial neural networks (ANN). We identified key RA markers, which can discriminate between healthy people and seropositive RA patients (serum containing autoantibodies) with accuracy of 83.3%. Combination of RA markers with glycan analysis provided much better discrimination accuracy of 92.5%. Immunoassays completely failed to identify seronegative RA patients (serum not containing autoantibodies), while glycan analysis correctly identified 43.8% of these patients. Further, we revealed other critical parameters for successful glycan analysis such as type of a sample, format of analysis and orientation of captured antibodies for glycan analysis.
Assuntos
Artrite Reumatoide/sangue , Inteligência Artificial , Glicômica , Polissacarídeos/sangue , Fator Reumatoide/sangue , Autoanticorpos/sangue , Biomarcadores/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
An extensive characterization of pristine and oxidized Ti3C2Tx (T: =O, -OH, -F) MXene showed that exposure of MXene to an anodic potential in the aqueous solution oxidizes the nanomaterial forming TiO2 layer or TiO2 domains with subsequent TiO2 dissolution by F- ions, making the resulting nanomaterial less electrochemically active compared to the pristine Ti3C2Tx. The Ti3C2Tx could be thus applied for electrochemical reactions in a cathodic potential window i.e. for ultrasensitive detection of H2O2 down to nM level with a response time of approx. 10 s. The manuscript also shows electrochemical behavior of Ti3C2Tx modified electrode towards oxidation of NADH and towards oxygen reduction reactions.
RESUMO
Screening serum for the presence of prostate specific antigen (PSA) belongs to the most common approach for the detection of prostate cancer. This review (with 156 refs.) addresses recent developments in PSA detection based on the use of various kinds of nanomaterials. It starts with an introduction into the field, the significance of testing for PSA, and on current limitations. A first main section treats electrochemical biosensors for PSA, with subsections on methods based on the use of gold electrodes, graphene or graphene-oxide, carbon nanotubes, hybrid nanoparticles, and other types of nanoparticles. It also covers electrochemical methods based on the enzyme-like activity of PSA, on DNA-, aptamer- and biofuel cell-based methods, and on the detection of PSA via its glycan part. The next main section covers optical biosensors, with subsections on methods making use of surface plasmon resonance (SPR), localized SPR and plasmonic ELISA-like schemes. This is followed by subsections on methods based on the use of fiber optics, fluorescence, chemiluminescence, Raman scattering and SERS, electrochemiluminescence and cantilever-based methods. The most sensitive biosensors are the electrochemical ones, with lowest limits of detection (down to attomolar concentrations), followed by mass cantilever sensing and electrochemilumenescent strategies. Optical biosensors show lower performance, but are still more sensitive compared to standard ELISA. The most commonly applied nanomaterials are metal and carbon-based ones and their hybrid composites used for different amplification strategies. The most attractive sensing schemes are summarized in a Table. The review ends with a section on conclusions and perspectives.
Assuntos
Técnicas Biossensoriais/métodos , Nanoestruturas/química , Antígeno Prostático Específico/análise , Neoplasias da Próstata/diagnóstico , Animais , Aptâmeros de Nucleotídeos/química , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/sangue , Técnicas Biossensoriais/instrumentação , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Desenho de Equipamento , Tecnologia de Fibra Óptica/instrumentação , Tecnologia de Fibra Óptica/métodos , Humanos , Luminescência , Masculino , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/sangue , Espectrometria de Fluorescência/instrumentação , Espectrometria de Fluorescência/métodosRESUMO
We describe a self-assembled monolayer (SAM) on a gold surface with a carboxybetaine ester functionality to control the interaction between DNA and gold nanoparticles via pH. The negatively charged phosphate backbone of DNA interacts with and adsorbs to the positively charged carboxybetaine esters on the SAM. DNA release can be achieved by the hydrolysis of carboxybetaine ester (CBE) to a zwitterionic carboxybetaine state. Furthermore, the adsorption of negatively charged citrate-capped gold nanoparticles to a SAM-modified plain gold surface can be controlled by the pH. The SAM based on carboxybetaine ester allows for the homogeneous adsorption of particles, whereas the SAM after hydrolysis at high pH repels AuNP adsorption. The antifouling surface properties of the surface modified with carboxybetaine were investigated with protein samples.
Assuntos
Nanopartículas Metálicas , Adsorção , DNA , Ouro , Concentração de Íons de Hidrogênio , Propriedades de SuperfícieRESUMO
In recent decades, it has become clear that most of human proteins are glycosylated and that protein glycosylation plays an important role in health and diseases. At present, simple, fast and inexpensive methods are sought for clinical applications and particularly for improved diagnostics of various diseases, including cancer. We propose a label- and reagent-free electrochemical method based on chronopotentiometric stripping (CPS) analysis and a hanging mercury drop electrode for the detection of interaction of sialylated protein biomarker a prostate specific antigen (PSA) with two important lectins: Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA). Incubation of PSA-modified electrode with specific SNA lectin resulted in an increase of CPS peak H of the complex as compared to this peak of individual PSA. By adjusting polarization current and temperature, PSA-MAA interaction can be either eliminated or distinguished from the more abundant PSA-SNA complex. CPS data were in a good agreement with the data obtained by complementary methods, namely surface plasmon resonance and fluorescent lectin microarray. It can be anticipated that CPS will find application in glycomics and proteomics.
Assuntos
Aglutininas/metabolismo , Condutividade Elétrica , Ácido N-Acetilneuramínico/metabolismo , Antígeno Prostático Específico/química , Antígeno Prostático Específico/metabolismo , Eletroquímica , Maackia/química , Sambucus nigra/químicaRESUMO
An impedimetric lectin biosensor for the detection of changes in the glycan structure of antibodies isolated from human serum is here correlated with the progression of rheumatoid arthritis (RA). The biosensor was built up from a mixed self-assembled monolayer (SAM) on gold consisting of two different thiolated zwitterionic derivatives, carboxybetaine and sulfobetaine, to resist nonspecific interactions. The carboxyl-terminated one was applied also for the covalent immobilization of lectin Ricinus communis agglutinin I (RCA-I). The process of building a bioreceptive layer was optimized and characterized using a diverse range of techniques. Impedimetric assays were integrated on a chip consisting of eight gold working electrodes, which is an important step toward the achievement of a moderate level of multiplexing for the analysis of human serum samples. At the end, the results obtained by the impedimetric analysis of immunoglobulins G (IgGs) isolated from serum samples were compared with those of two other standard bioanalytical methods employing lectins, that is, lectin microarrays (MAs) and enzyme-linked lectin binding assays (ELLBAs). The impedimetric results agreed very well with the DAS28 index (RA disease activity score 28), suggesting that impedimetric assays could be used for the development of a new diagnostic procedure sensitive to glycosylation changes in human IgGs and thus RA progression.