Multiplexed flow cytometric approach for detection of anti-SARS-CoV-2 IgG, IgM and IgA using beads covalently coupled to the nucleocapsid protein.

Flow cytometry has emerged as a promising technique for detection of SARS-CoV-2 antibodies. In this study, we developed an innovative strategy for simultaneous detection of immunoglobulin G (IgG), IgM and IgA. The SARS-CoV-2 nucleocapsid protein was covalently bound to functional beads surface applying sulpho-SMCC chemistry. BUV395 anti-IgG, BB515 anti-IgM, biotinylated anti-IgA1/IgA2 and BV421 streptavidin were used as fluorophore conjugated secondary antibodies. Serum and antibodies reaction conditions were optimized for each antibody isotype detection and a multiplexed detection assay was developed. This new cell-free assay efficiently discriminate COVID-19 negative and positive samples. The simultaneous detection of IgG, IgM and IgA showed a sensitivity of 88·5-96·2% and specificity of 100%. This novel strategy opens a new avenue for flow cytometry-based diagnosis.

Interleukin-18 (rs187238) and glucose transporter 4 (rs5435) polymorphisms in Euro-Brazilians with type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease with a strong genetic component that has been associated with several genetic loci. Interleukin 18 (IL-18) is a potent proinflammatory cytokine, which is involved in the innate and adaptive immune responses, and in the pathogenesis of various diseases including T1D. Glucose transporter 4 (GLUT4) is known to be an insulin-responsive glucose transporter and has been associated with various diseases, including diabetes mellitus. We investigated the association of the polymorphisms rs187238 (IL-18) and rs5435 (GLUT4) in a case-control study in Euro-Brazilians with T1D (N = 136) and healthy subjects (N = 144). Real-time PCR with TaqMan® fluorescent probes were applied for genotyping. All polymorphisms were in Hardy-Weinberg equilibrium. The minor allele frequencies for the G-allele (rs187238; IL-18) in healthy and T1D groups were 28.5% [95%CI = 23-34%] vs 31.6% [95%CI = 26-37%], P = 0.416, and for the T-allele (rs5435, GLUT4) were 33% [95%CI = 28-39] vs 27% [95%CI = 23-33%], P = 0.167, respectively. Genotype comparisons for both polymorphisms showed no significant differences (P > 0.05). The polymorphisms rs187238 and rs5435 were not associated with T1D in the studied population. The minor allele frequencies for both polymorphisms were similar to those of other Caucasian populations.

A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry.

The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots.

A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry

The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots.

Rapid quantification of rice root-associated bacteria by flow cytometry.

UNLABELLED: To understand the mechanism of plant-bacterium interaction, it is critical to enumerate epiphytic bacteria colonizing the roots of the host. We developed a new approach, based on flow cytometry, for enumerating these bacteria and used it with rice plants, 7 and 20 days after colonization with Herbaspirillum rubrisubalbicans and Azospirillum brasilense. The results were compared with those obtained with the traditional plate count method. Both methods gave similar numbers of H. rubrisubalbicans associated with rice roots (c. 10(9) CFU g(-1) ). However, flow cytometry gave a number of viable cells of rice-associated A. brasilense that was approx. 10-fold greater than that obtained with the plate count method. These results suggest that the plate count method can underestimate epiphytic populations. Flow cytometry has the additional advantage that it is more precise and much faster than the plate count method. SIGNIFICANCE AND IMPACT OF THE STUDY: Determination of precise number of root-associated bacteria is critical for plant-bacteria interaction studies. We developed a flow cytometry approach for counting bacteria and compared it with the plate count method. Our flow cytometry assay solves two major limitations of the plate count method, namely that requires long incubation times of up to 48 h and only determines culturable cells. This flow cytometry assay provides an efficient, precise and fast tool for enumerating epiphytic cells.

Targeting the multidrug ABCG2 transporter with flavonoidic inhibitors: in vitro optimization and in vivo validation.

This review describes the breast cancer resistance protein ABCG2 through its structure, functional roles and involvement in cell multidrug resistance, especially in cancer cells resistance to chemotherapeutics. The different types of known inhibitors are described, some being non-selective, since they also bind to other targets, and others being quite specific such as flavonoids. The different classes of active flavonoids and other polyphenols are described, some as plant natural compounds, but most of them being prepared and derivatized through medicinal chemistry. Quantitative structure-activity relationships of the ability of flavones, chalcones, xanthones, acridones and various benzopyrane/benzofurane derivatives to inhibit ABCG2-mediated drug efflux have led to pharmacophores and molecular models allowing to optimize the available hit compounds and to design new-generation lead compounds. Interestingly, inhibitory flavonoids are quite specific for ABCG2 versus ABCB1 and ABCC1, and appear either non-competitive or partially competitive towards mitoxantrone efflux. Most compounds do not inhibit ATPase activity, and are assumed not to be transported themselves by the transporter. Some acridones, firstly optimized in vitro as potent inhibitors, are indeed efficient in vivo, against human xenografts in SCID mice, more efficiently than gefitinib taken as a control. Future developments should open the way to more efficient/targeted modulators including (i) the potential interest of bimodulation by combining two different inhibitors, (ii) computer-assisted ligand-based drug design for getting more potent and more specific inhibitors, (iii) structure-based drug design from ABCG2 molecular models allowing in silico screening and docking of new inhibitors.

cDNA cloning and mitochondrial import of the beta-subunit of the human electron-transfer flavoprotein.

We have isolated a cDNA clone which encodes the entire beta-subunit of human electron-transferring flavoprotein (ETF) by screening an expression library from human liver using polyclonal antibodies against porcine ETF. This cDNA encodes a protein of 255 amino-acid residues with a predicted molecular mass of 27,877 Da which shows a high degree of similarity with partial amino-acid sequences obtained from both rat liver and Paracoccus denitrificans beta-ETF. Northern-blot analysis shows that the human beta-ETF mRNA is approximately 1 kb in size and is abundant in liver, heart and skeletal muscle. Incubation with intact mitochondria indicates that the cDNA-encoded beta-ETF polypeptide contains the information necessary to reach the mitochondrial matrix. These data are in agreement with previous experiments suggesting that beta-ETF, unlike the majority of nuclear-encoded mitochondrial matrix proteins, does not have a cleavable leader peptide. Furthermore, when valinomycin is added to the incubation mixture, the import is abolished, thus demonstrating that it is an energy-dependent process. Interestingly, the sequence analysis of beta-ETF protein identifies a 26.3% identity with the Fix A gene product of the nitrogen-fixing bacterium Azorhizobium caulinodans.