Assessment of Escherichia coli B with enhanced permeability to fluorochromes for flow cytometric assays of bacterial cell function.

Background Flow cytometry has become a choice methodology for microbiological research. However, functional cytometric assays in live bacteria are still limited. This is due, in part, to the cell wall impairing penetration of vital dyes in bacteria, thus imposing permeabilization procedures. These manipulations may affect cell physiology, provoke cell aggregation or lysis, and they are time-consuming. Escherichia coli B strains have been used for mutagenic assays because of an altered lipopolysaccharide that provokes increased membrane permeability. We assessed the use of these strains as possible alternatives for flow cytometric assays to avoid the permeabilization steps. Methods Suspensio…

A Predictive Model for Automatic Detection of Social Isolation in Older Adults

Social isolation is a problem that is accentuated in the stage of old age. This condition puts at risk the physical and mental integrity of older adults. This paper presents a predictive model for the automatic detection of social isolation in older adults. The predictive model was implemented in a mobile application that monitors communication and mobility activities performed by an older adult. The system also considers demographic aspects to improve the prediction results. The mobile application was also generated for a caregiver who is responsible for receiving notifications about the specific level of social isolation of the older adult. The predictive model was evaluated using an expe…

Lung tumorspheres reveal cancer stem cell-like properties and a score with prognostic impact in resected non-small-cell lung cancer

AbstractThe high resistance against current therapies found in non-small-cell lung cancer (NSCLC) has been associated to cancer stem-like cells (CSCs), a population for which the identification of targets and biomarkers is still under development. In this study, primary cultures from early-stage NSCLC patients were established, using sphere-forming assays for CSC enrichment and adherent conditions for the control counterparts. Patient-derived tumorspheres showed self-renewal and unlimited exponential growth potentials, resistance against chemotherapeutic agents, invasion and differentiation capacities in vitro, and superior tumorigenic potential in vivo. Using quantitative PCR, gene express…

Nitic oxide promotes strong cytotoxicity of phenolic compounds against escherichia coli. The influence of antioxidant defenses

[EN] The induction of mutagenic and cytotoxic effects by simple phenolics, including catechol (CAT), 3,4dihydroxyphenylacetic acid (DOPAC), hydroquinone (HQ), and 2,5-dihydroxyphenylacetic (homogentisic) acid (HGA), appears to occur through an oxidative mechanism based on the ability of these compounds to undergo autoxidation, leading to quinone formation with the production of reactive oxygen species. This is supported by the detection of such adverse effects in plate assays using Escherichia coli tester strains deficient in the OxyR function, but not in OxyR(+) strains. The OxyR protein is a redox-sensitive regulator of genes encoding antioxidant enzymes including catalase and alkyl hydro…

Foxp3 Silencing with Antisense Oligonucleotide Improves Immunogenicity of an Adjuvanted Recombinant Vaccine against Sporothrix schenckii

Made available in DSpace on 2021-06-25T10:56:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-04-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Background: In recent years, there has been great interest in developing molecular adjuvants based on antisense oligonucleotides (ASOs) targeting immunosuppressor pathways with inhibitory effects on regulatory T cells (Tregs) to improve immunogenicity and vaccine efficacy. We aim to evaluate the immunostimulating effect of 2′OMe phosphorothioated Foxp3-targeted ASO in an antifungal adjuvanted recombinant vaccine. Methods: The uptake kinetics of Foxp3 ASO, its cyto-toxicity and its ability to deplete Tregs were evaluated in…

Multiparametric characterization by flow cytometry of flow-sorted subpopulations of a human hepatoma cell line useful for drug research

Background Primary cultured hepatocytes are the closest model to the liver for drug research. However, to overcome its limited availability, the search for hepatic cell lines as an alternative to primary cultures is a matter of current interest. In particular, highly differentiated hepatocellular carcinomas have been proposed as in vitro tools for routine experiments in hepatotoxicity and drug metabolism. Methods Cell populations were selected by fluorescence-activated cell sorting based on low and high relative expressions of P-glycoprotein. These cell lines were characterized after 21 days in culture by multiparametric analysis with flow cytometry providing direct information on key cellu…

Expression of chemokine receptor CXCR3, CXCR4, and CXCR7 and their respective ligands in rhabdomyosarcoma

Detection of oxidative mutagenesis by isoniazid and other hydrazine derivatives in Escherichia coli WP2 tester strain IC203, deficient in OxyR: strong protective effects of rat liver S9

Abstract Strain IC203, deficient in the OxyR function, was sensitive to both cytotoxic and mutagenic effects of isoniazid (INH) whereas its parent, WP2 uvrA /pKM101, was resistant to these effects. Four other hydrazine compounds, hydrazine hydrate (HZH), phenylhydrazine (PHZ), hydralazine (HLZ) and nialamide (NLD), were mutagenic in WP2 uvrA /pKM101. Increases in mutagenicity were observed in IC203 for HZH and PHZ but not for HLZ and NLD. Growth inhibition zones by HZH, PHZ and NLD were larger in IC203 than in WP2 uvrA /pKM101. The enhancements in the effects of INH, HZH and PHZ in IC203 with respect to its oxyR + parent are considered to be caused by the production of reactive oxygen speci…

Functional assays of oxidative stress using genetically engineered Escherichia coli strains.

Oxidative stress may be induced in bacteria by exogenous biocidal agents and is involved in endogenous metabolism. The oxyR operon is a main sensor of oxidative stress and oxyR-deficient bacteria show enhanced sensitivity to oxidative stress and increased accumulation of intracellular reactive oxygen species (ROS). Flow cytometric functional assays in bacteria are limited by the impaired penetration of vital dyes trough the cell wall. Escherichia coli B WP2 strains possess an altered cell-wall lipopolysaccharide that leads to increased membrane permeability. Flow cytometric analysis of WP2 strains is a convenient alternative for cytometric assays of bacterial function. This unit presents pr…

The Relevance of Flow Cytometry for Biochemical Analysis

Flow cytometry (FCM) allows the simultaneous measurement of multiple fluorescences and light scatter induced by illumination of single cells or microscopic particles in suspension, as they flow rapidly through a sensing area. In some systems, individual cells or particles may be sorted according to the properties exhibited. By using appropriate fluorescent markers, FCM is unique in that multiple structural and functional parameters can be quantified simultaneously on a single-particle basis, whereas up to thousands of biological particles per second may be examined. FCM is increasingly used for basic, clinical, biotechnological, and environmental studies of biochemical relevance. In this cr…