Simple analytical devices suitable for the analysis of various biochemical and immunechemical markers are highly desirable and can provide laboratory diagnoses outside standard hospitals. This study focuses on constructing an easily reproducible do-it-yourself ELISA plate reader biosensor device, assembled from generally available and inexpensive parts. The colorimetric biosensor was based on standard 96-well microplates, 3D-printed parts, and a smartphone camera as a detector was utilized here as a tool to replace the ELISA method, and its function was illustrated in the assay of TNFα as a model immunochemical marker. The assay provided a limit of detection of 19 pg/mL when the B channel of the RGB color model was used for calibration. The assay was well correlated with the ELISA method, and no significant matrix effect was observed for standard biological samples or interference of proteins expected in a sample. The results of this study will inform the development of simple analytical devices easily reproducible by 3D printing and found on generally available electronics.
- MeSH
- 3D tisk * MeSH
- biosenzitivní techniky * MeSH
- ELISA * MeSH
- kolorimetrie MeSH
- lidé MeSH
- TNF-alfa * analýza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Urine test strips for urinalysis are a common diagnostic tool with minimal costs and are used in various situations including homecare and hospitalization. The coloration scaled by the naked eye is simple, but it is suitable for semiquantitative analysis only. In this paper, a colorimetric assay is developed based on a smartphone digital camera and urine test strips. Assays of pH, albumin, glucose, and lipase activity were performed as a tool for the diagnosis of aciduria, alkaluria, glycosuria, proteinuria, and leukocyturia. The RGB color channels were analyzed in the colorimetric assay, and the assay exerted good sensitivity, and all the particular diagnoses proved to be reliable. The limits of detection for glucose (0.11 mmol/L), albumin (0.15 g/L), and lipase (2.50 U/μL) were low enough to cover the expected physiological concentration, and the range for pH was also satisfactory. The urine test strips with a camera as an output detector proved applicability to spiked urine samples, and the results were also well in comparison to the standard assays which confirms the practical relevance of the presented findings.
- Publikační typ
- časopisecké články MeSH
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known for its multifunctionality in several pathogenic bacteria. Our previously reported data suggest that the GAPDH homologue of Francisella tularensis, GapA, might also be involved in other processes beyond metabolism. In the present study, we explored GapA's potential implication in pathogenic processes at the host cell level. Using immunoelectron microscopy, we demonstrated the localization of this bacterial protein inside infected macrophages and its peripheral distribution in bacterial cells increasing with infection time. A quantitative proteomic approach based on stable isotope labeling of amino acids in cell culture (SILAC) combined with pull-down assay enabled the identification of several of GapA's potential interacting partners within the host cell proteome. Two of these partners were further confirmed by alternative methods. We also investigated the impact of gapA deletion on the transcription of selected cytokine genes and the activation of the main signaling pathways. Our results show that ∆gapA-induced transcription of genes encoding several cytokines whose expressions were not affected in cells infected with a fully virulent wild-type strain. That might be caused, at least in part, by the detected differences in ERK/MAPK signaling activation. The experimental observations together demonstrate that the F. tularensis GAPDH homologue is directly implicated in multiple host cellular processes and, thereby, that it participates in several molecular mechanisms of pathogenesis.
Biosensors containing cholinesterase are analytical devices suitable for the assay of neurotoxic compounds. In the research on biosensors, a new platform has appeared some years ago. It is the digital photography and scoring of coloration (photogrammetry). In this paper, a colorimetric biosensor is constructed using 3D-printed multiwell pads treated with indoxylacetate as a chromogenic substrate and gold nanoparticles with the immobilized enzyme butyrylcholinesterase. A smartphone camera served for photogrammetry. The biosensor was tested for the assay of carbofuran and paraoxon ethyl as two types of covalently binding inhibitors: irreversible and pseudoirreversible. The biosensor exerted good sensitivity to the inhibitors and was able to detect carbofuran with a limit of detection for carbofuran 7.7 nmol/l and 17.6 nmol/l for paraoxon ethyl. A sample sized 25 μl was suitable for the assay lasting approximately 70 minutes. Up to 121 samples can be measured contemporary using one multiwell pad. The received data fully correlated with the standard spectrophotometry. The colorimetric biosensor exerts promising specifications and appears to be competitive to the other analytical procedures working on the principle of cholinesterase inhibition. Low-cost, simple, and portable design represent an advantage of the assay of the biosensor. Despite the overall simplicity, the biosensor can fully replace the standard spectroscopic methods.
- Publikační typ
- časopisecké články MeSH
It is usual for information to be unavailable regarding the molecular composition of extracts from herbs or animal tissues that are popular in folk medicine. Here, we present analysis of the alcohol-ether extract from bovine tissue analogous to the basic substance used in such commercial products as Retisin, Imuregen, Actovegin, and Solcoseryl. The tested extract contains a whole spectrum of free amino acids, small proteins and oligopeptides of molecular weight up to 10 kDa, various nucleotides, and a small amount of phospholipids. Among the molecules that can explain some biological activities of the extract were identified those of taurine (2-aminoethanesulfonic acid, a derivative of the amino acid cysteine), several defensins, and bactericidal hemoglobin fragments known as hemocidins. All those molecules identified are natural components of bovine tissues, and a substantial number of them might be biologically active in vivo. Others are sources of readily available nutrients.
- Klíčová slova
- Juvenil,
- MeSH
- lidé MeSH
- potravní doplňky analýza MeSH
- tkáňové extrakty MeSH
- Check Tag
- lidé MeSH
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can serve as biochemical markers of various pathologies like liver disfunction and poisonings by nerve agents. Ellman's assay is the standard spectrophotometric method to measure cholinesterase activity in clinical laboratories. The authors present a new colorimetric test to assess AChE and BChE activity in biological samples using chromogenic reagents, treated 3D-printed measuring pads and a smartphone camera as a signal detector. Multiwell pads treated with reagent substrates 2,6-dichlorophenolindophenyl acetate, indoxylacetate, ethoxyresorufin and methoxyresorufin were prepared and tested for AChE and BChE. In the experiments, 3D-printed pads containing indoxylacetate as a chromogenic substrate were optimal for analytical purposes. The best results were achieved using the red (R) channel, where the limit of detection was 4.05 µkat/mL for BChE and 4.38 µkat/mL for AChE using a 40 µL sample and a 60 min assay. The major advantage of this method is its overall simplicity, as samples are applied directly without any specific treatment or added reagents. The assay was also validated to the standard Ellman's assay using human plasma samples. In conclusion, this smartphone camera-based colorimetric assay appears to have practical applicability and to be a suitable method for point-of-care testing because it does not require specific manipulation, additional education of staff or use of sophisticated analytical instruments.
- MeSH
- acetylcholinesterasa * MeSH
- butyrylcholinesterasa * MeSH
- cholinesterasové inhibitory MeSH
- chytrý telefon MeSH
- kolorimetrie MeSH
- lidé MeSH
- spektrofotometrie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The gut microbiota is one of the modulators influencing its host's development, metabolism, as well as immunological, psychological, and cognitive abilities. The gut microbiota consortium influences enteroendocrine regulation, neurohormonal regulation, as well as natural immune regulation. Disruptions occurring in life can lead to dysbiosis that in turn influences the host homeostasis and/or disease. Targeted modulation of microbiota composition thus appears to be an appropriate intervention strategy in cases of certain specific health problems. Here, we demonstrate that application of the nutritional supplement Imuregen, which is a natural immune booster, modulates the Bacteroidetes/Firmicutes ratio in favor of the Bacteroidetes genera and causes no pathological changes to intestinal epithelium.
The DsbA homolog of Francisella tularensis was previously demonstrated to be required for intracellular replication and animal death. Disruption of the dsbA gene leads to a pleiotropic phenotype that could indirectly affect a number of different cellular pathways. To reveal the broad effects of DsbA, we compared fractions enriched in membrane proteins of the wild-type FSC200 strain with the dsbA deletion strain using a SILAC-based quantitative proteomic analysis. This analysis enabled identification of 63 proteins with significantly altered amounts in the dsbA mutant strain compared to the wild-type strain. These proteins comprise a quite heterogeneous group including hypothetical proteins, proteins associated with membrane structures, and potential secreted proteins. Many of them are known to be associated with F. tularensis virulence. Several proteins were selected for further studies focused on their potential role in tularemia's pathogenesis. Of them, only the gene encoding glyceraldehyde-3-phosphate dehydrogenase, an enzyme of glycolytic pathway, was found to be important for full virulence manifestations both in vivo and in vitro. We next created a viable mutant strain with deleted gapA gene and analyzed its phenotype. The gapA mutant is characterized by reduced virulence in mice, defective replication inside macrophages, and its ability to induce a protective immune response against systemic challenge with parental wild-type strain. We also demonstrate the multiple localization sites of this protein: In addition to within the cytosol, it was found on the cell surface, outside the cells, and in the culture medium. Recombinant GapA was successfully obtained, and it was shown that it binds host extracellular serum proteins like plasminogen, fibrinogen, and fibronectin.
- MeSH
- delece genu * MeSH
- faktory virulence analýza MeSH
- Francisella tularensis enzymologie imunologie patogenita MeSH
- glyceraldehyd-3-fosfátdehydrogenasy nedostatek metabolismus MeSH
- krevní proteiny metabolismus MeSH
- mikrobiální viabilita MeSH
- modely nemocí na zvířatech MeSH
- myši MeSH
- proteindisulfidisomerasy nedostatek MeSH
- proteom analýza MeSH
- salmonelová infekce u zvířat mikrobiologie patologie MeSH
- vazba proteinů MeSH
- virulence MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- galantamin * aplikace a dávkování terapeutické užití MeSH
- imunitní systém účinky léků MeSH
- imunoglobuliny analýza MeSH
- interleukin-2 analýza MeSH
- interleukin-4 analýza MeSH
- interleukin-6 analýza MeSH
- modely nemocí na zvířatech MeSH
- myši MeSH
- tumor nekrotizující faktory * analýza MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
Francisella tularensis is a facultative intracellular, gram-negative bacterium that induces apoptosis in macrophages and B cells. Here we show apoptotic pathways that are activated in the Ramos human B cell line in the course of F. tularensis infection. Live bacteria F. tularensis FSC200 activate caspases 8, 9 and 3, as well as Bid; release cytochrome c and apoptosis-inducing factor from mitochondria; and induce depolarization of mitochondrial membrane potential in the Ramos cell line, thus leading these cells to apoptosis. Unlike live bacteria, killed F. tularensis FSC200 bacteria activated only caspase 3, and did not cause apoptosis of Ramos cells as measured by annexin V. Killed bacteria also caused accumulation of anti-apoptotic protein Bclx(L) in mitochondrial membranes. Thus, live F. tularensis activates both caspase pathways (receptor-mediated and intrinsic) as well as caspase-independent mitochondrial death.
- MeSH
- apoptóza MeSH
- B-lymfocyty mikrobiologie MeSH
- buněčné linie MeSH
- cytochromy c metabolismus MeSH
- Francisella tularensis patogenita MeSH
- kaspasa 3 biosyntéza MeSH
- kaspasa 8 biosyntéza MeSH
- kaspasa 9 biosyntéza MeSH
- lidé MeSH
- membránový potenciál mitochondrií MeSH
- mitochondrie enzymologie fyziologie MeSH
- protein Bid biosyntéza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
