Nitrocellulose redox permanganometry: A simple method for reductive capacity assessment.
Ontology highlight
ABSTRACT:
SUBMITTER: Homolak J
PROVIDER: S-EPMC8718987 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The 6×-Histidine tag which is commonly used for purification of recombinant proteins was converted to a catalytic redox-active center by incorporation of Co(2+). Two examples of the biological activity of this engineered protein-derived cofactor are presented. After inactivation of the natural diheme cofactor of MauG, it was shown that the Co(2+)-loaded 6×His-tag could substitute for the hemes in the H2O2-driven catalysis of tryptophan tryptophylquinone biosynthesis. To further demonstrate that the Co(2+)-loaded 6×His-tag could mediate long range electron transfer, it was shown that addition of H2O2 to the Co(2+)-loaded 6×His-tagged Cu(1+) amicyanin oxidizes the copper site which is 20Å away. These results provide proof of principle for this simple method by which to introduce a catalytic redox-active site into proteins for potential applications in research and biotechnology.
| S-EPMC4285715 | biostudies-literature
Project description:OBJECTIVES:To determine if the capitohamate (CH) planimetry could be a reliable indicator of bone age, and to compare it with Greulich-Pyle (GP) method. METHODS:This retrospective study included 391 children (age, 1-180 months). Two reviewers manually measured the areas of the capitate and hamate on plain radiographs. CH planimetry was defined as the measurement of the sum of areas of the capitate and hamate. Two reviewers independently applied the CH planimetry and GP methods in 109 children whose heights were at the 50th percentile of the growth chart. RESULTS:There was a strong positive correlation between chronological age and CH planimetry measurement (right, r = 0.9702; left, r = 0.9709). There was no significant difference in accuracy between CH planimetry (84.39-84.46 %) and the GP method (85.15-87.66 %) (p ? 0.0867). The interobserver reproducibility of CH planimetry (precision, 4.42 %; 95 % limits of agreement [LOA], -10.5 to 13.4 months) was greater than that of the GP method (precision, 8.45 %; LOA, -29.5 to 21.1 months). CONCLUSIONS:CH planimetry may be a reliable method for bone age assessment. KEY POINTS:• Bone age assessment is important in the work-up of paediatric endocrine disorders. • Radiography of the left hand is widely used to estimate bone age. • Capitatohamate planimetry is a reliable and reproducible method for assessing bone age.
| S-EPMC5938295 | biostudies-literature
Project description:Background:Seed viability monitoring is very important in ex situ germplasm preservation to detect germplasm deterioration. This requires seed-, time- and labor- saving methods with high precision to assess seed germination as viability. Although the current non-invasive, rapid, sensing methods (NRSs) are time- and labor-saving, they lack the precision and simplicity which are the virtues of traditional germination. Moreover, they consume a considerable amount of seeds to adjust sensed signals to germination percentage, which disregards the seed-saving objective. This becomes particularly severe for rare or endangered species whose seeds are already scarce. Here we propose a new method that is precise, low-invasive, simple, and quick, which involves analyzing the pattern of dehiscence (seed coat rupture), followed by embryonic protrusion. Results:Dehiscence proved simple to identify. After the trial of 20 treatments from 3 rice varieties, we recognized that dehiscence percentage at the 48th hour of germination (D(48)) correlates significantly with germination rate for tested seed lots. In addition, we found that the final germination percentage corresponded to D(48) plus 5. More than 70% of the seeds survived post-dehiscence desiccation for storage. Hydrogen peroxide (1 mM) as the solution for imbibition could further improve the survival. The method also worked quicker than tetrazolium which is honored as a fast, traditional method, in detecting less vigorous but viable seeds. Conclusion:We demonstrated the comprehensive virtues of dehiscence method in assessing rice seed: it is more precise and easier to use than NRSs and is faster and more seed-saving than traditional methods. We anticipate modifications including artificial intelligence to extend our method to increasingly diverse circumstances and species.
| S-EPMC6085679 | biostudies-literature
Project description:Cells receive growth and survival stimuli through their attachment to an extracellular matrix (ECM). Overcoming the addiction to ECM-induced signals is required for anchorage-independent growth, a property of most malignant cells. Detachment from ECM is associated with enhanced production of reactive oxygen species (ROS) owing to altered glucose metabolism. Here we identify an unconventional pathway that supports redox homeostasis and growth during adaptation to anchorage independence. We observed that detachment from monolayer culture and growth as anchorage-independent tumour spheroids was accompanied by changes in both glucose and glutamine metabolism. Specifically, oxidation of both nutrients was suppressed in spheroids, whereas reductive formation of citrate from glutamine was enhanced. Reductive glutamine metabolism was highly dependent on cytosolic isocitrate dehydrogenase-1 (IDH1), because the activity was suppressed in cells homozygous null for IDH1 or treated with an IDH1 inhibitor. This activity occurred in absence of hypoxia, a well-known inducer of reductive metabolism. Rather, IDH1 mitigated mitochondrial ROS in spheroids, and suppressing IDH1 reduced spheroid growth through a mechanism requiring mitochondrial ROS. Isotope tracing revealed that in spheroids, isocitrate/citrate produced reductively in the cytosol could enter the mitochondria and participate in oxidative metabolism, including oxidation by IDH2. This generates NADPH in the mitochondria, enabling cells to mitigate mitochondrial ROS and maximize growth. Neither IDH1 nor IDH2 was necessary for monolayer growth, but deleting either one enhanced mitochondrial ROS and reduced spheroid size, as did deletion of the mitochondrial citrate transporter protein. Together, the data indicate that adaptation to anchorage independence requires a fundamental change in citrate metabolism, initiated by IDH1-dependent reductive carboxylation and culminating in suppression of mitochondrial ROS.
| S-EPMC4860952 | biostudies-literature
Project description:Background: The objective and accurate assessment of children's sedentary and physical behavior is important for investigating their relation to health. The purpose of this study is to validate a simple and robust method for the identification of sitting, standing, walking, running and biking performed by preschool children, children and adolescents in the age from 3 to 16 years from a single thigh-worn accelerometer. Method: A total of 96 children were included in the study and all subjects followed a structured activity protocol performed in the subject's normal kindergarten or school environment. Thigh acceleration was measured using the Axivity AX3 (Axivity, Newcastle, UK) device. Method development and accuracy was evaluated by equally dividing the subjects into a development and test group. Results: The sensitivity and specificity for identifying sitting and standing was above 99.3% and for walking and running above 82.6% for all age groups. The sensitivity and specificity for identifying biking was above 85.8% for children and adolescents and above 64.8% for the preschool group using running bikes. Conclusion: The accurate assessment of sitting, standing, walking, running and biking from thigh acceleration and with children in the age range of 3 to 16 is valid, although not with preschool children using running bikes.
| S-EPMC7401882 | biostudies-literature
Project description:N-glycolylneuraminic acid (Neu5Gc) is an immunogenic sugar of dietary origin that metabolically incorporates into diverse native glycoconjugates in humans. Anti-Neu5Gc antibodies are detected in all human sera, though with variable levels and epitope-recognition profiles. These antibodies likely play a role in several inflammation-mediated pathologies including cardiovascular diseases and cancer. In cancer, they have dualistic and opposing roles, either stimulating or repressing disease, as a function of their dose, and some of these antibodies serve as carcinoma biomarkers. Thus, anti-Neu5Gc antibodies may signify risk of inflammation-mediated diseases, and changes in their levels could potentially be used to monitor disease progression and/or response to therapy. Currently, it is difficult to determine levels of anti-Neu5Gc antibodies in individual human samples because these antibodies recognize multiple Neu5Gc-epitopes. Here we describe a simple and specific method for detection and overall estimation of human anti-Neu5Gc antibodies. We exploit the difference between two mouse models that differ only by Neu5Gc-presence (wild-type) or Neu5Gc-absence (Cmah(-/-) knockout). We characterize mouse serum from both strains by HPLC, lectin and mass-spectrometry analysis and show the target Neu5Gc-epitopes. We then use Cmah(-/-) knockout sera to inhibit all non-Neu5Gc-reactivity followed by binding to wild-type sera to detect overall anti-Neu5Gc response in a single assay. We applied this methodology to characterize and quantify anti-Neu5Gc IgG and IgA in sera of patients with Kawasaki disease (KD) at various stages compared to controls. KD is an acute childhood febrile disease characterized by inflammation of coronary arteries that untreated may lead to coronary artery aneurysms with risk of thrombosis and myocardial infarction. This estimated response is comparable to the average of detailed anti-Neu5Gc IgG profile analyzed by a sialoglycan microarray. Both assays revealed an elevated response in acute KD patients with normal coronaries compared to patients with aneurysm or dilated coronaries. Implications of these findings are discussed.
| S-EPMC3592828 | biostudies-literature
Project description:Microbes have an astonishing capacity to transform their environments. Yet, the metabolic capacity of a single species is limited and the vast majority of microorganisms form complex communities and join forces to exhibit capabilities far exceeding those achieved by any single species. Such enhanced metabolic capacities represent a promising route to many medical, environmental, and industrial applications and call for the development of a predictive, systems-level understanding of synergistic microbial capacity. Here we present a comprehensive computational framework, integrating high-quality metabolic models of multiple species, temporal dynamics, and flux variability analysis, to study the metabolic capacity and dynamics of simple two-species microbial ecosystems. We specifically focus on detecting emergent biosynthetic capacity--instances in which a community growing on some medium produces and secretes metabolites that are not secreted by any member species when growing in isolation on that same medium. Using this framework to model a large collection of two-species communities on multiple media, we demonstrate that emergent biosynthetic capacity is highly prevalent. We identify commonly observed emergent metabolites and metabolic reprogramming patterns, characterizing typical mechanisms of emergent capacity. We further find that emergent secretion tends to occur in two waves, the first as soon as the two organisms are introduced, and the second when the medium is depleted and nutrients become limited. Finally, aiming to identify global community determinants of emergent capacity, we find a marked association between the level of emergent biosynthetic capacity and the functional/phylogenetic distance between community members. Specifically, we demonstrate a "Goldilocks" principle, where high levels of emergent capacity are observed when the species comprising the community are functionally neither too close, nor too distant. Taken together, our results demonstrate the potential to design and engineer synthetic communities capable of novel metabolic activities and point to promising future directions in environmental and clinical bioengineering.
| S-EPMC4084645 | biostudies-literature
Project description:Fluorescent tagged ligands are commonly used to determine binding to proteins. However, bound and free ligand concentrations are not directly determined. Instead the response in a fluorescent ligand titration experiment is considered to be proportional to the extent of binding and, therefore, the maximum value of binding is scaled to the total protein concentration. Here, a simple model-free method is presented to be performed in two steps. In the first step, normalized bound and free spectra of the ligand are determined. In the second step, these spectra are used to fit composite spectra as the sum of individual components or linear spectral summation. Using linear spectral summation, free and bound 1-Anilinonaphthalene-8-Sulfonic Acid (ANS) fluorescent ligand concentrations are directly calculated to determine ANS binding to tear lipocalin (TL), an archetypical ligand binding protein. Error analysis shows that the parameters that determine bound and free ligand concentrations were recovered with high certainty. The linear spectral summation method is feasible when fluorescence intensity is accompanied by a spectral shift upon protein binding. Computer simulations of the experiments of ANS binding to TL indicate that the method is feasible when the fluorescence spectral shift between bound and free forms of the ligand is just 8 nm. Ligands tagged with environmentally sensitive fluorescent dyes, e.g., dansyl chromophore, are particularly suitable for this method.
| S-EPMC3945049 | biostudies-literature
Project description:The direct transformation of racemic feedstock materials to valuable enantiopure compounds is of significant importance for sustainable chemical synthesis. Toward this goal, the radical mechanism has proven uniquely effective in stereoconvergent carbon-carbon bond forming reactions. Here we report a mechanistically distinct redox-enabled strategy for an efficient enantioconvergent coupling of pyrroles with simple racemic secondary alcohols. In such processes, chirality is removed from the substrate via dehydrogenation and reinstalled in the catalytic reduction of a key stabilized cationic intermediate. This strategy provides significant advantage of utilizing simple pyrroles to react with feedstock alcohols without the need for leaving group incorporation. This overall redox-neutral transformation is also highly economical with no additional reagent nor waste generation other than water. In our studies, oxime-derived iridacycle complexes are introduced, which cooperate with a chiral phosphoric acid to enable heteroarylation of alcohols, accessing a wide range of valuable substituted pyrroles in high yield and enantioselectivity.
| S-EPMC8376995 | biostudies-literature
Project description:BACKGROUND:A functional cure of chronic hepatitis B (CHB) is feasible, but a clear view of the intrahepatic viral dynamics in each patient is needed. Intrahepatic covalently closed circular DNA (cccDNA) is the stable form of the viral genome in infected cells, and represents the ideal marker of parenchymal colonization. Its relationships with easily accessible peripheral parameters need to be elucidated in order to avoid invasive procedures in patients. OBJECTIVES:The goal of this study was to design, set up, and validate a reliable and straightforward method for the quantification of the cccDNA and total DNA of the hepatitis B virus (HBV) in a variety of clinical samples. PATIENTS AND METHODS:Clinical samples from a cohort of CHB patients, including liver biopsies in some, were collected for the analysis of intracellular HBV molecular markers using novel molecular assays. RESULTS:A plasmid construct, including sequences from the HBV genome and from the human gene hTERT, was generated as an isomolar multi-standard for HBV quantitation and normalization to the cellular contents. The specificity of the real-time assay for the cccDNA was assessed using Dane particles isolated on a density gradient. A comparison of liver tissue from 6 untreated and 6 treated patients showed that the treatment deeply reduced the replicative capacity (total DNA/cccDNA), but had limited impact on the parenchymal colonization. The peripheral blood mononuclear cells (PBMCs) and granulocytes from the treated and untreated patients were also analyzed. CONCLUSIONS:A straightforward method for the quantification of intracellular HBV molecular parameters in clinical samples was developed and validated. The widespread use of such versatile assays could better define the prognosis of CHB, and allow a more rational approach to time-limited tailored treatment strategies.
| S-EPMC5111393 | biostudies-literature