Improvement of the chemical and physical stability of the EC1 domain of E-cadherin by blocking its disulfide-mediated dimerization

The EC1 protein has an important role in the E-cadherin mediated cell-cell adhesion in epithelial and endothelial tissues. It may be used as modulator of cellular adhesion to improve paracellular delivery of macromolecules. EC1 undergoes oxidation of its Cys residue to form disulfide-linked covalent dimers. These dimers associate to form physical oligomers. The dimerization and oligomerization also lead to hydrolysis of the Asp93-Pro94 peptide bond and precipitation. To be able to study the cell adhesion-modulating activity of EC1, it is important that we block its disulfide-dimerization and subsequent oligomerization. The strategies used to block the dimerization were addition of dithiothreitol to the EC1 solution, modification of the Cys thiol with iodoacetamide and polyethylene glycol. These derivatives were studied for their stability using HPLC, CD and fluorescence spectroscopy. All strategies applied showed improvement in the stability of EC1. The PEGylated EC1 showed the best stability

Improving the Stability of the EC1 Domain of E-cadherin by Thiol Alkylation of the Cysteine Residue

The objective of this work was to improve chemical and physical stability of the EC1 protein derived from the extracellular domain of E-cadherin. In solution, the EC1 protein has been shown to form a covalent dimer via a disulfide bond formation followed by physical aggregation and precipitation. To improve solution stability of the EC1 protein, the thiol group of the Cys13 residue in EC1 was alkylated with iodoacetate, iodoacetamide, and maleimide-PEG-5000 to produce thioether derivatives called EC1-IA, EC1-IN, and EC1-PEG. The physical and chemical stabilities of the EC1 derivatives and the parent EC1 were evaluated at various pHs (3.0, 7.0, and 9.0) and temperatures (0, 3, 70 °C). The structural characteristics of each molecule were analyzed by circular dichroism (CD) and fluorescence spectroscopy and the derivatives have similar secondary structure as the parent EC1 protein at pH 7.0. Both EC1-IN and EC1-PEG derivatives showed better chemical and physical stability profiles than did the parent EC1 at pH 7.0. EC1-PEG had the best stability profile compared to EC1-IN and EC1 in solution under various conditions

The role of covalent dimerization on the physical and chemical stability of the EC1 domain of human E-cadherin

The objective of this work was to evaluate the solution stability of the EC1 domain of E-cadherin under various conditions. The EC1 domain was incubated at various temperatures (4, 37, and 70 °C) and pH values (3.0, 7.0, and 9.0). At pH 9.0 and 37 or 70 °C, a significant loss of EC1 was observed due to precipitation and a hydrolysis reaction. The degradation was suppressed upon addition of DTT, suggesting that the formation of EC1 dimer facilitated the EC1 degradation. At 4 °C and various pH values, the EC1 secondary and tertiary showed changes upon incubation up to 28 days, and DTT prevented any structural changes upon 28 days of incubation. Molecular dynamics simulations indicated that the dimer of EC1 has higher mobility than does the monomer; this higher mobility of the EC1 dimer may contribute to instability of the EC1 domain

Worldwide trends in underweight and obesity from 1990 to 2022: a pooled analysis of 3663 population-representative studies with 222 million children, adolescents, and adults

Background Underweight and obesity are associated with adverse health outcomes throughout the life course. We estimated the individual and combined prevalence of underweight or thinness and obesity, and their changes, from 1990 to 2022 for adults and school-aged children and adolescents in 200 countries and territories. Methods We used data from 3663 population-based studies with 222 million participants that measured height and weight in representative samples of the general population. We used a Bayesian hierarchical model to estimate trends in the prevalence of different BMI categories, separately for adults (age ≥20 years) and school-aged children and adolescents (age 5–19 years), from 1990 to 2022 for 200 countries and territories. For adults, we report the individual and combined prevalence of underweight (BMI 2 SD above the median). Findings From 1990 to 2022, the combined prevalence of underweight and obesity in adults decreased in 11 countries (6%) for women and 17 (9%) for men with a posterior probability of at least 0·80 that the observed changes were true decreases. The combined prevalence increased in 162 countries (81%) for women and 140 countries (70%) for men with a posterior probability of at least 0·80. In 2022, the combined prevalence of underweight and obesity was highest in island nations in the Caribbean and Polynesia and Micronesia, and countries in the Middle East and north Africa. Obesity prevalence was higher than underweight with posterior probability of at least 0·80 in 177 countries (89%) for women and 145 (73%) for men in 2022, whereas the converse was true in 16 countries (8%) for women, and 39 (20%) for men. From 1990 to 2022, the combined prevalence of thinness and obesity decreased among girls in five countries (3%) and among boys in 15 countries (8%) with a posterior probability of at least 0·80, and increased among girls in 140 countries (70%) and boys in 137 countries (69%) with a posterior probability of at least 0·80. The countries with highest combined prevalence of thinness and obesity in school-aged children and adolescents in 2022 were in Polynesia and Micronesia and the Caribbean for both sexes, and Chile and Qatar for boys. Combined prevalence was also high in some countries in south Asia, such as India and Pakistan, where thinness remained prevalent despite having declined. In 2022, obesity in school-aged children and adolescents was more prevalent than thinness with a posterior probability of at least 0·80 among girls in 133 countries (67%) and boys in 125 countries (63%), whereas the converse was true in 35 countries (18%) and 42 countries (21%), respectively. In almost all countries for both adults and school-aged children and adolescents, the increases in double burden were driven by increases in obesity, and decreases in double burden by declining https://researchonline.ljmu.ac.uk/images/research_banner_face_lab_290.jpgunderweight or thinness. Interpretation The combined burden of underweight and obesity has increased in most countries, driven by an increase in obesity, while underweight and thinness remain prevalent in south Asia and parts of Africa. A healthy nutrition transition that enhances access to nutritious foods is needed to address the remaining burden of underweight while curbing and reversing the increase in obesity

A Comparative Study of Apical Root Resorption during Intrusion of Maxillary Anterior Teeth Treated with Intrusion Arch and MiniImplants-A CBCT Study

Background: To evaluate and compare the relationship between the amount of root resorption after the intrusion of maxillary anterior teeth using two different intrusion mechanics i.e. Intrusion arch (Utility arch) and Miniimplants through CBCT scans. 20 patients randomly selected were divided into 2 groups: Group A: 10 patients with intrusion arch mechanics and Group B: 10 patients with mini-implant mechanics. Pre- and post-intrusion sectional CBCT scans of maxillary anterior teeth were taken. Changes in volumetric measurements (in mm3 ), linear measurements (in mm), and angular measurements (in degree) between pre and post-intrusion scans were measured for each incisor in both groups using paired t-tests. An independent t-test was used for comparing the change in all three parameters between both groups. Pearson’s correlation test was done for correlating the amount of root resorption with the amount of intrusion for each incisor in both groups. Results: Changes in all three parameters between pre-and post-treatment CBCT scans for all incisors for each group and between both groups were statistically significant. Pearson’s correlation shows a statistically nonsignificant correlation for all incisors in both groups except lateral incisors in group B. Conclusion: Clinically significant root resorption is observed with the intrusion of maxillary anterior teeth either with utility arch or mini-implants. The amount of root resorption and range of intrusion is more with miniimplants, while the amount of proclination is more with the use of utility arch. More amount of root resorption is seen in lateral incisors than in central incisors in both groups

Mechanism of horseradish peroxidase-catalyzed heme oxidation and polymerization (β-hematin formation)

Horseradish peroxidase (HRP) catalyzes the polymerization of free heme (β-hematin formation) through its oxidation. Heme when added to HRP compound II (Fe^IV = O) causes spectral shift from 417 nm (Compound II) to 402 nm (native, Fe ^III) indicating that heme may be oxidized via one-electron transfer. Direct evidence for one-electron oxidation of heme by HRP intermediates is provided by the appearance of an E.s.r signal of a 5,5-dimethyl-1-pyrroline N-oxide (spin trap)-heme radical adduct (a1^H=14.75 G, a2^H=4.0 G) in E.s.r studies. Heme-polymerization by HRP is inhibited by spin trap indicating that one-electron oxidation product of heme ultimately leads to the formation of heme-polymer. HRP, when incubated with diethyl pyrocarbonate (DEPC), a histidine specific reagent, shows concentration dependent loss of heme-polymerization indicating the role of histidine residues in the process. We suggest that HRP catalyzes the formation of heme-polymer through one-electron oxidation of free heme

Clotrimazole inhibits hemoperoxidase of Plasmodium falciparum and induces oxidative stress: proposed antimalarial mechanism of clotrimazole

The mechanism of antimalarial activity of clotrimazole was studied placing emphasis on its role in inhibiting hemoperoxidase for inducing oxidative stress in Plasmodium falciparum. Clotrimazole, in the presence of H₂O₂, causes irreversible inactivation of the enzyme, and the inactivation follows pseudo-first order kinetics, consistent with a mechanism-based (suicide) mode. The pseudo-first order kinetic constants are k_i= 2.85 μm, k_inact = 0.9 min^-1, and t½ = 0.77 min. The one-electron oxidation product of clotrimazole has been identified by EPR spectroscopy as the 5,5′-dimethyl-1-pyrroline N-oxide (DMPO) adduct of the nitrogen-centered radical (aN = 15 G), and as DMPO protects against inactivation, this radical is involved in the inactivation process. Binding studies indicate that the clotrimazole oxidation product interacts at the heme moiety, and the heme-clotrimazole adduct has been dissociated from the inactivated enzyme and identified (m/z 1363) by mass analysis. We found that the inhibition of hemoperoxidase increases the accumulation of H2O2 in P. falciparum and causes oxidative stress. Furthermore, the inhibition of hemoperoxidase correlates well with the inhibition of parasite growth. The results described herein indicate that the antimalarial activity of clotrimazole might be due to the inhibition of hemoperoxidase and subsequent development of oxidative stress in P. falciparum