Engineering the Catalytic Properties of Two-Domain Laccase from Streptomyces griseoflavus Ac-993

Laccases catalyze the oxidation of substrates with the concomitant reduction of oxygen to water. Recently, we found that polar residues located in tunnels leading to Cu2 and Cu3 ions control oxygen entrance (His 165) and proton transport (Arg 240) of two-domain laccase (2D) from Streptomyces griseoflavus (SgfSL). In this work, we have focused on optimizing the substrate-binding pocket (SBP) of SgfSL while simultaneously adjusting the oxygen reduction process. SgfSL variants with three single (Met199Ala, Met199Gly, and Tyr230Ala) and three double amino acid residues substitutions (Met199Gly/His165Ala, His165Ala/Arg240His, Met199Gly/Arg240His) were constructed, purified, and investigated. Combination of substitutions in the SBP and in the tunnel leading to Cu2 ion (Met199Gly/Arg240His) increased SgfSL catalytic activity towards ABTS by 5-fold, and towards 2.6-DMP by 16-fold. The high activity of the Met199Gly/Arg240His variant can be explained by the combined effect of the SBP geometry optimization (Met199Gly) and increased proton flux via the tunnel leading to Cu2 ion (Arg240His). Moreover, the variant with Met199Gly and His165Ala mutations did not significantly increase SgfSL’s activity, but led to a drastic shift in the optimal pH of 2.6-DMP oxidation. These results indicate that His 165 not only regulates oxygen access, but it also participates in proton transport in 2D laccases.

SGT1 SGS-domain mutations impair interactions with the barley MLA6 immune receptor in association with loss of NLR protein

The Mla (Mildew resistance locus a) of barley (Hordeum vulgare L.) is an effective model for cereal immunity against fungal pathogens. Like many resistance proteins, variants of the MLA coiled-coil nucleotide-binding leucine-rich-repeat (CC-NLR) receptor require the HRS complex to function, which includes HSP90 (Heat Shock Protein 90), RAR1 (Required for Mla12 Resistance 1), and SGT1 (Suppressor of G-two allele of Skp1). However, functional analysis of Sgt1 has been particularly difficult as deletions are often lethal. Recently, we identified rar3 (Required for Mla6 resistance 3), an in-frame Sgt1ΔKL308-309 mutation in the SGS domain that alters resistance conferred by MLA, but without lethality. Here we use autoactive MLA6 and heterologous yeast-two-hybrid strains with stably integrated HvRar1 and HvHsp90, to determine that this mutation weakens, but doesn’t entirely disrupt, the interaction between SGT1 and MLA. This causes a concomitant reduction in MLA6 protein accumulation below the apparent threshold required for effective resistance. The ΔKL308-309 deletion had a lesser effect on intramolecular interactions than alanine or arginine substitutions, and MLA variants that display diminished interactions with SGT1 appear to be disproportionately affected by the SGT1ΔKL308-309 mutation. We hypothesize that those dimeric plant CC-NLRs that appear unaffected by Sgt1 silencing are those with the strongest intermolecular interactions with it. Combining our data with recent work in CC-NLRs, we propose a cyclical model of the MLA-HRS resistosome interactions.

Peculiarities of administration of medications to patients with circulatory system diseases within the framework of the federal program of preferential provision of medicines

Objective: to analyse of medicines prescriptions for federal beneficiaries with circulatory system diseases and to identify the main trends in the implementation of the federal program for the provision of necessary medicines in the Samara Region in 2014–2017.Material and methods. The authors reviewed the nomenclature of medicines used to treat circulatory system diseases (CSDs) and distributed to the population of the Samara Region as part of the federal program for the provision of necessary medicines for the period from 2014 to 2017. The methods of comparative, retrospective, logical, graphical and content analysis, the method of data grouping according to Anatomical Therapeutic Chemical classification and methods of descriptive statistics were used.Results. In 2014–2017 the share of financial costs for the purchase of medicines in the total budget of the federal program for the provision of necessary medicines in the Samara Region did not exceed 6%. Still, in physical terms (in terms of the number of packages) the share of medicines of this pharmacotherapeutic group in the federal program for the provision of necessary medicines averaged about 25%. Nomenclature of medicines of the analyzed group prescribed to federal beneficiaries in 2014–2017 ranged from 90 to 107 nomenclature items. The number of international nonproprietary names (INN) decreased from 51 in 2014 to 36 in 2017. During the period under review, there was a decrease in the acquisition cost of medicines for the treatment of circulatory system diseases and the weighted average cost of one package (by 63% and 53%, respectively).Conclusion. Perindopril-containing medicines occupied the maximum consumption volumes in monetary terms. There was a decrease in the average cost of one package in 2017, which may be associated with the replacement of original medicines with generics and a concomitant reduction in the number of INN within the subgroup.

Crystal structures of a dodecameric multicopper oxidase from Marinithermus hydrothermalis

Multicopper oxidases (MCOs) represent a diverse family of enzymes that catalyze the oxidation of either an organic or a metal substrate with concomitant reduction of dioxygen to water. These enzymes contain variable numbers of cupredoxin domains, two, three or six per subunit, and rely on four copper ions, a single type I copper and three additional copper ions organized in a trinuclear cluster (TNC), with one type II and two type III copper ions, to catalyze the reaction. Here, two crystal structures and the enzymatic characterization of Marinithermus hydrothermalis MCO, a two-domain enzyme, are reported. This enzyme decolorizes Congo Red dye at 70°C in the presence of high halide concentrations and may therefore be useful in the detoxification of industrial waste that contains dyes. In two distinct crystal structures, MhMCO forms the trimers seen in other two-domain MCOs, but differs from these enzymes in that four trimers interact to create a dodecamer. This dodecamer of MhMCO forms a closed ball-like structure and has implications for the sequestration of bound divalent metal ions as well as substrate accessibility. In each subunit of the dodecameric structures, a Trp residue, Trp351, located between the type I and TNC sites exists in two distinct conformations, consistent with a potential role in facilitating electron transfer in the enzyme.

Autoantibodies against IL-1-receptor-antagonist in multisystem inflammatory syndrome in children

Multisystem inflammatory syndrome in children (MIS-C or PIMS) is a rare but serious complication after an infection with SARS-CoV-2. A possible involvement of pathogenetically relevant autoantibodies has been discussed. Recently neutralizing autoantibodies against anti-inflammatory receptor antagonists progranulin (PGRN) and IL-1-receptor antagonist (IL-1-Ra) were discovered in adult patients with critical COVID-19.Plasma of an index case with severe PIMS/MIS-C was analyzed for autoantibodies against IL-1-Ra and PGRN. The study was extended by a case series of 12 additional patients. In addition to ELISA for of antibodies, IL-1-Ra plasma levels were determined and IL-1-Ra was analyzed by Western-blot and isoelectric focusing. Functional activity of the autoantibodies was examined in vitro with IL-1ß reporter assays.Antibodies against IL-1-Ra could be detected in 10 of 13 (76.9%) patients with PIMS/MIS-C, but not in controls. In contrast to critical COVID-19 in adults, no IL-1-Ra antibodies of the IgM class were detected in PIMS/MIS-C. IL-1-Ra-antibodies exclusively belonged to IgG1. No antibodies directed against PGRN were detected. Western blots and ELISA showed a concomitant reduction of free IL-1-Ra plasma levels in the presence of IL-1-Ra-antibodies. The antibodies inhibited IL-1-Ra function in IL-1ß reporter cell assays. Notably, an additional, hyperphosphorylated, transiently occurring atypical isoform of IL-1-Ra was observed in all IL-1-Ra autoantibody-positive patients.To conclude, IL-1-Ra autoantibodies were observed in high frequency in children with PIMS/MIS-C. They may represent a diagnostic and pathophysiologically relevant marker for PIMS/MIS-C. Their generation is likely to be triggered by an atypical, hyperphosphorylated isoform of IL-1-Ra.

Serum and Urinary Levels of Nitric Oxide and Uric Acid in Nigerian Children with Asthma

Background: Asthma is associated with increased production of reactive oxygen and nitrogen species and an alteration in the levels of antioxidants activities in the lung and blood. The increased production of the superoxide anion radicals contributes to airway remodelling and disease severity. Physiologically, the effect of increased free radical generation is eliminated by corresponding activities of a network of antioxidants. Presently, there is the dearth of information on the steady-state concentrations of nitric oxide (NO) and uric acid (UA) in children with asthma. The serum and urinary levels of NO and UA in children with asthma were thus determined in this study. Methodology: Fifty children consisting of 25 children with asthma and 25 age-matched apparently healthy children without asthma were enrolled into this study. Serum and urinary levels of NO and UA were determined using standard methods. Results: Serum levels of NO and UA were significantly higher while the urinary levels of NO and UA were significantly lower in children with asthma compared with the controls. There was no significant correlation between the serum ad urinary levels of NO and UA in children with asthma. Also, gender differences were not observed in the serum and urinary levels of NO and UA in children with asthma. Conclusion: Children with asthma have elevated serum levels of NO and UA accompanied with suboptimal urinary excretion. Therefore, children with asthma might benefit from routine renal function assessment owing to damages that can result from systemic accumulation of UA with concomitant reduction in its urinary excretion.

Macroevolutionary Trade-Offs and Trends in Life History Traits of Cephalopods Through a Comparative Phylogenetic Approach

One of the major mechanisms responsible for the animals’ fitness dynamics is fecundity. Fecundity as a trait does not evolve independently, and rather interacts with other traits such as body and egg size. Here, our aim was to correctly infer the macroevolutionary trade-offs between body length, egg length, and potential fecundity, using cephalopods as study model. The correlated evolution among those traits was inferred by comparative phylogenetic methods. Literature data on biological and reproductive traits (body length, egg length, and potential fecundity) was obtained for 90 cephalopod species, and comparative phylogenetic methods based on a previous molecular phylogeny were used to test the correlated evolution hypothesis. Additionally, we estimated the phylogenetic signal and fitted five different evolutionary models to each trait. All traits showed high phylogenetic signal, and the selected model suggested an evolutionary trend toward increasing body length, egg length, and fecundity in relation to the ancestral state. Evidence of correlated evolution between body length and fecundity was observed, although this relationship was not detected between body length and egg length. The robust inverse relationship between fecundity and egg length indicates that cephalopods evolved a directional selection that favored an increase of fecundity and a reduction of egg length in larger species, or an increase in egg length with the concomitant reduction of fecundity and body length in order to benefit offspring survival. The use of phylogenetic comparative methods allowed us to properly detect macroevolutionary trade-offs.

Technological Potential of a Lupin Protein Concentrate as a Nutraceutical Delivery System in Baked Cookies

Previous reports have shown that lupin protein extracts (LE) contain a polypeptide named deflamin with a potent matrix metalloproteinase (MMP)-9 inhibitory activity. The aim of our study was to develop an efficient delivery method for incorporating deflamin into cookies using different alternative flours. A lupin protein concentrate (10 g protein/100 g cookie dough) was added to gluten and gluten-free flours to produce savoury cookies, and its impacts on the physical properties of doughs and cookies, as well on the maintenance of deflamin’s anti-MMP-9 activity, were analysed. The results showed that the biochemical compositions of all cookies with LE presented higher protein and ash contents when compared to the control cookies. Rice, buckwheat and oat doughs were firmer than the others, whereas the addition of LE to kamut and buckwheat flours made cookies significantly firmer than the controls. Additionally, strong interactions between LE and several flours were observed, yielding different impacts on the MMP-9 bioactivity. Overall, the only flour that did not interfere with the desired nutraceutical activities was buckwheat, with 60% MMP-9 inhibitory activity and a concomitant reduction of colon cancer migration; hence, buckwheat flour was revealed to be a good vehicle to deliver bioactive deflamin, showing strong potential as a functional food to be used in preventive or curative approaches to gastrointestinal diseases.

Exogenous Nitric Oxide Reinforces Photosynthetic Efficiency, Osmolyte, Mineral Uptake, Antioxidant, Expression of Stress-Responsive Genes and Ameliorates the Effects of Salinity Stress in Wheat

Salinity stress is one of the major environmental constraints responsible for a reduction in agricultural productivity. This study investigated the effect of exogenously applied nitric oxide (NO) (50 μM and 100 μM) in protecting wheat plants from NaCl-induced oxidative damage by modulating protective mechanisms, including osmolyte accumulation and the antioxidant system. Exogenously sourced NO proved effective in ameliorating the deleterious effects of salinity on the growth parameters studied. NO was beneficial in improving the photosynthetic efficiency, stomatal conductance, and chlorophyll content in normal and NaCl-treated wheat plants. Moreover, NO-treated plants maintained a greater accumulation of proline and soluble sugars, leading to higher relative water content maintenance. Exogenous-sourced NO at both concentrations up-regulated the antioxidant system for averting the NaCl-mediated oxidative damage on membranes. The activity of antioxidant enzymes increased the protection of membrane structural and functional integrity and photosynthetic efficiency. NO application imparted a marked effect on uptake of key mineral elements such as nitrogen (N), potassium (K), and calcium (Ca) with a concomitant reduction in the deleterious ions such as Na+. Greater K and reduced Na uptake in NO-treated plants lead to a considerable decline in the Na/K ratio. Enhancing of salt tolerance by NO was concomitant with an obvious down-regulation in the relative expression of SOS1, NHX1, AQP, and OSM-34, while D2-protein was up-regulated.