Identification of an Amylomaltase from the Halophilic Archaeon Haloquadratum walsbyi by Functional Metagenomics: Structural and Functional Insights

Amylomaltases are prokaryotic 4-α-glucanotransferases of the GH77 family. Thanks to the ability to modify starch, they constitute a group of enzymes of great interest for biotechnological applications. In this work we report the identification, by means of a functional metagenomics screening of the crystallization waters of the saltern of Margherita di Savoia (Italy), of an amylomaltase gene from the halophilic archaeon Haloquadratum walsbyi, and its expression in Escherichia coli cells. Sequence analysis indicated that the gene has specific insertions yet unknown in homologous genes in prokaryotes, and present only in amylomaltase genes identified in the genomes of other H. walsbyi strains. The gene is not part of any operon involved in the metabolism of maltooligosaccharides or glycogen, as it has been found in bacteria, making it impossible currently to assign a precise role to the encoded enzyme. Sequence analysis of the H. walsbyi amylomaltase and 3D modelling showed a common structure with homologous enzymes characterized in mesophilic and thermophilic bacteria. The recombinant H. walsbyi enzyme showed starch transglycosylation activity over a wide range of NaCl concentrations, with maltotriose as the best acceptor substrate compared to other maltooligosaccharides. This is the first study of an amylomaltase from a halophilic microorganism.

Challenges and Opportunities in the Study of Innovation Ecosystems in the COVID-19 Pandemic Context

The process of knowledge construction, widely discussed in the literature, follows a common structure that encompasses transformation of data into information and then into knowledge, which converges social, technological, organizational, and strategic aspects. The advancement of information technologies and growing global research efforts in the health field has dynamically generated large datasets, thus providing potential innovative solutions to health problems, posing important challenges in selection and interpretation of useful information and possibilities. COVID-19 pandemic has intensified this data generation as results of global efforts, and cooperation has promoted a level of scientific production never experienced before concerning the overcoming of the pandemic. In this context, the search for an effective and safe vaccine that can prevent the spread of this virus has become a common goal of societies, governments, institutions, and companies. These collaborative efforts have contributed to speed up the development of these vaccines at an unprecedented pace in history.

Development of Generic Immuno-Magnetic Bead-Based Enzyme-Linked Immunoassay for Ustiloxins in Rice Coupled with Enrichment

Ustiloxins are a group of mycotoxins produced by rice false smut pathogen. Previous studies have shown that the false smut balls contain six types of ustiloxins, and these toxins are toxic to living organisms. Thus, immunoassay for on-site monitoring of ustiloxins in rice is urgently required. The current immunoassays are only for detecting single ustiloxin, and they cannot meet the demand for synchronous and rapid detection of the group toxins. Therefore, this study designed and synthesized a generic antigen with ustiloxin G as material based on the common structure of the mycotoxins. Ustiloxin G was conjugated to two carrier proteins including bovine serum albumin (BSA) and ovalbvmin (OVA) by carbon diimide method. The mice were immunized with ustiloxin-G-BSA to generate the antibody serum, which was further purified to obtain the generic antibody against ustiloxins. The conjugated ustiloxin G-OVA and generic antibodies were used for establishing the enzyme-linked immunosorbent assay (ELISA) for ustiloxin detection and optimizing experiment conditions. The characterization of the antibody showed that the semi-inhibitory concentrations (IC50) of ustiloxin A, B, and G were 0.53, 0.34, and 0.06 µg/mL, respectively, and that their corresponding cross-reactivities were 11.9%, 18.4%, and 100%, respectively. To increase ELISA detection efficiency, generic antibody was combined with magnetic beads to obtain sensitive and class-specific immune-magnetic beads. Based on these immuno-magnetic beads, a high-efficiency enzyme-linked immunoassay method was developed for ustiloxin detection, whose sensitivity to ustiloxin A, B, and G was improved to 0.15 µg/mL, 0.14 µg/mL, and 0.04 µg/mL, respectively. The method accuracy was evaluated by spiking ustiloxin G as standard, and the spiked samples were tested by the immune-magnetic bead-based ELISA. The result showed the ustiloxin G recoveries ranged from 101.9% to 116.4% and were accepted by a standard HPLC method, indicating that our developed method would be promising for on-site monitoring of ustiloxins in rice.

Recurrent high-impact mutations at cognate structural positions in class A G protein-coupled receptors expressed in tumors

G protein-coupled receptors (GPCRs) are the largest family of human proteins. They have a common structure and, signaling through a much smaller set of G proteins, arrestins, and effectors, activate downstream pathways that often modulate hallmark mechanisms of cancer. Because there are many more GPCRs than effectors, mutations in different receptors could perturb signaling similarly so as to favor a tumor. We hypothesized that somatic mutations in tumor samples may not be enriched within a single gene but rather that cognate mutations with similar effects on GPCR function are distributed across many receptors. To test this possibility, we systematically aggregated somatic cancer mutations across class A GPCRs and found a nonrandom distribution of positions with variant amino acid residues. Individual cancer types were enriched for highly impactful, recurrent mutations at selected cognate positions of known functional motifs. We also discovered that no single receptor drives this pattern, but rather multiple receptors contain amino acid substitutions at a few cognate positions. Phenotypic characterization suggests these mutations induce perturbation of G protein activation and/or β-arrestin recruitment. These data suggest that recurrent impactful oncogenic mutations perturb different GPCRs to subvert signaling and promote tumor growth or survival. The possibility that multiple different GPCRs could moonlight as drivers or enablers of a given cancer through mutations located at cognate positions across GPCR paralogs opens a window into cancer mechanisms and potential approaches to therapeutics.

Essential roles of buried phenylalanine in the structural stability of thioredoxin from a psychrophilic Arctic bacterium Sphingomonas sp.

Thioredoxin (Trx), a small redox protein, exhibits thermal stability at high temperatures regardless of its origin, including psychrophiles. Trxs have a common structure consisting of the central β-sheet flanked by an aliphatic cluster on one side and an aromatic cluster on the other side. Although the roles of aromatic amino acids in the folding and stability of proteins have been studied extensively, the contributions of aromatic residues to the stability and function of Trx, particularly Trxs from cold-adapted organisms, have not been fully elucidated. This study examined the roles of aromatic amino acids in the aromatic cluster of a Trx from the psychrophilic Arctic bacterium Sphingomonas sp. PAMC 26621 (SpTrx). The aromatic cluster of SpTrx was comprised of W11, F26, F69, and F80, in which F26 at the β2 terminus was buried inside. The substitution of tyrosine for F26 changed the SpTrx conformation substantially compared to that of F69 and F80. Further biochemical and spectroscopic investigations on F26 showed that the F26Y, F26W, and F26A mutants resulted in structural instability of SpTrx in both urea- and temperature-induced unfolding and lower insulin reduction activities. The Trx reductase (SpTR) showed lower catalytic efficiencies against F26 mutants compared to the wild-type SpTrx. These results suggest that buried F26 is essential for maintaining the active-site conformation of SpTrx as an oxidoreductase and its structural stability for interactions with SpTR at colder temperatures.

Implementation of methods and algorithms for geometric and structural modeling in the problems of parametric design of a ship's hull structure

В работе рассмотрены методология и алгоритмы геометрического и конструктивного моделирования судовых конструкций специализированного программного обеспечения и автоматизированных систем для проектирования конструкции корпуса судна в соответствии с требованиями Правил Российского морского Регистра судоходства и Гармонизированных общих Правил МАКО. Даны понятия геометрического и конструктивного моделирования. Показано место геометрического и конструктивного моделирования в общей структуре системы автоматизированного проектирования судовых конструкций, рассмотрены основы моделирования конструкций в виде плоского конструктивного сечения и трехмерного перекрытия. Описаны возможности специального программного обеспечения – редакторов геометрической и конструктивной модели. Рассмотрена структура специализированного геометрического ядра автоматизированной системы. Приведены примеры использования предлагаемых методов и алгоритмов в разработанном авторами программном обеспечении автоматизированных систем, обоснованы преимущества предлагаемых методов по сравнению с ранее известными решениями. The paper considers the methodology and algorithms for geometric and structural modeling of ship structures, which are used in specialized software of computer-aided systems for the design of the ship's hull structure in accordance with the requirements of the Russian maritime register of Shipping Rules and the IACS Harmonized Common Structure Rules. The concepts of geometric and constructive modeling are given. The place of geometric and structural modeling in the general structure of the computer-aided design of ship structures is shown, the basics of modeling structures in the form of a flat structural section and three-dimensional grillage are considered. The possibilities of special software - editors of geometric and structural models are described. The structure of a specialized geometric kernel of the computer-aided system is considered. Examples of using the proposed methods and algorithms in the developed by authors software of computer-aided systems are given, the advantages of the proposed methods are substantiated in comparison with previously known solutions.

An Exceptionally Stable and Widespread Hydrated Amorphous Calcium Carbonate Precipitated By the Dog Vomit Slime Mold Fuligo Septica (Myxogastria)

Biogenic amorphous calcium carbonate (ACC) is typically metastable and can rapidly transform through aging, dehydration, and/or heating to crystalline calcium carbonate. Gaining insight into its structure and properties is typically hampered by its tendency to crystallize over short time periods once isolated from the host organism, and also by the small quantities that are usually available for study. Here, we describe an exceptionally stable hydrated ACC (HACC) precipitated by the cosmopolitan slime mold, Fuligo septica (L.) F.H. Wigg. (1780). A single slime mold can precipitate up to one gram of HACC over the course of one night. Powder x-ray diffraction (XRD) patterns, transmission electron microscopy (TEM) images, infrared absorption (IR) spectra, and lack of optical birefringence are consistent with an amorphous material. XRD simulations supported by thermogravimetric (TG) and evolved gas analysis (EGA) data suggest an intimate association of organic matter with ~1-nm-sized ACC units that have monohydrocalcite- and calcite-like nano-structural properties. It is postulated that this association imparts the extreme stability of the HACC by preventing loss of H2O and subsequent crystallization. The composition, structure, and thermal behavior of the HACC precipitated by F. septica collected over 8000 km apart, and in markedly different environments, suggests a common structure, as well as similar biochemical and biomineralization mechanisms for the HACC formation.

Mnemonic Aids in Linguodidactic Discourse

The article examines modern English memory aids as part of linguodidactic discourse in teaching English, and the patterns of mnemonic devices represented by sentence, acronym, abbreviation and verse. The empirical material of the research incorporates 54 units. The focus is made on an extensive number of mnemonics compiled as a meaningful text and the patterns of the devices in the form of a sentence, and the mnemonic acronyms homonymous to general vocabulary, i. e. homoacronyms. At that, the mnemonic aids which are not similar to other words, are not so often used as mnemonic devices. The mnemonic devices in the paper have been studied on the basis of the English and Russian sources published in the 21st century.Mnemonic devices have become part of research only in recent years. Previously mnemonics were studied within the framework of pedagogical discourse in teaching Geography, Biology, Astronomy, History, and Music. In the present research the mnemonics related to teaching English were distributed by structure. Afterwards, mnemonic sentences and the mnemonics homonymous to the existing lexemes were identified. The analysis of the structures showed the domination of the mnemonics structured as sentences and homoacronyms. 54 units of mnemonics under study were presented in the form of 22 sentences, 3 verses, 17 acronyms and 12 abbreviations. The most common structure proved to be a sentence, while the least common one was verse. The mnemonics considered contained only 13 units which were not sentences or homoacronyms: 12 abbreviations and 1 acronym of primary nomination. The examination of the structures showed the domination of the mnemonics organized as a sentence or a homoacronym. The research confirms the assumptions made earlier on the frequent use of sentence mnemonics, which, as it turned out, exceed the shares of the other mnemonic patterns. In their turn, homoacronyms made up a larger share compared to acronyms and abbreviations with no reference to general English words or verse. Thus, we can consider these structures (sentences and homoacronyms) within English teaching as part of pedagogical discourse. To sum it up, while compiling mnemonic aids, preference should be given to sentences or homoforms based on the vocabulary, while verses and abbreviations might be used economically. The article has also revealed additional features of mnemonics, in particular applying asyndeton in acronyms and abbreviations, the average number of 3 or 4 components in a mnemonic aid. Studying such structures will contribute to examining shortened forms and functioning of mnemonics in linguodidactic discourse.

Synthesis of N-Glycans and Azasugars to Target Disease

<p>In this thesis two aspects of carbohydrate research will be discussed. First, the total synthesis of N-glycans found on allergens that are known to stimulate an allergic immune response and second, the synthesis of iminosugars in an attempt to extend the scope of the PGF-methodology. Asthma affects 235 million people worldwide, with New Zealand ranking among the highest in the world. Although there is a good understanding of how allergens trigger the immune system on a “macroscopic” level, how an allergen’s molecular structure causes such an allergic response remains unknown. Upon close review of carbohydrates present on the allergens that are known to give an allergic T helper (Th 2) immune response, a common structure has been identified. The structure consists of a complex type N-glycan made up of a pentasaccharide core (Man3(GlcNHAc)2), with additional 1,3-linked α-L-fucose and 1,2-linked D-xylose cappings. As part of a structure relationship study this heptasaccharide and structural derivatives thereof have been synthesised. The synthesis of these N-glycans will allow a more detailed study of the role of these defined structures in triggering an allergic immune response. The second part of this thesis focuses on the protecting group free (PGF) synthesis of iminosugars, which are potent glycosidase inhibitors and are currently used in the treatment of a variety of diseases. Synthetic strategies for the synthesis of iminosugars involve the use of protecting groups, which are necessary to block potential competing reactive centres within a molecule during the multistep synthesis. The disadvantage, however, is that the installation of protecting groups introduces additional steps to the total synthesis, which inevitably leads to lower yields and the generation of waste. Within our group, PGF methodologies have been developed which allow for the synthesis of a variety of iminosugars. The work presented in this thesis extends the scope of this methodology for the synthesis of an important class of iminosugars, the 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidines. For the purpose of introducing an additional hydroxymethyl group, a ketose starting material was required, and therefore an efficient Vasella/reductive amination reaction using ketoses was developed. Additionally, iodocyclisation and carbamate annulation of the intermediate alkenylamines provided successful entry to the 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidines, exemplified by the efficient 6-step synthesis of 2,5-dideoxy-2,5-imino-L-iditol and 2,5-dideoxy-2,5-imino-D-mannitol (DMDP).</p>

Synthesis of N-Glycans and Azasugars to Target Disease

<p>In this thesis two aspects of carbohydrate research will be discussed. First, the total synthesis of N-glycans found on allergens that are known to stimulate an allergic immune response and second, the synthesis of iminosugars in an attempt to extend the scope of the PGF-methodology. Asthma affects 235 million people worldwide, with New Zealand ranking among the highest in the world. Although there is a good understanding of how allergens trigger the immune system on a “macroscopic” level, how an allergen’s molecular structure causes such an allergic response remains unknown. Upon close review of carbohydrates present on the allergens that are known to give an allergic T helper (Th 2) immune response, a common structure has been identified. The structure consists of a complex type N-glycan made up of a pentasaccharide core (Man3(GlcNHAc)2), with additional 1,3-linked α-L-fucose and 1,2-linked D-xylose cappings. As part of a structure relationship study this heptasaccharide and structural derivatives thereof have been synthesised. The synthesis of these N-glycans will allow a more detailed study of the role of these defined structures in triggering an allergic immune response. The second part of this thesis focuses on the protecting group free (PGF) synthesis of iminosugars, which are potent glycosidase inhibitors and are currently used in the treatment of a variety of diseases. Synthetic strategies for the synthesis of iminosugars involve the use of protecting groups, which are necessary to block potential competing reactive centres within a molecule during the multistep synthesis. The disadvantage, however, is that the installation of protecting groups introduces additional steps to the total synthesis, which inevitably leads to lower yields and the generation of waste. Within our group, PGF methodologies have been developed which allow for the synthesis of a variety of iminosugars. The work presented in this thesis extends the scope of this methodology for the synthesis of an important class of iminosugars, the 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidines. For the purpose of introducing an additional hydroxymethyl group, a ketose starting material was required, and therefore an efficient Vasella/reductive amination reaction using ketoses was developed. Additionally, iodocyclisation and carbamate annulation of the intermediate alkenylamines provided successful entry to the 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidines, exemplified by the efficient 6-step synthesis of 2,5-dideoxy-2,5-imino-L-iditol and 2,5-dideoxy-2,5-imino-D-mannitol (DMDP).</p>