Microbial glucoamylases: structural and functional properties and biotechnological uses.

Glucoamylases (GAs) are one of the principal groups of enzymes involved in starch hydrolysis and belong to the glycosylhydrolase family. They are classified as exo-amylases due to their ability to hydrolyze α-1,4 glycosidic bonds from the non-reducing end of starch, maltooligosaccharides, and related substrates, releasing ß-D-glucose. Structurally, GAs possess a characteristic catalytic domain (CD) with an (α/α)6 fold and exhibit five conserved regions within this domain. The CD may or may not be linked to a non-catalytic domain with variable functions depending on its origin. GAs are versatile enzymes with diverse applications in food, biofuel, bioplastic and other chemical industries. Although fungal GAs are commonly employed for these purposes, they have limitations such as their low thermostability and an acidic pH requirement. Alternatively, GAs derived from prokaryotic organisms are a good option to save costs as they exhibit greater thermostability compared to fungal GAs. Moreover, a group of cold-adapted GAs from psychrophilic organisms demonstrates intriguing properties that make them suitable for application in various industries. This review provides a comprehensive overview of the structural and sequential properties as well as biotechnological applications of GAs in different industrial processes.

Metagenomic approach to infer rumen microbiome derived traits of cattle.

Ruminants enable the conversion of indigestible plant material into animal consumables, including dairy products, meat, and valuable fibers. Microbiome research is gaining popularity in livestock species because it aids in the knowledge of illnesses and efficiency processes in animals. In this study, we use WGS metagenomic data to thoroughly characterize the ruminal ecosystem of cows to infer positive and negative livestock traits determined by the microbiome. The rumen of cows from Argentina were described by combining different gene biomarkers, pathways composition and taxonomic information. Taxonomic characterization indicated that the two major phyla were Bacteroidetes and Firmicutes; in third place, Proteobacteria was highly represented followed by Actinobacteria; Prevotella, and Bacteroides were the most abundant genera. Functional profiling of carbohydrate-active enzymes indicated that members of the Glycoside Hydrolase (GH) class accounted for 52.2 to 55.6% of the total CAZymes detected, among them the most abundant were the oligosaccharide degrading enzymes. The diversity of GH families found suggested efficient hydrolysis of complex biomass. Genes of multidrug, macrolides, polymyxins, beta-lactams, rifamycins, tetracyclines, and bacitracin resistance were found below 0.12% of relative abundance. Furthermore, the clustering analysis of genera and genes that correlated to methane emissions or feed efficiency, suggested that the cows analysed could be regarded as low methane emitters and clustered with high feed efficiency reference animals. Finally, the combination of bioinformatic analyses used in this study can be applied to assess cattle traits difficult to measure and guide enhanced nutrition and breeding methods.

Two phosphoenolpyruvate carboxykinases with differing biochemical properties in Chlamydomonas reinhardtii.

Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the reversible reaction of decarboxylation and phosphorylation of oxaloacetate (OAA) to generate phosphoenolpyruvate (PEP) and CO2 playing mainly a gluconeogenic role in green algae. We found two PEPCK isoforms in Chlamydomonas reinhardtii and we cloned, purified and characterised both enzymes. ChlrePEPCK1 is more active as decarboxylase than ChlrePEPCK2. ChlrePEPCK1 is hexameric and its activity is affected by citrate, phenylalanine and malate, while ChlrePEPCK2 is monomeric and it is regulated by citrate, phenylalanine and glutamine. We postulate that the two PEPCK isoforms found originate from alternative splicing of the gene or regulated proteolysis of the enzyme. The presence of these two isoforms would be part of a mechanism to finely regulate the biological activity of PEPCKs.

Junctional instability in neuroepithelium and network hyperexcitability in a focal cortical dysplasia human model.

Focal cortical dysplasia is a highly epileptogenic cortical malformation with few treatment options. Here, we generated human cortical organoids from patients with focal cortical dysplasia type II. Using this human model, we mimicked some focal cortical dysplasia hallmarks, such as impaired cell proliferation, the presence of dysmorphic neurons and balloon cells, and neuronal network hyperexcitability. Furthermore, we observed alterations in the adherens junctions zonula occludens-1 and partitioning defective 3, reduced polarization of the actin cytoskeleton, and fewer synaptic puncta. Focal cortical dysplasia cortical organoids showed downregulation of the small GTPase RHOA, a finding that was confirmed in brain tissue resected from these patients. Functionally, both spontaneous and optogenetically-evoked electrical activity revealed hyperexcitability and enhanced network connectivity in focal cortical dysplasia organoids. Taken together, our findings suggest a ventricular zone instability in tissue cohesion of neuroepithelial cells, leading to a maturational arrest of progenitors or newborn neurons, which may predispose to cellular and functional immaturity and compromise the formation of neural networks in focal cortical dysplasia.

Multi-omics in mesial temporal lobe epilepsy with hippocampal sclerosis: Clues into the underlying mechanisms leading to disease.

Mesial temporal lobe epilepsy (MTLE) is one of the most common types of focal epilepsy in the adult population. MTLE is frequently associated with a specific histopathological lesion in the medial temporal structures, namely hippocampal sclerosis (HS). A significant proportion of patients with MTLE+HS have severe epilepsy, which is often resistant to clinical treatment. For these patients, surgical resection of the epileptogenic lesion can be performed. Our understanding of the underlying mechanisms leading to MTLE+HS has improved significantly over the past few decades. In this review, we aim to present and discuss the most recent findings regarding the genetic determinants of MTLE+HS. Furthermore, we will address studies about transcriptomics, proteomics, metabolomics, and epigenomic signatures of the tissue that is surgically removed from patients with refractory MTLE+HS and animal models of the disorder. We expect to provide an overview and a critical discussion of the findings, limitations, new approaches, and future directions for multi-omics studies in MTLE+HS.

Identification and characterization of ChlreSEX4, a novel glucan phosphatase from Chlamydomonas reinhardtii green alga.

Chlamydomonas reinhardtii is the best known unicellular green alga model which has long been used to investigate all kinds of cellular processes, including starch metabolism. Here we identified and characterized a novel enzyme, ChlreSEX4, orthologous to glucan phosphatase SEX4 from Arabidopsis thaliana, that is capable of binding and dephosphorylating amylopectin in vitro. We also reported that cysteine 224 and tryptophan 305 residues are critical for enzyme catalysis and substrate binding. Furthermore, we verified that ChlreSEX4 gene is expressed in vivo and that glucan phosphatase activity is measurable in Chlamydomonas protein extracts. In view of the results presented, we suggest ChlreSEX4 as a functional phosphoglucan phosphatase from C. reinhardtii. Our data obtained so far contribute to understanding the phosphoglucan phosphatases evolutionary process in the green lineage and their role in starch reversible phosphorylation. In addition, this allows to position Chlamydomonas as a potential tool to obtain starches with different degrees of phosphorylation for industrial or biotechnological purposes.

Administración crónica de los inhibidores de la bomba de protones: un problema de salud pública / Chronic administration of proton pump inhibitors: A problem of public health

Resumen En la actualidad se ha generalizado la administración de los inhibidores de la bomba de protones casi de manera universal, pero con especial insistencia en los pacientes hospitalizados. Su administración se ha considerado inocua y con efectos protectores; sin embargo, la administración indiscriminada de este grupo de fármacos tiene serias consecuencias para la salud.

Abstract Currently the use of proton pump inhibitors has become widespread almost universally but with special emphasis on hospitalized patients. Its use has been considered innocuous and with protective effects; however, the indiscriminate administration of this group of drugs has health consequences.

Identification and analysis of OsttaDSP, a phosphoglucan phosphatase from Ostreococcus tauri.

Ostreococcus tauri, the smallest free-living (non-symbiotic) eukaryote yet described, is a unicellular green alga of the Prasinophyceae family. It has a very simple cellular organization and presents a unique starch granule and chloroplast. However, its starch metabolism exhibits a complexity comparable to higher plants, with multiple enzyme forms for each metabolic reaction. Glucan phosphatases, a family of enzymes functionally conserved in animals and plants, are essential for normal starch or glycogen degradation in plants and mammals, respectively. Despite the importance of O. tauri microalgae in evolution, there is no information available concerning the enzymes involved in reversible phosphorylation of starch. Here, we report the molecular cloning and heterologous expression of the gene coding for a dual specific phosphatase from O. tauri (OsttaDSP), homologous to Arabidopsis thaliana LSF2. The recombinant enzyme was purified to electrophoretic homogeneity to characterize its oligomeric and kinetic properties accurately. OsttaDSP is a homodimer of 54.5 kDa that binds and dephosphorylates amylopectin. Also, we also determined that residue C162 is involved in catalysis and possibly also in structural stability of the enzyme. Our results could contribute to better understand the role of glucan phosphatases in the metabolism of starch in green algae.

Development of fast and simple chromogenic methods for glucan phosphatases in-gel activity assays.

Glucan phosphatases are essential for normal starch degradation in plants and glycogen metabolism in mammals. Here we develop two chromogenic methods for the detection of glucan phosphatase activity in situ after non denaturing poliacrylamide gel electrophoresis; one method uses pNPP and the second one applies BCIP/NBT. The assays are sensitive, fast, simple, reliable and cost-effective preventing the use of radioactive or fluorogenic compounds. Taking advantage of an efficient separation method combined with the reported assays it is possible to obtain information about oligomeric state of the active enzymes as well as to simultaneously detect glucan substrate binding and phosphatase activity.

Beyond the 'east-west' dichotomy: Global variation in cultural models of selfhood.

Markus and Kitayama's (1991) theory of independent and interdependent self-construals had a major influence on social, personality, and developmental psychology by highlighting the role of culture in psychological processes. However, research has relied excessively on contrasts between North American and East Asian samples, and commonly used self-report measures of independence and interdependence frequently fail to show predicted cultural differences. We revisited the conceptualization and measurement of independent and interdependent self-construals in 2 large-scale multinational surveys, using improved methods for cross-cultural research. We developed (Study 1: N = 2924 students in 16 nations) and validated across cultures (Study 2: N = 7279 adults from 55 cultural groups in 33 nations) a new 7-dimensional model of self-reported ways of being independent or interdependent. Patterns of global variation support some of Markus and Kitayama's predictions, but a simple contrast between independence and interdependence does not adequately capture the diverse models of selfhood that prevail in different world regions. Cultural groups emphasize different ways of being both independent and interdependent, depending on individualism-collectivism, national socioeconomic development, and religious heritage. Our 7-dimensional model will allow future researchers to test more accurately the implications of cultural models of selfhood for psychological processes in diverse ecocultural contexts. (PsycINFO Database Record

Individual and culture-level components of survey response styles: A multi-level analysis using cultural models of selfhood.

Variations in acquiescence and extremity pose substantial threats to the validity of cross-cultural research that relies on survey methods. Individual and cultural correlates of response styles when using 2 contrasting types of response mode were investigated, drawing on data from 55 cultural groups across 33 nations. Using 7 dimensions of self-other relatedness that have often been confounded within the broader distinction between independence and interdependence, our analysis yields more specific understandings of both individual- and culture-level variations in response style. When using a Likert-scale response format, acquiescence is strongest among individuals seeing themselves as similar to others, and where cultural models of selfhood favour harmony, similarity with others and receptiveness to influence. However, when using Schwartz's (2007) portrait-comparison response procedure, acquiescence is strongest among individuals seeing themselves as self-reliant but also connected to others, and where cultural models of selfhood favour self-reliance and self-consistency. Extreme responding varies less between the two types of response modes, and is most prevalent among individuals seeing themselves as self-reliant, and in cultures favouring self-reliance. As both types of response mode elicit distinctive styles of response, it remains important to estimate and control for style effects to ensure valid comparisons.

Influência da modalidade de reconto na avaliação do desempenho de escolares em compreensão leitora / Influence of retelling modality on reading comprehension assessment

Este estudo buscou estudar a influência da modalidade de reconto sobre a avaliação da compreensão leitora. Avaliaram-se 34 escolares do 2º ao 5º ano do Ensino Fundamental, sem queixas de leitura, agrupados em: 1) Grupo Reconto Oral: 19 crianças que recontaram oralmente texto narrativo e expositivo lidos; 2) Grupo Reconto Escrito: 15 que o fizeram por escrito. A análise dos recontos quantificou proposições de ideia central, detalhe, inferências e número de enlaces. Estabeleceu-se, também, o padrão de compreensão (3-0, do melhor para o pior desempenho). Resultados evidenciaram semelhanças entre as médias de desempenho dos grupos nas variáveis do reconto de texto narrativo. Para o expositivo, houve diferença para enlaces de primeiro nível (t = -2,114, p = 0,042), com maior média do Grupo reconto escrito. Concluiu-se, portanto, que a modalidade do reconto não influenciou a avaliação da compreensão de textos narrativos, mas o reconto escrito de textos expositivos favoreceu o aparecimento de enlaces entre ideias centrais.

The aim of the present study was to investigate the influence of the retelling strategy modality on the assessment of reading comprehension. Thirty-four 2nd-5th grade students (elementary school) without reading difficulties were evaluated and grouped as follows: Group 1. Oral retelling: students orally reconstructed narrative and expository texts they read (19 students); Group 2. Writing retelling: students retold these same stories in writing (15 students). The analysis conducted assessed retelling in terms of: proposition of central idea, details, inferences, and number of links. A performance reference standard (3-0) was also established from the best to the worst performance. The results obtained showed similarity between the average performance of the variables of the oral narrative text retelling groups. As for the expository texts, there were differences in the first level of links (t = -2.114, p = 0.042), and the highest average performance was found for the writing retelling group. It can be concluded that the retelling strategy modality did not influence the evaluation of the understanding of narrative texts, but the written retelling of expository texts influenced the connection of central ideas.

Influência da modalidade de reconto na avaliação do desempenho de escolares em compreensão leitora / Influence of retelling modality on reading comprehension assessment

Este estudo buscou estudar a influência da modalidade de reconto sobre a avaliação da compreensão leitora. Avaliaram-se 34 escolares do 2º ao 5º ano do Ensino Fundamental, sem queixas de leitura, agrupados em: 1) Grupo Reconto Oral: 19 crianças que recontaram oralmente texto narrativo e expositivo lidos; 2) Grupo Reconto Escrito: 15 que o fizeram por escrito. A análise dos recontos quantificou proposições de ideia central, detalhe, inferências e número de enlaces. Estabeleceu-se, também, o padrão de compreensão (3-0, do melhor para o pior desempenho). Resultados evidenciaram semelhanças entre as médias de desempenho dos grupos nas variáveis do reconto de texto narrativo. Para o expositivo, houve diferença para enlaces de primeiro nível (t = -2,114, p = 0,042), com maior média do Grupo reconto escrito. Concluiu-se, portanto, que a modalidade do reconto não influenciou a avaliação da compreensão de textos narrativos, mas o reconto escrito de textos expositivos favoreceu o aparecimento de enlaces entre ideias centrais.(AU)

The aim of the present study was to investigate the influence of the retelling strategy modality on the assessment of reading comprehension. Thirty-four 2nd-5th grade students (elementary school) without reading difficulties were evaluated and grouped as follows: Group 1. Oral retelling: students orally reconstructed narrative and expository texts they read (19 students); Group 2. Writing retelling: students retold these same stories in writing (15 students). The analysis conducted assessed retelling in terms of: proposition of central idea, details, inferences, and number of links. A performance reference standard (3-0) was also established from the best to the worst performance. The results obtained showed similarity between the average performance of the variables of the oral narrative text retelling groups. As for the expository texts, there were differences in the first level of links (t = -2.114, p = 0.042), and the highest average performance was found for the writing retelling group. It can be concluded that the retelling strategy modality did not influence the evaluation of the understanding of narrative texts, but the written retelling of expository texts influenced the connection of central ideas.(AU)

Cultural bases for self-evaluation: seeing oneself positively in different cultural contexts.

Several theories propose that self-esteem, or positive self-regard, results from fulfilling the value priorities of one's surrounding culture. Yet, surprisingly little evidence exists for this assertion, and theories differ about whether individuals must personally endorse the value priorities involved. We compared the influence of four bases for self-evaluation (controlling one's life, doing one's duty, benefitting others, achieving social status) among 4,852 adolescents across 20 cultural samples, using an implicit, within-person measurement technique to avoid cultural response biases. Cross-sectional and longitudinal analyses showed that participants generally derived feelings of self-esteem from all four bases, but especially from those that were most consistent with the value priorities of others in their cultural context. Multilevel analyses confirmed that the bases of positive self-regard are sustained collectively: They are predictably moderated by culturally normative values but show little systematic variation with personally endorsed values.

Improving the glycosyltransferase activity of Agrobacterium tumefaciens glycogen synthase by fusion of N-terminal starch binding domains (SBDs).

Glycogen and starch, the major storage carbohydrate in most living organisms, result mainly from the action of starch or glycogen synthases (SS or GS, respectively, EC 2.4.1.21). SSIII from Arabidopsis thaliana is an SS isoform with a particular modular organization: the C-terminal highly conserved glycosyltransferase domain is preceded by a unique specific region (SSIII-SD) which contains three in tandem starch binding domains (SBDs, named D1, D2 and D3) characteristic of polysaccharide degrading enzymes. N-terminal SBDs have a probed regulatory role in SSIII activity, showing starch binding ability and modulating the catalytic properties of the enzyme. On the other hand, GS from Agrobacterium tumefaciens has a simple primary structure organization, characterized only by the highly conserved glycosyltransferase domain and lacking SBDs. To further investigate the functional role of A. thaliana SSIII-SD, three chimeric proteins were constructed combining the SBDs from A. thaliana with the GS from A. tumefaciens. Recombinant proteins were expressed in and purified to homogeneity from Escherichia coli cells in order to be kinetically characterized. Furthermore, we tested the ability to restore in vivo glycogen biosynthesis in transformed E. coli glgA(-) cells, deficient in GS. Results show that the D3-GS chimeric enzyme showed increased capacity of glycogen synthesis in vivo with minor changes in its kinetics parameters compared to GS.

Regulation of Bacillus subtilis DesK thermosensor by lipids.

Temperature sensing is essential for the survival of living cells. The membrane-bound thermosensor DesK from Bacillus subtilis is a key representative of histidine kinases receptors able to remodel membrane lipid composition when the temperature drops below ~30°C. Although the receptor is well studied, a central issue remains: how does the compositional and functional diversity of the surrounding membrane modulate receptor function? Reconstituting full-length DesK into proteoliposomes of well-defined and controlled lipid composition represents a minimal synthetic approach to systematically address this question. Thus DesK has been reconstituted in a variety of phospholipid bilayers and its temperature-regulated autokinase activity determined as function of fatty acyl chain length, lipid head-group structure and phase preference. We show that the head group structure of lipids (both in vitro and in vivo) has little effect on DesK thermosensing, whereas properties determined by the acyl chain of lipids, such as membrane thickness and phase separation into coexisting lipid domains, exert a profound regulatory effect on kinase domain activation at low temperatures. These experiments suggest that the non-polar domain of glycerolipids is essential to regulate the allosteric structural transitions of DesK, by activating the autophosphorylation of the intracellular kinase domain in response to a decrease in temperature.

Polysaccharide-synthesizing glycosyltransferases and carbohydrate binding modules: the case of starch synthase III.

Glycosyltransferases (GTs) are a ubiquitous group of enzymes that catalyze the transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds. Nucleotide-sugars, lipid phosphate sugars and phosphate sugars can act as activated donor molecules while acceptor substrates involve carbohydrates, proteins, lipids, DNA and also, numerous small molecules (i. e. antibiotics, flavonols, steroids). GTs enzyme families are very ancient. They are founded in all the three domains of life and display three different folds (named GT-A, GTB and GT-C) which are a variant of a common α/ß scaffold. In addition, several GTs contain an associated non-catalytic carbohydrate binding module (CBM) that could be critical for enzyme activity. This work reviews the current knowledge on the GTs structures and functions and highlights those enzymes that contain CBMs, particularly starch binding domains (SBDs). In addition, we also focus on A. thaliana starch synthase III enzyme, from the GT-5 family. This protein has a GT-B fold, and contains in its N-terminal region three in tandem SBDs, which are essential for the regulation of enzyme activity.

A mitochondrial alkaline/neutral invertase isoform (A/N-InvC) functions in developmental energy-demanding processes in Arabidopsis.

Recent findings demonstrate that alkaline/neutral invertases (A/N-Invs), enzymes that catalyze the breakdown of sucrose into glucose and fructose, are essential proteins in plant life. The fact that different isoforms are present in multiple locations makes them candidates for the coordination of metabolic processes. In the present study, we functionally characterized the encoding gene of a novel A/N-Inv (named A/N-InvC) from Arabidopsis, which localizes in mitochondria. A/N-InvC is expressed in roots, in aerial parts (shoots and leaves) and flowers. A detailed phenotypic analysis of knockout mutant plants (invc) reveals an impaired growth phenotype. Shoot growth was severely reduced, but root development was not affected as reported for A/N-InvA mutant (inva) plants. Remarkably, germination and flowering, two energy demanding processes, were the most affected stages. The effect of exogenous growth regulators led us to suggest that A/N-InvC may be modulating hormone balance in relation to the radicle emergence. We also show that oxygen consumption is reduced in inva and invc in comparison with wild-type plants, indicating that both organelle isoenzymes may play a fundamental role in mitochondrion functionality. Taken together, our results emphasize the involvement of mitochondrial A/N-Invs in developmental processes and uncover the possibility of playing different roles for the two isoforms located in the organelle.

Immune-related gene expression in nurse honey bees (Apis mellifera) exposed to synthetic acaricides.

The mite Varroa destructor is an ectoparasite affecting honey bees worldwide. Synthetic acaricides have been among the principal tools available to beekeepers for its control, although several studies have shown its negative effects on honey bee physiology. Recent research suggests that those molecules strongly impact on immune signaling cascades and cellular immunity. In the present work, LC(50) in six-day-old bees were determined for the following acaricides: tau-fluvalinate, flumethrin, amitraz and coumaphos. According to this obtained value, a group of individuals was treated with each acaricide and then processed for qPCR analysis. Transcript levels for genes encoding antimicrobial peptides and immune-related proteins were assessed. Flumethrin increased the expression of hymenoptaecin when comparing treated and control bees. Significant differences were recorded between coumaphos and flumethrin treatments, while the first one reduced the expression of hymenoptaecin and abaecin, the last one up-regulated their expressions. No significant statistically changes were recorded in the expression levels of vitellogenin, lysozyme or glucose dehydrogenase among bees treated with acaricides and control bees. This work constitutes the first report, under laboratory conditions, about induction of immune related genes in response to synthetic miticides.

Culture and the distinctiveness motive: constructing identity in individualistic and collectivistic contexts.

The motive to attain a distinctive identity is sometimes thought to be stronger in, or even specific to, those socialized into individualistic cultures. Using data from 4,751 participants in 21 cultural groups (18 nations and 3 regions), we tested this prediction against our alternative view that culture would moderate the ways in which people achieve feelings of distinctiveness, rather than influence the strength of their motivation to do so. We measured the distinctiveness motive using an indirect technique to avoid cultural response biases. Analyses showed that the distinctiveness motive was not weaker-and, if anything, was stronger-in more collectivistic nations. However, individualism-collectivism was found to moderate the ways in which feelings of distinctiveness were constructed: Distinctiveness was associated more closely with difference and separateness in more individualistic cultures and was associated more closely with social position in more collectivistic cultures. Multilevel analysis confirmed that it is the prevailing beliefs and values in an individual's context, rather than the individual's own beliefs and values, that account for these differences.