Developmental arrest in Drosophila melanogaster caused by mitochondrial DNA replication defects cannot be rescued by the alternative oxidase

The xenotopic expression of the alternative oxidase AOX from the tunicate Ciona intestinalis in diverse models of human disease partially alleviates the phenotypic effects of mitochondrial respiratory chain defects. AOX is a non-proton pumping, mitochondrial inner membrane-bound, single-subunit enzyme that can bypass electron transport through the cytochrome segment, providing an additional site for ubiquinone reoxidation and oxygen reduction upon respiratory chain overload. We set out to investigate whether AOX expression in Drosophila could counteract the effects of mitochondrial DNA (mtDNA) replication defects caused by disturbances in the mtDNA helicase or DNA polymerase gamma. We observed that the developmental arrest imposed by either the expression of mutant forms of these enzymes or their knockdown was not rescued by AOX. Considering also the inability of AOX to ameliorate the phenotype of tko(25t), a fly mutant with mitochondrial translation deficiency, we infer that this alternative enzyme may not be applicable to cases of mitochondrial gene expression defects. Finding the limitations of AOX applicability will help establish the parameters for the future putative use of this enzyme in gene therapies for human mitochondrial diseases.Peer reviewe

Expression of Ciona intestinalis AOX causes male reproductive defects in Drosophila melanogaster

Background: Mitochondrial alternative respiratory-chain enzymes are phylogenetically widespread, and buffer stresses affecting oxidative phosphorylation in species that possess them. However, they have been lost in the evolutionary lineages leading to vertebrates and arthropods, raising the question as to what survival or reproductive disadvantages they confer. Recent interest in using them in therapy lends a biomedical dimension to this question. Methods: Here, we examined the impact of the expression of Ciona intestinalis alternative oxidase, AOX, on the reproductive success of Drosophila melanogaster males. Sperm-competition assays were performed between flies carrying three copies of a ubiquitously expressed AOX construct, driven by the a-tubulin promoter, and wild-type males of the same genetic background. Results: In sperm-competition assays, AOX conferred a substantial disadvantage, associated with decreased production of mature sperm. Sperm differentiation appeared to proceed until the last stages, but was spatially deranged, with spermatozoids retained in the testis instead of being released to the seminal vesicle. High AOX expression was detected in the outermost cell-layer of the testis sheath, which we hypothesize may disrupt a signal required for sperm maturation. Conclusions: AOX expression in Drosophila thus has effects that are deleterious to male reproductive function. Our results imply that AOX therapy must be developed with caution.Peer reviewe

Expression of Ciona intestinalis AOX causes male reproductive defects in Drosophila melanogaster

Background: Mitochondrial alternative respiratory-chain enzymes are phylogenetically widespread, and buffer stresses affecting oxidative phosphorylation in species that possess them. However, they have been lost in the evolutionary lineages leading to vertebrates and arthropods, raising the question as to what survival or reproductive disadvantages they confer. Recent interest in using them in therapy lends a biomedical dimension to this question. Methods: Here, we examined the impact of the expression of Ciona intestinalis alternative oxidase, AOX, on the reproductive success of Drosophila melanogaster males. Sperm-competition assays were performed between flies carrying three copies of a ubiquitously expressed AOX construct, driven by the a-tubulin promoter, and wild-type males of the same genetic background. Results: In sperm-competition assays, AOX conferred a substantial disadvantage, associated with decreased production of mature sperm. Sperm differentiation appeared to proceed until the last stages, but was spatially deranged, with spermatozoids retained in the testis instead of being released to the seminal vesicle. High AOX expression was detected in the outermost cell-layer of the testis sheath, which we hypothesize may disrupt a signal required for sperm maturation. Conclusions: AOX expression in Drosophila thus has effects that are deleterious to male reproductive function. Our results imply that AOX therapy must be developed with caution.Peer reviewe

Membrane Protein Production and Purification from Escherichia coli and Sf9 Insect Cells

A major obstacle to studying membrane proteins by biophysical techniques is the difficulty in producing sufficient amounts of materials for functional and structural studies. To overexpress the target membrane protein heterologously, especially an eukaryotic protein, a key step is to find the optimal host expression system and perform subsequent expression optimization. In this chapter, we describe protocols for screening membrane protein production using bacterial and insect cells, solubilization screening, large-scale production, and commonly used affinity chromatography purification methods. We discuss general optimization conditions, such as promoters and tags, and describe current techniques that can be used in any laboratory without specialized expensive equipment. Especially for insect cells, GFP fusions are particularly useful for localization and in-gel fluorescence detection of the proteins on SDS-PAGE. We give detailed protocols that can be used to screen the best expression and purification conditions for membrane protein study.Peer reviewe

VRK1 (Y213H) homozygous mutant impairs Cajal bodies in a hereditary case of distal motor neuropathy.

Background: Distal motor neuropathies with a genetic origin have a heterogeneous clinical presentation with overlapping features affecting distal nerves and including spinal muscular atrophies and amyotrophic lateral sclerosis. This indicates that their genetic background is heterogeneous. Patient and methods: In this work, we have identified and characterized the genetic and molecular base of a patient with a distal sensorimotor neuropathy of unknown origin. For this study, we performed whole-exome sequencing, molecular modelling, cloning and expression of mutant gene, and biochemical and cell biology analysis of the mutant protein. Results: A novel homozygous recessive mutation in the human VRK1 gene, coding for a chromatin kinase, causing a substitution (c.637T > C; p.Tyr213His) in exon 8, was detected in a patient presenting since childhood a progressive distal sensorimotor neuropathy and spinal muscular atrophy syndrome, with normal intellectual development. Molecular modelling predicted this mutant VRK1 has altered the kinase activation loop by disrupting its interaction with the C-terminal regulatory region. The p.Y213H mutant protein has a reduced kinase activity with different substrates, including histones H3 and H2AX, proteins involved in DNA damage responses, such as p53 and 53BP1, and coilin, the scaffold for Cajal bodies. The mutant VRK1(Y213H) protein is unable to rescue the formation of Cajal bodies assembled on coilin, in the absence of wild-type VRK1. Conclusion: The VRK1(Y213H) mutant protein alters the activation loop, impairs the kinase activity of VRK1 causing a functional insufficiency that impairs the formation of Cajal bodies assembled on coilin, a protein that regulates SMN1 and Cajal body formation.post-print2120 K

Flow Cytometry of Microencapsulated Colonies for Genetics Analysis of Filamentous Fungi

The analysis of filamentous fungi by flow cytometry has been impossible to date due to their filamentous nature and size. In this work, we have developed a method that combines single-spore microencapsulation and large-particle flow cytometry as a powerful alternative for the genetic analysis of filamentous fungi. Individual spores were embedded in monodisperse alginate microparticles and incubated in the appropriate conditions. Growth could be monitored by light or fluorescent microscopy and Complex Object Parametric Analyzer and Sorter large-particle flow cytometry. Microencapsulated Trichoderma and Aspergillus spores could germinate and grow inside the alginate capsules. Growth tests revealed that auxotrophic mutants required the appropriate nutrients and that pyrithiamine and glufosinate halted fungal growth of sensitive but not resistant strains. We used an Aspergillus nidulans, thermosensitive mutant in the cell-cycle regulator gene nimXCDK1 as proof-of-concept to the detection and identification of genetic phenotypes. Sorting of the microparticles containing the clonal fungal mycelia proved the power of this method to perform positive and/or negative selection during genetic screenings

Brazilian Version of the Foot Health Status Questionnaire (FHSQ-Br): Cross-Cultural Adaptation and Evaluation of Measurement Properties

OBJECTIVE: To conduct a cross-cultural adaptation of the Foot Health Status Questionnaire into Brazilian-Portuguese and to assess its measurement properties. INTRODUCTION: This instrument is an outcome measure with 10 domains with scores ranging from 0-100, worst to best, respectively. The translated instrument will improve the examinations and foot care of rheumatoid arthritis patients. METHODS: The questions were translated, back-translated, evaluated by a multidisciplinary committee and pre-tested (n = 40 rheumatoid arthritis subjects). The new version was submitted to a field test (n = 65) to evaluate measurement properties such as test-retest reliability, internal consistency and construct validity. The Health Assessment Questionnaire, Numeric Rating Scale for foot pain and Sharp/van der Heijde scores for foot X-rays were used to test the construct validity. RESULTS: The cross-cultural adaptation was completed with minor wording adaptations from the original instrument. The evaluation of measurement properties showed high reliability with low variation coefficients between interviews. The a-Cronbach coefficients varied from 0.468 to 0.855, while correlation to the Health Assessment Questionnaire and Numeric Rating Scale was statistically significant for five out of eight domains. DISCUSSION: Intra- and inter-observer correlations showed high reliability. Internal consistency coefficients were high for all domains, revealing higher values for less subjective domains. As for construct validity, each domain revealed correlations with a specific group of parameters according to what the domains intended to measure. CONCLUSION: The FHSQ was cross-culturally adapted, generating a reliable, consistent, and valid instrument that is useful for evaluating foot health in patients with rheumatoid arthritis

HodgeRank as a new tool to explore the structure of a social representation

Social representation theory is a branch of social psychology that aims to identify the framework of concepts, ideas, opinions, beliefs, or feelings shared by the individuals within a social group, regarding a social object. Two main problems arise in this theory. The first concerns the identification of the content of the representation, which is the set of cognitive elements shared by the group; the second concerns its structure, which is the way these elements are organized and related among themselves. It is desirable that the methods to address these problems be simple, in regards to the feasibility of the data collection, and reliable, in the sense that they should provide a clear picture of the content and the structure of the representation. No single method proposed in the literature until now fully satisfies these features at the same time. Here we propose the use of HodgeRank, a global ranking method based on the Hodge combinatorial theory, as a new tool to explore the structure of a social representation. In this proposal, the input data is the same as those required for the hierarchical word associations, which is the main method in the field of social representations. However, the HodgeRank provides richer results when compared to the usual approach to analysing this kind of data, based on the Vergés’ double-entry table. The main outcome of the HodgeRank is a graph, analogous to an electric circuit, from which some structural elements of the representation can already be identified. Moreover, the HodgeRank technique identifies the sources of inconsistencies between the global ranking and the aggregated answers within the social group. We interpret such inconsistencies in terms of the stability of the representation and use them to raise conjectures about the potential dynamics of the representation. We illustrate the application of this method in the study of a social representation of COVID-19 within a group of students and also within a group of faculty members from higher education institutions in Brazil

Intraclonal Genome Stability of the Metallo-beta-lactamase SPM-1-producing Pseudomonas aeruginosa ST277, an Endemic Clone Disseminated in Brazilian Hospitals

Carbapenems represent the mainstay therapy for the treatment of serious P aeruginosa infections. However, the emergence of carbapenem resistance has jeopardized the clinical use of this important class of compounds. The production of SPM-1 metallo-beta-lactamase has been the most common mechanism of carbapenem resistance identified in P. aeruginosa isolated from Brazilian medical centers. Interestingly, a single SPM-1-producing P. aeruginosa clone belonging to the ST277 has been widely spread within the Brazilian territory. In the current study, we performed a next-generation sequencing of six SPM-1-producing P. aeruginosa ST277 isolates. The core genome contains 5899 coding genes relative to the reference strain P. aeruginosa PAO1. A total of 26 genomic islands were detected in these isolates. We identified remarkable elements inside these genomic islands, such as copies of the bla(spM-1) gene conferring resistance to carbapenems and a type I-C CRISPR-Cas system, which is involved in protection of the chromosome against foreign DNA. In addition, we identified single nucleotide polymorphisms causing amino acid changes in antimicrobial resistance and virulence-related genes. Together,these factors could contribute to the marked resistance and persistence of the SPM-1-producing P aeruginosa ST277 clone. A comparison of the SPM-1-producing P. aeruginosa ST277 genomes showed that their core genome has a high level nucleotide similarity and synteny conservation. The variability observed was mainly due to acquisition of genomic islands carrying several antibiotic resistance genes.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Lab Nacl Comp Cient, Lab Bioinformat, Petropolis, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Internal Med, Lab Alerta,Div Infect Dis, Sao Paulo, BrazilLaboratório Alerta, Division of Infectious Diseases, Department of Internal Medicine, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, BrazilCNPq: 305535/2014-5CNPq: 302768/2011-4CNPq: 312864/2015-9FAPERJ: E-26/202.903/2016Web of Scienc

Development of new eco-friendly supports for immobilization of enzymes based on cellulose residues

The pulp and paper industry generates a high volume of solid wastes that are usually burned to obtain energy, directed to landfills, or incinerated. Among the wastes generated in this process is the paper sludge, a residue rich in cellulose with low lignin content making it a useful raw material to produce high-value products such as cheap immobilization supports. In the current work, paper sludge was activated using different functional groups (amino, epoxy, and aldehyde). The xylanase GH10 from Malbranchea pulchella was used as a model enzyme for the immobilization assays. The enzyme was efficiently immobilized through reversible immobilization on aminated support monoaminoethyl-N-ethyl (MANAE) and polyethyleneimine (PEI), achieving yields of more than 90 %. Furthermore, the yield and activity of the biocatalyst immobilized with paper sludge using groups glyoxyl and epoxy (irreversible immobilization) were higher than the enzyme immobilized on agarose supports. The biocatalyst immobilized on paper sludge-epoxy presented the best results, reaching 12.54 U.g-1 of support. Therefore, the use of paper sludge, such as backbone of different immobilization supports, was an efficient method and promising approach for the immobilization of enzymes such as xylanase. More studies are necessary to optimize the displayed potential for future applications as tests in other enzymes of different characteristics and their behavior with bifunctional reagents as the glutaraldehyde. Furthermore, the valorization of these residues in a biorefinery context holds great socio-economical relevance for Portugal and Brazil. Thus, our perspectives are the development of a hybrid biocatalyst using magnetics nanoparticles and paper sludge.info:eu-repo/semantics/publishedVersio