Evidence for inflammasome activation in the brain of mucopolysaccharidosis type II mice.

Hunter syndrome or mucopolysaccharidosis type II (MPS II) is an X-linked recessive disease caused by the deficiency of iduronate 2-sulfatase (IDS), leading to storage of undegraded heparan and dermatan sulfate. Patients with the severe form present neurological abnormalities, but the mechanisms of such alterations are unknown. Here, we hypothesized that the undegraded substances found in this disease could be recognized as damage-associated molecular patterns (DAMPS), leading to activation of the inflammasome. Brains from 2 and 5 months normal and MPS II mice were studied. We observed an increase in cathepsin B activity in the brain tissue and leakage of this enzyme from the lysosome to the cytoplasm in a MPS II neuronal cell line, which is a known activator of the inflammasome. Furthermore, Caspase-1 activity and IL-1-beta levels were elevated at 5 months, confirming that this pathway is indeed altered. Our results suggest that undegraded GAG activate the inflammasome pathway in MPS II and future studies could focus on blocking such pathway to better understand the role of this process to the pathogenesis of MPS II.

Comparison of different DNA preservation solutions for oral cytological samples.

OBJECTIVE: The objective of this study was to compare the DNA preservation capacity of buccal mucosa exfoliated cells when stored in different solutions under varying time and temperature conditions. DESIGN: DNA preservation solutions, including Dimethyl sulphoxide disodium-EDTA-saturated NaCl (DESS), Tris-EDTA-NaCl-Tween20 buffer (TENT), Nucleic Acid Preservation Buffer (NAP), and phosphate-buffered saline (PBS), were prepared. Buccal mucosa cells from a single patient were collected, dispensed into these solutions, and stored at room temperature (RT) and 4 °C for 24 h, 72 h, 30 days, 90 days, and 180 days. DNA was extracted using the salting-out method and the QIAamp DNA Mini Kit. DNA concentration and purity were determined using the QuBit device and NanoDrop, while DNA integrity was assessed using the Agilent 4200 TapeStation system. The ability to amplify the IFNA primer was also evaluated by PCR. RESULTS: The salting-out method yielded better concentration and purity results, with PBS, TENT, and DESS buffers demonstrating superior concentration values when stored at 4 °C, resulting in mean values exceeding 10 ng/µL for up to 30 days. DESS consistently exhibited the best integrity values over time for both temperature conditions. Amplification capacity was enhanced when samples were stored at 4 °C. When stored at RT, PBS achieved 100% amplification within 24 h. NAP yielded the poorest results. CONCLUSION: In the context of long-term preservation, the DESS buffer emerges as the most effective solution, maintaining requisite DNA quality and quantity standards for up to 30 days at RT and up to 3 months at 4 °C.

Novel human recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in a glyco-engineered Escherichia coli strain.

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disease caused by mutations in the gene encoding the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS), resulting in the accumulation of keratan sulfate (KS) and chondroitin-6-sulfate (C6S). Previously, it was reported the production of an active human recombinant GALNS (rGALNS) in E. coli BL21(DE3). However, this recombinant enzyme was not taken up by HEK293 cells or MPS IVA skin fibroblasts. Here, we leveraged a glyco-engineered E. coli strain to produce a recombinant human GALNS bearing the eukaryotic trimannosyl core N-glycan, Man3GlcNAc2 (rGALNSoptGly). The N-glycosylated GALNS was produced at 100 mL and 1.65 L scales, purified and characterized with respect to pH stability, enzyme kinetic parameters, cell uptake, and KS clearance. The results showed that the addition of trimannosyl core N-glycans enhanced both protein stability and substrate affinity. rGALNSoptGly was capture through a mannose receptor-mediated process. This enzyme was delivered to the lysosome, where it reduced KS storage in human MPS IVA fibroblasts. This study demonstrates the potential of a glyco-engineered E. coli for producing a fully functional GALNS enzyme. It may offer an economic approach for the biosynthesis of a therapeutic glycoprotein that could prove useful for MPS IVA treatment. This strategy could be extended to other lysosomal enzymes that rely on the presence of mannose N-glycans for cell uptake.

CNS-wide repopulation by hematopoietic-derived microglia-like cells corrects progranulin deficiency in mice.

Hematopoietic stem cell transplantation can deliver therapeutic proteins to the central nervous system (CNS) through transplant-derived microglia-like cells. However, current conditioning approaches result in low and slow engraftment of transplanted cells in the CNS. Here we optimized a brain conditioning regimen that leads to rapid, robust, and persistent microglia replacement without adverse effects on neurobehavior or hematopoiesis. This regimen combines busulfan myeloablation and six days of Colony-stimulating factor 1 receptor inhibitor PLX3397. Single-cell analyses revealed unappreciated heterogeneity of microglia-like cells with most cells expressing genes characteristic of homeostatic microglia, brain-border-associated macrophages, and unique markers. Cytokine analysis in the CNS showed transient inductions of myeloproliferative and chemoattractant cytokines that help repopulate the microglia niche. Bone marrow transplant of progranulin-deficient mice conditioned with busulfan and PLX3397 restored progranulin in the brain and eyes and normalized brain lipofuscin storage, proteostasis, and lipid metabolism. This study advances our understanding of CNS repopulation by hematopoietic-derived cells and demonstrates its therapeutic potential for treating progranulin-dependent neurodegeneration.

Improved engraftment and therapeutic efficacy by human genome-edited hematopoietic stem cells with Busulfan-based myeloablation.

Autologous hematopoietic stem cell transplantation using genome-edited cells can become a definitive therapy for hematological and non-hematological disorders with neurological involvement. Proof-of-concept studies using human genome-edited hematopoietic stem cells have been hindered by the low efficiency of engraftment of the edited cells in the bone marrow and their modest efficacy in the CNS. To address these challenges, we tested a myeloablative conditioning regimen based on Busulfan in an immunocompromised model of mucopolysaccharidosis type 1. Compared with sub-lethal irradiation, Busulfan conditioning enhanced the engraftment of edited CD34+ cells in the bone marrow, as well the long-term homing and survival of bone-marrow-derived cells in viscera, and in the CNS, resulting in higher transgene expression and biochemical correction in these organs. Edited cell selection using a clinically compatible marker resulted in a population with low engraftment potential. We conclude that conditioning can impact the engraftment of edited hematopoietic stem cells. Furthermore, Busulfan-conditioned recipients have a higher expression of therapeutic proteins in target organs, particularly in the CNS, constituting a better conditioning approach for non-hematological diseases with neurological involvement.

Genome editing in lysosomal disorders.

Lysosomal disorders are a group of heterogenous diseases caused by mutations in genes that encode for lysosomal proteins. With exception of some cases, these disorders still lack both knowledge of disease pathogenesis and specific therapies. In this sense, genome editing arises as a technique that allows both the creation of specific cell lines, animal models and gene therapy protocols for these disorders. Here we explain the main applications of genome editing for lysosomal diseases, with examples based on the literature. The ability to rewrite the genome will be of extreme importance to study and potentially treat these rare disorders.

Characterization of Human Recombinant N-Acetylgalactosamine-6-Sulfate Sulfatase Produced in Pichia pastoris as Potential Enzyme for Mucopolysaccharidosis IVA Treatment.

Mucopolysaccharidosis IVA (MPS IVA or Morquio A syndrome) is a lysosomal storage disease caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to lysosomal storage of keratan sulfate and chondroitin-6-sulfate. Currently, enzyme replacement therapy using an enzyme produced in CHO cells represents the main treatment option for MPS IVA patients. As an alternative, we reported the production of an active GALNS enzyme produced in the yeast Pichia pastoris (prGALNS), which showed internalization by cultured cells through a potential receptor-mediated process and similar post-translational processing as human enzyme. In this study, we further studied the therapeutic potential of prGALNS through the characterization of the N-glycosylation structure, in vitro cell uptake and keratan sulfate reduction, and in vivo biodistribution and generation of anti-prGALNS antibodies. Taken together, these results represent an important step in the development of a P. pastoris-based platform for production of a therapeutic GALNS for MPS IVA enzyme replacement therapy.

Identification of Ezetimibe and Pranlukast as Pharmacological Chaperones for the Treatment of the Rare Disease Mucopolysaccharidosis Type IVA.

Mucopolysaccharidosis type IVA (MPS IVA) is a rare disease caused by mutations in the gene encoding the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS). We report here two GALNS pharmacological chaperones, ezetimibe and pranlukast, identified by molecular docking-based virtual screening. These compounds bound to the active cavity of GALNS and increased its thermal stability as well as the production of recombinant GALNS in bacteria, yeast, and HEK293 cells. MPS IVA fibroblasts treated with these chaperones exhibited increases in GALNS protein and enzyme activity and reduced the size of enlarged lysosomes. Abnormalities in autophagy markers p62 and LC3B-II were alleviated by ezetimibe and pranlukast. Combined treatment of recombinant GALNS with ezetimibe or pranlukast produced an additive effect. Altogether, the results demonstrate that ezetimibe and pranlukast can increase the yield of recombinant GALNS and be used as a monotherapy or combination therapy to improve the therapeutic efficacy of MPS IVA enzyme replacement therapy.

[Thoughts on the preparation of nursing graduate students for undergraduate teaching]. / Reflexões sobre o preparo para a docência na pós-graduação em enfermagem.

It is noticeable the greater focus on scientific production in Graduate courses compared to the attention given to the preparation for teaching practices. This is a relevant issue considering the importance of having Masters and Doctors also prepared to form professionals in their area. This article discusses some aspects of the preparation of Graduate level students enrolled in the Teaching Improvement Program (Programa de Aperfeiçoamento de Ensino, PAE), which the University of São Paulo offers. It also describes the experience of students who participate in the program, emphasizing its contribution to the development of competences for college teaching in Nursing.

Implantes dentários na reabilitação oral de pacientes com periodontite avançada: um relato de caso / Dental implants for oral rehabilitation of patients with advanced periodontitis: a case report

Introdução: Os implantes osseointegráveis e sua evolução ao longo dos anos têm se consolidado como a principal alternativa para a reabilitação oral de pacientes total ou parcialmente desdentados. Este trabalho tem como objetivo relatar um caso de um paciente do gênero masculino, 41 anos que compareceu ao ambulatório com queixa principal de mobilidade dentária e estética prejudicada. Relato de caso: Ao exame intrabucal, observou-se extensa recessão gengival, perda de dentes, lesão de furca e presença de mobilidade grau 3 ou 4. Após pactuação com o paciente, foi estabelecida a reabilitação da arcada superior com instalação de prótese total fixa metaloplástica implantossuportada. Na primeira etapa do tratamento, executou-se exodontia múltipla e instalação imediata de prótese total tradicional removível superior. Optou-se pela instalação de seis implantes do tipo cone-morse. Considerações Finais: O paciente encontra-se sob acompanhamento ambulatorial e respondendo positivamente em virtude de ter atendido suas expectativas estéticas e funcionais... (AU)

Introduction: The advances in osseointegrated implants over the year have consolidated these devices as the main alternative for oral rehabilitation of totally or partially edentulous patients. The objective of this study was to report the case of a 41-year-old male patient who attended the outpatient clinic complaining mainly of tooth mobility and compromised esthetics. Intraoral examination revealed extensive gingival recession, tooth loss, a furcation defect, and the presence of grade 3 or 4 mobility. Case report: After consent of the patient, rehabilitation of the upper arch was established, which consisted of the installation of an implant-supported metal/plastic fixed complete dental prosthesis. In the first step of treatment, multiple teeth were extracted and a conventional removable complete upper denture was immediately installed. Six cone morse implants were chosen for installation. Final considerations: The patient is under outpatient follow-up and responds positively since his esthetic and functional expectations were met... (AU)

Reflexões sobre o preparo para a docência na pós-graduação em enfermagem / Thoughts on the preparation of nursing graduate students for undergraduate teaching

É evidente nos cursos de pós-graduação stricto sensu o enfoque principal na formação para a pesquisa científica e a menor atenção ao preparo para a docência. Trata-se de uma questão relevante, tendo em vista a importância da capacitação de mestres e doutores também para a formação de profissionais da sua área. O presente texto aborda aspectos do preparo de pós-graduandos em enfermagem para a docência, por meio do Programa de Aperfeiçoamento de Ensino (PAE), oferecido pela Universidade de São Paulo. Relata, ainda, a experiência de alunas que participaram desse Programa, evidenciando a sua contribuição no desenvolvimento de competências para o exercício da docência universitária em enfermagem.

Ser comissário de bordo: os significados destes profissionais a cerca das adversidades no trabalho / Being a flight attendant: representations concerning work adversities

A atividade profissional do Comissário de bordo vem sendo vinculada a duas características ao mesmo tempo opostas e igualmente importantes enquanto marcas distintivas da profissão. Uma delas, fruto do imaginário social, vinculada ao "glamour", à aventura, à liberdade; a outra vinculada à realidade de um trabalho que isola o sujeito de seu grupo social, o submete a condições ambientais e circadianas extremamente desfavoráveis à saúde. Com a finalidade de investigar como os sujeitos que desempenham esta atividade profissional apreendem estas duas características e reagem a ela, os objetivos deste estudo foram conhecer significados de um grupo de comissários de bordo a cerca de "ser Comissário", conhecer as adversidades relacionadas ao trabalho identificadas por este grupo, além de analisar a inter relação entre significado e adversidade. Para tanto, optou-se pelo método qualitativo de pesquisa e análise, mais especificamente a Hermenêutica. Os dados, foram obtidos por meio de uma entrevista semi-estruturada feita com 9 funcionários ativos de uma mesma companhia aérea. Nesta entrevista, alem de informações relativas a dados de identificação pessoal, os sujeitos foram argüidos a partir da pergunta central: "O que significa para você ser Comissário de bordo?". Após a análise das falas, foram identificados quatro núcleos de significados. Os dois primeiros referem-se aos significados que a profissão representa para os sujeitos e foram denominados de O Comissário de bordocomo ser que cuida e O Comissário de bordo como ser capaz de realizar-se. O terceiro núcleo versa sobre As implicações negativas da profissão sob a ótica dos sujeitos. Este núcleo foi subdivido em duas categorias identificadas como Dificuldades inerentes à profissão e Prejuízos decorrentes da profissão. O quarto núcleo surgiu espontaneamente nas falas dos sujeitos, e tratou dos Atributos necessários para ser Comissário de bordo. ) A inter relação entre os significados apreendidos e os prejuízos referidos pelos sujeitos deste grupo, revelou a presença de sentimentos de desilusão, frustração e até depressão, possivelmente resultantes da vivência da realidade cotidiana inconciliável com uma idealização fantasiada.

Flight attendant occupation has brought about two different but important representations. One of them, as a result of social representation, it is related to "glamour", adventure, freedom; the other, due to its actual nature, to a kind of work that isolates the subject from their social group, that submits them to environmental and circadian conditions extremely unfavorable to health. Aiming at investigating how the subjects who have this profession apprehend both characteristics and react to them, the objectives of this study were to know the representations for a group of flight attendants concerning "being a flight attendant", to know adversities related to work and to understand the inter-relationship between representations and adversities. In order to do that, it was adopted the qualitative method for research and analysis, specifically Hermeutics. The data were obtained through semi-structured interview with nine subjects. Besides the information related to personal data, the subjects were interviewed based on the central the question: "What does it mean for you to be a flight attendant?" After analyzing the answers, it was identified four cores. Two of them related to representations about the profession that were denominated Flight Attendant is the one who cares and Flight attendant is the one who can feel realized. The third one is referring to Negative implication concerning the profession according to the subjects. This core was subdivided into twocategories identified as Difficulties inherited from the profession and Harms from the profession. The fourth core came up with the answers and covered the necessary attributions in order to be a flight attendant. The inter-relationship between the meanings and the harms reported by the subjects of study revealed the presence of some feelings such as disillusions, frustration and even depression. These feelings are probably resulted from the every day routine that does not match the fantasized idealization.