ABSTRACT
OBJECTIVE: Evaluate the assimilation capacity and ease of handling of the Fox plane accessory by dentistry students, through a questionnaire about the experience in using this device compared to the conventional one. Its intention is to minimize possible interpretation errors and challenges that the traditional method determines. METHODS: After approval by the Research Ethics Committee, registration at Sistema Nacional de Informações sobre Ética em Pesquisa/National Information System on Research Ethics (SISNEP) and signing of the free and informed consent form, 51 undergraduate students treating patients in need of complete dentures at Instituto de Ciência e Tecnologia/Institute of Science and Technology (ICT) Unesp in São José dos Campos completed a questionnaire to evaluate the technical ease of use and provider acceptance. The obtained data were submitted to statistical analysis, evaluating technical ease and acceptance by operators. RESULTS: The results were analyzed using descriptive and inferential statistics using the Jamovi 2.2.5 software. From the responses, the frequency was obtained for each question in the questionnaire, then the weighted mean was calculated, average rating and percentage. With the results of these analyses it was possible to measure the level of satisfaction of the participants in relation to the use of the device. CONCLUSION: It was concluded that most students have difficulty using the conventional Fox plane and that the accessory facilitated the process of determining the superior orientation plane, and its ease of use.
Subject(s)
Students, Dental , Humans , Students, Dental/psychology , Surveys and Questionnaires , Denture, Complete , Male , Female , Equipment Design , Young AdultABSTRACT
Obesity is defined as abnormal or excessive fat accumulation that may impair health and is a risk factor for developing other diseases, such as type 2 diabetes and cardiovascular disorder. Obesity is also associated with structural and functional alterations in the brain, and this condition has been shown to increase the risk of Alzheimer's disease. However, while obesity has been associated with neurodegenerative processes, its impact on brain cell composition remains to be determined. In the current study, we used the isotropic fractionator method to determine the absolute composition of neuronal and non-neuronal cells in different brain regions of the genetic mouse models of obesity Lepob/ob and LepRNull/Null . Our results show that 10- to 12-month-old female Lepob/ob and LepRNull/Null mice have reduced neuronal number and density in the hippocampus compared to C57BL/6 wild-type mice. Furthermore, LepRNull/Null mice have increased density of non-neuronal cells, mainly glial cells, in the hippocampus, frontal cortex and hypothalamus compared to wild-type or Lepob/ob mice, indicating enhanced inflammatory responses in different brain regions of the LepRNull/Null model. Collectively, our findings suggest that obesity might cause changes in brain cell composition that are associated with neurodegenerative and inflammatory processes in different brain regions of female mice.
ABSTRACT
Macroptilium (Benth.) Urb. is a neotropical legume genus from the subtribe Phaseolinae. The investigated species present a stable chromosome number (2n = 22), but differ in their karyotype formulae, suggesting the presence of chromosome rearrangements. In this work, we comparatively analysed the karyotypes of six species (Macroptilium atropurpureum, Macroptilium bracteatum, Macroptilium erythroloma, Macroptilium gracile, Macroptilium lathyroides, and Macroptilium martii) from the two main clades that form the genus. Heterochromatin distribution was investigated with chromomycin A3 (CMA)/4',6-diamidino-2-phenylindole (DAPI) staining and fluorescent in situ hybridization was used to localize the 5S and 35S ribosomal DNA (rDNA) sites. Single copy bacterial artificial chromosomes (BACs) previously mapped in the related genera Phaseolus L. and Vigna Savi were used to establish chromosome orthologies and to investigate possible rearrangements among species. CMA+/DAPI- bands were observed, mostly associated with rDNA sites. Additional weak, pericentromeric bands were observed on several chromosomes. Although karyotypes were similar, species could be differentiated mainly by the number and position of the 5S and 35S rDNA sites. BAC markers demonstrated conserved synteny of the main rDNA sites on orthologous chromosomes 6 and 10, as previously observed for Phaseolus and Vigna. The karyotypes of the six species could be differentiated, shedding light on its karyotype evolution.
Subject(s)
Phaseolus , In Situ Hybridization, Fluorescence , Karyotyping , Karyotype , Phaseolus/genetics , DNA, Ribosomal/genetics , Chromosome BandingABSTRACT
Non-destructive fluorophore diffusion across cell membranes to provide an unbiased fluorescence intensity readout is critical for quantitative imaging applications in live cells and tissues. Commercially available small-molecule fluorophores have been engineered for biological compatibility, imparting high water solubility by modifying rhodamine and cyanine dye scaffolds with multiple sulfonate groups. The resulting net negative charge, however, often renders these fluorophores cell-membrane-impermeant. Here we report the design and development of our biologically compatible, water-soluble and cell-membrane-permeable fluorophores, termed OregonFluor (ORFluor). By adapting previously established ratiometric imaging methodology using bio-affinity agents, it is now possible to use small-molecule ORFluor-labelled therapeutic inhibitors to quantitatively visualize their intracellular distribution and protein target-specific binding, providing a chemical toolkit for quantifying drug target availability in live cells and tissues.
Subject(s)
Fluorescent Dyes , Water , Fluorescent Dyes/chemistry , Rhodamines/chemistryABSTRACT
Objective: The study aimed to develop a cell phone application entitled: "Total Prosthesis Manual Rehabilitating smiles", as an auxiliary teaching tool for teachers and students through digital technology, with a smartphone. Material and Methods: The tool was structured on the "Application Factory website", which allows the creation of mobile applications in different formats, with broad and interactive features on IOS and Android platforms. The expository format of the content is in slide format, containing descriptive theory and images about the stages of making a complete prosthesis; from necessary materials, photos and descriptive guidance of the steps. Results: The application is a complementary teaching resource to assist undergraduate and postgraduate students and professionals working in the area of complete prosthetics. The theoretical and practical content selected for the application covered all stages of understanding, development and possible complications associated with the manufacture of a complete bimaxillary prosthesis, from planning, impressions, models, orientation plans, tooth assembly, adaptations and delivery. Conclusion: The application provided a low-cost, expandable and easy-to-use teaching resource for teaching complete dentures. It is essential to develop various analyzes such as user experience tests, application effectiveness, development of new technologies and improvement of techniques, so that their potential for enriching learning in complete dentures and dentistry in general can be verified.(AU)
Objetivo: O estudo teve como objetivo desenvolver um aplicativo de celular intitulado: "Manual de Prótese Total Reabilitando sorrisos", como uma ferramenta didática auxiliar para professores e alunos por meio da tecnologia digital, com smartphone. Material e Métodos: A ferramenta foi estruturada no "site da Fábrica de Aplicativos", que permite a criação de aplicativos móveis em diversos formatos, com recursos amplos e interativos nas plataformas IOS e Android. A modalidade expositiva do conteúdo é em formato de slides, contendo teoria descritiva e imagens sobre as etapas de confecção de uma prótese total; a partir de materiais necessários, fotos e orientação descritiva das etapas. Resultados: O aplicativo é um recurso didático complementar para auxiliar estudantes de graduação, pós-graduação e profissionais que atuam na área de prótese total. O conteúdo teórico e prático selecionado para a aplicação visou todas as etapas de compreensão, desenvolvimento e possíveis complicações associadas à confecção de uma prótese total bimaxilar, desde o planejamento, moldagens, maquetes, planos de orientação, montagem dos dentes, adaptações e entrega. Conclusão: O aplicativo trouxe um recurso didático de baixo custo, expansível e fácil de usar para o ensino de próteses totais. É fundamental desenvolver diversas análises como testes de experiência do usuário, eficácia de aplicação, desenvolvimento de novas tecnologias e aprimoramento de técnicas, de forma que possa ser verificado seu potencial de enriquecimento do aprendizado em prótese total e odontologia em geral(AU)
Subject(s)
Humans , Educational Technology , Denture, Complete , Education, Dental , Mobile Applications , Education, Distance , Dentists/educationABSTRACT
In their natural form, antibodies are always in an "on-state" and are capable of binding to their targets. This leads to undesirable interactions in a wide range of therapeutic, analytical, and synthetic applications. Modulating binding kinetics of antibodies to turn them from an "off-state" to an "on-state" with temporal and spatial control can address this. Here we demonstrate a method to modulate binding activity of antibodies in a predictable and reproducible way. We designed a blocking construct that uses both covalent and non-covalent interactions with the antibody. The construct consisted of a Protein L protein attached to a flexible linker ending in a blocking-peptide designed to interact with the antibody binding site. A mutant Protein L was developed to enable photo-triggered covalent crosslinking to the antibody at a specific location. The covalent bond anchored the linker and blocking peptide to the antibody light chain keeping the blocking peptide close to the antibody binding site. This effectively put the antibody into an "off-state". We demonstrate that protease-cleavable and photocleavable moieties in the tether enable controlled antibody activation to the "on-state" for anti-FLAG and cetuximab antibodies. Protein L can bind a range of antibodies used therapeutically and in research for wide applicability.
Subject(s)
Antibodies , Peptides , Binding Sites, Antibody , KineticsABSTRACT
Tumor protein p63 (TP63) is a member of the TP53 protein family that are important for development and in tumor suppression. Unlike TP53, TP63 is rarely mutated in cancer, but instead different TP63 isoforms regulate its activity. TA isoforms (TAp63) act as tumor suppressors, whereas ΔN isoforms are strong drivers of squamous or squamous-like cancers. Many of these tumors become addicted to ΔN isoforms and removal of ΔN isoforms result in cancer cell death. Furthermore, some TP53 conformational mutants (TP53CM) gain the ability to interact with TAp63 isoforms and inhibit their antitumorigenic function, while indirectly promoting tumorigenic function of ΔN isoforms, but the exact mechanism of TP63-TP53CM interaction is unclear. The changes in the balance of TP63 isoform activity are crucial to understanding the transition between normal and tumor cells. Here, we modeled TP63-TP53CM complex using computational approaches. We then used our models to design peptides to disrupt the TP63-TP53CM interaction and restore antitumorigenic TAp63 function. In addition, we studied ΔN isoform oligomerization and designed peptides to inhibit its oligomerization and reduce their tumorigenic activity. We show that some of our peptides promoted cell death in a TP63 highly expressed cancer cell line, but not in a TP63 lowly expressed cancer cell line. Furthermore, we performed kinetic-binding assays to validate binding of our peptides to their targets. Our computational and experimental analyses present a detailed model for the TP63-TP53CM interaction and provide a framework for potential therapeutic peptides for the elimination of TP53CM cancer cells.
Subject(s)
Carcinoma, Squamous Cell , Tumor Suppressor Protein p53 , Humans , Tumor Suppressor Protein p53/genetics , Protein Isoforms/metabolism , Cell Line, Tumor , Carcinogenesis , Carcinoma, Squamous Cell/metabolism , Computer Simulation , Transcription Factors/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
Utilizing an intersectional framework (Crenshaw, 1989) and socioecological theory (Bronfenbrenner, 1979), we investigated the gendered racialized messages Asian American women receive growing up, otherwise known as gendered racial socialization, from their family, peers, and mass media. Consensual Qualitative Research was used to analyze interview data from 12 second-generation Asian American women. The results demonstrated that (a) family socialization messages included gendered racial discrimination, body image and physical appearance, marital/dating attitudes, role responsibilities and expectations, and academic/work expectations, (b) peer socialization included oppressive messages (e.g., physical objectification, denial of identity, lack of presence) and affirming messages (e.g., positive self-concept messages), and (c) mass media socialization included oppressive messages (e.g., lack of representation, stereotypical depictions), and affirming messages (e.g., messages about empowerment). These messages impacted Asian American women's views on their body image and physical appearance, self-esteem, career/work, mental health, and critical consciousness. Implications and findings of the need to dismantle interlocking oppressive forces are discussed. (PsycInfo Database Record (c) 2022 APA, all rights reserved).
Subject(s)
Racism , Socialization , Asian , Female , Gender Identity , Humans , Self ConceptABSTRACT
BACKGROUND: Senescent cells accumulate in tissues over time as part of the natural ageing process and the removal of senescent cells has shown promise for alleviating many different age-related diseases in mice. Cancer is an age-associated disease and there are numerous mechanisms driving cellular senescence in cancer that can be detrimental to recovery. Thus, it would be beneficial to develop a senolytic that acts not only on ageing cells but also senescent cancer cells to prevent cancer recurrence or progression. METHODS: We used molecular modelling to develop a series of rationally designed peptides to mimic and target FOXO4 disrupting the FOXO4-TP53 interaction and releasing TP53 to induce apoptosis. We then tested these peptides as senolytic agents for the elimination of senescent cells both in cell culture and in vivo. FINDINGS: Here we show that these peptides can act as senolytics for eliminating senescent human cancer cells both in cell culture and in orthotopic mouse models. We then further characterized one peptide, ES2, showing that it disrupts FOXO4-TP53 foci, activates TP53 mediated apoptosis and preferentially binds FOXO4 compared to TP53. Next, we show that intratumoural delivery of ES2 plus a BRAF inhibitor results in a significant increase in apoptosis and a survival advantage in mouse models of melanoma. Finally, we show that repeated systemic delivery of ES2 to older mice results in reduced senescent cell numbers in the liver with minimal toxicity. INTERPRETATION: Taken together, our results reveal that peptides can be generated to specifically target and eliminate FOXO4+ senescent cancer cells, which has implications for eradicating residual disease and as a combination therapy for frontline treatment of cancer. FUNDING: This work was supported by the Cancer Early Detection Advanced Research Center at Oregon Health & Science University.
Subject(s)
Antineoplastic Agents/chemistry , Cell Cycle Proteins/chemistry , Drug Design , Forkhead Transcription Factors/chemistry , Models, Molecular , Peptides/chemistry , Senotherapeutics/chemistry , Tumor Suppressor Protein p53/chemistry , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Cycle Proteins/metabolism , Cellular Senescence/drug effects , Disease Models, Animal , Female , Forkhead Transcription Factors/metabolism , Humans , Male , Melanoma , Mice , Molecular Docking Simulation , Molecular Dynamics Simulation , Peptides/pharmacology , Protein Conformation , Senotherapeutics/pharmacology , Structure-Activity Relationship , Tumor Suppressor Protein p53/metabolism , Xenograft Model Antitumor AssaysABSTRACT
OBJECTIVE: The aim of this study was to address the first cases of TOETVA done in Brazil, by TOETVA-Bra study group, regarding safety and complications. METHODS: Series of the first 93 TOETVAs cases in Brazil. All authors except LPK, AJG JOR and RPT received TOETVA training including cadaveric hands-on in Thailand or United States (Johns Hopkins Medicine) during 2017. After they came back to Brazil and started doing their first TOETVA cases in the cities of Rio de Janeiro, Sao Paulo and Chapecó they agreed to collaborate and gather data using an online spreadsheet. All patients were submitted to the technique described by Anuwong. RESULTS: A total of 93 patients underwent TOETVA. Most patients (58.1%) were submitted to total thyroidectomy and 59.1% had benign disease. Two patients (2.2%) needed conversion to open surgery. Five patients (9.3%) developed transient hypoparathyroidism and there were 3 (2.0%) temporary recurrent laryngeal nerve palsy. There was one (0.7%) permanent unilateral palsy. Twenty patients had some sort of complication, 16.1% were minor and 5.4% were major. A total of 73 patients (78.5%) had an uneventful recovery. CONCLUSION: The technique is reproducible with a low complication rate. While further studies are needed to confirm equivalency, early efforts suggest that TOETVA is not inferior to traditional open thyroidectomy in appropriately selected patients.
Subject(s)
Hypoparathyroidism , Thyroidectomy , Brazil , Endoscopy , Humans , Learning Curve , Thyroidectomy/adverse effectsABSTRACT
ABSTRACT Background: The aim of this study was to address the first cases of TOETVA done in Brazil, by TOETVA-Bra study group, regarding safety and complications. Materials and Methods: Series of the first 93 TOETVAs cases in Brazil. All authors except LPK, AJG JOR and RPT received TOETVA training including cadaveric hands-on in Thailand or United States (Johns Hopkins Medicine) during 2017. After they came back to Brazil and started doing their first TOETVA cases in the cities of Rio de Janeiro, Sao Paulo and Chapecó they agreed to collaborate and gather data using an online spreadsheet. All patients were submitted to the technique described by Anuwong. Results: A total of 93 patients underwent TOETVA. Most patients (58.1%) were submitted to total thyroidectomy and 59.1% had benign disease. Two patients (2.2%) needed conversion to open surgery. Five patients (9.3%) developed transient hypoparathyroidism and there were 3 (2.0%) temporary recurrent laryngeal nerve palsy. There was one (0.7%) permanent unilateral palsy. Twenty patients had some sort of complication, 16.1% were minor and 5.4% were major. A total of 73 patients (78.5%) had an uneventful recovery. Conclusion: The technique is reproducible with a low complication rate. While further studies are needed to confirm equivalency, early efforts suggest that TOETVA is not inferior to traditional open thyroidectomy in appropriately selected patients.
Subject(s)
Humans , Thyroidectomy/adverse effects , Hypoparathyroidism , Brazil , Endoscopy , Learning CurveABSTRACT
Digital dentistry has gained space in several dental specialties. It is possible to achieve excellent results with the digital workflow, which combines the efficiency of the restorative material with a greater marginal adaptation. This study aimed to report a clinical case through the digital workflow, with a faster and clinically acceptable prosthetic resolution. In this clinical case report, digital workflow allowed a faster and clinically acceptable prosthetic resolution. A 45-year-old female patient reported cementation failure of the prosthetic crown on tooth 14. As it was a vital tooth, the tooth received a total crown preparation. In the same clinical session, the patient's mouth was scanned then a capture software obtained a virtual model. After, the design software planned a digital "diagnostic wax-up", so a leucitic ceramic was chosen for the rehabilitation. The ceramic block was milled and receive stain and glaze, dispensing the prosthesis laboratory. Then, the adhesive cementation was performed with a dual-polymerized resin cement. The final crown had ideal adaptation, with no need for interproximal and occlusal adjustments, with an excellent marginal fit. Within the limitations of this study, this case report showed that the digital workflow allowed a favorable result in a shorter working time, which brought back function and aesthetics, without the need for interproximal and occlusal adjustments. (AU)
A odontologia digital vem ganhando espaço em diversas especialidades odontológicas. Com o fluxo de trabalho digital, é possível alcançar excelentes resultados na reabilitação protética, combinando a eficiência do material restaurador com a adaptação marginal proporcionada pela odontologia digital. O objetivo desse estudo foi relatar um caso clínico através do fluxo de trabalho digital, com uma resolução protética mais rápida e clinicamente aceitável. Paciente do sexo feminino, 45 anos, relatou falha de cimentação da coroa protética do dente 26. Por ser um dente vital, o dente recebeu um preparo de coroa total e os dentes foram escaneados e um software de captura obteve um modelo virtual. Posteriormente, o software de projeto planejou um "enceramento diagnóstico" digital, sendo escolhida uma cerâmica leucítica para a reabilitação. O bloco cerâmico foi fresado e recebeu acabamento, maquiagem e glaze pelo próprio dentista, dispensando um técnico laboratorial de prótese dentária. Em seguida, foi realizada cimentação adesiva definitiva. Este relato de caso mostra que, dentro das limitações desse estudo, o fluxo digital permite um resultado favorável em um menor tempo de trabalho, devolvendo a função e estética, sem necessidade de ajustes interproximais e oclusais (AU)
Subject(s)
Humans , Female , Adult , Dental Prosthesis , Computer-Aided Design , Esthetics, DentalABSTRACT
No tocante ao ensino teórico-prático em prótese fixa docentes utilizam diversos recursos didáticos no intuito de implementar estratégias de como realizar tecnicamente preparos (desgastes) dentais coronários visando a reposição de estruturas dentais por próteses, a fim de formar um conceito e fixar a imagem de um preparo ideal; conteúdo esse que deve ser aplicado e refinado na prática odontológica tanto laboratorial quanto clinicamente. O presente estudo teve como objetivo validar duas ferramentas novas no ensino e aprendizagem do conteúdo da disciplina de Prótese Fixa, a partir das principais dificuldades dos alunos em realizar um preparo. Participaram da pesquisa 61 alunos da graduação em Odontologia. Duas metodologias foram avaliadas quanto a sua eficácia na melhora do preparo final realizado pelo aluno: 1) Aplicativo de celular contendo um banco de dados com os principais preparos coronários e, 2) Um manequim com esquema de cores que auxilia a visualizar e calibrar os alunos na quantidade de redução coronária nos preparos. Como metodologia os alunos realizaram o preparo de um dente íntegro antes e após o uso das ferramentas didáticas do aplicativo e manequim. Os preparos foram escaneados e um software de análise de deformidade de superfície avaliou a diferença entre o preparo inicial e o valor de referência, preparo realizado após a aplicação da metodologia x valor de referência, podendo assim ser inferida a possível melhora no resultado da aplicação da metodologia comparativamente ao conceito teórico de um preparo ideal. Os dados computados foram analisados estatisticamente pelo test t com nível de significância estabelecido foi de p<0,05. A metodologia do aplicativo de celular mostrou poucas diferenças estatísticas quando comparado ao método do manequim com esquema de cores. O manequim esclareceu e mostrou resultados otimistas nas 3 das principais dificuldades dos alunos relatadas no questionário inicial, quantidade de desgaste, término cervical e inclinação e angulação dos preparos. Ambas as metodologias, respectivamente aplicativo e manequim, foram aprovadas 96,77% e 98,39% dos alunos e validadas como metodologias auxiliares ao ensino em PPF e odontologia pelo seu conteúdo teórico e prática associada (AU)
With regard to theoretical-practical teaching in fixed prostheses, teachers use several teaching resources in order to implement strategies on how to technically perform coronal dental preparations (wear) in order to replace dental structures with prostheses, in order to form a concept and fix the image of an ideal preparation; content that must be applied and refined in dental practice both laboratory and clinically. The present study aimed to validate two new tools in the teaching and learning of the content of the Fixed Prosthesis subject, based on the main difficulties of the students in carrying out a preparation. Sixty-one undergraduate students in Dentistry participated in the research. Two methodologies were evaluated for their effectiveness in improving the final preparation performed by the student: 1) Mobile application containing a database with the main coronary preparations and, 2) A mannequin with a color scheme that helps to visualize and calibrate students on the amount of coronary reduction in the preparations. As a methodology, the students prepared an intact tooth before and after using the didactic tools of the application and dummy. The preparations were scanned and a surface deformity analysis software evaluated the difference between the initial preparation and the reference value, preparation carried out after the application of the methodology x reference value, thus being able to infer the possible improvement in the result of the application of the methodology compared to the theoretical concept of an ideal preparation. The computed data were statistically analyzed by the t test with a significance level of p<0.05. The cell phone application methodology showed few statistical differences when compared to the color scheme dummy method. The dummy clarified and showed optimistic results in the 3 of the main difficulties of the students reported in the initial questionnaire, amount of wear, cervical end and inclination and angulation of the preparations. Both methodologies, respectively application and mannequin, were approved by 96.77% and 98.39% of students and validated as auxiliary methodologies for teaching in PPF and dentistry for their theoretical content and associated practice (AU)
Subject(s)
Teaching , Dentistry , Denture, Partial, Fixed , Mobile Applications , LearningABSTRACT
CONTEXT: Students can have some issues in the comprehension and execution of coronal preparations in fixed partial prosthesis (FPP). Some issues pertain to the amount of reduction, the inclination, and the positioning, all of which are important for the execution of an ideal preparation while respecting the required biomechanical principles. OBJECTIVE: The present study's aim was to evaluate the main problems experienced by graduation students regarding coronal preparation in FPP and to suggest teaching skills to help students and professors. DESIGN: A total of 87 students, who were enrolled in the 3rd year of Dentistry at the FPP course - in the Institute of Science and Technology, São Paulo State University, between 2017 and 2018, participated in the study. Two methodologies were developed, applied, and validated: first, a mannequin with a scheme of colors to help students visualize the coronal preparation; second, a comparison of different didactic methods. RESULTS: Only 54.02% of the students answered the questionnaire, and the main problem identified by the respondents was the amount of reduction required (78.2%). In the second place, 50.9% of the students stated that they had problems with the inclination and angle of the preparation. The mannequin method with color schemes was approved by 91.5%. The favorite didactic method was a live demonstration (face to face), with 61.8%. In the second place, 47.3% of the students stated that live projection was also adequate. CONCLUSIONS: Both auxiliary didactic methods were approved by the students and they reported that it helped them to visualize the amount of reduction necessary and the sequence of the preparation. Hence, it was concluded that the mannequin with color schemes and the live projection were approved as auxiliary didactic resources for teaching FPP.
Subject(s)
Dental Implants , Humans , Manikins , Students , Surveys and Questionnaires , Teaching , UniversitiesABSTRACT
Viral vectors are extensively purified for use in biomedical research, in order to separate biologically active virus particles and to eliminate production related impurities that are assumed to be detrimental to the host. For recombinant adeno-associated virus (rAAV) vectors this is typically accomplished using density gradient-based methods, which are tedious and require specialized ultracentrifugation equipment. In order to streamline the preparation of rAAV vectors for pilot and small animal studies, we recently devised a simple ultrafiltration approach that permits rapid virus concentration and partial removal of production-related impurities. Here we show that systemic administration of such rapidly prepared (RP) rAAV8 vectors in mice is safe and efficiently transduces the liver. Across a range of doses, delivery of RP rAAV8-CMV-eGFP vector induced enhanced green fluorescent protein (eGFP) expression in liver that was comparable to that obtained from a conventional iodixanol gradient-purified (IP) vector. Surprisingly, no liver inflammation or systemic cytokine induction was detected in RP rAAV injected animals, revealing that residual impurities in the viral vector preparation are not deleterious to the host. Together, these data demonstrate that partially purified rAAV vector can be safely and effectively administered in vivo. The speed and versatility of the RP method and lack of need for cumbersome density gradients or expensive ultracentrifuge equipment will enable more widespread use of RP prepared rAAV vectors, such as for pilot liver gene transfer studies.
Subject(s)
Dependovirus/isolation & purification , Genetic Vectors/administration & dosage , Genetic Vectors/isolation & purification , Liver , Transduction, Genetic , Ultrafiltration , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral , Cell Line , Dependovirus/genetics , Dependovirus/immunology , Gene Expression , Gene Transfer Techniques , Genes, Reporter , Genetic Therapy , Genetic Vectors/adverse effects , Genetic Vectors/genetics , Hepatocytes/metabolism , Humans , Liver/metabolism , Male , Mice , Models, Animal , Transgenes , Ultracentrifugation , Ultrafiltration/methods , Viral Load , Virus ReplicationABSTRACT
O primeiro contato de alunos de graduação com a disciplina de Prótese Parcial Fixa ocorre em aulas teóricas e posteriormente em práticas laboratoriais. Na teoria os docentes utilizam de diversas formas para elucidar um preparo de Prótese Parcial Fixa, fotos e vídeos no passo a passo, guias de silicone, guias de redução, marcações coloridas e até mesmo demonstrações ao vivo. Utilizam- se desses métodos a fim de formar um conceito e fixar uma imagem de um preparo ideal obedecendo aos princípios mecânicos e biológicos das reduções estruturais em prótese fixa. O presente estudo teve como objetivo avaliar as principais dificuldades dos alunos de graduação nos preparos coronários em Prótese Parcial Fixa e sugerir ferramentas de ensino para auxiliar aluno e professor, a elucidar e calibrar os alunos nos desgastes coronários. Participaram da pesquisa ao todo 87 alunos matriculados do 3° ano integral, dos anos 2017 e 2018 da disciplina de Prótese Parcial Fixa, da graduação em Odontologia do ICT. Foi desenvolvido um aplicativo de celular com um banco de dados dos principais preparos coronários e com a possibilidade de comparação do preparo finalizado através de fotos. Também foram desenvolvidas, aplicadas e validadas 02 metodologias: Um manequim com esquema de cores que auxiliou o aluno a visualizar os preparos coronários e uma demonstração prática projetada comparou os métodos de ensino teórico e prático. 78,2% dos alunos relatam a quantidade de redução como a principal dificuldade. O método do manequim com esquema de cores foi preferência de técnica por 91,5% dos alunos. Ambos os métodos auxiliares didáticos avaliados foram aprovados pelos alunos, em 100% no método do manequim e 98,2% na vídeo aula. Respectivamente, os métodos auxiliaram na visualização da quantidade de redução e na sequência do preparo. Conclui-se que tanto o manequim com esquema de cores e a projeção ao vivo quanto o se mostraram eficazes e aprovados como recursos didáticos auxiliares no ensino de Prótese Parcial Fixa e o desenvolvimento do aplicativo de celular como ferramenta no ensino odontológico expande as possibilidades do uso de tecnologias de ensino na odontologia(AU)
The first contact of undergraduate students with the discipline of Partial Fixed Prosthesis occurs in theory classes and later in laboratory practices. In theory, teachers use a variety of ways to elucidate a Fixed Partial Prosthesis preparation, step-by-step photos and videos, silicone guides, reduction guides, color markers and even live demonstrations. These methods are used to form a concept and to fix an image of an ideal preparation obeying the mechanical and biological principles of the structural reductions in fixed prosthesis. The present study had as objective to evaluate the main difficulties of undergraduate students in coronary arteries in Fixed Partial Prosthesis and to suggest teaching tools to help students and teachers to elucidate and calibrate students in coronary wear. A total of 87 enrolled students from the 3rd full year, from the years 2017 and 2018 of the discipline of Partial Fixed Prosthesis, from the graduation in Dentistry from the ICT participated in the research. A cellular application was developed with a database of the main coronary preparations and the possibility of comparing the finished preparation with photos. Two methodologies were also developed, applied and validated: a color design mannequin that helped the student to visualize the coronary preparations and a practical demonstration designed to compare the methods of theory and practical teaching. 78.2% of the students report the amount of reduction as the main difficulty. The method of color design mannequin was a technique preference for 91.5% of the students. Both didactic methods were approved by the students, 100% in the color design mannequin and 98.2% in the video lesson. Respectively, the methods aided in visualizing the amount of reduction and the preparation sequence. It was concluded that both the color-blind mannequin and the live projection were effective and approved as auxiliary didactic resources in the teaching of Partial Fixed Prosthesis and the development of the cellular application as a tool in dental education expands the possibilities of use of teaching technologies in dentistry(AU)
Subject(s)
Humans , Teaching/trends , Dentistry/classification , Denture, Partial, Fixed/statistics & numerical data , Learning/classificationABSTRACT
There is keen interest to define gene therapies aimed at restoration of auditory and vestibular function in the diseased or damaged mammalian inner ear. A persistent limitation of regenerative medical strategies that seek to correct or modify gene expression in the sensory epithelia of the inner ear involves efficacious delivery of a therapeutic genetic construct. Our approach is to define methodologies that enable fetal gene transfer to the developing mammalian inner ear in an effort to correct defective gene expression during formation of the sensory epithelia or during early postnatal life. Conceptually, the goal is to atraumatically introduce the genetic construct into the otocyst-staged mouse inner ear and transfect otic progenitors that give rise to sensory hair cells and supporting cells. Our long-term goal is to define therapeutic interventions for congenital deafness and balance disorders with the expectation that the approach may also be exploited for therapeutic intervention postnatally.In the inaugural volume of this series, we introduced electroporation-mediated gene transfer to the developing mouse inner ear that encompassed our mouse survival surgery and transuterine microinjection protocols (Brigande et al., Methods Mol Biol 493:125-139, 2009). In this chapter, we first briefly update our use of sodium pentobarbital anesthesia, our preferred anesthetic for mouse ventral laparotomy, in light of its rapidly escalating cost. Next, we define a rapid, cost-effective method to produce recombinant adeno-associated virus (rAAV) for efficient gene transfer to the developing mouse inner ear. Our immediate goal is to provide a genetic toolkit that will permit the definition and validation of gene therapies in mouse models of human deafness and balance disorders.
Subject(s)
Deafness/therapy , Dependovirus/genetics , Ear, Inner/embryology , Genetic Engineering/economics , Adjuvants, Anesthesia/administration & dosage , Animals , Deafness/genetics , Disease Models, Animal , Gene Transfer Techniques , Genetic Therapy , HEK293 Cells , Humans , Mice , Pentobarbital/administration & dosageABSTRACT
Advancement in immunogen selection and vaccine design that will rapidly elicit a protective Ab response is considered critical for HIV vaccine protective efficacy. Vaccine-elicited Ab responses must therefore have the capacity to prevent infection by neutralization-resistant phenotypes of transmitted/founder (T/F) viruses that establish infection in humans. Most vaccine candidates to date have been ineffective at generating Abs that neutralize T/F or early variants. In this study, we report that coimmunizing rhesus macaques with HIV-1 gp160 DNA and gp140 trimeric protein selected from native envelope gene sequences (envs) induced neutralizing Abs against Tier 2 autologous viruses expressing cognate envelope (Env). The Env immunogens were selected from envs emerging during the earliest stages of neutralization breadth developing within the first 2 years of infection in two clade B-infected human subjects. Moreover, the IgG responses in macaques emulated the targeting to specific regions of Env known to be associated with autologous and heterologous neutralizing Abs developed within the human subjects. Furthermore, we measured increasing affinity of macaque polyclonal IgG responses over the course of the immunization regimen that correlated with Tier 1 neutralization. In addition, we report firm correlations between Tier 2 autologous neutralization and Tier 1 heterologous neutralization, as well as overall TZM-bl breadth scores. Additionally, the activation of Env-specific follicular helper CD4 T cells in lymphocytes isolated from inguinal lymph nodes of vaccinated macaques correlated with Tier 2 autologous neutralization. These results demonstrate the potential for native Env derived from subjects at the time of neutralization broadening as effective HIV vaccine elements.
Subject(s)
Antibodies, Neutralizing/immunology , HIV Antibodies/immunology , HIV Infections/immunology , HIV-1/immunology , env Gene Products, Human Immunodeficiency Virus/immunology , Animals , Antibody Affinity/immunology , Antibody Specificity/immunology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Disease Models, Animal , Epitopes/immunology , Immunization , Immunization Schedule , Immunoglobulin G/blood , Immunoglobulin G/immunology , Lymphoid Tissue/immunology , Macaca mulatta , Neutralization Tests , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , VaccinationABSTRACT
Developing a vaccine that overcomes the diversity of HIV-1 is likely to require a strategy that directs antibody (Ab) responses toward conserved regions of the viral Envelope (Env). However, the generation of neutralizing Abs (NAbs) targeting these regions through vaccination has proven to be difficult. One conserved region of particular interest is the membrane proximal external region (MPER) of Env located within the gp41 ectodomain. In order to direct the immune response to this region, the MPER and gp41 ectodomain were expressed separately as N-terminal fusions to the E2 protein of Geobacillus stearothermophilus. The E2 protein acts as a scaffold by self-assembling into 60-mer particles, displaying up to 60 copies of the fused target on the surface. Rabbits were immunized with E2 particles displaying MPER and/or the gp41 ectodomain in conjunction with DNA encoding full-length gp160. Only vaccines including E2 particles displaying MPER elicited MPER-specific Ab responses. NAbs were elicited after two immunizations that largely targeted the V3 loop. To overcome V3 immunodominance in the DNA component, E2 particles displaying MPER were used in conjunction with gp160 DNA lacking hypervariable regions V2, V3, or combined V1V2V3. All rabbits had HIV binding Ab responses and NAbs following the second vaccination. Using HIV-2/HIV-1 MPER chimeric viruses as targets, NAbs were detected in 12/16 rabbits after three immunizations. Low levels of NAbs specific for Tier 1 and 2 viruses were observed in all groups. This study provides evidence that co-immunizing E2 particles displaying MPER and gp160 DNA can focus Ab responses toward conserved regions of Env.
