Pseudotyping exosomes for enhanced protein delivery in mammalian cells.

Exosomes are cell-derived nanovesicles that hold promise as living vehicles for intracellular delivery of therapeutics to mammalian cells. This potential, however, is undermined by the lack of effective methods to load exosomes with therapeutic proteins and to facilitate their uptake by target cells. Here, we demonstrate how a vesicular stomatitis virus glycoprotein (VSVG) can both load protein cargo onto exosomes and increase their delivery ability via a pseudotyping mechanism. By fusing a set of fluorescent and luminescent reporters with VSVG, we show the successful targeting and incorporation of VSVG fusions into exosomes by gene transfection and fluorescence tracking. We subsequently validate our system by live cell imaging of VSVG and its participation in endosomes/exosomes that are ultimately released from transfected HEK293 cells. We show that VSVG pseudotyping of exosomes does not affect the size or distributions of the exosomes, and both the full-length VSVG and the VSVG without the ectodomain are shown to integrate into the exosomal membrane, suggesting that the ectodomain is not required for protein loading. Finally, exosomes pseudotyped with full-length VSVG are internalized by multiple-recipient cell types to a greater degree compared to exosomes loaded with VSVG without the ectodomain, confirming a role of the ectodomain in cell tropism. In summary, our work introduces a new genetically encoded pseudotyping platform to load and enhance the intracellular delivery of therapeutic proteins via exosome-based vehicles to target cells

Spectral imaging in preclinical research and clinical pathology.

Spectral imaging methods are attracting increased interest from researchers and practitioners in basic science, pre-clinical and clinical arenas. A combination of better labeling reagents and better optics creates opportunities to detect and measure multiple parameters at the molecular and cellular level. These tools can provide valuable insights into the basic mechanisms of life, and yield diagnostic and prognostic information for clinical applications. There are many multispectral technologies available, each with its own advantages and limitations. This chapter will present an overview of the rationale for spectral imaging, and discuss the hardware, software and sample labeling strategies that can optimize its usefulness in clinical settings

A Review on the Applications of Crowdsourcing in Human Pathology

The advent of the digital pathology has introduced new avenues of diagnostic medicine. Among them, crowdsourcing has attracted researchers' attention in the recent years, allowing them to engage thousands of untrained individuals in research and diagnosis. While there exist several articles in this regard, prior works have not collectively documented them. We, therefore, aim to review the applications of crowdsourcing in human pathology in a semi-systematic manner. We firstly, introduce a novel method to do a systematic search of the literature. Utilizing this method, we, then, collect hundreds of articles and screen them against a pre-defined set of criteria. Furthermore, we crowdsource part of the screening process, to examine another potential application of crowdsourcing. Finally, we review the selected articles and characterize the prior uses of crowdsourcing in pathology

Validation of digital microscopy in the histopathological diagnoses of oral diseases

Orientador: Alan Roger dos Santos SilvaDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: A microscopia digital (MD) expandiu-se nos últimos anos em ambientes educacionais e profissionais para interconsulta, telepatologia, armazenamento e relatórios anatomopatológicos, colocando sistemas whole slide imaging (WSI) na posição privilegiada de dispositivos inovadores para interpretação de diagnósticos primários, aplicação previamente concebida com receio. Esta é uma consequência direta da falta de regulamentação desses dispositivos. É necessário reunir evidências sobre o desempenho da MD, para estabelecer se esta tecnologia pode ser usada para fornecer diagnóstico primário com segurança. O primeiro capítulo apresentado no presente estudo teve como objetivo fornecer informações sobre o desempenho de sistemas WSI, avaliando concordância intra-observador como melhor evidência. Uma busca eletrônica nas bases Scopus, MEDLINE/PubMed e Embase foi conduzida. As características metodológicas, a concordância entre a microscopia convencional (MC) e a MD e as razões para a ocorrência de diagnósticos discordantes foram analisadas. Um total de 13 artigos foram incluídos. As concordâncias intra-observadores variaram de 90% a 98,3% (intervalo de confiança de ? = 0,8-0,98). A dificuldade do caso foi o principal motivo de discordância (46,15%), seguido por dificuldades na identificação de microrganismos (15,38%). 58,84% enfatizam que o desempenho do método digital não está relacionado com a ocorrência de discordâncias. Apenas 25% das discordâncias tinham diagnósticos preferenciais por WSI. 15,38% dos estudos incluídos apresentaram alto risco de viés devido à seleção da amostra e 15,38% devido à ausência de especificação de um limiar de positividade. Todos os estudos foram classificados como baixa risco em relação à aplicabilidade. Esta revisão sistemática demonstrou uma alta concordância entre os diagnósticos por WSI e CLM. É possível confirmar que essa tecnologia pode ser usada para fornecer diagnóstico primário em várias especialidades da patologia humana. O segundo capítulo apresentado neste estudo teve como objetivo validar um sistema WSI para fins de diagnóstico de doenças bucais, utilizando a variabilidade intraobservador como a principal forma de análise. Setenta (n = 70) lâminas de vidro coradas em H&E de biópsias orais foram escaneadas pelo Aperio Digital Pathology System (Aperio Technologies Inc., Vista, CA, EUA) em uma magnificação de 20x. Dois patologistas experientes analisaram cegamente todos os casos com MLC e, após 3 meses de washout, com WSI. Informações clínicas foram fornecidas em ambas as análises. A concordância intraobservador entre os métodos foi de 97% para ambos os patologistas. Entre os casos discordantes, a maioria dos diagnósticos preferidos foi por MLC. Ambos os patologistas tiveram as mesmas discordâncias em diferentes casos. A dificuldade de alguns casos, que possibilitou interpretações controversas, e a pouca quantidade de tecido para análise foram consideradas razões principais de desacordo em detrimento dos métodos de diagnóstico. O valor de tempo (mediana) foi maior apenas com MLC para um patologista e, a melhoria do tempo com WSI está relacionada com o melhor fluxo de trabalho provido pelo sistema WSI. Os valores máximos de tempo ocorreram em casos discordantes e em outros casos considerados difíceis. Este estudo fornece evidências originais de um alto desempenho do sistema WSI para fins de diagnóstico na prática clínica, patologia de rotina e diagnóstico primário no campo da patologia oralAbstract: Digital microscopy (DM) has expanded recently in professional settings for interconsultations, telepathology, storage and routine reporting, what puts whole-slide imaging (WSI) systems in the privileged position of innovative devices for interpretation of primary diagnoses, application previously conceived with fear. This is a direct consequence of the lack of regulation of these devices. It is necessary to assemble evidence regarding the performance of the DM, in order to establish whether this technology can be used to provide primary diagnosis. The first chapter presented in this study aimed to provide information regarding the performance of whole slide imaging (WSI) devices, evaluating intraobserver agreement as the best evidence to elucidate whether digital microscopy (DM) is reliable for primary diagnostic purposes. Scopus, MEDLINE/PubMed and Embase were searched electronically. The methodological characteristics, the intraobserver agreement between conventional light microscopy (CLM) and WSI and the reasons for discordant diagnoses were analysed. Thirteen articles were included. The intraobserver agreements showed an excellent concordance, with values ranging from 90% to 98,3%, (? coef?cient range 0.8¿0.98). Challenging cases were the main reasons for disagreements (46.15%) followed by difficulties in the identification of microorganisms (15.38%). 58,84% emphasize that the performance of the digital method is not related to the occurrence of disagreements. Only 25% of discordant cases had preferred WSI diagnosis. 15.38% presented high risk of bias due to unclear sample selection, and 15.38% due to the absence of specification of a threshold. Regarding to applicability, all studies were classified as a low concern. This systematic review showed a high concordance between diagnoses achieved by using WSI and CLM. These studies were also optimally designed to validate WSI for general clinical use and, most importantly, it is possible to confirm that this technology can be used to provide primary diagnosis in several specialties of human pathology.Second chapter of this study intended to validate a WSI system for diagnostic purposes of oral diseases, using the intraobserver variability as the primary form of analysis. Seventy (n = 70) H&E-stained glass slides of oral biopsies were scanned by the Aperio Digital Pathology System (Aperio Technologies Inc., Vista, CA, USA) at a magnification of 20x. Two experienced pathologists blindly analysed all cases with CLM and, after 3 months washout, with WSI. Clinical information was provided in both analyses. The intraobserver agreement between CLM and WSI system diagnoses was 97% for both pathologists. Among discordances, the majority of preferred diagnoses were by CLM. Both pathologists had the same discordances in different cases. Difficult cases, which allowed controversial interpretations, and the lack of tissue for analyses, were considered main reasons for disagreement rather than the diagnostic methods. Median time was higher only in CLM for one pathologist and the improvement of time in WSI was related to better workflow of WSI. Time outliers occurred in discordant cases and other difficult cases. This study provides original evidence for the high-performance of WSI for diagnostic purposes in clinical practice, routine pathology and primary diagnosis in the field of oral pathologyMestradoEstomatologiaMestra em Estomatopatologia33003033009P4CAPE

Imaging Sensors and Applications

In past decades, various sensor technologies have been used in all areas of our lives, thus improving our quality of life. In particular, imaging sensors have been widely applied in the development of various imaging approaches such as optical imaging, ultrasound imaging, X-ray imaging, and nuclear imaging, and contributed to achieve high sensitivity, miniaturization, and real-time imaging. These advanced image sensing technologies play an important role not only in the medical field but also in the industrial field. This Special Issue covers broad topics on imaging sensors and applications. The scope range of imaging sensors can be extended to novel imaging sensors and diverse imaging systems, including hardware and software advancements. Additionally, biomedical and nondestructive sensing applications are welcome