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1.
Front Neurol ; 15: 1386654, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38817550

RESUMO

Calvarial bone marrow has been found to be central in the brain immune response, being connected to the dura through channels which allow leukocyte trafficking. Temporal bone marrow is thought to play important roles in relation to the inner ear, but is still largely uncharacterized, given this bone complex anatomy. We characterized the geometry and connectivity of rat temporal bone marrow using lightsheet imaging of cleared samples and microCT. Bone marrow was identified in cleared tissue by cellular content (and in particular by the presence of megakaryocytes); since air-filled cavities are absent in rodents, marrow clusters could be recognized in microCT scans by their geometry. In cleared petrosal bone, autofluorescence allowed delineation of the otic capsule layers. Within the endochondral layer, bone marrow was observed in association to the cochlear base and vestibule, and to the cochlear apex. Cochlear apex endochondral marrow (CAEM) was a separated cluster from the remaining endochondral marrow, which was therefore defined as "vestibular endochondral marrow" (VEM). A much larger marrow island (petrosal non-endochondral marrow, PNEM) extended outside the otic capsule surrounding semicircular canal arms. PNEM was mainly connected to the dura, through bone channels similar to those of calvarial bone, and only a few channels were directed toward the canal periosteum. On the contrary, endochondral bone marrow was well connected to the labyrinth through vascular loops (directed to the spiral ligament for CAEM and to the bony labyrinth periosteum for VEM), and to dural sinuses. In addition, CAEM was also connected to the tensor tympani fossa of the middle ear and VEM to the endolymphatic sac. Endochondral marrow was made up of small lobules connected to each other and to other structures by channels lined by elongated macrophages, whereas PNEM displayed larger lobules connected by channels with a sparse macrophage population. Our data suggest that the rat inner ear is surrounded by bone marrow at the junctions with middle ear and brain, most likely with "customs" role, restricting pathogen spread; a second marrow network with different structural features is found within the endochondral bone layer of the otic capsule and may play different functional roles.

2.
Nat Commun ; 15(1): 2679, 2024 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-38538644

RESUMO

In 2015, we launched the mesoSPIM initiative, an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of such microscopes. Here, we introduce the next-generation mesoSPIM ("Benchtop") with a significantly increased field of view, improved resolution, higher throughput, more affordable cost, and simpler assembly compared to the original version. We develop an optical method for testing detection objectives that enables us to select objectives optimal for light-sheet imaging with large-sensor cameras. The improved mesoSPIM achieves high spatial resolution (1.5 µm laterally, 3.3 µm axially) across the entire field of view, magnification up to 20×, and supports sample sizes ranging from sub-mm up to several centimeters while being compatible with multiple clearing techniques. The microscope serves a broad range of applications in neuroscience, developmental biology, pathology, and even physics.


Assuntos
Microscopia , Neurociências , Microscopia/métodos
3.
Nat Biotechnol ; 42(1): 65-71, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36997681

RESUMO

Imaging large, cleared samples requires microscope objectives that combine a large field of view (FOV) with a long working distance (WD) and a high numerical aperture (NA). Ideally, such objectives should be compatible with a wide range of immersion media, which is challenging to achieve with conventional lens-based objective designs. Here we introduce the multi-immersion 'Schmidt objective' consisting of a spherical mirror and an aspherical correction plate as a solution to this problem. We demonstrate that a multi-photon variant of the Schmidt objective is compatible with all homogeneous immersion media and achieves an NA of 1.08 at a refractive index of 1.56, 1.1-mm FOV and 11-mm WD. We highlight its versatility by imaging cleared samples in various media ranging from air and water to benzyl alcohol/benzyl benzoate, dibenzyl ether and ethyl cinnamate and by imaging of neuronal activity in larval zebrafish in vivo. In principle, the concept can be extended to any imaging modality, including wide-field, confocal and light-sheet microscopy.


Assuntos
Telescópios , Animais , Imersão , Microscopia/métodos , Peixe-Zebra
4.
Mol Cancer Ther ; 23(4): 507-519, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38159110

RESUMO

The small-molecule inhibitor of ataxia telangiectasia and Rad3-related protein (ATR), elimusertib, is currently being tested clinically in various cancer entities in adults and children. Its preclinical antitumor activity in pediatric malignancies, however, is largely unknown. We here assessed the preclinical activity of elimusertib in 38 cell lines and 32 patient-derived xenograft (PDX) models derived from common pediatric solid tumor entities. Detailed in vitro and in vivo molecular characterization of the treated models enabled the evaluation of response biomarkers. Pronounced objective response rates were observed for elimusertib monotherapy in PDX, when treated with a regimen currently used in clinical trials. Strikingly, elimusertib showed stronger antitumor effects than some standard-of-care chemotherapies, particularly in alveolar rhabdomysarcoma PDX. Thus, elimusertib has strong preclinical antitumor activity in pediatric solid tumor models, which may translate to clinically meaningful responses in patients.


Assuntos
Antineoplásicos , Neoplasias , Criança , Humanos , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Biomarcadores , Linhagem Celular Tumoral
5.
Psychol Methods ; 28(5): 1005-1028, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37471017

RESUMO

Latent state-trait (LST) models are increasingly applied in psychology. Although existing LST models offer many possibilities for analyzing variability and change, they do not allow researchers to relate time-varying or time-invariant covariates, or a combination of both, to loading, intercept, and factor variance parameters in LST models. We present a general framework for the inclusion of nominal and/or continuous time-varying and time-invariant covariates in LST models. The new framework builds on modern LST theory and Bayesian moderated nonlinear factor analysis and is termed moderated nonlinear LST (MN-LST) framework. The MN-LST framework offers new modeling possibilities and allows for a fine-grained analysis of trait change, person-by-situation interaction effects, as well as inter- or intraindividual variability. The new MN-LST approach is compared to alternative modeling strategies. The advantages of the MN-LST approach are illustrated in an empirical application examining dyadic coping in romantic relationships. Finally, the advantages and limitations of the approach are discussed, and practical recommendations are provided. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

6.
Angew Chem Int Ed Engl ; 62(42): e202309274, 2023 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-37515774

RESUMO

The chemistry of alkynyl triazenes is an emerging field for organic chemists and especially acid-induced nucleophilic functionalizations, either directly, or after a prior reaction towards aromatic triazenes under extrusion of nitrogen, paved the way for fruitful strategies. In contrast, the chemical behavior of alkynyl triazenes upon irradiation with light is still unknown. Herein we present the first photoactivation of alkynyl triazenes that triggers an uncommon reactivity pattern involving the cleavage of the N1-N2 bond of the triazene moiety resulting in a unique approach to cyanocarbenes from a readily available, stable, and insensitive precursor. This allows to access various nitrile compounds without the use of a toxic cyanating agent by exploiting the reactivity pattern of carbenes. By variation of the reaction conditions and light sources, different substitution patterns can be obtained selectively in good yields under mild and metal-free conditions, thus introducing the alkynyl triazene unit as a photo accessible methylene nitrile synthon. Using this synthon, subclasses like α-alkoxynitriles, α-aminonitriles and α-cyanohydrazones become easily available. These exhibit synthetically valuable substitution patterns for the synthesis of pharmaceuticals, intermediates for total synthesis and amino acid synthesis.

7.
Res Social Adm Pharm ; 19(5): 807-820, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36858904

RESUMO

BACKGROUND: Brief interventions for lifestyle behaviour change are effective health promotion interventions. Primary care settings, including pharmacies, are the most frequently visited healthcare facilities and are well placed to provide brief health interventions. However, despite the evidence-based and policy guidance, barriers to brief interventions have limited their implementation. OBJECTIVES: This study aimed to explore the usability and acceptability of HealthEir, a blended model with digital, print, and communication elements designed to support the delivery of brief health-promoting interventions in pharmacy practice. METHODS: Usability tests using a think aloud protocol and set tasks (to assess usability) and semi-structured interviews (to assess acceptability) were conducted with fifteen patients and twelve pharmacists. Usability data were analysed deductively using Nielsen's five quality components of usability as a framework. Acceptability data were analysed using inductive thematic analysis. FINDINGS: All participants found HealthEir straightforward to use and completed the required tasks without assistance. A small number of patient and pharmacist participants identified opportunity for improvement of the digital element. Acceptability was also high, with individual comments from participants identifying opportunity for improvements to HealthEir, and two patients identifying privacy or safety concerns. The majority of comments regarding acceptability reflected wider social and cultural challenges such as perceptions of the roles of pharmacists and pharmacies and the low priority of preventative health interventions. CONCLUSION: HealthEir has been demonstrated to be a usable and acceptable support that has the potential to overcome barriers to delivery of brief interventions in pharmacies. Usability and acceptability testing identified minor changes that may further improve its design prior to piloting and implementation.


Assuntos
Serviços Comunitários de Farmácia , Assistência Farmacêutica , Farmácias , Humanos , Intervenção em Crise , Pesquisa Qualitativa , Farmacêuticos , Atenção Primária à Saúde
8.
Nat Protoc ; 18(4): 1197-1242, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36697871

RESUMO

Homeostatic and pathological phenomena often affect multiple organs across the whole organism. Tissue clearing methods, together with recent advances in microscopy, have made holistic examinations of biological samples feasible. Here, we report the detailed protocol for nanobody(VHH)-boosted 3D imaging of solvent-cleared organs (vDISCO), a pressure-driven, nanobody-based whole-body immunolabeling and clearing method that renders whole mice transparent in 3 weeks, consistently enhancing the signal of fluorescent proteins, stabilizing them for years. This allows the reliable detection and quantification of fluorescent signal in intact rodents enabling the analysis of an entire body at cellular resolution. Here, we show the high versatility of vDISCO applied to boost the fluorescence signal of genetically expressed reporters and clear multiple dissected organs and tissues, as well as how to image processed samples using multiple fluorescence microscopy systems. The entire protocol is accessible to laboratories with limited expertise in tissue clearing. In addition to its applications in obtaining a whole-mouse neuronal projection map, detecting single-cell metastases in whole mice and identifying previously undescribed anatomical structures, we further show the visualization of the entire mouse lymphatic system, the application for virus tracing and the visualization of all pericytes in the brain. Taken together, our vDISCO pipeline allows systematic and comprehensive studies of cellular phenomena and connectivity in whole bodies.


Assuntos
Encéfalo , Imageamento Tridimensional , Camundongos , Animais , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Solventes/química , Neuritos , Corantes
9.
EMBO Mol Med ; 15(1): e16789, 2023 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-36382364

RESUMO

Many efforts targeting amyloid-ß (Aß) plaques for the treatment of Alzheimer's Disease thus far have resulted in failures during clinical trials. Regional and temporal heterogeneity of efficacy and dependence on plaque maturity may have contributed to these disappointing outcomes. In this study, we mapped the regional and temporal specificity of various anti-Aß treatments through high-resolution light-sheet imaging of electrophoretically cleared brains. We assessed the effect on amyloid plaque formation and growth in Thy1-APP/PS1 mice subjected to ß-secretase inhibitors, polythiophenes, or anti-Aß antibodies. Each treatment showed unique spatiotemporal Aß clearance, with polythiophenes emerging as a potent anti-Aß compound. Furthermore, aligning with a spatial-transcriptomic atlas revealed transcripts that correlate with the efficacy of each Aß therapy. As observed in this study, there is a striking dependence of specific treatments on the location and maturity of Aß plaques. This may also contribute to the clinical trial failures of Aß-therapies, suggesting that combinatorial regimens may be significantly more effective in clearing amyloid deposition.


Assuntos
Doença de Alzheimer , Microscopia , Camundongos , Animais , Camundongos Transgênicos , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/tratamento farmacológico , Encéfalo/metabolismo , Placa Amiloide/tratamento farmacológico , Modelos Animais de Doenças , Precursor de Proteína beta-Amiloide , Presenilina-1/farmacologia
10.
bioRxiv ; 2023 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-38168219

RESUMO

In 2015, we launched the mesoSPIM initiative (www.mesospim.org), an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of light-sheet microscopy. Here, we introduce the next-generation mesoSPIM ("Benchtop") with significantly increased field of view, improved resolution, higher throughput, more affordable cost and simpler assembly compared to the original version. We developed a new method for testing objectives, enabling us to select detection objectives optimal for light-sheet imaging with large-sensor sCMOS cameras. The new mesoSPIM achieves high spatial resolution (1.5 µm laterally, 3.3 µm axially) across the entire field of view, a magnification up to 20x, and supports sample sizes ranging from sub-mm up to several centimetres, while being compatible with multiple clearing techniques. The new microscope serves a broad range of applications in neuroscience, developmental biology, and even physics.

11.
Nat Protoc ; 17(9): 2025-2053, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35831614

RESUMO

Light-sheet fluorescence microscopy is a rapidly growing technique that has gained tremendous popularity in the life sciences owing to its high-spatiotemporal resolution and gentle, non-phototoxic illumination. In this protocol, we provide detailed directions for the assembly and operation of a versatile light-sheet fluorescence microscopy variant, referred to as axially swept light-sheet microscopy (ASLM), that delivers an unparalleled combination of field of view, optical resolution and optical sectioning. To democratize ASLM, we provide an overview of its working principle and applications to biological imaging, as well as pragmatic tips for the assembly, alignment and control of its optical systems. Furthermore, we provide detailed part lists and schematics for several variants of ASLM that together can resolve molecular detail in chemically expanded samples, subcellular organization in living cells or the anatomical composition of chemically cleared intact organisms. We also provide software for instrument control and discuss how users can tune imaging parameters to accommodate diverse sample types. Thus, this protocol will serve not only as a guide for both introductory and advanced users adopting ASLM, but as a useful resource for any individual interested in deploying custom imaging technology. We expect that building an ASLM will take ~1-2 months, depending on the experience of the instrument builder and the version of the instrument.


Assuntos
Imageamento Tridimensional , Software , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos
12.
Nat Biomed Eng ; 6(9): 1031-1044, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35835994

RESUMO

Deposits of amyloid-ß (Aß) in the brains of rodents can be analysed by invasive intravital microscopy on a submillimetre scale, or via whole-brain images from modalities lacking the resolution or molecular specificity to accurately characterize Aß pathologies. Here we show that large-field multifocal illumination fluorescence microscopy and panoramic volumetric multispectral optoacoustic tomography can be combined to longitudinally assess Aß deposits in transgenic mouse models of Alzheimer's disease. We used fluorescent Aß-targeted probes (the luminescent conjugated oligothiophene HS-169 and the oxazine-derivative AOI987) to transcranially detect Aß deposits in the cortex of APP/PS1 and arcAß mice with single-plaque resolution (8 µm) and across the whole brain (including the hippocampus and the thalamus, which are inaccessible by conventional intravital microscopy) at sub-150 µm resolutions. Two-photon microscopy, light-sheet microscopy and immunohistochemistry of brain-tissue sections confirmed the specificity and regional distributions of the deposits. High-resolution multiscale optical and optoacoustic imaging of Aß deposits across the entire brain in rodents thus facilitates the in vivo study of Aß accumulation by brain region and by animal age and strain.


Assuntos
Peptídeos beta-Amiloides , Placa Amiloide , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Oxazinas , Placa Amiloide/patologia
13.
Int J Infect Dis ; 117: 130-136, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34022333

RESUMO

INTRODUCTION: Latin America accounts for one-quarter of global COVID-19 cases and one-third of deaths. Inequalities in the region lead to barriers to the best use of diagnostic tests during the pandemic. There is a need for simplified guidelines that consider the region's limited health resources, international guidelines, medical literature, and local expertise. METHODS: Using a modified Delphi method, 9 experts from Latin American countries developed a simplified algorithm for COVID-19 diagnosis on the basis of their answers to 24 questions related to diagnostic settings, and discussion of the literature and their experiences. RESULTS: The algorithm considers 3 timeframes (≤7 days, 8-13 days, and ≥14 days) and presents diagnostic options for each. SARS-CoV-2 real- time reverse transcription-polymerase chain reaction is the test of choice from day 1 to 14 after symptom onset or close contact, although antigen testing may be used in specific circumstances, from day 5 to 7. Antibody assays may be used for confirmation, usually after day 14; however, if clinical suspicion is very high, but other tests are negative, these assays may be used as an adjunct to decision-making from day 8 to 13. CONCLUSION: The proposed algorithm aims to support COVID-19 diagnosis decision-making in Latin America.


Assuntos
COVID-19 , SARS-CoV-2 , Algoritmos , COVID-19/diagnóstico , Teste para COVID-19 , Consenso , Humanos , América Latina/epidemiologia
14.
Development ; 148(21)2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34739029

RESUMO

Genome editing simplifies the generation of new animal models for congenital disorders. However, the detailed and unbiased phenotypic assessment of altered embryonic development remains a challenge. Here, we explore how deep learning (U-Net) can automate segmentation tasks in various imaging modalities, and we quantify phenotypes of altered renal, neural and craniofacial development in Xenopus embryos in comparison with normal variability. We demonstrate the utility of this approach in embryos with polycystic kidneys (pkd1 and pkd2) and craniofacial dysmorphia (six1). We highlight how in toto light-sheet microscopy facilitates accurate reconstruction of brain and craniofacial structures within X. tropicalis embryos upon dyrk1a and six1 loss of function or treatment with retinoic acid inhibitors. These tools increase the sensitivity and throughput of evaluating developmental malformations caused by chemical or genetic disruption. Furthermore, we provide a library of pre-trained networks and detailed instructions for applying deep learning to the reader's own datasets. We demonstrate the versatility, precision and scalability of deep neural network phenotyping on embryonic disease models. By combining light-sheet microscopy and deep learning, we provide a framework for higher-throughput characterization of embryonic model organisms. This article has an associated 'The people behind the papers' interview.


Assuntos
Aprendizado Profundo , Desenvolvimento Embrionário/genética , Fenótipo , Animais , Anormalidades Craniofaciais/embriologia , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/patologia , Modelos Animais de Doenças , Processamento de Imagem Assistida por Computador , Camundongos , Microscopia , Mutação , Redes Neurais de Computação , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/patologia , Doenças Renais Policísticas/embriologia , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/patologia , Proteínas de Xenopus/genética , Xenopus laevis
15.
Dalton Trans ; 50(42): 15296-15309, 2021 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-34636381

RESUMO

The di(hydroperoxy)adamantane adducts of water (1) and phosphine oxides p-Tol3PO·(HOO)2C(C9H14) (2), o-Tol3PO·(HOO)2C(C9H14) (3), and Cy3PO·(HOO)2C(C9H14) (4), as well as a CH2Cl2 adduct of a phosphole oxide dimer (8), have been created and investigated by multinuclear NMR spectroscopy, and by Raman and IR spectroscopy. The single crystal X-ray structures for 1-4 and 8 are reported. The IR and 31P NMR data are in accordance with strong hydrogen bonding of the di(hydroperoxy)adamantane adducts. The Raman ν(O-O) stretching bands of 1-4 prove that the peroxo groups are present in the solids. Selected di(hydroperoxy)alkane adducts, in combination with AlCl3 as catalyst, have been applied for the direct oxidative esterification of n-nonyl aldehyde, benzaldehyde, p-methylbenzaldehyde, p-bromobenzaldehyde, and o-hydroxybenzaldehyde to the corresponding methyl esters. The esterification takes place in an inert atmosphere, under anhydrous and oxygen-free conditions, within a time frame of 45 minutes to 5 hours at room temperature. Hereby, two oxygen atoms per adduct assembly are active with respect to the quantitative transformation of the aldehyde into the ester.

16.
Front Cell Dev Biol ; 9: 692617, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34395426

RESUMO

The choroid plexus (CP) acts as a regulated gate between blood and cerebrospinal fluid (CSF). Despite its simple histology (a monostratified cuboidal epithelium overlying a vascularized stroma), this organ has remarkably complex functions several of which involve local interaction with cells located around ventricle walls. Our knowledge of CP structural organization is mainly derived from resin casts, which capture the overall features but only allow reconstruction of the vascular pattern surface, unrelated to the overlying epithelium and only loosely related to ventricular location. Recently, CP single cell atlases are starting to emerge, providing insight on local heterogeneities and interactions. So far, however, few studies have described CP spatial organization at the mesoscale level, because of its fragile nature and deep location within the brain. Here, using an iDISCO-based clearing approach and light-sheet microscopy, we have reconstructed the normal rat hindbrain CP (hCP) macro- and microstructure, using markers for epithelium, arteries, microvasculature, and macrophages, and noted its association with 4th ventricle-related neurovascular structures. The hCP is organized in domains associated to a main vessel (fronds) which carry a variable number of villi; the latter are enclosed by epithelium and may be flat (leaf-like) or rolled up to variable extent. Arteries feeding the hCP emerge from the cerebellar surface, and branch into straight arterioles terminating as small capillary anastomotic networks, which run within a single villus and terminate attaching multiple times to a large tortuous capillary (LTC) which ends into a vein. Venous outflow mostly follows arterial pathways, except for the lateral horizontal segment (LHS) and the caudal sagittal segment. The structure of fronds and villi is related to the microvascular pattern at the hCP surface: when LTCs predominate, leaflike villi are more evident and bulge from the surface; different, corkscrew-like villi are observed in association to arterioles reaching close to the CP surface with spiraling capillaries surrounding them. Both leaf-like and corkscrew-like villi may reach the 4th ventricle floor, making contact points at their tip, where no gap is seen between CP epithelium and ependyma. Contacts usually involve several adjacent villi and may harbor epiplexus macrophages. At the junction between medial (MHS) and lateral (LHS) horizontal segment, arterial supply is connected to the temporal bone subarcuate fossa, and venous outflow drains to a ventral vein which exits through the cochlear nuclei at the Luschka foramen. These vascular connections stabilize the hCP overall structure within the 4th ventricle but make MHS-LHS joint particularly fragile and very easily damaged when removing the brain from the skull. Even in damaged samples, however, CP fronds (or isolated villi) often remain strongly attached to the dorsal cochlear nucleus (DCN) surface; in these fronds, contacts are still present and connecting "bridges" may be seen, suggesting the presence of real molecular contacts rather than mere appositions.

17.
Res Social Adm Pharm ; 17(12): 2036-2043, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34229952

RESUMO

Challenges faced by health systems have become increasingly complex, and expanding the range of methodological options available via interdisciplinary collaboration is important to enable researchers to address them. As complexity increases, it can be more difficult to ensure solutions remain patient-centered. Human-centered design is an approach that focuses on engaging with and understanding the needs of all services users while retaining a systems perspective. Therefore, design professionals skilled in these approaches are increasingly collaborating within health systems in pharmacy and health research teams. This methodological paper considers the potential contribution of human-centered design approaches to optimising development, implementation, and sustainability of patient-centered interventions in pharmacy and health services research. It provides an overview of human-centered design principles and their application, and outlines the emerging roles of design professionals in pharmacy and health services research. It focuses on three key human-centered design methods that can most readily be used by pharmacy and health services researchers. Journey mapping, prototyping, and user testing are discussed in detail. Journey mapping enables holistic visualisation of patient experience from practical and emotional perspectives. It may be used to visualize current practice or model potential future services, and can be informed by quantitative and qualitative data derived from both primary and secondary research. Prototyping facilitates exploration of interventions such as new services quickly and at low-cost. Health services researchers can utilize prototypes for services, processes, experiences, physical objects, environments, spaces, or digital tools for example. Formative evaluation and user testing supports rapid iteration of prototypes to ensure that they meet patient and healthcare professional needs. Finally, challenges with interdisciplinary collaboration and strategies to maximize the potential of using human-centered design approaches in pharmacy and health services research to address complex challenges, enhance practice and deliver benefits for service users, patients, and health systems are discussed.


Assuntos
Assistência Farmacêutica , Farmácias , Farmácia , Pessoal de Saúde , Pesquisa sobre Serviços de Saúde , Humanos
18.
Nat Protoc ; 16(6): 2732-2748, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34021294

RESUMO

Tissue clearing has become a powerful technique for studying anatomy and morphology at scales ranging from entire organisms to subcellular features. With the recent proliferation of tissue-clearing methods and imaging options, it can be challenging to determine the best clearing protocol for a particular tissue and experimental question. The fact that so many clearing protocols exist suggests there is no one-size-fits-all approach to tissue clearing and imaging. Even in cases where a basic level of clearing has been achieved, there are many factors to consider, including signal retention, staining (labeling), uniformity of transparency, image acquisition and analysis. Despite reviews citing features of clearing protocols, it is often unknown a priori whether a protocol will work for a given experiment, and thus some optimization is required by the end user. In addition, the capabilities of available imaging setups often dictate how the sample needs to be prepared. After imaging, careful evaluation of volumetric image data is required for each combination of clearing protocol, tissue type, biological marker, imaging modality and biological question. Rather than providing a direct comparison of the many clearing methods and applications available, in this tutorial we address common pitfalls and provide guidelines for designing, optimizing and imaging in a successful tissue-clearing experiment with a focus on light-sheet fluorescence microscopy (LSFM).


Assuntos
Técnicas de Preparação Histocitológica , Microscopia de Fluorescência , Animais , Humanos
19.
Front Synaptic Neurosci ; 13: 643138, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33867967

RESUMO

Projections from the lateral habenula (LHb) control ventral tegmental area (VTA) neuronal populations' activity and both nuclei shape the pathological behaviors emerging during cocaine withdrawal. However, it is unknown whether cocaine withdrawal modulates LHb neurotransmission onto subsets of VTA neurons that are part of distinct neuronal circuits. Here we show that, in mice, cocaine withdrawal, drives discrete and opposing synaptic adaptations at LHb inputs onto VTA neurons defined by their output synaptic connectivity. LHb axons innervate the medial aspect of VTA, release glutamate and synapse on to dopamine and non-dopamine neuronal populations. VTA neurons receiving LHb inputs project their axons to medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and lateral hypothalamus (LH). While cocaine withdrawal increases glutamate release from LHb onto VTA-mPFC projectors, it reduces presynaptic release onto VTA-NAc projectors, leaving LHb synapses onto VTA-to-LH unaffected. Altogether, cocaine withdrawal promotes distinct adaptations at identified LHb-to-VTA circuits, which provide a framework for understanding the circuit basis of the negative states emerging during abstinence of drug intake.

20.
Neuron ; 109(1): 135-148.e6, 2021 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-33159842

RESUMO

In the neocortex, each sensory modality engages distinct sensory areas that route information to association areas. Where signal flow converges for maintaining information in short-term memory and how behavior may influence signal routing remain open questions. Using wide-field calcium imaging, we compared cortex-wide neuronal activity in layer 2/3 for mice trained in auditory and tactile tasks with delayed response. In both tasks, mice were either active or passive during stimulus presentation, moving their body or sitting quietly. Irrespective of behavioral strategy, auditory and tactile stimulation activated distinct subdivisions of the posterior parietal cortex, anterior area A and rostrolateral area RL, which held stimulus-related information necessary for the respective tasks. In the delay period, in contrast, behavioral strategy rather than sensory modality determined short-term memory location, with activity converging frontomedially in active trials and posterolaterally in passive trials. Our results suggest behavior-dependent routing of sensory-driven cortical signals flow from modality-specific posterior parietal cortex (PPC) subdivisions to higher association areas.


Assuntos
Percepção Auditiva/fisiologia , Aprendizagem por Discriminação/fisiologia , Memória de Curto Prazo/fisiologia , Neocórtex/fisiologia , Tato/fisiologia , Estimulação Acústica/métodos , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neocórtex/química , Optogenética/métodos , Estimulação Física/métodos , Transdução de Sinais/fisiologia
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