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1.
J Microsc ; 294(3): 397-410, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38691400

RESUMEN

In the dynamic landscape of scientific research, imaging core facilities are vital hubs propelling collaboration and innovation at the technology development and dissemination frontier. Here, we present a collaborative effort led by Global BioImaging (GBI), introducing international recommendations geared towards elevating the careers of Imaging Scientists in core facilities. Despite the critical role of Imaging Scientists in modern research ecosystems, challenges persist in recognising their value, aligning performance metrics and providing avenues for career progression and job security. The challenges encompass a mismatch between classic academic career paths and service-oriented roles, resulting in a lack of understanding regarding the value and impact of Imaging Scientists and core facilities and how to evaluate them properly. They further include challenges around sustainability, dedicated training opportunities and the recruitment and retention of talent. Structured across these interrelated sections, the recommendations within this publication aim to propose globally applicable solutions to navigate these challenges. These recommendations apply equally to colleagues working in other core facilities and research institutions through which access to technologies is facilitated and supported. This publication emphasises the pivotal role of Imaging Scientists in advancing research programs and presents a blueprint for fostering their career progression within institutions all around the world.


Asunto(s)
Investigadores , Humanos , Movilidad Laboral , Investigación Biomédica/métodos , Selección de Profesión
2.
ArXiv ; 2024 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-38351940

RESUMEN

Together with the molecular knowledge of genes and proteins, biological images promise to significantly enhance the scientific understanding of complex cellular systems and to advance predictive and personalized therapeutic products for human health. For this potential to be realized, quality-assured image data must be shared among labs at a global scale to be compared, pooled, and reanalyzed, thus unleashing untold potential beyond the original purpose for which the data was generated. There are two broad sets of requirements to enable image data sharing in the life sciences. One set of requirements is articulated in the companion White Paper entitled "Enabling Global Image Data Sharing in the Life Sciences," which is published in parallel and addresses the need to build the cyberinfrastructure for sharing the digital array data (arXiv:2401.13023 [q-bio.OT], https://doi.org/10.48550/arXiv.2401.13023). In this White Paper, we detail a broad set of requirements, which involves collecting, managing, presenting, and propagating contextual information essential to assess the quality, understand the content, interpret the scientific implications, and reuse image data in the context of the experimental details. We start by providing an overview of the main lessons learned to date through international community activities, which have recently made considerable progress toward generating community standard practices for imaging Quality Control (QC) and metadata. We then provide a clear set of recommendations for amplifying this work. The driving goal is to address remaining challenges, and democratize access to common practices and tools for a spectrum of biomedical researchers, regardless of their expertise, access to resources, and geographical location.

3.
Microsc Microanal ; 29(2): 616-634, 2023 04 05.
Artículo en Inglés | MEDLINE | ID: mdl-37749742

RESUMEN

This article outlines a global study conducted by the Association of Biomedical Resource Facilities (ABRF) Light Microscopy Research Group (LMRG). The results present a novel 3D tissue-like biologically relevant standard sample that is affordable and straightforward to prepare. Detailed sample preparation, instrument-specific image acquisition protocols and image analysis methods are presented and made available to the community. The standard consists of sub-resolution and large well characterized relative intensity fluorescence microspheres embedded in a 120 µm thick 3D gel with a refractive index of 1.365. The standard allows the evaluation of several properties as a function of depth. These include the following: 1) microscope resolution with automated analysis of the point-spread function (PSF), 2) automated signal-to-noise ratio analysis, 3) calibration and correction of fluorescence intensity loss, and 4) quantitative relative intensity. Results demonstrate expected refractive index mismatch dependent losses in intensity and resolution with depth, but the relative intensities of different objects at similar depths are maintained. This is a robust standard showing reproducible results across laboratories, microscope manufacturers and objective lens types (e.g., magnification, immersion medium). Thus, these tools will be valuable for the global community to benchmark fluorescence microscopes and will contribute to improved scientific rigor and reproducibility.


Asunto(s)
Procesamiento de Imagen Asistido por Computador , Reproducibilidad de los Resultados , Microscopía Fluorescente/métodos
4.
bioRxiv ; 2023 Aug 17.
Artículo en Inglés | MEDLINE | ID: mdl-37398479

RESUMEN

Antibodies are critical reagents to detect and characterize proteins. It is commonly understood that many commercial antibodies do not recognize their intended targets, but information on the scope of the problem remains largely anecdotal, and as such, feasibility of the goal of at least one potent and specific antibody targeting each protein in a proteome cannot be assessed. Focusing on antibodies for human proteins, we have scaled a standardized characterization approach using parental and knockout cell lines (Laflamme et al., 2019) to assess the performance of 614 commercial antibodies for 65 neuroscience-related proteins. Side-by-side comparisons of all antibodies against each target, obtained from multiple commercial partners, demonstrates that: i) more than 50% of all antibodies failed in one or more tests, ii) yet, ~50-75% of the protein set was covered by at least one high-performing antibody, depending on application, suggesting that coverage of human proteins by commercial antibodies is significant; and iii) recombinant antibodies performed better than monoclonal or polyclonal antibodies. The hundreds of underperforming antibodies identified in this study were found to have been used in a large number of published articles, which should raise alarm. Encouragingly, more than half of the underperforming commercial antibodies were reassessed by the manufacturers, and many had alterations to their recommended usage or were removed from the market. This first such study helps demonstrate the scale of the antibody specificity problem but also suggests an efficient strategy toward achieving coverage of the human proteome; mine the existing commercial antibody repertoire, and use the data to focus new renewable antibody generation efforts.

5.
Sci Rep ; 13(1): 4223, 2023 03 14.
Artículo en Inglés | MEDLINE | ID: mdl-36918704

RESUMEN

In mesenchymal cell motility, several migration patterns have been observed, including directional, exploratory and stationary. Two key members of the Rho-family of GTPases, Rac and Rho, along with an adaptor protein called paxillin, have been particularly implicated in the formation of such migration patterns and in regulating adhesion dynamics. Together, they form a key regulatory network that involves the mutual inhibition exerted by Rac and Rho on each other and the promotion of Rac activation by phosphorylated paxillin. Although this interaction is sufficient in generating wave-pinning that underscores cellular polarization comprised of cellular front (high active Rac) and back (high active Rho), it remains unclear how they interact collectively to induce other modes of migration detected in Chinese hamster Ovary (CHO-K1) cells. We previously developed a six-variable (6V) reaction-diffusion model describing the interactions of these three proteins (in their active/phosphorylated and inactive/unphosphorylated forms) along with other auxiliary proteins, to decipher their role in generating wave-pinning. In this study, we explored, through computational modeling and image analysis, how differences in timescales within this molecular network can potentially produce the migration patterns in CHO-K1 cells and how switching between migration modes could occur. To do so, the 6V model was reduced to an excitable 4V spatiotemporal model possessing three different timescales. The model produced not only wave-pinning in the presence of diffusion, but also mixed-mode oscillations (MMOs) and relaxation oscillations (ROs). Implementing the model using the Cellular Potts Model (CPM) produced outcomes in which protrusions in the cell membrane changed Rac-Rho localization, resulting in membrane oscillations and fast directionality variations similar to those observed experimentally in CHO-K1 cells. The latter was assessed by comparing the migration patterns of experimental with CPM cells using four metrics: instantaneous cell speed, exponent of mean-square displacement ([Formula: see text]-value), directionality ratio and protrusion rate. Variations in migration patterns induced by mutating paxillin's serine 273 residue were also captured by the model and detected by a machine classifier, revealing that this mutation alters the dynamics of the system from MMOs to ROs or nonoscillatory behaviour through variation in the scaled concentration of an active form of an adhesion protein called p21-Activated Kinase 1 (PAK). These results thus suggest that MMOs and adhesion dynamics are the key mechanisms regulating CHO-K1 cell motility.


Asunto(s)
Proteínas de Unión al GTP rac , Proteínas de Unión al GTP rho , Animales , Cricetinae , Paxillin/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Células CHO , Especies Reactivas de Oxígeno/metabolismo , Cricetulus , Movimiento Celular/fisiología , Proteínas de Unión al GTP rac/metabolismo , Polaridad Celular
10.
Nat Methods ; 18(12): 1489-1495, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34862503

RESUMEN

For quality, interpretation, reproducibility and sharing value, microscopy images should be accompanied by detailed descriptions of the conditions that were used to produce them. Micro-Meta App is an intuitive, highly interoperable, open-source software tool that was developed in the context of the 4D Nucleome (4DN) consortium and is designed to facilitate the extraction and collection of relevant microscopy metadata as specified by the recent 4DN-BINA-OME tiered-system of Microscopy Metadata specifications. In addition to substantially lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training purposes.


Asunto(s)
Metadatos , Microscopía Confocal/instrumentación , Microscopía Confocal/métodos , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Aplicaciones Móviles , Lenguajes de Programación , Programas Informáticos , Animales , Línea Celular , Biología Computacional/métodos , Humanos , Procesamiento de Imagen Asistido por Computador , Ratones , Reconocimiento de Normas Patrones Automatizadas , Control de Calidad , Reproducibilidad de los Resultados , Interfaz Usuario-Computador , Flujo de Trabajo
11.
Curr Protoc ; 1(10): e304, 2021 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-34662501
13.
Curr Protoc ; 1(9): e243, 2021 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-34516049

RESUMEN

Traditional arc-based light sources and Light Emitting Diodes (LEDs) are described, and the pros and cons of these sources with respect to fluorescence microscopy are discussed. For multi-color applications, arc-based light sources offer white light ranging from the ultraviolet (UV) to the infrared (IR), while LEDs come in a range of colors spanning the same wavelengths. The power of traditional arc-based sources is controlled with neutral density (ND) filters, reducing power across the entire range of wavelengths, while LED-based sources can be controlled directly by modulating current passing through the electronics. Similarly, arc-based sources use physical shutters to control sample exposure to light in a range of tens to hundreds of milliseconds (ms), while LEDs can be turned ON/OFF electronically in <1 ms. The complexity of comparing and measuring light power on the sample, due to normalization of available light source spectra and complex power measurements, is discussed. The superiority of LEDs for stability of light power output is covered. Direct coupling of light sources to the microscope is more cost effective and leads to higher available light power. Various options for setting up multi-color imaging, including high-speed imaging with multiple LEDs and a triple cube, are described. A brief introduction to lasers, with suggested further reading, is included in this article. Finally, the smaller environmental footprint of LEDs relative to arc-based light sources is highlighted. © 2021 Wiley Periodicals LLC.


Asunto(s)
Microscopía Fluorescente
14.
Autism Res ; 14(12): 2663-2676, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34545706

RESUMEN

The COVID-19 pandemic has had a significant impact on the mental health and wellbeing of the world's population, with particularly negative effects on vulnerable populations, including autistic people. Although some consensus regarding specific impact on aspects of wellbeing and mental health in autism is starting to emerge, it is unclear whether the pandemic has increased suicide risk. The goals of this study were to examine (a) potential associations between COVID-19 impact and depression, personal wellbeing, and suicide risk factors in Australian autistic adults and (b) age and gender effects. The COVID-19 Impact Scale (CIS), Personal Wellbeing Index, Patient Health Questionnaire, and the Suicide Behavior Questionnaire, Revised (SBQ-R), were administered to 111 autistic adults aged 20 to 71 years during the second wave of the COVID-19 pandemic in Australia. COVID-19 impact showed small associations with poorer personal wellbeing (r = -0.224, p = 0.023, [-0.409, -0.016]) and higher depressive symptoms (r = 0.268, p = 0.006, [0.056, 0.445]) and was not associated with the SBQ-R suicide risk score (r = 0.081, p = 0.418, [-0.118, 0.264). No significant effects were identified for age. Although model results were similar for women and men, the strength of the associations between personal wellbeing and depression (z = -2.16, p = 0.015), and depression and SBQ-R suicide risk (z = 1.961, p = 0.025), were stronger in women than in men. Qualitative analysis of an open response question from the CIS suggested that the pandemic had both positive and negative impacts on participants. The COVID-19 pandemic has had a large impact on the mental health and wellbeing of the world's population, particularly vulnerable populations such as autistic people. It is not known if these impacts on mental health and wellbeing have increased suicide risk. Our findings suggest that the impact of the COVID-19 pandemic may be associated with poorer wellbeing and higher depression, but is not associated with suicide risk. Overall, autistic people reported both positive and negative impacts of the pandemic on their lives.


Asunto(s)
Trastorno del Espectro Autista , Trastorno Autístico , COVID-19 , Suicidio , Adulto , Australia/epidemiología , Depresión/epidemiología , Femenino , Humanos , Masculino , Pandemias , Factores de Riesgo , SARS-CoV-2
15.
Nat Commun ; 12(1): 4697, 2021 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-34349123

RESUMEN

Polarized epithelial cells can organize into complex structures with a characteristic central lumen. Lumen formation requires that cells coordinately orient their polarity axis so that the basolateral domain is on the outside and apical domain inside epithelial structures. Here we show that the transmembrane aminopeptidase, CD13, is a key determinant of epithelial polarity orientation. CD13 localizes to the apical membrane and associates with an apical complex with Par6. CD13-deficient cells display inverted polarity in which apical proteins are retained on the outer cell periphery and fail to accumulate at an intercellular apical initiation site. Here we show that CD13 is required to couple apical protein cargo to Rab11-endosomes and for capture of endosomes at the apical initiation site. This role in polarity utilizes the short intracellular domain but is independent of CD13 peptidase activity.


Asunto(s)
Antígenos CD13/metabolismo , Polaridad Celular , Células Epiteliales/citología , Epitelio/crecimiento & desarrollo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antígenos CD13/química , Antígenos CD13/genética , Células CACO-2 , Membrana Celular/metabolismo , Endocitosis , Endosomas/metabolismo , Células Epiteliales/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Dominios Proteicos , Proteínas de Unión al GTP rab/metabolismo
16.
J Microsc ; 284(1): 56-73, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34214188

RESUMEN

A modern day light microscope has evolved from a tool devoted to making primarily empirical observations to what is now a sophisticated , quantitative device that is an integral part of both physical and life science research. Nowadays, microscopes are found in nearly every experimental laboratory. However, despite their prevalent use in capturing and quantifying scientific phenomena, neither a thorough understanding of the principles underlying quantitative imaging techniques nor appropriate knowledge of how to calibrate, operate and maintain microscopes can be taken for granted. This is clearly demonstrated by the well-documented and widespread difficulties that are routinely encountered in evaluating acquired data and reproducing scientific experiments. Indeed, studies have shown that more than 70% of researchers have tried and failed to repeat another scientist's experiments, while more than half have even failed to reproduce their own experiments. One factor behind the reproducibility crisis of experiments published in scientific journals is the frequent underreporting of imaging methods caused by a lack of awareness and/or a lack of knowledge of the applied technique. Whereas quality control procedures for some methods used in biomedical research, such as genomics (e.g. DNA sequencing, RNA-seq) or cytometry, have been introduced (e.g. ENCODE), this issue has not been tackled for optical microscopy instrumentation and images. Although many calibration standards and protocols have been published, there is a lack of awareness and agreement on common standards and guidelines for quality assessment and reproducibility. In April 2020, the QUality Assessment and REProducibility for instruments and images in Light Microscopy (QUAREP-LiMi) initiative was formed. This initiative comprises imaging scientists from academia and industry who share a common interest in achieving a better understanding of the performance and limitations of microscopes and improved quality control (QC) in light microscopy. The ultimate goal of the QUAREP-LiMi initiative is to establish a set of common QC standards, guidelines, metadata models and tools, including detailed protocols, with the ultimate aim of improving reproducible advances in scientific research. This White Paper (1) summarizes the major obstacles identified in the field that motivated the launch of the QUAREP-LiMi initiative; (2) identifies the urgent need to address these obstacles in a grassroots manner, through a community of stakeholders including, researchers, imaging scientists, bioimage analysts, bioimage informatics developers, corporate partners, funding agencies, standards organizations, scientific publishers and observers of such; (3) outlines the current actions of the QUAREP-LiMi initiative and (4) proposes future steps that can be taken to improve the dissemination and acceptance of the proposed guidelines to manage QC. To summarize, the principal goal of the QUAREP-LiMi initiative is to improve the overall quality and reproducibility of light microscope image data by introducing broadly accepted standard practices and accurately captured image data metrics.


Asunto(s)
Microscopía , Estándares de Referencia , Reproducibilidad de los Resultados
19.
Psychiatr Clin North Am ; 43(4): 735-743, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33127005

RESUMEN

There is a moderate degree of comorbidity between autism and eating disorders, particularly anorexia nervosa in female individuals. Research indicates that up to 30% of patients with anorexia are autistic, or display high levels of autistic traits. Frequently, an autism diagnosis is secondary to an eating disorder diagnosis, which brings concomitant issues into treatment efficacy and outcomes for both conditions. Less is known about comorbidity with other eating disorder subtypes. Autistic traits can impede standard approaches to eating disorder treatment. Treatment options and settings may need to be modified to better accommodate autistic female individuals.

20.
Nat Protoc ; 15(5): 1878, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32313255

RESUMEN

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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