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1.
J Microsc ; 294(3): 397-410, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38691400

RESUMO

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.


Assuntos
Pesquisadores , Humanos , Mobilidade Ocupacional , Pesquisa Biomédica/métodos , Escolha da Profissão
3.
Elife ; 122023 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-37530405

RESUMO

A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in Mycobacterium tuberculosis during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded 'mycobacterial mutasome' - minimally comprising DnaE2 polymerase and ImuA' and ImuB accessory proteins - remain elusive. Following exposure of mycobacteria to DNA damaging agents, we observe that DnaE2 and ImuB co-localize with the DNA polymerase III ß subunit (ß clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an imuBAAAAGG mutant containing a disrupted ß clamp-binding motif abolishes ImuB-ß clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this ß clamp-binding antibiotic collapses pre-formed ImuB-ß clamp complexes. These observations establish the essentiality of the ImuB-ß clamp interaction for mutagenic DNA repair in mycobacteria, identifying the mutasome as target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance.


Assuntos
Proteínas de Bactérias , Mycobacterium tuberculosis , Proteínas de Bactérias/química , Mycobacterium tuberculosis/genética , Mutagênese , Reparo do DNA , Antituberculosos/farmacologia
4.
Nature ; 620(7976): 1117-1125, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37587339

RESUMO

PIEZOs are mechanosensitive ion channels that convert force into chemoelectric signals1,2 and have essential roles in diverse physiological settings3. In vitro studies have proposed that PIEZO channels transduce mechanical force through the deformation of extensive blades of transmembrane domains emanating from a central ion-conducting pore4-8. However, little is known about how these channels interact with their native environment and which molecular movements underlie activation. Here we directly observe the conformational dynamics of the blades of individual PIEZO1 molecules in a cell using nanoscopic fluorescence imaging. Compared with previous structural models of PIEZO1, we show that the blades are significantly expanded at rest by the bending stress exerted by the plasma membrane. The degree of expansion varies dramatically along the length of the blade, where decreased binding strength between subdomains can explain increased flexibility of the distal blade. Using chemical and mechanical modulators of PIEZO1, we show that blade expansion and channel activation are correlated. Our findings begin to uncover how PIEZO1 is activated in a native environment. More generally, as we reliably detect conformational shifts of single nanometres from populations of channels, we expect that this approach will serve as a framework for the structural analysis of membrane proteins through nanoscopic imaging.


Assuntos
Canais Iônicos , Membrana Celular/metabolismo , Fluorescência , Canais Iônicos/química , Canais Iônicos/metabolismo , Modelos Moleculares , Movimento , Conformação Proteica , Análise de Célula Única
6.
J Microsc ; 2023 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-36779652

RESUMO

Microscopy core facilities are increasingly utilised research resources, but they are generally only available to users within the host institution. Such localised access misses an opportunity to facilitate research across a broader user base. Here, we present the model of an open-access microscopy facility, using the Advanced Imaging Center (AIC) at Howard Hughes Medical Institute Janelia Research Campus as an example. The AIC has pioneered a model whereby advanced microscopy technologies and expertise are made accessible to researchers on a global scale. We detail our experiences in addressing the considerable challenges associated with this model for those who may be interested in launching an open-access imaging facility. Importantly, we focus on how this model can empower researchers, particularly those from resource-constrained settings.

7.
J Cell Sci ; 135(6)2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35319069

RESUMO

Fluorescence microscopy images should not be treated as perfect representations of biology. Many factors within the biospecimen itself can drastically affect quantitative microscopy data. Whereas some sample-specific considerations, such as photobleaching and autofluorescence, are more commonly discussed, a holistic discussion of sample-related issues (which includes less-routine topics such as quenching, scattering and biological anisotropy) is required to appropriately guide life scientists through the subtleties inherent to bioimaging. Here, we consider how the interplay between light and a sample can cause common experimental pitfalls and unanticipated errors when drawing biological conclusions. Although some of these discrepancies can be minimized or controlled for, others require more pragmatic considerations when interpreting image data. Ultimately, the power lies in the hands of the experimenter. The goal of this Review is therefore to survey how biological samples can skew quantification and interpretation of microscopy data. Furthermore, we offer a perspective on how to manage many of these potential pitfalls.


Assuntos
Biologia , Luz , Anisotropia , Microscopia de Fluorescência/métodos , Fotodegradação
9.
J Cell Sci ; 133(21)2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33154172

RESUMO

One of the challenges in modern fluorescence microscopy is to reconcile the conventional utilization of microscopes as exploratory instruments with their emerging and rapidly expanding role as a quantitative tools. The contribution of microscopy to observational biology will remain enormous owing to the improvements in acquisition speed, imaging depth, resolution and biocompatibility of modern imaging instruments. However, the use of fluorescence microscopy to facilitate the quantitative measurements necessary to challenge hypotheses is a relatively recent concept, made possible by advanced optics, functional imaging probes and rapidly increasing computational power. We argue here that to fully leverage the rapidly evolving application of microscopes in hypothesis-driven biology, we not only need to ensure that images are acquired quantitatively but must also re-evaluate how microscopy-based experiments are designed. In this Opinion, we present a reverse logic that guides the design of quantitative fluorescence microscopy experiments. This unique approach starts from identifying the results that would quantitatively inform the hypothesis and map the process backward to microscope selection. This ensures that the quantitative aspects of testing the hypothesis remain the central focus of the entire experimental design.


Assuntos
Óptica e Fotônica , Projetos de Pesquisa , Microscopia de Fluorescência
10.
Cancers (Basel) ; 12(6)2020 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-32560230

RESUMO

A challenge in cancer research is the definition of reproducible, reliable, and practical models, which reflect the effects of complex treatment modalities and the heterogeneous response of patients. Proton beam radiotherapy (PBRT), relative to conventional photon-based radiotherapy, offers the potential for iso-effective tumor control, while protecting the normal tissue surrounding the tumor. However, the effects of PBRT on the tumor microenvironment and the interplay with newly developed chemo- and immunotherapeutic approaches are still open for investigation. This work evaluated thin-cut tumor slice cultures (TSC) of head and neck cancer and organotypic brain slice cultures (OBSC) of adult mice brain, regarding their relevance for translational radiooncology research. TSC and OBSC were treated with PBRT and investigated for cell survival with a lactate dehydrogenase (LDH) assay, DNA repair via the DNA double strand break marker γH2AX, as well as histology with regards to morphology. Adult OBSC failed to be an appropriate model for radiobiological research questions. However, histological analysis of TSC showed DNA damage and tumor morphological results, comparable to known in vivo and in vitro data, making them a promising model to study novel treatment approaches in patient-derived xenografts or primary tumor material.

11.
Front Mol Biosci ; 4: 75, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29184888

RESUMO

Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB), an infectious disease which results in approximately 10 million incident cases and 1.4 million deaths globally each year, making it the leading cause of mortality from infection. An effective frontline combination chemotherapy exists for TB; however, this regimen requires the administration of four drugs in a 2 month long intensive phase followed by a continuation phase of a further 4 months with two of the original drugs, and is only effective for the treatment of drug-sensitive TB. The emergence and global spread of multidrug-resistant (MDR) as well as extensively drug-resistant (XDR) strains of M. tuberculosis, and the complications posed by co-infection with the human immunodeficiency virus (HIV) and other co-morbidities such as diabetes, have prompted urgent efforts to develop shorter regimens comprising new compounds with novel mechanisms of action. This demands that researchers re-visit cellular pathways and functions that are essential to M. tuberculosis survival and replication in the host but which are inadequately represented amongst the targets of current anti-mycobacterial agents. Here, we consider the DNA replication and repair machinery as a source of new targets for anti-TB drug development. Like most bacteria, M. tuberculosis encodes a complex array of proteins which ensure faithful and accurate replication and repair of the chromosomal DNA. Many of these are essential; so, too, are enzymes in the ancillary pathways of nucleotide biosynthesis, salvage, and re-cycling, suggesting the potential to inhibit replication and repair functions at multiple stages. To this end, we provide an update on the state of chemotherapeutic inhibition of DNA synthesis and related pathways in M. tuberculosis. Given the established links between genotoxicity and mutagenesis, we also consider the potential implications of targeting DNA metabolic pathways implicated in the development of drug resistance in M. tuberculosis, an organism which is unusual in relying exclusively on de novo mutations and chromosomal rearrangements for evolution, including the acquisition of drug resistance. In that context, we conclude by discussing the feasibility of targeting mutagenic pathways in an ancillary, "anti-evolution" strategy aimed at protecting existing and future TB drugs.

12.
Ther Innov Regul Sci ; 51(4): 431-438, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30227058

RESUMO

BACKGROUND: Typography significantly influences the legibility and usability of patient information. This study investigated the implementation and changes of different typographic subjects in package leaflets used in the European Union. METHODS: A randomly selected sample of all German package leaflets investigated in 2005 was reanalyzed in 2015 for different important and predefined typographic subjects. RESULTS: The 138 package leaflets revisited in 2015 showed significant increases in word count (average 2551 words), font size (1.43 mm x-height, 2.0 mm cap-height), line spacing (3.13 mm), use of text attributes (for example, lists in 94.2% and bold print in 83.2% package leaflet's body text), use of light-condensed or condensed font (34.1%), and use of portrait format (81.9%) ( P ≤ .015). Otherwise, line length significantly decreased to on average 62.6 characters per line ( P = .012) and the classes of fonts used remained almost unchanged. CONCLUSION: To achieve any further increase of package leaflet font size and other typographic improvements, a significant decrease in text volume is essential. To this end, replacing the current 840-word QRD template with a 200-word version would allow optimization of typography in all package leaflets, without deleting information essential for patients or incurring any unfavorable format increase.

13.
Nat Struct Mol Biol ; 20(5): 582-8, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23542154

RESUMO

During protein synthesis, the ribosome translates nucleotide triplets in single-stranded mRNA into polypeptide sequences. Strong downstream mRNA secondary structures, which must be unfolded for translation, can slow or even halt protein synthesis. Here we used single-molecule fluorescence resonance energy transfer to determine reaction rates for specific steps within the elongation cycle as the Escherichia coli ribosome encounters stem-loop or pseudoknot mRNA secondary structures. Downstream stem-loops containing 100% GC base pairs decrease the rates of both tRNA translocation within the ribosome and deacylated tRNA dissociation from the ribosomal exit site (E site). Downstream stem-loops or pseudoknots containing both GC and AU pairs also decrease the rate of tRNA dissociation, but they have little effect on tRNA translocation rate. Thus, somewhat unexpectedly, unfolding of mRNA secondary structures is more closely coupled to E-site tRNA dissociation than to tRNA translocation.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Cinética , Modelos Biológicos , Modelos Moleculares
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