STED nanoscopy allows for the direct observation of dynamic processes in living cells and tissues with diffraction-unlimited resolution. Although fluorescent proteins can be used for STED imaging, these labels are often outperformed in photostability by organic fluorescent dyes. This feature is especially crucial for time-lapse imaging. Unlike fluorescent proteins, organic fluorophores cannot be genetically fused to a target protein but require different labeling strategies. To achieve simultaneous imaging of more than one protein in the interior of the cell with organic fluorophores, bioorthogonal labeling techniques and cell-permeable dyes are needed. In addition, the fluorophores should preferentially emit in the red spectral range to reduce the potential phototoxic effects that can be induced by the STED light, which further restricts the choice of suitable markers. In this work, we selected five different cell-permeable organic dyes that fulfill all of the above requirements and applied them for SPIEDAC click labeling inside living cells. By combining click-chemistry-based protein labeling with other orthogonal and highly specific labeling methods, we demonstrate two-color STED imaging of different target structures in living specimens using different dye pairs. The excellent photostability of the dyes enables STED imaging for up to 60 frames, allowing the observation of dynamic processes in living cells over extended time periods at super-resolution.
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http://dx.doi.org/10.3390/cells13080683 | DOI Listing |
PLoS Pathog
January 2025
Strategic Area: Protecting Crops and the Environment, Rothamsted Research, Harpenden, Hertfordshire, United Kingdom.
Filamentous plant pathogenic fungi pose significant threats to global food security, particularly through diseases like Fusarium Head Blight (FHB) and Septoria Tritici Blotch (STB) which affects cereals. With mounting challenges in fungal control and increasing restrictions on fungicide use due to environmental concerns, there is an urgent need for innovative control strategies. Here, we present a comprehensive analysis of the stage-specific infection process of Fusarium graminearum in wheat spikes by generating a dual weighted gene co-expression network (WGCN).
View Article and Find Full Text PDFAllergol Immunopathol (Madr)
January 2025
Department of Geriatric Medicine, Qinghai University Affiliated Hospital, Xining, Qinghai, China.
The main goal of this investigation is to find out how solute carrier family 27 member 3 (SLC27A3) is expressed in the lung tissue of mice with chronic obstructive pulmonary disease (COPD), and how it relates to lung function. A model of COPD was established by exposing organisms to cigarette smoke, followed by investigating the role of SLC27A3 in COPD through experiments conducted both in living organisms and in laboratory settings. Knockout mice lacking SLC27A3 were produced through siRNA transfection to investigate lung function and inflammatory response, using methods such as hematoxylin-eosin staining and enzyme-linked immunosorbent assay.
View Article and Find Full Text PDFElife
January 2025
Eikon Therapeutics Inc, Hayward, United States.
The regulation of cell physiology depends largely upon interactions of functionally distinct proteins and cellular components. These interactions may be transient or long-lived, but often affect protein motion. Measurement of protein dynamics within a cellular environment, particularly while perturbing protein function with small molecules, may enable dissection of key interactions and facilitate drug discovery; however, current approaches are limited by throughput with respect to data acquisition and analysis.
View Article and Find Full Text PDFOdontology
January 2025
Department of Stomatology, Shanghai Fengxian District Central Hospital, No.6600 Nanfeng Highway, Shanghai, 201400, China.
Pulpitis seriously affects people's living standards and dental health, so identifying effective therapeutic targets is crucial for pulpitis. The research aimed to explore the underlying regulatory mechanism of LINC01094 and miR-340-5p in pulpitis. The study involved a total of 173 subjects (97 pulpitis and 76 healthy individuals).
View Article and Find Full Text PDFAlzheimers Dement
December 2024
Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Background: The nuclear clearance and cytoplasmic aggregation of splicing repressor TAR DNA/RNA-binding protein-43 (TDP-43) occur in approximately 50% of Alzheimer's disease (AD) cases and about 45% of frontotemporal dementia (FTD). However, it is not clear how early such mechanism occurs in AD and FTD as there is no method of detecting TDP-43 dysregulation in living individuals. Since the loss of nuclear TDP-43 leads to cryptic exon inclusion, we propose that cryptic exon-encoded peptides may be detected in patient biofluids as biomarkers of TDP-43 loss of function.
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