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The tissue diagnosis of amyloidosis and confirmation of fibril protein type, which are crucial for clinical management, have traditionally relied on Congo red (CR) staining followed by immunohistochemistry (IHC) using fibril protein specific antibodies. However, amyloid IHC is qualitative, non‐standardised, requires operator expertise, and not infrequently fails to produce definitive results. More recently, laser dissection mass spectrometry (LDMS) has been developed as an alternative method to characterise amyloid in tissue sections. We sought to compare these techniques in a real world setting. During 2017, we performed LDMS on 640 formalin‐fixed biopsies containing amyloid (CR+ve) comprising all 320 cases that could not be typed by IHC (IHC−ve) and 320 randomly selected CR+ve samples that had been typed (IHC+ve). In addition, we studied 60 biopsies from patients in whom there was a strong suspicion of amyloidosis, but in whom histology was non‐diagnostic (CR–ve). Comprehensive clinical assessments were conducted in 532 (76%) of cases. Among the 640 CR+ve samples, 602 (94%) contained ≥2 of 3 amyloid signature proteins (ASPs) on LDMS (ASP+ve) supporting the presence of amyloid. A total of 49 of the 60 CR‐ve samples were ASP–ve; 7 of 11 that were ASP+ve were glomerular. The amyloid fibril protein was identified by LDMS in 255 of 320 (80%) of the IHC–ve samples and in a total of 545 of 640 (85%) cases overall. The LDMS and IHC techniques yielded discordant results in only 7 of 320 (2%) cases. CR histology and LDMS are corroborative for diagnosis of amyloid, but LDMS is superior to IHC for confirming amyloid type.

To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab) fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph) and acetylated H3K9 (H3K9ac). These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye:protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green), Cy3 (red), and Cy5 or CF640 (far-red).

This protocol describes the use of TurboID and split-TurboID in proximity labeling applications for mapping protein–protein interactions and subcellular proteomes in live mammalian cells. TurboID is an engineered biotin ligase that uses ATP to convert biotin into biotin–AMP, a reactive intermediate that covalently labels proximal proteins. Optimized using directed evolution, TurboID has substantially higher activity than previously described biotin ligase–related proximity labeling methods, such as BioID, enabling higher temporal resolution and broader application in vivo. Split-TurboID consists of two inactive fragments of TurboID that can be reconstituted through protein–protein interactions or organelle–organelle interactions, which can facilitate greater targeting specificity than full-length enzymes alone. Proteins biotinylated by TurboID or split-TurboID are then enriched with streptavidin beads and identified by mass spectrometry. Here, we describe fusion construct design and characterization (variable timing), proteomic sample preparation (5–7 d), mass spectrometric data acquisition (2 d), and proteomic data analysis (1 week). This protocol describes proximity labeling approaches using TurboID and split-TurboID, which can be used for mapping protein–protein interactions and organelle proteomes in live mammalian cells with nanometer spatial resolution.

AimsTo compare the efficacy and safety of 3% diquafosol ophthalmic solution with those of 0.1% sodium hyaluronate ophthalmic solution in patients with dry eye in China and Singapore.MethodsA total of 497 patients with dry eye (Schirmer's test, 5 mm; fluorescein and RB score, 3 points) from China and Singapore were randomised to receive either diquafosol ophthalmic solution (diquafosol) or sodium hyaluronate ophthalmic solution (HA) at 1:1 ratio. The fluorescein staining scores and rose bengal (RB) subjective symptom scores and tear film breakup time were evaluated before treatment and 2 and 4 weeks after start of treatment.ResultsIn the diquafosol group, changes in fluorescein and RB scores compared with baseline at week 4 or at the time of discontinuation were −2.1±1.5 and −2.5±2.0, respectively. Compared with the HA group, changes in fluorescein score were non-inferior and changes in RB score were superior (p=0.019). In addition, diquafosol and HA improved tear film breakup time by 1.046±1.797 and 0.832±1.775 s, respectively (no significant intergroup difference). Adverse event onset rates were 16.3% (40 of 246 subjects) and 10.0% (25 of 251 subjects) in the diquafosol group and HA group, respectively, with borderline significant intergroup differences (p=0.046), while adverse drug reaction incidence rates were 12.2% (30 of 246 subjects) and 6.0% (15 of 251 subjects), respectively (p=0.019). Only mild adverse drug reactions (>2%) in the form of eye discharge, itching or irritation were observed.ConclusionsDiquafosol improved fluorescein staining score in a manner similar to HA, and significantly improved RB score compared with HA.Trial registration numberNCT01101984.

Improved expansion microscopy method preserves signal from fluorescent proteins and antibodies using off-the-shelf reagents. Expansion microscopy (ExM) enables imaging of preserved specimens with nanoscale precision on diffraction-limited instead of specialized super-resolution microscopes. ExM works by physically separating fluorescent probes after anchoring them to a swellable gel. The first ExM method did not result in the retention of native proteins in the gel and relied on custom-made reagents that are not widely available. Here we describe protein retention ExM (proExM), a variant of ExM in which proteins are anchored to the swellable gel, allowing the use of conventional fluorescently labeled antibodies and streptavidin, and fluorescent proteins. We validated and demonstrated the utility of proExM for multicolor super-resolution (∼70 nm) imaging of cells and mammalian tissues on conventional microscopes.

The ability to isolate rare live cells within a heterogeneous population based solely on visual criteria remains technically challenging, due largely to limitations imposed by existing sorting technologies. Here, we present a new method that permits labeling cells of interest by attaching streptavidin-coated magnetic beads to their membranes using the lasers of a confocal microscope. A simple magnet allows highly specific isolation of the labeled cells, which then remain viable and proliferate normally. As proof of principle, we tagged, isolated, and expanded individual cells based on three biologically relevant visual characteristics: i) presence of multiple nuclei, ii) accumulation of lipid vesicles, and iii) ability to resolve ionizing radiation-induced DNA damage foci. Our method constitutes a rapid, efficient, and cost-effective approach for isolation and subsequent characterization of rare cells based on observable traits such as movement, shape, or location, which in turn can generate novel mechanistic insights into important biological processes. When scientists use microscopes to look at cells, they often want to then isolate certain cells based on how these look like. For example, researchers may want to select cells with specific shapes, movements or division rates, because these visual clues give important information about how the cells may be behaving in the body. However, it remains difficult to precisely pick a few live cells within a bigger sample. To address this problem, Binan et al. created a new approach, called single cell magneto-optical capture (scMOCa), to set aside specific cells within a larger population. The technique uses the lasers present on confocal microscopes to attach tiny metallic beads to the surface of chosen cell. Then, a magnetic field is applied to gently pull the cell to a new location. The method is cheap – it relies on commonly available research tools – and it works on a broad variety of cells. In the future, scMOCa could be used to capture and then grow cells that can only be recognized by how they look or behave, which will help to study them in greater details.

INTRODUCTION:Lugol chromoendoscopy (LCE) is valuable, cost-effective, and widely used in early esophageal cancer screening, yet it suffers from low compliance because of adverse events after LCE. In addition, the reflux of iodine during iodine staining in the upper esophagus brings the risk of bucking and aspiration. We introduced a new model called distance countdown (DC) aimed to reduce reflux during iodine staining in upper esophageal LCE.METHODS:In this randomized controlled trial, 204 patients were randomized into the DC and No-DC groups. The primary end point was the difference in the incidence of positive starch reagent reaction (iodine solution reflux) between the 2 groups. The secondary end points were the comparisons of the incidence of other adverse events after LCE between the 2 groups.RESULTS:The rate of iodine solution reflux was 1.0% in the DC group and 26.5% in the No-DC group (P < 0.001). Furthermore, the incidences of bucking between the 2 groups were 1.0% and 9.8% (P = 0.005). LCE satisfaction rates were 78.4% and 76.5% in the DC and No-DC groups (P = 0.363), respectively. Concerning symptoms after LCE, incidences of sore throat, pharyngeal discomfort or odor, bitter taste, and heartburn were also reduced in the DC group (all P < 0.05).DISCUSSION:Adding DC as an auxiliary effect during LCE would reduce the risk of iodine solution reflux, as well as other adverse events after LCE. Implementing this measure could be beneficial in improving the safety of LCE in early esophageal cancer screening.

Despite rapid developments in single cell sequencing, sample-specific batch effects, detection of cell multiplets, and experimental costs remain outstanding challenges. Here, we introduce Cell Hashing, where oligo-tagged antibodies against ubiquitously expressed surface proteins uniquely label cells from distinct samples, which can be subsequently pooled. By sequencing these tags alongside the cellular transcriptome, we can assign each cell to its original sample, robustly identify cross-sample multiplets, and “super-load” commercial droplet-based systems for significant cost reduction. We validate our approach using a complementary genetic approach and demonstrate how hashing can generalize the benefits of single cell multiplexing to diverse samples and experimental designs.

Evaluation of tissues is a common and important aspect of translational research studies. Labeling techniques such as immunohistochemistry can stain cells/tissues to enhance identification of specific cell types, cellular activation states, and protein expression. While qualitative evaluation of labeled tissues has merit, use of semiquantitative and quantitative scoring approaches can greatly enhance the rigor of the tissue data. Adhering to key principles for reproducible scoring can enhance the quality and reproducibility of the tissue data so as to maximize its biological relevance and scientific impact.

In DNA-PAINT, transient binding of dye-labeled oligonucleotides to their target strands creates the ‘blinking’ required for stochastic nanoscopy. This protocol describes how to apply DNA-PAINT, from sample preparation to data processing. Super-resolution techniques have begun to transform biological and biomedical research by allowing researchers to observe structures well below the classic diffraction limit of light. DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) offers an easy-to-implement approach to localization-based super-resolution microscopy, owing to the use of DNA probes. In DNA-PAINT, transient binding of short dye-labeled ('imager') oligonucleotides to their complementary target ('docking') strands creates the necessary 'blinking' to enable stochastic super-resolution microscopy. Using the programmability and specificity of DNA molecules as imaging and labeling probes allows researchers to decouple blinking from dye photophysics, alleviating limitations of current super-resolution techniques, making them compatible with virtually any single-molecule-compatible dye. Recent developments in DNA-PAINT have enabled spectrally unlimited multiplexing, precise molecule counting and ultra-high, molecular-scale (sub-5-nm) spatial resolution, reaching ∼1-nm localization precision. DNA-PAINT can be applied to a multitude of in vitro and cellular applications by linking docking strands to antibodies. Here, we present a protocol for the key aspects of the DNA-PAINT framework for both novice and expert users. This protocol describes the creation of DNA origami test samples, in situ sample preparation, multiplexed data acquisition, data simulation, super-resolution image reconstruction and post-processing such as drift correction, molecule counting (qPAINT) and particle averaging. Moreover, we provide an integrated software package, named Picasso, for the computational steps involved. The protocol is designed to be modular, so that individual components can be chosen and implemented per requirements of a specific application. The procedure can be completed in 1–2 d.