Labeling Tool Research Articles

A straightforward approach for bioorthogonal labeling of proteins and organelles in live mammalian cells, using a short peptide tag

BackgroundIn the high-resolution microscopy era, genetic code expansion (GCE)-based bioorthogonal labeling offers an elegant way for direct labeling of proteins in live cells with fluorescent dyes. This labeling approach is currently not broadly used in live-cell applications, partly because it needs to be adjusted to the specific protein under study.ResultsWe present a generic, 14-residue long, N-terminal tag for GCE-based labeling of proteins in live mammalian cells. Using this tag, we generated a library of GCE-based organelle markers, demonstrating the applicability of the tag for labeling a plethora of proteins and organelles. Finally, we show that the HA epitope, used as a backbone in our tag, may be substituted with other epitopes and, in some cases, can be completely removed, reducing the tag length to 5 residues.ConclusionsThe GCE-tag presented here offers a powerful, easy-to-implement tool for live-cell labeling of cellular proteins with small and bright probes.

Tissue clearing and its applications inneuroscience.

State-of-the-art tissue-clearing methods provide subcellular-level optical access to intact tissues from individual organs and even to some entire mammals. When combined with light-sheet microscopy and automated approaches to image analysis, existing tissue-clearing methods can speed up and may reduce the cost of conventional histology by several orders of magnitude. In addition, tissue-clearing chemistry allows whole-organ antibody labelling, which can be applied even to thick human tissues. By combining the most powerful labelling, clearing, imaging and data-analysis tools, scientists are extracting structural and functional cellular and subcellular information on complex mammalian bodies and large human specimens at an accelerated pace. The rapid generation of terabyte-scale imaging data furthermore creates a high demand for efficient computational approaches that tackle challenges in large-scale data analysis and management. In this Review, we discuss how tissue-clearing methods could provide an unbiased, system-level view of mammalian bodies and human specimens and discuss future opportunities for the use of these methods in human neuroscience.

Performance Improvement in Aerospace Production Through Lean Manufacturing Implementation

The high demand in aerospace industry require on going improvement to meet its costumer. Prototyping is a form of effort to ensure the quality of airplanes. However, several problems rose in this phase but two of them are significant in disrupting the production system. They are the spare parts tardiness and unnecessary motion leading a high lead time. The objective of this article is to develop lean action plan to reduce lead time in airplane prototyping. The method applied in this paper is divided into three major phases. Firstly, evaluate current system performance using Value Stream Mapping (VSM). Secondly, determine the dominant waste by cooperating waste finding checklist. Finally, develop a lean action plan. Most waste occurred in production activities. Waste of motion is the highest among other waste with the score of 44 followed by waiting with the score of 24. The third highest waste is excess processing with the score of 2. The causes of these wastes are lack of facilities such as machines and tools and inefficient operators in picking and mixing sealant. The lean action plans are to add machines and tools for labelling, adding sealant room and to change the production plant lay out. By doing so, the lead time can be reduced up to 0,25 hours.

Generation of an Induced Pluripotent Stem Cell Line with the Constitutive EGFP Reporter.

The discovery of induced pluripotent stem cell (iPSC) technology has provided a versatile platform for basic science research and regenerative medicine. With the rise of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) systems and the ease at which they can be utilized for gene editing, creating genetically modified iPSCs has never been more advantageous for studying both organism development and potential clinical applications. However, to better understand the behavior and true therapeutic potential of iPSCs and iPSC-derived cells, a tool for labeling and monitoring these cells in vitro and in vivo is needed. Here, we describe a protocol that provides a straightforward method for introducing a stable, highly expressed fluorescent protein into iPSCs using the CRISPR/Cas9 system and a standardized donor vector. The approach involves the integration of the EGFP transgene into the transcriptionally active adeno-associated virus integration site 1 (AAVS1) locus through homology directed repair. The knockin of this transgene results in the generation of iPSC lines with constitutive expression of the EGFP protein that also persists in differentiated iPSCs. These EGFP-labeled iPSC lines are ideal for assessing iPSC differentiation in vitro and evaluating the distribution of iPSC-derived cells in vivo after transplantation into model animals.

In Vitro Labeling Mesenchymal Stem Cells with Superparamagnetic Iron Oxide Nanoparticles: Efficacy and Cytotoxicity.

Mesenchymal stem cell (MSC) therapy has emerged as a potential therapeutic option for several diseases due to their unique properties of releasing important bioactive factors. Despite the advances in stem cell therapy, it is still difficult to accurately determine the mechanisms of cell activities after in vivo transplantation. The application of noninvasive cell tracking approaches is important to determine tissue distribution and the lifetime of stem cells following their injection, which consequently provides knowledge about the mechanisms of stem cell tissue repair. Superparamagnetic iron oxide nanoparticles (SPION)can provide a very useful tool for labeling and tracking stem cells by magnetic resonance imaging without causing toxic cellular effects and do not elicit any other side effects. Here we describe how to use SPIONs to label mesenchymal stem cells and evaluate efficacy and potential cytotoxicity in vitro.

AI-IDS: Application of Deep Learning to Real-Time Web Intrusion Detection

Deep Learning has been widely applied to problems in detecting various network attacks. However, no cases on network security have shown applications of various deep learning algorithms in real-time services beyond experimental conditions. Moreover, owing to the integration of high-performance computing, it is necessary to apply systems that can handle large-scale traffic. Given the rapid evolution of web-attacks, we implemented and applied our Artificial Intelligence-based Intrusion Detection System (AI-IDS). We propose an optimal convolutional neural network and long short-term memory network (CNN-LSTM) model, normalized UTF-8 character encoding for Spatial Feature Learning (SFL) to adequately extract the characteristics of real-time HTTP traffic without encryption, calculating entropy, and compression. We demonstrated its excellence through repeated experiments on two public datasets (CSIC-2010, CICIDS2017) and fixed real-time data. By training payloads that analyzed true or false positives with a labeling tool, AI-IDS distinguishes sophisticated attacks, such as unknown patterns, encoded or obfuscated attacks from benign traffic. It is a flexible and scalable system that is implemented based on Docker images, separating user-defined functions by independent images. It also helps to write and improve Snort rules for signature-based IDS based on newly identified patterns. As the model calculates the malicious probability by continuous training, it could accurately analyze unknown web-attacks.

Evaluation of measurement techniques for modelling buildings in energy simulation and labelling tool

The dwellings built between 1945 and 1980 have the largest energy demand in the EU, which by 2009 represented 70% of the final energy use in buildings. A great portion of these dwellings have not been retrofitted and most of them were not built with any energy efficiency measures, since most of the energy regulations were implemented after the oil crisis in the 1970s. The current renovation rate of residential buildings has not reached targeted goals, due to the numerous barriers that arise in the renovation process. The evaluation and labelling of existing residential buildings represent a big challenge, and the lack of geometric information on buildings is one of the main issues hindering an assessment through simulations. Currently, there is no scientific literature that focuses on improving this task. However, the emergence of new technologies from different fields may streamline the geometric data gathering with the modelling task and greatly improve both accuracy and workload. This study focuses on the revision of geometry measurement techniques, based on the application and quantification of the benefits and barriers that these techniques represent for their use in the building simulation and labelling. The techniques tested were Hololens, handheld laser scanner and handheld laser distance measurer. The evaluation considers time, cost and accuracy as well the tasks related to the post process of the data in BIM, which is not mandatory for building simulation, but it provided multiple benefits.

Synthesis and evaluation of Nα,Nε-diacetyl-l-lysine-inositol conjugates as cancer-selective probes for metabolic engineering of GPIs and GPI-anchored proteins.

Two myo-inositol derivatives having an Nα,Nε-diacetyl-l-lysine (Ac2Lys) moiety linked to the inositol 1-O-position through a self-cleavable linker and a metabolically stable 2-azidoethyl group linked to the inositol 3-O- and 4-O-positions, respectively, were designed and synthesized. The Ac2Lys moiety blocking the inositol 1-O-position required for GPI biosynthesis was expected to be removable by a combination of two enzymes, histone deacetylase (HDAC) and cathepsin L (CTSL), abundantly expressed in cancer cells, but not in normal cells, to transform these inositol derivatives into biosynthetically useful products with a free 1-O-position. As a result, it was found that these inositol derivatives could be incorporated into the glycosylphosphatidylinositol (GPI) biosynthetic pathway by cancer cells, but not by normal cells, to express azide-labeled GPIs and GPI-anchored proteins on cell surfaces. Consequently, this study has established a novel strategy and new molecular tools for selective metabolic labeling of cancer cells, which should be useful for various biological studies and applications.

The TV p Regularized Mumford-Shah Model for Image Labeling and Segmentation

The Mumford-Shah model is an important tool for image labeling and segmentation, which pursues a piecewise smooth approximation of the original image and the boundaries with the shortest length. In contrast to previous efforts, which use the total variation regularization to measure the total length of the boundaries, we build up a novel piecewise smooth Mumford-Shah model by utilizing a non-convex $\ell ^{p}$ regularity term for $p\in (0,1)$ , which can well preserve sharp edges and eliminate geometric staircasing effects. We present optimization algorithms with convergence verification, where all subproblems can be solved by either the closed-form solution or fast Fourier transform (FFT). The method is compared to piecewise constant labeling algorithm and several state-of-the-art piecewise smooth Mumford-Shah models based on image decomposition approximations. Both labeling and segmentation results on synthetic and real images confirm the robustness and efficiency of the proposed method.

Determination of the glycoprotein specificity of lectins on cell membranes through oxidative proteomics.

The cell membrane is composed of a network of glycoconjugates including glycoproteins and glycolipids that presents a dense matrix of carbohydrates playing critical roles in many biological processes. Lectin-based technology has been widely used to characterize glycoconjugates in tissues and cell lines. However, their specificity toward their putative glycan ligand and sensitivity in situ have been technologically difficult to study. Additionally, because they recognize primarily glycans, the underlying glycoprotein targets are generally not known. In this study, we employed lectin proximity oxidative labeling (Lectin PROXL) to identify cell surface glycoproteins that contain glycans that are recognized by lectins. Commonly used lectins were modified with a probe to produce hydroxide radicals in the proximity of the labeled lectins. The underlying polypeptides of the glycoproteins recognized by the lectins are oxidized and identified by the standard proteomic workflow. As a result, approximately 70% of identified glycoproteins were oxidized in situ by all the lectin probes, while only 5% of the total proteins were oxidized. The correlation between the glycosites and oxidation sites demonstrated the effectiveness of the lectin probes. The specificity and sensitivity of each lectin were determined using site-specific glycan information obtained through glycomic and glycoproteomic analyses. Notably, the sialic acid-binding lectins and the fucose-binding lectins had higher specificity and sensitivity compared to other lectins, while those that were specific to high mannose glycans have poor sensitivity and specificity. This method offers an unprecedented view of the interactions of lectins with specific glycoproteins as well as protein networks that are mediated by specific glycan types on cell membranes.

Endomembrane Systems are Reorganized by ORF3a and Membrane (M) of SARS-CoV-2

The endomembrane reticulum (ER) is largely reorganized by SARS-CoV-2 infection, however, detailed molecular mechanisms of ER remodeling by the virus are still not fully understood. Here, we introduce a “plug and playable” proximity labeling tool (TurboID-GBP) for interactome mapping of GFP-tagged ORF3a and membrane (M) proteins of SARS-CoV-2. Through the mass spectrometric identification of the biotinylated lysine residue (K+226 Da) on the viral proteins using TurboID, 117 and 191 proteins were robustly determined as interactomes of ORF3a and M, respectively, and many of them overlap with the mitochondrial-associated membrane (MAM) proteome, including RNF5 (E3 ubiquitin ligase). We also found that MAM formation and secreted proteome profiles were largely affected by the expression of ORF3a. Our results imply that SARS-CoV-2 may utilize MAM as a replication site, and we can suggest novel antiviral treatments for the blockade of viral replication in host cells. Funding: This work was supported by the National Research Foundation of Korea (NRF-,2019R1A2C3008463) and the Organelle Network Research Center (NRF-,2017R1A5A1015366). H.W.R. was supported by Creative-Pioneering Researchers Program through Seoul National University. J.Y.M. and M.J. are supported by KBRI basic research program through Korea Brain Research Institute funded by Ministry of Science and ICT (Information & Communication Technology) (20-BR-01-09). EM data were acquired at Brain Research Core Facilities in KBRI. J.-S.K. are supported by IBS-R008-D1 of the Institute for Basic Science from the Ministry of Science and ICT of Korea. Conflict of Interest: The authors declare no competing interests.

Intrinsic Buffer Hydroxyl Radical Dosimetry Using Tris(hydroxymethyl)aminomethane.

Fast photochemical oxidation of proteins (FPOP) is a powerful covalent labeling tool that uses hydroxyl radicals generated by laser flash photolysis of hydrogen peroxide to footprint protein surfaces. Because radical production varies with many experimental parameters, hydroxyl radical dosimeters have been introduced to track the effective radical dosage experienced by the protein analyte. FPOP experiments performed using adenine optical radical dosimetry containing protein in Tris buffer demonstrated unusual dosimetry behavior. We have investigated the behavior of Tris under oxidative conditions in detail. We find that Tris can act as a novel gain-of-signal optical hydroxyl radical dosimeter in FPOP experiments. This new dosimeter is also amenable to inline real-time monitoring, thereby allowing real-time adjustments to compensate for differences in samples for their quenching ability.

Identification of a Small Probe That Can Be Conjugated to Proteins by Proximity Labeling.

Proximity labeling has been used to study protein-protein interactions and can also be used as a protein labeling tool. We developed a novel 14-amino acid peptide substrate for the proximity-labeling enzyme PafA. The N terminus of the peptide can be modified with biotin or fluorophores, which allows various chemical moieties to be ligated to the target protein. We used PafA-mediated peptide labeling to label antibodies with a biotin tag without affecting the antigen binding capacity of the antibody. Similar strategies can be used for other types of protein labeling in the future.

Imaging the adenovirus infection cycle.

Incoming adenoviruses seize control of cytosolic transport mechanisms to relocate their genome from the cell periphery to specialized sites in the nucleoplasm. The nucleus is the site for viral gene expression, genome replication, and the production of progeny for the next round of infection. By taking control of the cell, adenoviruses also suppress cell-autonomous immunity responses. To succeed in their production cycle, adenoviruses rely on well-coordinated steps, facilitated by interactions between viral proteins and cellular factors. Interactions between virus and host can impose remarkable morphological changes in the infected cell. Imaging adenoviruses has tremendously influenced how we delineate individual steps in the viral life cycle, because it allowed the development of specific optical markers to label these morphological changes in space and time. As technology advances, innovative imaging techniques and novel tools for specimen labeling keep uncovering previously unseen facets of adenovirus biology emphasizing why imaging adenoviruses is as attractive today as it was in the past. This review will summarize past achievements and present developments in adenovirus imaging centered on fluorescence microscopy approaches.

Direct in Vitro Selection of Trans-Acting Ribozymes for Posttranscriptional, Site-Specific, and Covalent Fluorescent Labeling of RNA.

General and efficient tools for site-specific fluorescent or bioorthogonal labeling of RNA are in high demand. Here, we report direct in vitro selection, characterization, and application of versatile trans-acting 2'-5' adenylyl transferase ribozymes for covalent and site-specific RNA labeling. The design of our partially structured RNA pool allowed for in vitro evolution of ribozymes that modify a predetermined nucleotide in cis (i.e., intramolecular reaction) and can then be easily engineered for applications in trans (i.e., in an intermolecular setup). The resulting ribozymes are readily designed for specific target sites in small and large RNAs and accept a wide variety of N6-modified ATP analogues as small-molecule substrates. The most efficient new ribozyme (FH14) shows excellent specificity toward its target sequence also in the context of total cellular RNA.

The Bioorthogonal Isonitrile-Chlorooxime Ligation.

Bioorthogonal reactions are valuable tools for the selective labeling and imaging of natural products and proteins. Here, we present the reaction between isonitriles and chlorooximes as a ligation that proceeds quickly (k ≈ 1 M-1 s-1) and with high chemoselectivity in an aqueous environment. Imaging of metabolically labeled cell surface glycans underlined the tolerance of the ligation to common functional groups in cellular systems. Live-cell dual-labeling experiments revealed that the isonitrile-chlorooxime ligation is orthogonal to the strain-promoted azide-alkyne cycloaddition.

PH-Responsive Carboxymethylcellulose Nanoparticles for 68Ga-WBC Labeling in PET Imaging.

Carboxymethylcellulose (CMC) is a well-known pharmaceutical polymer, recently gaining attention in the field of nanomedicine, especially as a polyelectrolyte agent for the formation of complexes with oppositely charged macromolecules. Here, we report on the application of pH-sensitive pharmaceutical grade CMC-based nanoparticles (NP) for white blood cells (WBC) PET imaging. In this context and as an alternative to 99mTc-HMPAO SPECT labeling, the use of 68Ga3+ as PET radionuclide was investigated since, at early time points, it could provide the greater spatial resolution and patient convenience of PET tomography over SPECT clinical practices. Two operator-friendly kit-type formulations were compared, with the intention of radiolabeling within a short time (10 min), under mild conditions (physiological pH, room temperature) and in agreement with the actual clinically applied guidelines. NP were labeled by directly using 68Ga3+ eluted in HCL 0.05 N, from hospital suited 68Ge/68Ga generator and in absence of chelator. The first kit type approach involved the application of 68Ga3+ as an ionotropic gelation agent for in-situ forming NP. The second kit type approach concerned the re-hydration of a proper freeze-dried injectable NP powder. pH-sensitive NP with 250 nm average diameter and 80% labeling efficacy were obtained. The NP dispersant medium, including a cryoprotective agent, was modulated in order to optimize the Zeta potential value (−18 mV), minimize the NP interaction with serum proteins and guarantee a physiological environment for WBC during NP incubation. Time-dependent WBC radiolabeling was correlated to NP uptake by using both confocal and FT-IR microscopies. The ready to use lyophilized NP formulation approach appears promising as a straightforward 68Ga-WBC labeling tool for PET imaging applications.

Stable isotope labeling of metal/metal oxide nanomaterials for environmental and biological tracing.

Engineered nanomaterials (NMs) are often compositionally indistinguishable from their natural counterparts, and thus their tracking in the environment or within the biota requires the development of appropriate labeling tools. Stable isotope labeling has become a well-established such tool, developed to assign 'ownership' or a 'source' to engineered NMs, enabling their tracing and quantification, especially in complex environments. A particular methodological challenge for stable isotope labeling is to ensure that the label is traceable in a range of environmental or biological scenarios but does not induce modification of the properties of the NM or lose its signal, thus retaining realism and relevance. This protocol describes a strategy for stable isotope labeling of several widely used metal and metal oxide NMs, namely ZnO, CuO, Ag, and TiO2, using isotopically enriched precursors, namely 67Zn or 68Zn metal, 65CuCl2, 107Ag or 109Ag metal, and 47TiO2 powder. A complete synthesis requires 1-8 d, depending on the type of NM, the precursors used, and the synthesis methods adopted. The physicochemical properties of the labeled particles are determined by optical, diffraction, and spectroscopic techniques for quality control. The procedures for tracing the labels in aquatic (snail and mussel) and terrestrial (earthworm) organisms and for monitoring the environmental transformation of labeled silver (Ag) NMs are also described. We envision that this labeling strategy will be adopted by industry to facilitate applications such as nanosafety assessments before NMs enter the market and environment, as well as for product authentication and tracking.

A Data Set for Airborne Maritime Surveillance Environments

This paper presents a data set with surveillance imagery over the sea captured by a small size UAV. This data set presents the object examples ranging from cargo ships, small boats, life rafts to hydrocarbon slick. The video sequences were captured using different types of cameras, at different heights, and different perspectives. The data set also contains thousands of labels with positions of objects of interest. This was only possible to achieve with the labeling tool also described in this paper. Additionally, using standard evaluation frameworks, we establish a baseline of results using algorithms developed by the authors, which are better adapted to the maritime environment.

EuroFlow Lymphoid Screening Tube (LST) data base for automated identification of blood lymphocyte subsets

In recent years the volume and complexity of flow cytometry data has increased substantially. This has led to a greater number of identifiable cell populations in a single measurement. Consequently, new gating strategies and new approaches for cell population definition are required. Here we describe how the EuroFlow Lymphoid Screening Tube (LST) reference data base for peripheral blood (PB) samples was designed, constructed and validated for automated gating of the distinct lymphoid (and myeloid) subsets in PB of patients with chronic lymphoproliferative disorders (CLPD). A total of 46 healthy/reactive PB samples which fulfilled pre-defined technical requirements, were used to construct the LST-PB reference data base. In addition, another set of 92 PB samples (corresponding to 10 healthy subjects, 51 B-cell CLPD and 31 T/NK-cell CLPD patients), were used to validate the automated gating and cell-population labeling tools with the Infinicyt software.An overall high performance of the LST-PB data base was observed with a median percentage of alarmed cellular events of 0.8% in 10 healthy donor samples and of 44.4% in CLPD data files containing 49.8% (range: 1.3–96%) tumor cells. The higher percent of alarmed cellular events in every CLPD sample was due to aberrant phenotypes (75.6% cases) and/or to abnormally increased cell counts (86.6% samples). All 18 (22%) data files that only displayed numerical alterations, corresponded to T/NK-cell CLPD cases which showed a lower incidence of aberrant phenotypes (41%) vs B-cell CLPD cases (100%). Comparison between automated vs expert-bases manual classification of normal (r2 = 0.96) and tumor cell populations (rho = 0.99) showed a high degree of correlation.In summary, our results show that automated gating of cell populations based on the EuroFlow LST-PB data base provides an innovative, reliable and reproducible tool for fast and simplified identification of normal vs pathological B and T/NK lymphocytes in PB of CLPD patients.