Indirect Labeling Research Articles

Comparative Characteristics of Immunochromatographic Test Systems for Tylosin Antibiotic in Meat Products.

Tylosin (TYL) is a macrolide antibiotic widely used in animal husbandry. Due to associated health risks, there is a demand for sensitive methods for mass screening of TYL in products of animal origin. This article describes the development of lateral flow immunoassays (LFIAs) for TYL detection using direct (anti-TYL antibodies conjugated with nanoparticles) and indirect antibody labeling (anti-species antibodies conjugated with nanoparticles and combined with native anti-TYL antibodies). The choice of LFIA conditions, such as concentrations of hapten-protein conjugates, specific antibodies, and gold nanoparticle (GNP) conjugates with antibodies, as well as incubation time of reagents and the concentration of detergent in the sample buffer, is presented. The achieved limits of TYL detection using LFIAs with indirect labeling were 0.8 ng/mL (visual) and 0.07 ng/mL (instrumental), compared to 4 ng/mL (visual) and 0.4 ng/mL (instrumental) for the case of direct labeling. The sensitivity of the LFIA using the indirect format was up to seven times higher, allowing the determination of the target analyte at low concentrations. TYL detection in ground meat using LFIA with indirect antibody labeling ranged from 76-119%.

Comparison between USPIOs and SPIOs for Multimodal Imaging of Extracellular Vesicles Extracted from Adipose Tissue-Derived Adult Stem Cells.

Adipose tissue-derived adult stem (ADAS) cells and extracellular vesicle (EV) therapy offer promising avenues for treating neurodegenerative diseases due to their accessibility and potential for autologous cell transplantation. However, the clinical application of ADAS cells or EVs is limited by the challenge of precisely identifying them in specific regions of interest. This study compares two superparamagnetic iron oxide nanoparticles, differing mainly in size, to determine their efficacy for allowing non-invasive ADAS tracking via MRI/MPI and indirect labeling of EVs. We compared a USPIO (about 5 nm) with an SPIO (Resovist®, about 70 nm). A physicochemical characterization of nanoparticles was conducted using DLS, TEM, MRI, and MPI. ADAS cells were labeled with the two nanoparticles, and their viability was assessed via MTT assay. MRI detected labeled cells, while TEM and Prussian Blue staining were employed to confirm cell uptake. The results revealed that Resovist® exhibited higher transversal relaxivity value than USPIO and, consequently, allows for detection with higher sensitivity by MRI. A 200 µgFe/mL concentration was identified as optimal for ADAS labeling. MPI detected only Resovist®. The findings suggest that Resovist® may offer enhanced detection of ADAS cells and EVs, making it suitable for multimodal imaging. Preliminary results obtained by extracting EVs from ADAS cells labeled with Resovist® indicate that EVs retain the nanoparticles, paving the way to an efficient and multimodal detection of EVs.

Preliminary Research of Radiolabeled Atezolizumab for the Noninvasive Evaluation of TNBC PD-L1 Expression InVivo.

Programmed death-ligand 1 (PD-L1) expression is related to the efficacy and prognosis in triple-negative breast cancer. This study employed an indirect labeling method to synthesize [125I]PI-Atezolizumab. The invitro stability of [125I]PI-Atezolizumab was assessed through incubation in phosphate buffered saline and fetal bovine serum, revealing sustained stability. Specific binding of [125I]PI-Atezolizumab to MDA-MB-231 cells expressing humanized PD-L1 was assessed through invitro incubation, yielding a Kd value comparable to that of Atezolizumab. This suggests that the labeling process did not compromise the affinity of the Atezolizumab to PD-L1. Subsequently, pharmacokinetic studies were conducted in normal mice and biodistribution experiments in tumor-bearing mice. A comparison of the biodistribution results between [125I]PI-Atezolizumab and 125I-labeled Atezolizumab indicated better invivo stability for the former. Single photon emission computed tomography (SPECT)/CT imaging further confirmed the targeted specificity of [125I]PI-Atezolizumab for PD-L1 in MDA-MB-231 xenografts, which were validated by immunohistochemistry staining. This research underscores the utility of [125I]PI-Atezolizumab, prepared via indirect labeling, for monitoring PD-L1 in triple-negative breast cancer models.

Sortase-Mediated Site-Specific Conjugation to Prepare Fluorine-18-Labeled Nanobodies.

Single-domain antibodies, or nanobodies (Nbs), are promising biomolecules for use in molecular imaging due to their excellent affinity, specificity, and fast clearance from the blood. Given their short blood half-life, pairing Nbs with short-lived imaging radioisotopes is desirable. Because fluorine-18 (18F) is routinely used for clinical imaging, it is an attractive radioisotope for Nbs. We report a novel sortase-based, site-specific 18F-labeling method applied to three nanobodies. Labeled nanobodies were synthesized either by a two-step indirect radiolabeling method in one pot or by a one-step direct labeling method using a sortase-mediated conjugation of either the radiolabeled chelator (H-GGGK((±)-Al[18F]FH3RESCA)-NH2) or the unlabeled chelator (H-GGGK((±)-H3RESCA)-NH2) followed by labeling with Al[18F]F, respectively. The overall radiochemical yields were 15-43% (n = 22, decay-corrected) in 70 min (indirect labeling) and 23-58% (n = 12, decay-corrected) in 50 min (direct labeling). The radiochemical purities of the labeled nanobodies prepared by both methods were >98% with a specific activity of 400-600 Ci/mmol (n = 22) for each of the three Nbs tested and exhibited excellent stability profiles under physiological conditions. This simple, site-specific, reproducible, and generalizable 18F-labeling method to prepare nanobodies (Nb-Al[18F]F-RESCA) or other low molecular weight biomolecules can easily be adopted in various settings for preclinical and clinical studies.

Magnetic Metalloimmunoassays: Influence of Particle Shape and Size on Galvanic Exchange-Based Electrochemical Detection

The electrooxidation of silver (Ag) offers improved signal sharpness and intensity, enabling precise measurements and enhanced resolution in electrochemical sensing. Leveraging silver nanoparticles (AgNPs) as indirect labels in electrochemical assays provides a distinct advantage for detecting various biomarkers at clinically relevant concentrations. In our recent work, we have introduced an electrochemical approach that involves a galvanic exchange (GE) reaction between electrodeposited gold and AgNPs, followed by anodic stripping voltammetry (ASV), with potential applications in immunosensing. In this assay protocol, specific monoclonal antibodies immobilize the biomarker of interest separately on AgNPs and magnetic beads. Subsequently, magnetic bead-AgNP conjugates are attracted to the electrode surface by a magnetic force. However, to achieve the expected analytical performance of the bioassay, it is imperative to characterize the composition, size, shape, and surface modifications of the AgNPs. Consequently, our research focuses on investigating the electrochemical properties of a model bioconjugate formed between magnetic microbeads (MB) and AgNPs through Streptavidin-biotin interactions under various assay conditions. Notably, our recent studies reveal that conducting the GE cycle for three repetitions significantly enhances the Ag signal, yielding an improvement of approximately two-fold or greater compared to previously reported methods. Previous studies have shown that altering the shape of AgNPs, transitioning from nanospheres to other forms like nanocubes and nanodisks enhances both the limit of detection and the range of analyte detection. However, despite these enhancements, spherical AgNPs remain the most stable and easily synthesized option, characterized by excellent homogeneity. Recently, we compared AgNPs with diameters ranging from 20 to 100 nm paired with magnetic microbeads with diameters spanning from 50 nm to 3 µm to identify the optimal combination. Preliminary results indicate that the Mb (3 µm)-AgNP (50 nm) conjugate exhibits the highest Ag charge and Ag collection efficiency. Nonetheless, critical knowledge gaps persist regarding the fundamental understanding of the electrogenerated GE process and we are continually working on refining the electrochemical and assay parameters to develop a robust universal platform for GE-based electrochemical immunosensing.

Hetero-Diels-Alder and CuAAC Click Reactions for Fluorine-18 Labeling of Peptides: Automation and Comparative Study of the Two Methods.

Radiolabeled peptides are valuable tools for diagnosis or therapies; they are often radiofluorinated using an indirect approach based on an F-18 prosthetic group. Herein, we are reporting our results on the F-18 radiolabeling of three peptides using two different methods based on click reactions. The first one used the well-known CuAAC reaction, and the second one is based on our recently reported hetero-Diels-Alder (HDA) using a dithioesters (thia-Diels-Alder) reaction. Both methods have been automated, and the 18F-peptides were obtained in similar yields and synthesis time (37-39% decay corrected yields by both methods in 120-140 min). However, to obtain similar yields, the CuAAC needs a large amount of copper along with many additives, while the HDA is a catalyst and metal-free reaction necessitating only an appropriate ratio of water/ethanol. The HDA can therefore be considered as a minimalist method offering easy access to fluorine-18 labeled peptides and making it a valuable additional tool for the indirect and site-specific labeling of peptides or biomolecules.

A Useful Method for in situ Indirect Radiolabeling of Carbonyl-Containing Biomolecules

[125I] 2-(2,3-diiodo-4-hydroxyphenyl)-1,3,4-oxadiazole was synthesized (48% yield) and acid-hydrolyzed in situ with direct derivatization with p-nitro benzaldehyde to obtain the required 125I hydrazone (89% yield). A blood clearance kinetic study was performed on the 125I oxadiazole in a Sprague-Dawley rat and a biphasic behaviour was obtained. This study shows that 125I oxadiazole has potential to be metabolically stable and enough to allow imaging and tracking for a minimum of 2 h, making it a viable option for in situ indirect labeling of carbonyl containing macromolecules and keto steroids.

Antibody elution with 2-me/SDS solution: Uses for multi-layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexus

Indirect immunofluorescence is usually restricted to 3-5 markers per preparation, limiting analysis of coexistence. A solution containing 2-mercaptoethanol and sodium dodecyl sulfate (2-ME/SDS) can elute indirect immunofluorescence labelling (i.e. primary antisera followed by fluorophore-conjugated secondary antisera) and has been used for sequential staining of sections. The aim of this study was to test whether 2-ME/SDS is effective for eluting indirect immunofluorescent staining (with primary antisera visualised by fluorophore-coupled secondary antisera) in wholemount preparations. We also analysed how 2-ME/SDS may work and used this understanding to devise additional uses for immunofluorescence in the nervous system. 2-ME/SDS appears to denature unfixed proteins (including antisera used as reagents) but has much less effect on antigenicity of formaldehyde-fixed epitopes. Moieties linked by strong biotin-streptavidin bonds are highly resistant to elution by 2-ME/SDS. Two primary antisera raised in the same species can be applied without spurious cross-reactivity, if a specific order of labelling is followed. The first primary antiserum is followed by a biotinylated secondary, then a tertiary of fluorophore-conjugated streptavidin. The preparation is then exposed to 2-ME/SDS, which has minimal impact on labelling by the first primary/secondary/tertiary combination. However, when this is followed by a second primary antiserum (raised in the same species), followed by a fluorophore-conjugated secondary antiserum, the intervening 2-ME/SDS exposure prevents cross-reactivity between primary and secondary antisera of the two layers. A third property of 2-ME/SDS is that it reduces lipofuscin autofluorescence, although it also raises background fluorescence and strongly enhances autofluorescence of erythrocytes. In summary, 2-ME/SDS is easy to use, cost-effective and does not require modified primary antisera. It can be used as the basis of a multi-layer immunohistochemistry protocol and allows 2 primary antisera raised in the same species to be used together.

The effect of aquaporin-4 inhibition on cerebrospinal fluid-tissue water exchange in mouse brain detected by magnetization transfer indirect spin labeling MRI.

The fluid transport of cerebrospinal fluid (CSF) and interstitial fluid in surrounding tissues plays an important role in the drainage pathway that facilitates waste clearance from the brain. This pathway is known as the glymphatic or perivascular system, and its functions are dependent on aquaporin-4 (AQP4). Recently, magnetization transfer indirect spin labeling (MISL) magnetic resonance imaging (MRI) has been proposed as a noninvasive and noncontrast-enhanced method for detecting water exchange between CSF and brain tissue. In this study, we first optimized the MISL sequence at preclinical 3 T MRI, and then studied the correlation of MISL in CSF with magnetization transfer (MT) in brain tissue, as well as the altered water exchange under AQP4 inhibition, using C57BL/6 mice. Results showed a strong correlation of MISL signal with MT signal. With the AQP4 inhibitor, we observed a significant decrease in MISL value (P < 0.05), suggesting that the hampered AQP4 activity led to decreased water exchange between CSF and brain tissue or the impairment of the glymphatic function. Overall, our findings demonstrate the potential application of MISL in assessing brain water exchange at 3 T MRI and its potential clinical translation.

Placement and acidification of biowastes: Potential strategies for improving phosphorus use efficiency

This study aimed to improve the phosphorus (P) use efficiency of various biowastes. To achieve this, we proposed the combination of placement and the acidification of biowastes for the development of P-efficient biobased fertilizers. Seven biowastes were used as P-fertilizers, including two meat and bone meals (MBM), two solid fractions of biogas digestate (BGF), and three sewage sludges (SS). Two acidification approaches were evaluated: pre-treatment with sulfuric acid (73.5 g sulfuric acid per kg fresh weight) and co-application with ammonium sulfate (15 mg N per kg soil). The treated materials were placed close to winter wheat (Triticum aestivum cv. Benchmark) seeds in a 42-day pot experiment in a growth chamber. The P use efficiency was compared to the corresponding untreated material homogeneously mixed or similarly placed in the soil. A 33P indirect labeling method was used to differentiate P uptake from soil and fertilizer. The negative control showed a shoot dry matter of 0.42 g ( ± 0.05) and a total P uptake of 0.62 ( ± 0.06) mg per plant. The placement of acidified sewage sludge did not show significant differences in shoot dry matter and P uptake compared to the negative control. On the other hand, the placement of acidified MBM significantly increased plant growth (MBM I: 2.93 ± 0.46; MBM II: 1.78 ± 0.09 g) and P uptake (MBM I: 7.38 ± 1.10; MBM II: 6.21 ± 0.53 mg) compared to the untreated MBM placed and the negative control. Similarly, the placement of acidified BGF showed promising results, with a 3-fold increase in P uptake from the fertilizer compared to untreated BGF mixed with the soil (7.74 ± 1.47 vs. 2.71 ± 0.26 mg). Therefore, our results demonstrate that the placement of acidified MBM and BGF can increase the P use efficiency of these biowastes and may provide a basis for formulating P-efficient biobased fertilizers using these materials.

Advances in CAR T Cell Therapy for Non-Small Cell Lung Cancer.

Since its first approval by the FDA in 2017, tremendous progress has been made in chimeric antigen receptor (CAR) T cell therapy, the adoptive transfer of engineered, CAR-expressing T lymphocyte. CAR T cells are all composed of three main elements: an extracellular antigen-binding domain, an intracellular signaling domain responsible for T cell activation, and a hinge that joins these two domains. Continuous improvement has been made in CARs, now in their fifth generation, particularly in the intracellular signaling domain responsible for T cell activation. CAR T cell therapy has revolutionized the treatment of hematologic malignancies. Nonetheless, the use of CAR T cell therapy for solid tumors has not attained comparable levels of success. Here we review the challenges in achieving effective CAR T cell therapy in solid tumors, and emerging CAR T cells that have shown great promise for non-small cell lung cancer (NSCLC). A growing number of clinical trials have been conducted to study the effect of CAR T cell therapy on NSCLC, targeting different types of surface antigens. They include epidermal growth factor receptor (EGFR), mesothelin (MSLN), prostate stem cell antigen (PSCA), and mucin 1 (MUC1). Potential new targets such as erythropoietin-producing hepatocellular carcinoma A2 (EphA2), tissue factor (TF), and protein tyrosine kinase 7 (PTK7) are currently under investigation in clinical trials. The challenges in developing CAR T for NSCLC therapy and other approaches for enhancing CAR T efficacy are discussed. Finally, we provide our perspective on imaging CAR T cell action by reviewing the two main radionuclide-based CAR T cell imaging techniques, the direct labeling of CAR T cells or indirect labeling via a reporter gene.

The Fluorescence in situ hybridization (FISH): types and application: a review

The macromolecule identification method known as fluorescence in situ hybridization (FISH) is regarded as a recent development in the study of cytology. It was first created as a physical mapping method to identify genes inside chromosomes. Later, biological and medical studies benefited from the precision and adaptability of FISH. This attractive method offers a level of resolution in between chromosomal studies and DNA analysis. A hybridizing DNA probe used in FISH can either be directly or indirectly labeled. Fluorescent nucleotides are employed in direct labeling, whereas reporter molecules used in indirect labeling are afterwards recognized through affinity molecules, fluorescent antibodies, or other means. An excessively high number of chromosomes in a cell, unique gene fusions, or the loss of a chromosomal segment or an entire chromosome are examples of genetic disorders that can be detected with FISH. It is also used in a variety of scientific projects, including gene mapping and the discovery of fresh oncogenes. This article examines the FISH idea, its use, and its benefits in medical research.

Multiplex immunofluorescence staining of coverslip-mounted paraffin-embedded tissue sections.

Animal and human tissues are used extensively in physiological and pathophysiological research. Due to both ethical considerations and low availability, it is essential to maximize the use of these tissues. Therefore, the aim was to develop a new method allowing for multiplex immunofluorescence (IF) staining of kidney sections in order to reuse the same tissue section multiple times. The paraffin-embedded kidney sections were placed onto coated coverslips and multiplex IF staining was performed. Five rounds of staining were performed where each round consisted of indirect antibody labelling, imaging on a widefield epifluorescence microscope, removal of the antibodies using a stripping buffer, and then re-staining. In the final round, the tissue was stained with hematoxylin/eosin. Using this method, tubular segments in the nephron, blood vessels, and interstitial cells were labeled. Furthermore, by placing the tissue on coverslips, confocal-like resolution was obtained using a conventional widefield epifluorescence microscope and a 60x oil objective. Thus, using standard reagents and equipment, paraffin-embedded tissue was used for multiplex IF staining with increased Z-resolution. In summary, this method offers time-saving multiplex IF staining and allows for the retrieval of both quantitative and spatial expressional information of multiple proteins and subsequently for an assessment of the tissue morphology. Due to the simplicity and integrated effectivity of this multiplex IF protocol, it holds the potential to supplement standard IF staining protocols and maximize use of tissue.

Seven-colour multiplex immunochemistry/immunofluorescence and whole slide imaging of frozen sections

Multiplex Immunochemistry/Immunofluorescence (mIHC/IF) aims to visualise multiple biomarkers in a single tissue section and is especially powerful when used on slide scanners coupled with digital analysis tools. mIHC/IF is commonly employed in immuno-oncology to characterise features of the tumour microenvironment (TME) and correlate them with clinical parameters to guide prognostication and therapy. However, mIHC/IF can be applied to a wide range of organisms in any physiological or disease context. Recent innovation has extended the number of markers that can be detected using slide scanners well beyond the 3–4 markers typically reported in traditional fluorescence microscopy. However, these methods often require sequential antibody staining and stripping, and are not compatible with frozen tissue sections.Using fluorophore-conjugated antibodies, we have established a simple mIHC/IF imaging workflow that enables simultaneous staining and detection of seven markers in a single section of frozen tissue. Coupled with automated whole slide imaging and digital quantification, our data efficiently revealed the tumour-immune complexity in metastatic melanoma. Computational image analysis quantified the immune and stromal cell populations present in the TME as well as their spatial interactions. This imaging workflow can also be performed with an indirect labelling panel consisting of primary and secondary antibodies. Our new methods, combined with digital quantification, will provide a valuable tool for high-quality mIHC/IF assays in immuno-oncology research and other translational studies, especially in circumstances where frozen sections are required for detection of particular markers, or for applications where frozen sections may be preferred, such as spatial transcriptomics.

Extending the Range of SLIM-Labeling Applications: From Human Cell Lines in Culture to Caenorhabditis elegans Whole-Organism Labeling.

The simple light isotope metabolic-labeling technique relies on the in vivo biosynthesis of amino acids from U-[12C]-labeled molecules provided as the sole carbon source. The incorporation of the resulting U-[12C]-amino acids into proteins presents several key advantages for mass-spectrometry-based proteomics analysis, as it results in more intense monoisotopic ions, with a better signal-to-noise ratio in bottom-up analysis. In our initial studies, we developed the simple light isotope metabolic (SLIM)-labeling strategy using prototrophic eukaryotic microorganisms, the yeasts Candida albicans and Saccharomyces cerevisiae, as well as strains with genetic markers that lead to amino-acid auxotrophy. To extend the range of SLIM-labeling applications, we evaluated (i) the incorporation of U-[12C]-glucose into proteins of human cells grown in a complex RPMI-based medium containing the labeled molecule, considering that human cell lines require a large number of essential amino-acids to support their growth, and (ii) an indirect labeling strategy in which the nematode Caenorhabditis elegans grown on plates was fed U-[12C]-labeled bacteria (Escherichia coli) and the worm proteome analyzed for 12C incorporation into proteins. In both cases, we were able to demonstrate efficient incorporation of 12C into the newly synthesized proteins, opening the way for original approaches in quantitative proteomics.

Radiolabelling of Polyclonally Expanded Human Regulatory T Cells (Treg) with 89Zr-oxine for Medium-Term In Vivo Cell Tracking.

Regulatory T cells (Tregs) are a promising candidate cell therapy to treat autoimmune diseases and aid the longevity of transplanted solid organs. Despite increasing numbers of clinical trials using human Treg therapy, important questions pertaining to their in vivo fate, distribution, and function remain unanswered. Treg accumulation in relevant tissues was found to be crucial for Treg therapy efficacy, but existing blood-borne biomarkers are unlikely to accurately reflect the tissue state. Non-invasive Treg tracking by whole-body imaging is a promising alternative and can be achieved by direct radiolabelling of Tregs and following the radiolabelled cells with positron emission tomography (PET). Our goal was to evaluate the radiolabelling of polyclonal Tregs with 89Zr to permit their in vivo tracking by PET/CT for longer than one week with current preclinical PET instrumentation. We used [89Zr]Zr(oxinate)4 as the cell-labelling agent and achieved successful radiolabelling efficiency of human Tregs spanning 0.1-11.1 Bq 89Zr/Treg cell, which would be compatible with PET tracking beyond one week. We characterized the 89Zr-Tregs, assessing their phenotypes, and found that they were not tolerating these intracellular 89Zr amounts, as they failed to survive or expand in a 89Zr-dose-dependent manner. Even at 0.1 Bq 89Zr per Treg cell, while 89Zr-Tregs remained functional as determined by a five-day-long effector T cell suppression assay, they failed to expand beyond day 3 in vitro. Moreover, PET imaging revealed signs of 89Zr-Treg death after adoptive transfer in vivo. In summary, 89Zr labelling of Tregs at intracellular radioisotope amounts compatible with cell tracking over several weeks did not achieve the desired outcomes, as 89Zr-Tregs failed to expand and survive. Consequently, we conclude that indirect Treg labelling is likely to be the most effective alternative method to satisfy the requirements of this cell tracking scenario.

Tracking tools of extracellular vesicles for biomedical research.

Imaging of extracellular vesicles (EVs) will facilitate a better understanding of their biological functions and their potential as therapeutics and drug delivery vehicles. In order to clarify EV-mediated cellular communication in vitro and to track the bio-distribution of EV in vivo, various strategies have been developed to label and image EVs. In this review, we summarized recent advances in the tracking of EVs, demonstrating the methods for labeling and imaging of EVs, in which the labeling methods include direct and indirect labeling and the imaging modalities include fluorescent imaging, bioluminescent imaging, nuclear imaging, and nanoparticle-assisted imaging. These techniques help us better understand the mechanism of uptake, the bio-distribution, and the function of EVs. More importantly, we can evaluate the pharmacokinetic properties of EVs, which will help promote their further clinical application.

Fully automated 18F-fluorination of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) for indirect labelling of nanobodies

N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB), a widely used labeling agent to introduce the 4-[18F]fluorobenzoyl-prosthetic group, is normally obtained in three consecutive steps from [18F]fluoride ion. Here, we describe an efficient one-step labeling procedure of [18F]SFB starting from a tin precursor. This method circumvents volatile radioactive side-products and simplifies automatization. [18F]SFB was obtained after HPLC purification in a yield of 42 + 4% and a radiochemical purity (RCP) > 99% (n = 6). In addition, we investigate the automation of the coupling of [18F]SFB to a nanobody (cAbBcII10, targeting β-lactamase enzyme) and purification by size exclusion chromatography (PD-10 desalting column) to remove unconjugated reagent. Production and use of [18F]SFB were implemented on a radiosynthesis unit (Neptis®). The fully automated radiosynthesis process including purification and formulation required 160 min of synthesis time. [18F]SFB-labeled nanobody was obtained in a yield of 21 + 2% (activity yield 12 + 1% non-decay corrected) and a radiochemical purity (RCP) of > 95% (n = 3). This approach simplifies [18F]SFB synthesis to one-step, enhances the yield in comparison to the previous report and enables the production of radiolabeled nanobody on the same synthesis module.

Localization and functions of native and eGFP-tagged capsid proteins in HIV-1 particles.

In infectious HIV-1 particles, the capsid protein (CA) forms a cone-shaped shell called the capsid, which encases the viral ribonucleoprotein complex (vRNP). Following cellular entry, the capsid is disassembled through a poorly understood process referred to as uncoating, which is required to release the reverse transcribed HIV-1 genome for integration into host chromatin. Whereas single virus imaging using indirect CA labeling techniques suggested uncoating to occur in the cytoplasm or at the nuclear pore, a recent study using eGFP-tagged CA reported uncoating in the nucleus. To delineate the HIV-1 uncoating site, we investigated the mechanism of eGFP-tagged CA incorporation into capsids and the utility of this fluorescent marker for visualizing HIV-1 uncoating. We find that virion incorporated eGFP-tagged CA is effectively excluded from the capsid shell, and that a subset of the tagged CA is vRNP associated. These results thus imply that eGFP-tagged CA is not a direct marker for capsid uncoating. We further show that native CA co-immunoprecipitates with vRNP components, providing a basis for retention of eGFP-tagged and untagged CA by sub-viral complexes in the nucleus. Moreover, we find that functional viral replication complexes become accessible to integrase-interacting host factors at the nuclear pore, leading to inhibition of infection and demonstrating capsid permeabilization prior to nuclear import. Finally, we find that HIV-1 cores containing a mixture of wild-type and mutant CA interact differently with cytoplasmic versus nuclear pools of the CA-binding host cofactor CPSF6. Our results suggest that capsid remodeling (including a loss of capsid integrity) is the predominant pathway for HIV-1 nuclear entry and provide new insights into the mechanism of CA retention in the nucleus via interaction with vRNP components.

Taking molecular pathology to the next level: Whole slide multicolor confocal imaging with the Pannoramic Confocal digital pathology scanner.

The emergence and fast advance of digital pathology allows the acquisition, digital storage, interactive recall and analysis of morphology at the tissue level. When applying immunohistochemistry, it also affords the correlation of morphology with the expression of one or two specific molecule of interest. The rise of fluorescence pathology scanners expands the number of detected molecules based on multiplex labeling. The Pannoramic Confocal (created by 3DHistech, Hungary) is a first-of-the-kind digital pathology scanner that affords not only multiplexed fluorescent detection on top of conventional transmission imaging, but also confocality. We have benchmarked this scanner in terms of stability, precision, light efficiency, linearity and sensitivity. X-Y stability and relocalisation precision were well below resolution limit (≤50 nm). Light throughput in confocal mode was 4-5 times higher than that of a point scanning confocal microscope, yielding similar calculated confocal intensities but with the potential for improving signal to noise ratio or scan speed. Response was linear with R2 ≥ 0.9996. Calibrated measurements showed that using indirect labeling ≥2000 molecules per cell could be well detected and imaged on the cell surface. Both standard-based and statistical post-acquisition flatfield corrections are implemented. We have also measured the point spread function (PSF) of the instrument. The dimensions of the PSF are somewhat larger and less symmetric than of the theoretical PSF of a conventional CLSM, however, the spatial homogeneity of these parameters allows for obtaining a specific system PSF for each optical path and using it for optional on-the-fly deconvolution. In conclusion, the Pannoramic Confocal provides sensitive, quantitative widefield and confocal detection of multiplexed fluorescence signals, with optical sectioning and 3D reconstruction, in addition to brightfield transmission imaging. High speed scanning of large samples, analysis of tissue heterogeneity, and detection of rare events open up new ways for quantitatively analyzing tissue sections, organoid cultures or large numbers of adherent cells.