Direct Labeling Research Articles

Experimental study of 131I-caerin 1.1 and 131I-c(RGD)2 for internal radiation therapy of esophageal cancer xenografts

ObjectiveThe aim of this study was to analyze and compare the therapeutic effects of 131I-caerin 1.1 and 131I-c(RGD)2 on TE-1 esophageal cancer cell xenografts. Methods(1) The in vitro antitumor effects of the polypeptides caerin 1.1 and c(RGD)2 were verified by MTT and clonogenic assays. 131I-caerin 1.1 and 131I-c(RGD)2 were prepared by chloramine-T (Ch-T) direct labeling, and their basic properties were measured. The binding and elution of 131I-caerin 1.1, 131I-c(RGD)2, and Na131I (control group) in esophageal cancer TE-1 cells were studied through cell binding and elution assays. (2) The antiproliferative effect and cytotoxicity of 131I-caerin 1.1, 131I-c(RGD)2, Na131I, caerin 1.1 and c(RGD)2 on TE-1 cells were detected by Cell Counting Kit-8 (CCK-8) assay. (3) A nude mouse esophageal cancer (TE-1) xenograft model was established to study and compare the efficacy of 131I-caerin 1.1 and 131I-c(RGD)2 in internal radiation therapy for esophageal cancer. Results(1) Caerin 1.1 inhibited the in vitro proliferation of TE-1 cells in a concentration-dependent manner, with an IC50 of 13.00 µg/mL. The polypeptide c(RGD)2 had no evident inhibitory effect on the in vitro proliferation of TE-1 cells. Therefore, the antiproliferative effects of caerin 1.1 and c(RGD)2 on esophageal cancer cells were significantly different (P < 0.05). The clonogenic assay showed that the clonal proliferation of TE-1 cells decreased as the concentration of caerin 1.1 increased. Compared with the control group (drug concentration of 0 µg/mL), the caerin 1.1 group showed significantly lower clonal proliferation of TE-1 cells (P < 0.05). (2) The CCK-8 assay showed that 131I-caerin 1.1 inhibited the in vitro proliferation of TE-1 cells, while 131I-c(RGD)2 had no evident inhibitory effect on proliferation. The two polypeptides showed significantly different antiproliferative effects on esophageal cancer cells at higher concentrations (P < 0.05). Cell binding and elution assays showed that 131I-caerin 1.1 stably bound to TE-1 cells. The cell binding rate of 131I-caerin 1.1 was 15.8 % ± 1.09 % at 24 h and 6.95 % ± 0.22 % after 24 h of incubation and elution. The cell binding rate of 131I-c(RGD)2 was 0.06 % ± 0.02 % at 24 h and 0.23 % ± 0.11 % after 24 h of incubation and elution. (3) In the in vivo experiment, 3 days after the last treatment, the tumor sizes of the phosphate-buffered saline (PBS) group, caerin 1.1 group, c(RGD)2 group, 131I group, 131I-caerin 1.1 group, and 131I-c(RGD)2 group were 68.29 ± 2.67 mm3, 61.78 ± 3.58 mm3, 56.67 ± 5.65 mm3, 58.88 ± 1.71 mm3, 14.40 ± 1.38 mm3, and 60.14 ± 0.47 mm3, respectively. Compared with the other treatment groups, the 131I-caerin 1.1 group had significantly smaller tumor sizes (P < 0.001). After treatment, the tumors were isolated and weighed. The tumor weights in the PBS group, caerin 1.1 group, c(RGD)2 group, 131I group, 131I-caerin 1.1 group, and 131I-c(RGD)2 group were 39.50 ± 9.54 mg, 38.25 ± 5.38 mg, 38.35 ± 9.53 mg, 28.25 ± 8.50 mg, 9.50 ± 4.43 mg, and 34.75 ± 8.06 mg, respectively. The tumor weights in the 131I-caerin 1.1 group were significantly lighter than those in the other groups (P < 0.01). Conclusion131I-caerin 1.1 has tumor-targeting properties, is capable of targeted binding to TE-1 esophageal cancer cells, can be stably retained in tumor cells, and has an evident cytotoxic killing effect, while 131I-c(RGD)2 has no evident cytotoxic effect. 131I-caerin 1.1 better suppressed tumor cell proliferation and tumor growth than pure caerin 1.1, 131I-c(RGD)2, and pure c(RGD)2.

First-passage time analysis of diffusion-controlled reactions in single-molecule detection

Single-molecule detection (SMD) aims to achieve the ultimate limit-of-detection (LOD) in biosensing. This method detects a countable number of targeted analyte molecules in solution, where the dynamics of molecule diffusion, capturing, identification and delivery greatly impact the SMD's efficiency and accuracy. In this study, we adopt the first-passage time method to investigate the diffusion-controlled reaction process in SMD. We analyze the influence of detection conditions on incubation time and the expected coefficient of variation (CV) under three SMD molecule capturing strategies, including solid-phase capturing (one-dimensional solid-liquid interface fixation), liquid-phase magnetic bead (MB) capturing, and liquid-phase direct fluorescence pair labeling. We find that inside a finite-sized reaction chamber, a finite average reaction time exists in all three capturing strategies, while the liquid-phase strategies are in general more efficient than the solid-phase approaches. CV can be estimated by averaging first-passage time solely in all three strategies, and the CV reduction is achievable given an extended reaction time. To further enable zeptomolar detection, extra treatments, such as adopting liquid-phase fluorescence pairs with high diffusion rates to label the molecule, or designing specific sensing devices with large effective sensing areas would be required. This framework provides solid theoretical support to guide the design of SMD sensing strategies and sensor structures to achieve desired measurement time and CV.

Consumers' preferences and willingness to pay for coffee sustainability labels

AbstractConsumers are displaying an increasing awareness for the social and environmental impact of coffee production, and this is also reflected in their willingness to pay for sustainably produced coffee. For example, the Direct Trade label, which highlights a direct relationship between roasters and farmers, has gained popularity to strengthen the industry's efforts to make coffee production more sustainable. This leads to the question of whether consumers prefer coffee carrying this label. Hence, this research analyzes consumer preferences and willingness to pay for coffee labels representing sustainability efforts, such as the Direct Trade label. We informed participants about the efforts reflected by each sustainability label and utilized a hypothetical online choice experiment to determine US consumers' preferences and willingness to pay for coffee sustainability labels. The highest willingness to pay was found for coffee carrying both the Fair Trade and Organic labels, and the Direct Trade and Organic labels. The key findings of this study demonstrate that consumers are willing to pay more for sustainability labels that communicate efforts on solving social issues, followed by labels that highlight environmental issues. [EconLit Citation: C9].

Identifying optimal channels and features for multi-participant motor imagery experiments across a participant's multi-day multi-class EEG data.

The concept of the brain-computer interface (BCI) has become one of the popular research topics of recent times because it allows people to express their thoughts and control different applications and devices without actual movement. The communication between the brain and the computer or a machine is generally provided through Electroencephalogram (EEG) signals because they are cost-effective and easy to implement in normal life, not just in healthcare facilities. On the other hand, they are hard to process efficiently due to their nonlinearity and noisy nature. Thus, the field of BCI and EEG needs constant work and improvement. This paper focuses on generalizing the most efficient EEG channels and the most significant features of motor imagery (MI) signals by analyzing the recordings of one participant obtained over 20 different days. Because the classification performance usually decreases with an increasing number of class labels, we have realized the study by analyzing the signals through a new paradigm consisting of multi-class directional labels: right, left, forward, and backward. Afterward, the results are tested on EEG data obtained from 5 participants to see if the results are consistent with each other. The average accuracy of binary and multi-class classification using the Ensemble Subspace Discriminant classifier was found as 87.39 and 61.44%, respectively, with the most efficient 3-channel combination for daily BCI evaluation of one participant. On the other hand, the average accuracy of binary and multi-class classification was found as 71.84 and 50.42%, respectively, for 5 participants, with the most efficient channel combination of 4, where the first three are the same as the daily performance of one participant. During signal processing, the outliers of the signals were discarded by considering the channels separately. An algorithm was developed to dismiss the inconsistent samples within the classes. A novel adaptive filtering approach, correlation-based adaptive variational mode decomposition (CBAVMD), was proposed. The feature selection was realized based on the standard deviation values of the features between the classes. The paradigm based on the direction movements was found to be most effective, especially for binary classification of right and left directions. The generalization of effective channels and features was found to be generally successful.

Automated Synthesis of [18F]Flumazenil Application in GABAA Receptor Neuroimaging Availability for Rat Model of Anxiety.

Clinical studies have demonstrated that the γ-aminobutyric acid type A (GABAA) receptor complex plays a central role in the modulation of anxiety. Conditioned fear and anxiety-like behaviors have many similarities at the neuroanatomical and pharmacological levels. The radioactive GABA/BZR receptor antagonist, fluorine-18-labeled flumazenil, [18F]flumazenil, behaves as a potential PET imaging agent for the evaluation of cortical damage of the brain in stroke, alcoholism, and for Alzheimer disease investigation. The main goal of our study was to investigate a fully automated nucleophilic fluorination system, with solid extraction purification, developed to replace traditional preparation methods, and to detect underlying expressions of contextual fear and characterize the distribution of GABAA receptors in fear-conditioned rats by [18F]flumazenil. A carrier-free nucleophilic fluorination method using an automatic synthesizer with direct labeling of a nitro-flumazenil precursor was implemented. The semi-preparative high-performance liquid chromatography (HPLC) purification method (RCY = 15-20%) was applied to obtain high purity [18F]flumazenil. Nano-positron emission tomography (NanoPET)/computed tomography (CT) imaging and ex vivo autoradiography were used to analyze the fear conditioning of rats trained with 1-10 tone-foot-shock pairings. The anxiety rats had a significantly lower cerebral accumulation (in the amygdala, prefrontal cortex, cortex, and hippocampus) of fear conditioning. Our rat autoradiography results also supported the findings of PET imaging. Key findings were obtained by developing straightforward labeling and purification procedures that can be easily adapted to commercially available modules for the high radiochemical purity of [18F]flumazenil. The use of an automatic synthesizer with semi-preparative HPLC purification would be a suitable reference method for new drug studies of GABAA/BZR receptors in the future.

Novel electrochemical immunosensing platform based on magnetite-antibody conjugate as a direct signal label: design and application for Salmonella typhimurium antigen determination

A novel electrochemical sensing platform based on Fe3O4-antibody conjugate was developed for the rapid and sensitive determination of Salmonella typhimurium antigen in model suspensions. The conjugates Fe3O4-antibody were synthesized via carbodiimide cross-linking and characterized by infrared spectroscopy and cyclic voltammetry. The electrochemical properties of Fe3O4 magnetic nanoparticles in water were investigated; the possible mechanism of direct electrochemical reduction was proposed and justified as an analytical signal. The design of an electrochemical sensing platform with magnetically controlled flows has been proposed. Under the most appropriate conditions, the developed portable device was provided the linear response as I (µA) = (7.20 ± 0.35) × (–lg CAg) − (23.77 ± 1.95) in the concentration range 1 × 10−7–1 × 10−4 mg/mL. The detection limit was 390 ng/mL and was comparable to other works.

Method for direct measuring antibody affinity in biological sample.

We are reporting a new method to measure the affinity of specific antibodies in biological sample without the need of purification, or direct labeling. It was used to measure the affinity of anti-RBD (SARS-CoV2) IgG in plasma from Covid-19 recovered patient and vaccinated populations. Among the vaccinated population, we also measured the affinities of vaccine-induced antibodies against the RBD of the ancestral SARS-Cov-2 virus and the Omicron variants. The method has provided a quantitative way to evaluate antibodies produced by vaccination. The new method involves a classical Solution phase Equilibrium Titration step and an Imaging based Detection step (SET-ID). In the Solution phase Titration step, the antibody was kept at constant concentration (lower than the expected KD value) and mixed with various concentrations of antigen solution; In the Imaging Detection step, the amount of free antibody (not bound to antigen) at each antigen concentration was probed with small amount of microparticles coated with antigen and then the labeled secondary antibody. In this presentation, we will discuss the critical experimental condition, validation, and limitations. For verification, the binding curves of an antibody were compared using a well-established fluorescence spectroscopic-based method and the SET-ID method. The incubation time and the number of microparticles used in the second step were evaluated experimentally and theoretically to ensure minimal perturbation to the solution phase equilibrium, while the fraction of free antibody presented in the solution can still be measured by the signal on the microparticles.

A review on antibody of aducanumab- reduces the progression of Alzheimer disease

Alzheimer&#39;s disease which is the most common form of dementia and affecting millions of people, including family members who act as caretakers. This harmful disease is believed to account for 8% of total US healthcare spending, with health care workers costs amounting to approximately $29 billion. Alzheimer. The initial monoclonal antibody, to newly receive US FDA accelerated approval as moderate Alzheimer disease is aducanumab. Future therapies could be the first chemical approved to treat AD since 2003. The amyloid (A) pathway is becoming involved in the toxic of Alzheimer disease as it has evolved over time from a pathogen to a disease. - Biological structure. The approval of aducanumab is based on the reduction of the Amyload in the brain, which serves as a direct label for this signaling pathway. The fully human IgG1 monoclonal antibody aducanumab (BIIB-037) specifically binds to aggregated form A, inhibiting its matrix function and helping to clear plaque. There are three human trials that ended prematurely. Epidemiology, pathophysiology, and risk factors are discussed in this review, and presently aducanumab is the only drug approved for FDA that slows disease progression.

The Virus-Induced Cytopathic Effect.

The cytopathic effect comprises the set of cellular alterations produced by a viral infection. It is of great relevance since it constitutes a direct marker of infection. Likewise, these alterations are often virus-specific which makes them a phenotypic marker for many viral species. All these characteristics have been used to complement the study of the dynamics of virus-cell interactions through the kinetic study of the progression of damage produced by the infection. Various approaches have been used to monitor the cytopathic effect, ranging from light microscopy, immunofluorescence assays, and direct labeling with fluorescent dyes, to plaque assay for the characterization of the infection over time. Here we address the relevance of the study of cytopathic effect and describe different experimental alternatives for its application.

Visualization of Exosome Release and Uptake During Cell Migration Using the Live Imaging Reporter pHluorin_M153R-CD63.

Exosome secretion and uptake regulate cell migration through autocrine and paracrine mechanisms. Monitoring exosome secretion and uptake during cell migration is critical for investigation of these mechanisms. Exosomes can be visualized by direct labeling with fluorescent dyes or by tagging intrinsic markers with fluorescent proteins for live imaging. Due to several limitations of fluorescent dye-labeled exosomes, we created two bright genetically encoded reporters of exosome secretion, pHluorin_M153R-CD63 and pHluorin_M153R-CD63-mScarlet. Here, we describe how to visualize secretion and uptake of exosomes labeled with these pH-sensitive and pH-insensitive fluorescent protein-tagged exosomal markers during cell migration using time-lapse fluorescent microscopy.

Late-stage peptide labeling with near-infrared fluorogenic nitrobenzodiazoles by manganese-catalyzed C-H activation.

Late-stage diversification of structurally complex amino acids and peptides provides tremendous potential for drug discovery and molecular imaging. Specifically, labeling peptides with fluorescent tags is one of the most important methods for visualizing their mode of operation. Despite major recent advances in the field, direct molecular peptide labeling by C-H activation is largely limited to dyes with relatively short emission wavelengths, leading to high background signals and poor signal-to-noise ratios. In sharp contrast, here we report on the fluorescent labeling of peptides catalyzed by non-toxic manganese(i) via C(sp2)-H alkenylation in chemo- and site-selective manners, providing modular access to novel near-infrared (NIR) nitrobenzodiazole-based peptide fluorogenic probes.

Direct fluorescence labelling of NO inside plant cells.

Nitric oxide (NO) plays a key role in regulating plant growth, enhances nutrient uptake, and activates disease and stress tolerance mechanisms in most plants. NO is marked as a potential tool for improving the yield and quality of horticultural crop species. Research on NO in plant species can provide an abundance of valuable information regarding this. Hence, we have prepared a simple chemosensor (NPO) for the detection of endogenous NO in chickpea saplings. NPO selectively interacts with NO as determined through a chemodosimetric method to clearly show both the colorimetric and fluorometric changes. After the interaction with NO, the colorless NPO turns yellow as observed by the naked eye and shows bright cyan-blue fluorescence under a UV lamp. The 1 : 1 stoichiometric ratio between NPO and NO is determined from Job's plot resulting in a stable diazeniumdiolate product. The interaction mechanism is well established by absorption, fluorescence titration, NMR titration, HRMS, and DFT calculations. This method has successfully been employed in the plant's root and stem systems to label NO. Confocal microscopy images might help us to understand the endogenous NO generation and the mechanism that happens inside plant tissues.

Efficient and Practical Radiosynthesis of Novel [131I]-Xanthine and [131I]-Hypoxanthine

Natural products (NPs) have been the basis for the discovery and development of pharmacologically relevant drug-related molecules, including radiopharmaceuticals. Xanthine (3,7-dihydropurine-2,6-dione) and hypoxanthine (1,9-dihydro-6H-purin-6-one) are purine-based natural heterocyclic alkaloids that are generally found in some plants, animals, and the human body (e.g., muscle tissue, blood, and urine). The purpose of this study was to label xanthine and hypoxanthine with radioactive iodine-131 (a theranostic radionuclide) by a direct labeling method using chloramine-T as an oxidizing agent. Several experiments were performed to optimize the labeling efficiency by changing reaction conditions, including the ratio of starting material and chloramine-T, pH, solvent, temperature, and reaction time. Overall, labeling at acidic conditions in dimethyl sulfoxide (DMSO) resulted in considerable low radiochemical yields (RCYs) (&lt; 4.0 %), and therefore the focus was shifted to exploit the alkaline reaction conditions. The optimized reaction condition: pH (10.5-11.0), xanthine:chloramine-T ratio (1:2), reaction temperature (27 ºC), and reaction time (30 min), provided [ 131 I]-xanthine with a RCY of 65.8 ± 0.1 %. After purification with extraction using chloroform (CHCl 2 ), the radiochemical purity (RCP) of 95.1 % was achieved, as indicated by radio-thin layer chromatography (radio-TLC) analysis. In addition, the labeling of hypoxanthine was accomplished in a maximum 60.3 ± 0.2 % RCY, and after purification a RCP of 94.2 % was obtained. The present results provide an efficient and practical labeling method for xanthine and hypoxanthine with iodine-131, suggesting that these radiolabeled compounds can be further investigated in in vitro and in vivo studies for their theranostics potential.

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.

DIGE Analysis of Animal Tissues.

Two-dimensional difference gel electrophoresis (2D-DIGE) is an acrylamide gel electrophoresis-based technique for protein separation and quantification in complex mixtures. The technique addresses some of the drawbacks of conventional 2D polyacrylamide gel electrophoresis (2D-PAGE), offering improved sensitivity, more limited experimental variation, and accurate within-gel matching. 2D-DIGE is based on direct labeling of proteins with isobaric fluorescent dyes (known as CyDyes: Cy2, Cy3, and Cy5) prior to isoelectric focusing (IEF). Here, up to two samples and a reference pool (internal standard) can be mixed and loaded onto IEF for first dimension prior to SDS (sodium dodecyl sulfate)-PAGE separation in the second dimension. After the electrophoretic run, the gel is imaged at the specific excitation wavelength for each dye, in sequence, and gel scans are recorded separately. For each individual protein spot, intensities recorded at the different wavelengths are integrated and the ratio between volumes normalized to that of the internal standard. This provides an immediate appreciation of protein amount variations under the different conditions tested. In addition, proteins of interest can still be excised and identified with conventional mass spectrometric techniques and further analyzed by other biochemical methods. In this chapter, we describe application of this methodology to separation and quantitation of protein mixtures from porcine muscle exudate, collected following centrifugation of muscle specimens (centrifugal drip) for the characterization of quality parameters of importance in meat industry.

O-01 - [99mTc]Tc-(η6-pharm)2]+ synthesis with direct labelling of pharmaceuticals in water

O-01 - [99mTc]Tc-(η6-pharm)2]+ synthesis with direct labelling of pharmaceuticals in water

Direct labeling of milk cells without centrifugation for counting total and differential somatic cells using flow cytometry.

Somatic cell count (SCC) in milk is an essential indicator for defining and managing udder health. However, analyzing differential SCC (dSCC) can be helpful in determining the type or evolution stage of mastitis. A high abundance of polymorphonuclear cells (PMN) is associated with acute mastitis; however, the status of a chronic disease is less well characterized. A method capable of analyzing SCC and dSCC can prove to be a helpful tool for monitoring the status of evolution of mastitis disease in a better way. Therefore, a new direct-flow cytometry method was developed to count and differentiate somatic cells in milk without the steps of centrifugation or washing, avoiding variabilities that occur due to enrichment or loss of specific cell types. In this new method, SCC is analyzed using the method of DNA staining with Hoechst stain, whereas dSCC are analyzed using specific antibodies targeting 2 main cell types associated with mastitis: PMN cells and antigen-presenting cells, which are associated with innate and adaptive immunity. Equivalent SCC values were obtained between the new method and the routine ISO 13366-2 method in a comparison of 240 raw milk samples. Furthermore, dSCC results were confirmed by microscopy after May-Gründwald-Giemsa staining in 165 quarter milk samples from healthy and diseased cows. The method was verified with fluorescence microscopy on the 2 targeted cell types and in raw milk samples. The newly developed method is independent of any instrument and can be further designed to differentiate other cell types and animal species by selecting appropriate antibodies.

Selective RNA Labeling by RNA-Compatible Type II Restriction Endonuclease and RNA-Extending DNA Polymerase.

RNAs not only offer valuable information regarding our bodies but also regulate cellular functions, allowing for their specific manipulations to be extensively explored for many different biological and clinical applications. In particular, rather than temporary hybridization, permanent labeling is often required to introduce functional tags to target RNAs; however, direct RNA labeling has been revealed to be challenging, as native RNAs possess unmodifiable chemical moieties or indefinable dummy sequences at the ends of their strands. In this work, we demonstrate the combinatorial use of RNA-compatible restriction endonucleases (REs) and RNA-extending polymerases for sequence-specific RNA cleavage and subsequent RNA functionalization. Upon the introduction of complementary DNAs to target RNAs, Type II REs, such as AvrII and AvaII, could precisely cut the recognition site in the RNA-DNA heteroduplexes with exceptionally high efficiency. Subsequently, the 3′ ends of the cleaved RNAs were selectively and effectively modified when Therminator DNA polymerase template-dependently extended the RNA primers with a variety of modified nucleotides. Based on this two-step RNA labeling, only the target RNA could be chemically labeled with the desired moieties, such as bioconjugation tags or fluorophores, even in a mixture of various RNAs, demonstrating the potential for efficient and direct RNA modifications.

Evaluation of different 89Zr-labeled synthons for direct labeling and tracking of white blood cells and stem cells in healthy athymic mice

Cell based therapies are evolving as an effective new approach to treat various diseases. To understand the safety, efficacy, and mechanism of action of cell-based therapies, it is imperative to follow their biodistribution noninvasively. Positron-emission-tomography (PET)-based non-invasive imaging of cell trafficking offers such a potential. Herein, we evaluated and compared three different ready-to-use direct cell radiolabeling synthons, [89Zr]Zr-DFO-Bn-NCS, [89Zr]Zr-Hy3ADA5-NCS, and [89Zr]Zr-Hy3ADA5-SA for PET imaging-based trafficking of white blood cells (WBCs) and stem cells (SCs) up to 7 days in athymic nude mice. We compared the degree of 89Zr complexation and percentage of cell radiolabeling efficiencies with each. All three synthons, [89Zr]Zr-DFO-Bn-NCS, [89Zr]Zr-Hy3ADA5-NCS, and [89Zr]Zr-Hy3ADA5-SA, were successfully prepared, and used for radiolabeling of WBCs and SCs. The highest cell radiolabeling yield was found for [89Zr]Zr-DFO-Bn-NCS, followed by [89Zr]Zr-Hy3ADA5-NCS, and [89Zr]Zr-Hy3ADA5-SA. In terms of biodistribution, WBCs radiolabeled with [89Zr]Zr-DFO-Bn-NCS or [89Zr]Zr-Hy3ADA5-NCS, were primarily accumulated in liver and spleen, whereas SCs radiolabeled with [89Zr]Zr-DFO-Bn-NCS or [89Zr]Zr-Hy3ADA5-NCS were found in lung, liver and spleen. A high bone uptake was observed for both WBCs and SCs radiolabeled with [89Zr]Zr-Hy3ADA5-SA, suggesting in-vivo instability of [89Zr]Zr-Hy3ADA5-SA synthon. This study offers an appropriate selection of ready-to-use radiolabeling synthons for noninvasive trafficking of WBCs, SCs and other cell-based therapies.

Direct Capsid Labeling of Infectious HIV-1 by Genetic Code Expansion Allows Detection of Largely Complete Nuclear Capsids and Suggests Nuclear Entry of HIV-1 Complexes via Common Routes.

ABSTRACTThe cone-shaped mature HIV-1 capsid is the main orchestrator of early viral replication. After cytosolic entry, it transports the viral replication complex along microtubules toward the nucleus. While it was initially believed that the reverse transcribed genome is released from the capsid in the cytosol, recent observations indicate that a high amount of capsid protein (CA) remains associated with subviral complexes during import through the nuclear pore complex (NPC). Observation of postentry events via microscopic detection of HIV-1 CA is challenging, since epitope shielding limits immunodetection and the genetic fragility of CA hampers direct labeling approaches. Here, we present a minimally invasive strategy based on genetic code expansion and click chemistry that allows for site-directed fluorescent labeling of HIV-1 CA, while retaining virus morphology and infectivity. Thereby, we could directly visualize virions and subviral complexes using advanced microscopy, including nanoscopy and correlative imaging. Quantification of signal intensities of subviral complexes revealed an amount of CA associated with nuclear complexes in HeLa-derived cells and primary T cells consistent with a complete capsid and showed that treatment with the small molecule inhibitor PF74 did not result in capsid dissociation from nuclear complexes. Cone-shaped objects detected in the nucleus by electron tomography were clearly identified as capsid-derived structures by correlative microscopy. High-resolution imaging revealed dose-dependent clustering of nuclear capsids, suggesting that incoming particles may follow common entry routes.