Labeling Efficiency Research Articles

Targeted recovery of male cells in a male and female same-cell mixture.

DNA mixture deconvolution in the forensic DNA community has been addressed in a variety of ways. "Front-end" methods that separate the cellular components of mixtures can provide a significant benefit over computational methods as there is no need to rely on models with inherent uncertainty to generate conclusions. Historically, cell separation methods have been investigated but have been largely ineffective due to high cost, unreliability, and the lack of proper instrumentation. However, the last decade has given rise to more innovative technology that can target and recover cells more effectively. This study focuses on the development and optimization of a method to selectively label and recover male cells in a mixture of male and female epithelial cells using a Y-chromosome labeling kit with DEPArray™ technology, whereby male cells are labeled and recovered into a single extraction-ready tube. Labeling efficiency was tested using freshly collected and aged buccal swabs where 70%-75% and 38% of male cells were labeled, respectively, with less than 1% false positives. DEPArray™ detection was assessed using single buccal epithelial cells where approximately 80% of labeled cells were identified as male. Mixtures (1:1, 1:10, male to female) yielded profiles that were predominantly single source male or those in which the male component was more easily interpreted. The male-specific labeling method was demonstrated to be both robust and reliable when used on freshly collected cells. While the DEPArray™ meditated detection and recovery had notable limitations, it still improved the interpretation of the male component in same-cell mixtures in more recently collected samples.

Less Is More: Label Recommendation for Weakly Supervised Point Cloud Semantic Segmentation

Weak supervision has proven to be an effective strategy for reducing the burden of annotating semantic segmentation tasks in 3D space. However, unconstrained or heuristic weakly supervised annotation forms may lead to suboptimal label efficiency. To address this issue, we propose a novel label recommendation framework for weakly supervised point cloud semantic segmentation. Distinct from pre-training and active learning, the label recommendation framework consists of three stages: inductive bias learning, recommendations for points to be labeled, and point cloud semantic segmentation learning. In practice, we first introduce the point cloud upsampling task to induct inductive bias from structural information. During the recommendation stage, we present a cross-scene clustering strategy to generate centers of clustering as recommended points. Then we introduce a recommended point positions attention module LabelAttention to model the long-range dependency under sparse annotations. Additionally, we employ position encoding to enhance the spatial awareness of semantic features. Throughout the framework, the useful information obtained from inductive bias learning is propagated to subsequent semantic segmentation networks in the form of label positions. Experimental results demonstrate that our framework outperforms weakly supervised point cloud semantic segmentation methods and other methods for labeling efficiency on S3DIS and ScanNetV2, even at an extremely low label rate.

MICA: Towards Explainable Skin Lesion Diagnosis via Multi-Level Image-Concept Alignment

Black-box deep learning approaches have showcased significant potential in the realm of medical image analysis. However, the stringent trustworthiness requirements intrinsic to the medical field have catalyzed research into the utilization of Explainable Artificial Intelligence (XAI), with a particular focus on concept-based methods. Existing concept-based methods predominantly apply concept annotations from a single perspective (e.g., global level), neglecting the nuanced semantic relationships between sub-regions and concepts embedded within medical images. This leads to underutilization of the valuable medical information and may cause models to fall short in harmoniously balancing interpretability and performance when employing inherently interpretable architectures such as Concept Bottlenecks. To mitigate these shortcomings, we propose a multi-modal explainable disease diagnosis framework that meticulously aligns medical images and clinical-related concepts semantically at multiple strata, encompassing the image level, token level, and concept level. Moreover, our method allows for model intervention and offers both textual and visual explanations in terms of human-interpretable concepts. Experimental results on three skin image datasets demonstrate that our method, while preserving model interpretability, attains high performance and label efficiency for concept detection and disease diagnosis. The code is available at https://github.com/Tommy-Bie/MICA.

Improving STED microscopy with SUPPOSe: enhancing resolution from a single-image

Here we apply the SUPPOSe algorithm on images acquired using Stimulated Emission Depletion (STED) microscopy with the aim of improving the resolution limit achieved. We processed images of the nuclear pore complex (NPC) from cell lines in which the Nup96 nucleoporin was endogenously labeled. This reference protein forms a ring whose diameter is ∼107 nm with 8 corners ∼42 nm apart from each other. The stereotypic arrangement of proteins in the NPC has been used as reference structures to characterize the performance of a variety of microscopy techniques. STED microscopy images resolve the ring arrangement but not the eightfold symmetry of the NPC. After applying the SUPPOSe algorithm to the STED images, we were able to solve the octagonal structure of the NPC. After processing 562 single NPC, the average radius of the NPC was found to be R = 54.2 ± 2.9 nm, being consistent with the theoretical distances of this structure. To verify that the solutions obtained are compatible with a NPC-type geometry, we rotate the solutions to optimally fit an eightfold-symmetric pattern and we count the number of corners that contain at least one localization. Fitting a probabilistic model to the histogram of the number of bright corners gives an effective labeling efficiency of 31%, which is in agreement with the values reported in for other cell lines and ligands used in Single Molecule Localization microscopy, showing that SUPPOSe can reliably retrieve sub-resolution, nanoscale objects from single acquisitions even in noisy conditions.

In-section Click-iT detection and super-resolution CLEM analysis of nucleolar ultrastructure and replication in plants

Correlative light and electron microscopy (CLEM) is an important tool for the localisation of target molecule(s) and their spatial correlation with the ultrastructural map of subcellular features at the nanometre scale. Adoption of these advanced imaging methods has been limited in plant biology, due to challenges with plant tissue permeability, fluorescence labelling efficiency, indexing of features of interest throughout the complex 3D volume and their re-localization on micrographs of ultrathin cross-sections. Here, we demonstrate an imaging approach based on tissue processing and embedding into methacrylate resin followed by imaging of sections by both, single-molecule localization microscopy and transmission electron microscopy using consecutive CLEM and same-section CLEM correlative workflow. Importantly, we demonstrate that the use of a particular type of embedding resin is not only compatible with single-molecule localization microscopy but shows improvements in the fluorophore blinking behavior relative to the whole-mount approaches. Here, we use a commercially available Click-iT ethynyl-deoxyuridine cell proliferation kit to visualize the DNA replication sites of wild-type Arabidopsis thaliana seedlings, as well as fasciata1 and nucleolin1 plants and apply our in-section CLEM imaging workflow for the analysis of S-phase progression and nucleolar organization in mutant plants with aberrant nucleolar phenotypes.

Photoactivatable Xanthone (PaX) Dyes Enable Quantitative, Dual Color, and Live-Cell MINFLUX Nanoscopy.

The single-molecule localization concept MINFLUX has triggered a reevaluation of the features of fluorophores for attaining nanometer-scale resolution. MINFLUX nanoscopy benefits from temporally controlled fluorescence ("on"/"off") photoswitching. Combined with an irreversible switching behavior, the localization process is expected to turn highly efficient and quantitative data analysis simple. The potential in the recently reported photoactivable xanthone (PaX) dyes is recognized to extend the list of molecular switches used for MINFLUX with 561nm excitation beyond the fluorescent protein mMaple. The MINFLUX localization success rates of PaX560, PaX+560, and mMaple are quantitatively compared by analyzing the effective labeling efficiency of endogenously tagged nuclear pore complexes. The PaX dyes prove to be superior to mMaple and on par with the best reversible molecular switches routinely used in single-molecule localization microscopy. Moreover, the rationally designed PaX595 is introduced for complementing PaX560 in dual color 561nm MINFLUX imaging based on spectral classification and the deterministic, irreversible, and additive-independent nature of PaX photoactivation is showcased in fast live-cell MINFLUX imaging. The PaX dyes meet the demands of MINFLUX for a robust readout of each label position and fill the void of reliable fluorophores dedicated to 561nm MINFLUX imaging.

Formulation and In-vivo Characterization of 177Lu-tin-colloid as a Radiosynovectomy Agent.

Arthritis is an inflammatory disorder that affects one or more joints of the body for various reasons, including autoimmune disorders, trauma, or infection. In many cases, traditional long-term treatment with various drug combinations (NSAIDs, diseasemodifying antirheumatic drugs, systemic corticosteroids, etc.) can provide relief, but many joints require additional local treatment. Radiosynovectomy (RSV) is an alternative method to current treatment options. Both the global supply shortage of 90Y in recent years and the increasing use of 177Lu-labeled radiopharmaceuticals in the field of nuclear medicine have made it possible to develop 177Lu-labeled microparticles and test them in small groups as RSV agents. This study aimed to develop the 177Lu labeled tin colloid formulation and demonstrate its invivo characterization. Particle size, shape, and labelling efficiency of the four formulations developed were determined. The formula with the highest labelling efficiency was selected for further studies. The quality of the formulation was evaluated based on radionuclidic, radiochemical, and microbial purity. In-vitro stability was evaluated by determining the labelling efficiency. In-vitro stability was tested in PBS and synovial fluid. The biological characterization was assessed using SPECT/CT after injecting the formulation into the normal knee joints of the rabbits. Aggregated colloidal particles were spherical with a particle size of <5 μm. Labelling efficiency and radiochemical purity were >95 and 97.65% (Rf=0.2), respectively. The formulation was stable in vitro for up to 72 hours, both in PBS and synovial fluid. The formulation was homogeneously distributed in the joint at 0 and 1 hour after injection, and radioactivity- related involvement and inguinal lymph node involvement due to possible leakage were not detected in the late period. No pyrogenic/allergic side effects were observed during this period. 177Lu-tin-colloid was successfully prepared under optimized reaction conditions with high binding efficiency and radiochemical purity. The radiolabeled colloid was found to be stable in-vitro both in PBS and synovial fluid at room temperature. Serial PCET/CT images revealed that the activity was completely retained within the synovial cavity, with no activity leakage out of the joint until 48 hours after the injection. With the support of the results from further clinical studies, it may be possible for the formulation to enter clinical use.

SPD: Semi-Supervised Learning and Progressive Distillation for 3-D Detection.

Current learning-based 3-D object detection accuracy is heavily impacted by the annotation quality. It is still a challenge to expect an overall high detection accuracy for all classes under different scenarios given the dataset sparsity. To mitigate this challenge, this article proposes a novel method called semi-supervised learning and progressive distillation (SPD), which uses semi-supervised learning (SSL) and knowledge distillation to improve label efficiency. The SPD uses two big backbones to hand the unlabeled/labeled input data augmented by the periodic IO augmentation (PA). Then the backbones are compressed using progressive distillation (PD). Precisely, PA periodically shifts the data augmentation operations between the input and output of the big backbone, aiming to improve the network's generalization of the unseen and unlabeled data. Using the big backbone can benefit from large-scale augmented data better than the small one. And two backbones are trained by the data scale and ratio-sensitive loss (data-loss). It solves the over-flat caused by the large-scale unlabeled data from PA and helps the big backbone prevent overfitting on the limited-scale labeled data. Hence, using the PA and data loss during SSL training dramatically improves the label efficiency. Next, the trained big backbone set as the teacher CNN is progressively distilled to obtain a small student model, referenced as PD. PD mitigates the problem that student CNN performance degrades when the gap between the student and the teacher is oversized. Extensive experiments are conducted on the indoor datasets SUN RGB-D and ScanNetV2 and outdoor dataset KITTI. Using only 50% labeled data and a 27% smaller model size, SPD performs 0.32 higher than the fully supervised VoteNetqi2019deep which is adopted as our backbone. Besides, using only 2% labeled data, compared to the other fully supervised backbone PV-RCNNshi2020pv, SPD accomplishes a similar accuracy (84.1 and 84.83) and 30% less inference time.

Visualizing the trans-synaptic arrangement of synaptic proteins by expansion microscopy.

High fidelity synaptic neurotransmission in the millisecond range is provided by a defined structural arrangement of synaptic proteins. At the presynapse multi-epitope scaffolding proteins are organized spatially at release sites to guarantee optimal binding of neurotransmitters at receptor clusters. The organization of pre- and postsynaptic proteins in trans-synaptic nanocolumns would thus intuitively support efficient information transfer at the synapse. Visualization of these protein-dense regions as well as the minute size of protein-packed synaptic clefts remains, however, challenging. To enable efficient labeling of these protein complexes, we developed post-gelation immunolabeling expansion microscopy combined with Airyscan super-resolution microscopy. Using ~8-fold expanded samples, Airyscan enables multicolor fluorescence imaging with 20-40 nm spatial resolution. Post-immunolabeling of decrowded (expanded) samples provides increased labeling efficiency and allows the visualization of trans-synaptic nanocolumns. Our approach is ideally suited to investigate the pathological impact on nanocolumn arrangement e.g., in limbic encephalitis with autoantibodies targeting trans-synaptic leucine-rich glioma inactivated 1 protein (LGI1).

Method development for 14C-labeling of IgG antibodies in preparation for clinical trials

Abstract Objectives Carbon-14 (14C) labeling is a standard technology for tracing molecules and providing their pharmacokinetic profiles. However, its primary focus has been on small molecules, with limited application to biomacromolecules. Particularly in the development of new biological entities (NBE), the utilization of microdosing with a 14C-labeled biomacromolecule proves beneficial in the early stages of drug development, contributing to significant time and cost savings. This study investigates the 14C-labeling of antibody and explores the stability of 14C-labeled antibody under various storage conditions. Methods and results In this study, the utilization of 14C-formaldehyde for labeling target antibodies at various molar ratios revealed a direct correlation between labeling efficiency and the quantity of 14C-formaldehyde applied: 1.5 mol/mol for 14C-labeled antibody with the use of 10 equivalents of 14C-formaldehyde, 3.8 mol/mol for 14C-labeled antibody with the use of 10 equivalents of 14C-formaldehyde, and 10.5 mol/mol for 14C-labeled antibody with the use of 60 equivalents of 14C-formaldehyde. All the reaction conditions exhibited no antibody degradation, as evidenced by the absence of a significant change in HPLC purity compared to the unlabeled antibody. Stability tests revealed that all groups maintained their purities over a 4-week period at both − 75 ± 10 °C and 5 ± 3 °C. Given safety concerns related to internal radiation exposure in potential human subjects during microdosing, this study established optimal conditions for employing 14C-labeled antibodies. Therefore, it is optimized that 10 equivalents of 14C-formaldehyde can be used for 14C-antibody labeling through reductive amination, storing the antibodies at 5 ± 3 °C, and assigning a storage period of 4 weeks. Conclusion The findings from this study offer valuable insights into the effective application of 14C-labeling in microdosing studies, especially for larger molecules such as antibodies.

Diazobutanone-assisted isobaric labelling of phospholipids and sulfated glycolipids enables multiplexed quantitative lipidomics using tandem mass spectrometry.

Mass spectrometry-based quantitative lipidomics is an emerging field aiming to uncover the intricate relationships between lipidomes and disease development. However, quantifying lipidomes comprehensively in a high-throughput manner remains challenging owing to the diverse lipid structures. Here we propose a diazobutanone-assisted isobaric labelling strategy as a rapid and robust platform for multiplexed quantitative lipidomics across a broad range of lipid classes, including various phospholipids and glycolipids. The diazobutanone reagent is designed to conjugate with phosphodiester or sulfate groups, while accommodating various functional groups on different lipid classes, enabling subsequent isobaric labelling for high-throughput multiplexed quantitation. Our method demonstrates excellent performance in terms of labelling efficiency, detection sensitivity, quantitative accuracy and broad applicability to various biological samples. Finally, we performed a six-plex quantification analysis of lipid extracts from lean and obese mouse livers. In total, we identified and quantified 246 phospholipids in a high-throughput manner, revealing lipidomic changes that may be associated with obesity in mice.

Highly Sensitive Labeling, Clickable Functionalization, and Glycoengineering of the MUC1 Neighboring System.

This study introduces a novel wash-type affinity-primed proximity labeling (WAPL) strategy for labeling and surface engineering of the MUC1 protein neighboring system. The strategy entails the utilization of peroxidase in conjunction with a MUC1-selective aptamer, facilitating targeted binding to MUC1 and inducing covalent labeling of the protein neighboring system. This study reveals a novel finding that the WAPL strategy demonstrates superior labeling efficiency in comparison to nonwash-type affinity-primed proximity labeling, marking the first instance of such observations. The WAPL strategy provides signal amplification by converting a single recognition event into multiple covalent labeling events, thereby improving the detection sensitivity for subtle changes in MUC1. The WAPL platform employs two levels of labeling upgrades, modifying the biotin handles of the conventional labeling substrate, biotin-phenol. The first level involves a range of clickable molecules, facilitating dibenzoazacyclooctynylation, alkynylation, and trans-cyclooctenylation of the protein neighboring system. The second level utilizes lactose as a post-translational modification model, enabling rapid and reliable glycoengineering of the MUC1 neighboring system while remaining compatible with cell-based assays. The implementation of the WAPL strategy in protein neighboring systems has resulted in the establishment of a versatile platform that can effectively facilitate diverse monitoring and regulation techniques. This platform offers valuable insights into the regulation of relevant signaling pathways and promotes the advancement of novel therapeutic approaches, thereby bringing substantial implications for human health.

Development of the Purification Process of Gallium-68 Eluted from Germanium-68/Gallium-68 Generator

Objective: 68Ga has a half-life of 68 minutes, with 89% of its decay is through positron emission. It is available from generator systems and possesses suitable property for labeling radioligands. These aspects make 68Ga a promising tracer for positron emission tomography (PET) imaging. This study aims to develop the purification process of the 68Ga eluates from 68Ge/68Ga generator after its recommended shelf-life and ensuring the quality through the radiolabeling process. Materials and Methods: In this study, we explored the development of a purification method for 68Ga eluted from a68Ge/68Ga generator before radiolabeling was investigated. Cation and anion exchange chromatography techniques were combined to remove trace amounts of competing metal ion impurities. Post-purification, the eluate’s metal contents were analyzed using inductively coupled plasma atomic emission spectroscopy (ICP-AES). Breakthrough of 68Ge was measured using a multi-channel analyzer (MCA) spectrometer with high-purity germanium (HPGe) radiation detectors. Additionally, the radiochemical purity of 68Ga-NOTA-RGD was analyzed by high-performance liquid chromatography (HPLC). Results: Metal impurities including Fe(II), Zn(II) and Al(III) were reduced by 61%, 38% and 44% respectively. The 68Ge breakthrough was approximately ~10–3%. The labeling efficiency with NOTA-RGD, a tracer for angiogenesis imaging, resulted in an average yield of 68Ga-NOTA-RGD (not corrected for decay) of around 50%, with aradiochemical purity by HPLC of approximately 98%–99%. Conclusion: Cation exchange in combination with anion exchange chromatography was thus proven to be an efficient method for purification of the 68Ga eluate from a 68Ge/68Ga generator prior to labeling the 68Ga PET radiotracer.

Development of Small HN Linked Radionuclide Iodine-125 for Nanocarrier Image Tracing in Mouse Model.

Radionuclides have important roles in clinical tumor radiotherapy as they are used to kill tumor cells or as imaging agents for drug tracing. The application of radionuclides has been developing as an increasing number of nanomaterials are used to deliver radionuclides to tumor areas to kill tumor cells. However, promoting the efficient combination of radionuclides and nanocarriers (NCs), enhancing radionuclide loading efficiency, and avoiding environmental pollution caused by radionuclide overuse are important challenges that hinder their further development. In the present study, a new small molecule compound (3-[[(2S)-2-hydroxy-3-(4-hydroxyphenyl)-1-carbonyl] amino]-alanine, abbreviation: HN, molecular formula: C12H16N2O5) was synthesized as a linker between radionuclide iodine-125 (125I) and NCs to enable a more efficient binding between NCs and radionuclides. In vitro evidence indicated that the linker was able to bind 125I with higher efficiency (labeling efficiency >80%) than that of tyrosine, as well as various NCs, such as cellulose nanofibers, metal oxide NCs, and graphene oxide. Single-photon emission computed tomography/computed tomography imaging demonstrated the biological distribution of 125I-labeled NCs in different organs/tissues after administration in mice. These results showed an improvement in radionuclide labeling efficiency for nanocarriers and provided an approach for nanocarrier image tracing.

Abstract B043: Radiolabelled Trastuzumab Fab as a theranostic agent for HER2 expressing breast cancer

Abstract Breast cancer is the most common cause of mortality among women worldwide. Among all subtypes, HER2-positive breast cancers are highly aggressive and have poor prognosis. Surgical resection is choice of treatment. However, surgery may not be possible if disease is widespread. Trastuzumab, a monoclonal antibody, is globally used for immunotherapy. It is a large sized molecule (141kDa) and show slow pharmacokinetics and high liver uptake. We have explored trastuzumab fragment as a radio-theranostic agent. Trastuzumab Fab was generated using Papain enzyme tagged beads, incubation time 18 to 20 h at 850 rpm in phosphate buffer. Fab were collected with ultrafiltration device, cut off 30 kDa, in phosphate buffer pH 8. Quantification of Fab was done at 280 nm. Fab characterization was done using MALDI-TOF and SDS-PAGE. To enable radiolabelling of Fab with Ga-68 and Lu-177, Fab was conjugated with bifunctional chelator, DTPA. Fab-DTPA was separated by micro bio-spin P-6 chromatography columns and the average number of DTPA/Fab were calculated. Affinity of Fab conjugated Fab-DTPA for HER2 receptors was studied using the biolayer interferometry technique (BLI). Radiolabelling of Fab with Ga-68 for PET imaging and Lu-177 for therapy was optimized. Quality control included radiochemical purity using TLC in sodium citrate (pH 5) mobile phase, pyrogenicity using PTS, and sterility by tryptic soy broth culture media. Receptor affinity and apoptosis were studied with HER2 positive SKBR-3 cells using flow cytometry assay. The SKBR-3 cell (106) were cultured in 96 well plates and incubated with a known amount of Lu-177-DTPA-Fab (0- 18.5 MBq). First in human Ga-68 Fab PET/CT and Lu-177-Fab gamma camera images were acquired after obtaining clearance from Institutional Ethics Committee and inform signed consent. Patients were recruited with histopathological proven HER2 positive breast cancer and uptake on F-18 FDG PET/CT. Ga-68-DTPA-Fab (3.5-5 mCi) and 10-15 mCi Lu-177 DTPA-Fab was injected and images were acquired at 2/3 h and up to 7 days respectively. Dominant Fab peak at 45kDa on MALD-TOF, single band on SDS-PAGE (Non-reduced or reduced) have been obtained. Two DTPA/Fab were conjugated and confirmed by MALDI-TOF. BLI data showed Kd value of trastuzumab, Fab, and conjugated DTPA-Fab as 0.2 nM, 0.57 nM, and 20 nM for HER2. High labelling efficiency (≥98%) of Lu-177-DTPA-Fab and Ga-68-DTPA-Fab was achieved. Apoptosis (%) was 1.5%- 42.5% in SKBR-3 cells treated with Lu-177-DTPA-Fab. Both formulations were found sterile and pyrogen-free. Kidney and bladder activity of Ga-68/Lu-177-DTPA-Fab demonstrate renal clearance. Blood pool activity of both tracers were high. However, it was decreased, and lesion uptake was increased with time. All the lesions on Ga-68-DTPA-Fab, same as in F-18 FDG PET/CT, were picked up accept those near heart. Good lesion retention of Lu-177-DTPA-Fab was observed showing suitability for therapy. Fab-DTPA has potential to target HER2 receptor. It could be radiolabelled with Ga-68 for PET imaging and Lu-177 for targeted therapy. Citation Format: Jaya Shukla, Yogesh Rathore, Komalpreet Kaur, Santosh Irrinky, Rajender Kumar, Amanjit Bal, Bhagwant R Mittal. Radiolabelled Trastuzumab Fab as a theranostic agent for HER2 expressing breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B043.

Unraveling Interaction of Rhenium-188 Microspheres with Primary Hepatic Cancer Cell: A Breakthrough Study.

Introduction: Hepatocellular carcinoma is a prevalent contributor to global mortality rates. The main palliative treatments are trans-arterial chemoembolization and selective intra-arterial radionuclide therapy. Methods: A novel freeze-dried nonradioactive microsphere kit formulation has been developed, and the behavior and therapeutic potential of 188Re microspheres have been assessed. The microspheres were labeled with fluorescein isothiocyanate (FITC) and 188ReO4-. The uptake of FITC microspheres by HepG2 cells was examined at various time intervals. The impact of 188Re microspheres on cell viability and the mode of cell death were investigated with HepG2 cells using MTT and Annexin FITC-V/propidium iodide (PI) apoptosis assay. Results: The labeling efficiency of microspheres was more than 99% with FITC and 188ReO4-. The maximum uptake of FITC microspheres by HepG2 cells was achieved at 6 h. The exposure to 188Re microspheres has shown a decrease in cellular viability from 77.81% ± 0.015% to 42.03% ± 0.148% at 192 h of incubation (∼11 half-lives). The cellular uptake of 188Re microspheres was 0.255-0.901 MBq. These values were concordant with Annexin FITC-V/PI apoptosis assay. At 192 h, 53.28% ± 0.01% of cells entered the apoptotic phase after treatment with 188Re microspheres, and only 39.34% ± 0.02% of cells remained viable. However, in the cells treated with 188ReO4- alone, 74.86% ± 0.005% of cells were viable, and only 24.75% ± 0.577% of cells were in the early apoptotic phase at 192 h. Conclusion: The data revealed that 188Re microspheres treatment led to significant growth inhibition in HepG2 cells compared with 188ReO4-.

SpSrtA-Catalyzed Isopeptide Ligation on Lysine Residues.

Sortase-mediated ligation (SML) is widely used for protein bioconjugation. However, the sortase used in this strategy typically recognizes only the N-terminal oligoglycine, which is absent in most natural proteins. To broaden the spectrum of substrates compatible with SML, we focus on a novel sortase, sortase A from Streptococcus pneumoniae (SpSrtA), known for its expanded substrate specificity (N-terminal glycine, alanine, and serine). We present the first evidence showing that the reported SpSrtA mutant (SpSrtA*) can modify lysine residues in itself and other proteins. The modification sites of SpSrtA* were identified through LC-MS/MS analysis. Moreover, we discovered an optimal lysine-containing peptide tag by fusing it onto sfGFP, resulting in a labeling efficiency of 57%. Inspired by this, we applied the method to modify proteins on microorganism surfaces up to 13.5-fold. To enhance labeling efficiency, we fused the SpSrtA* onto a surface protein and achieved a 2.64-fold improvement. We further developed a high-throughput yeast display screening method for the directed evolution of SpSrtA*, achieving a 10-fold improvement in the labeling efficiency of this surface protein. Our study provides a novel strategy for modifying the lysine residues that will be a powerful addition to the protein bioconjugation toolbox.

A mathematical model for velocity-selective arterial spin labeling.

To present a theoretical framework that rigorously defines and analyzes key concepts and quantities for velocity selective arterial spin labeling (VSASL). An expression for the VSASL arterial delivery function is derived based on (1) labeling and saturation profiles as a function of velocity and (2) physiologically plausible approximations of changes in acceleration and velocity across the vascular system. The dependence of labeling efficiency on the amplitude and effective bolus width of the arterial delivery function is defined. Factors that affect the effective bolus width are examined, and timing requirements to minimize quantitation errors are derived. The model predicts that a flow-dependent negative bias in the effective bolus width can occur when velocity selective inversion (VSI) is used for the labeling module and velocity selective saturation (VSS) is used for the vascular crushing module. The bias can be minimized by choosing a nominal labeling cutoff velocity that is lower than the nominal cutoff velocity of the vascular crushing module. The elements of the model are specified in a general fashion such that future advances can be readily integrated. The model can facilitate further efforts to understand and characterize the performance of VSASL and provide critical theoretical insights that can be used to design future experiments and develop novel VSASL approaches.

Development of a rapid and ultrasensitive magnetic chemiluminescence immunoassay for the detection of adiponectin and its clinical application

Adiponectin (ADPN), which serum/plasma adiponectin levels are closely associated with insulin resistance and type 2 diabetes, and lower adiponectin levels predict an increased risk of diabetes, is a strong indicator of diabetes risk in people at high risk of diabetes in different races. Using the unique principle and performance advantages of chemiluminescence immunoassay (CLIA), an ADPN-CLIA method with high sensitivity, high specificity and wide detection range was established based on the principle of two-steps method of sandwich-type, with the magnetic particles (MPs) as the solid phase carrier and acridinium ester (AE) as the chemiluminescence reaction system. The selection of the main raw materials required, the preparation conditions of MPs-coated antibodies, the methods of AE-labeled antibodies, sample requirements and reaction modes were optimized and evaluated. AE labeling experiment was successfully performed with the labeling efficiency of 8.366 and the antibody utilization rate of 96.8%. The chemiluminescent immunoassay for ADPN had a good linear relationship from 0 ng/mL to 250 ng/mL (R2 =0.9993), with the detection limit of 0.05 ng/mL. The coefficient of variation (CV) of intra-assay and inter-assay precision were both less than 5% respectively. The recovery rates for accuracy were from 91.26% to 107.46%. The comparison experiment of 80 clinical serum samples between the developed ADPN-CLIA with the immunoturbidimetry showed that the correlation coefficient was 0.956, and the Bland-Altman analysis showed that the limits of agreement were − 0.364 and 0.433.

Proximity labeling expansion microscopy (PL-ExM) evaluates interactome labeling techniques.

Understanding protein-protein interactions (PPIs) through proximity labeling has revolutionized our comprehension of cellular mechanisms and pathology. Various proximity labeling techniques, such as HRP, APEX, BioID, TurboID, and μMap, have been widely used to biotinylate PPIs or organelles for proteomic profiling. However, the variability in labeling precision and efficiency of these techniques often results in limited reproducibility in proteomic detection. We address this persistent challenge by introducing proximity labeling expansion microscopy (PL-ExM), a super-resolution imaging technique that combines expansion microscopy with proximity labeling techniques. PL-ExM enabled up to 17 nm resolution with microscopes widely available, providing visual comparison of the labeling precision, efficiency, and false positives of different proximity labeling methods. Our mass spectrometry proteomic results confirmed that PL-ExM imaging is reliable in guiding the selection of proximity labeling techniques and interpreting the proteomic results with new spatial information.