A simple procedure for preparing biotinylated immunoglobulin M from hybridoma culture medium.

We previously reported a chromatography system for purifying immunoglobulin M (IgM) using N,N,N',N'-ethylenediaminetetrakis(methylenephosphonic acid)-modified zirconia particles that selectively absorb immunoglobulins. Here, we report a simple procedure for preparing biotinylated IgM from hybridoma culture medium using this zirconia-based chromatography system. The culture medium of an IgM-producing hybridoma cell line was used as the starting sample solution, and the IgM in the medium was concentrated and partially purified by zirconia chromatography. Next, 9-(biotinamido)-4,7-dioxanonanoic acid N-succinimidyl ester was added to react with the proteins in the sample. Subsequently, only the biotinylated IgM was isolated by Capto Core 400 polishing column chromatography. The entire process was easy to perform, could be completed within 2 h, and provided highly pure biotin-labeled IgM. This procedure is expected to be applicable to the labeling of IgM with various compounds and drugs.

Development and Validation of a Proximity Labeling Fusion Protein Construct to Identify the Protein-Protein Interactions of Transcription Factors.

Dynamic interactions between transcription factors govern changes in gene expression that mediate changes in cell state accompanying injury response and regeneration. Transcription factors frequently function as obligate dimers whose activity is often modulated by post-translational modifications. These critical and often transient interactions are not easily detected by traditional methods to investigate protein-protein interactions. This chapter discusses the design and validation of a fusion protein involving a transcription factor tethered to a proximity labeling ligase, APEX2. In this technique, proteins are biotinylated within a small radius of the transcription factor of interest, regardless of time of interaction. Here we discuss the validations required to ensure proper functioning of the transcription factor proximity labeling tool and the sample preparation of biotinylated proteins for mass spectrometry analysis of putative protein interactors.

Adaptor protein Abelson interactor 1 in homeostasis and disease.

Dysregulation of Abelson interactor 1 (ABI1) is associated with various states of disease including developmental defects, pathogen infections, and cancer. ABI1 is an adaptor protein predominantly known to regulate actin cytoskeleton organization processes such as those involved in cell adhesion, migration, and shape determination. Linked to cytoskeleton via vasodilator-stimulated phosphoprotein (VASP), Wiskott-Aldrich syndrome protein family (WAVE), and neural-Wiskott-Aldrich syndrome protein (N-WASP)-associated protein complexes, ABI1 coordinates regulation of various cytoplasmic protein signaling complexes dysregulated in disease states. The roles of ABI1 beyond actin cytoskeleton regulation are much less understood. This comprehensive, protein-centric review describes molecular roles of ABI1 as an adaptor molecule in the context of its dysregulation and associated disease outcomes to better understand disease state-specific protein signaling and affected interconnected biological processes.

Cerebral perfusion in patients with unilateral internal carotid artery occlusion by dual post-labeling delays arterial spin labeling imaging.

BACKGROUND: Global and regional cerebral blood flow (CBF) changes in patients with unilateral internal carotid artery occlusion (ICAO) are unclear when the dual post-labeling delays (PLD) arterial spin labeling (ASL) magnetic resonance imaging (MRI) technique is used. Manual delineation of regions of interest for CBF measurement is time-consuming and laborious. AIM: To assess global and regional CBF changes in patients with unilateral ICAO with the ASL-MRI perfusion technique. METHODS: Twenty hospitalized patients with ICAO and sex- and age-matched controls were included in the study. Regional CBF was measured by Dr. Brain's ASL software. The present study evaluated differences in global, middle cerebral artery (MCA) territory, anterior cerebral artery territory, and Alberta Stroke Program Early Computed Tomography Score (ASPECTS) regions (including the caudate nucleus, lentiform nucleus, insula ribbon, internal capsule, and M1-M6) and brain lobes (including frontal, parietal, temporal, and insular lobes) between ICAO patients and controls at PLD 1.5 s and PLD 2.5 s. RESULTS: When comparing CBF between ICAO patients and controls, the global CBF in ICAO patients was lower at both PLD 1.5 s and PLD 2.5 s; the CBF on the occluded side was lower in 15 brain regions at PLD 1.5 s, and it was lower in 9 brain regions at PLD 2.5 s; the CBF in the contralateral hemisphere was lower in the caudate nucleus and internal capsule at PLD 1.5 s and in M6 at PLD 2.5 s. The global CBF in ICAO patients was lower at PLD 1.5 s than at PLD 2.5 s. The ipsilateral CBF at PLD 1.5 s was lower than that at PLD 2.5 s in 15 regions, whereas the contralateral CBF was lower at PLD 1.5 s than at PLD 2.5 s in 12 regions. The ipsilateral CBF was lower than the contralateral CBF in 15 regions at PLD 1.5 s, and in M6 at PLD 2.5 s. CONCLUSION: Unilateral ICAO results in hypoperfusion in the global and MCA territories, especially in the ASPECTS area. Dual PLD settings prove more suitable for accurate CBF quantification in ICAO.

Cerebrospinal fluid flow in small-breed dogs with idiopathic epilepsy observed using time-spatial labeling inversion pulse images: a preliminary study.

Cerebrospinal fluid (CSF) circulation diseases, such as hydrocephalus and syringomyelia, are common in small-breed dogs. In human patients with CSF circulation diseases, time-spatial labeling inversion pulse (time-SLIP) sequence performed to evaluate CSF flow before and after treatment allows visualization of the restoration of CSF movement. However, studies evaluating CSF flow using the time-SLIP method in small-breed dogs are limited. Therefore, the present study aimed to evaluate intracranial CSF flow on time-SLIP images in small-breed dogs with idiopathic epilepsy, as an alternative model to healthy dogs. Time-SLIP images were obtained at two sites: 1) the mesencephalic aqueduct (MA) area (third ventricle, MA, and brain-base subarachnoid space [SAS]) and 2) the craniocervical junction area (fourth ventricle, brainstem, and cervical spinal cord SAS) to allow subsequent evaluation of the rostral and caudal CSF flow using subjective and objective methods. In total, six dogs were included. Caudal flow at the MA and brain-base SAS and rostral flow in the brainstem SAS were subjectively and objectively observed in all and 5/6 dogs, respectively. Objective evaluation revealed that a significantly smaller movement of the CSF, assessed as the absence of CSF flow by subjective evaluation, could be detected in some areas. In small-breed dogs, the MA, brain-base, and brainstem SAS would be appropriate areas for evaluating CSF movement, either in the rostral or caudal flows on time-SLIP images. In areas where CSF movement cannot detected by subjective methods, an objective evaluation should be conducted.

Cell Type-Specific Profiles and Developmental Trajectories of Transcriptomes in Primate Prefrontal Layer 3 Pyramidal Neurons: Implications for Schizophrenia.

OBJECTIVE: In schizophrenia, impaired working memory is associated with transcriptome alterations in layer 3 pyramidal neurons (L3PNs) in the dorsolateral prefrontal cortex (DLPFC). Distinct subtypes of L3PNs that send axonal projections to the DLPFC in the opposite hemisphere (callosal projection [CP] neurons) or the parietal cortex in the same hemisphere (ipsilateral projection [IP] neurons) play critical roles in working memory. However, how the transcriptomes of these L3PN subtypes might shift during late postnatal development when working memory impairments emerge in individuals later diagnosed with schizophrenia is not known. The aim of this study was to characterize and compare the transcriptome profiles of CP and IP L3PNs across developmental transitions from prepuberty to adulthood in macaque monkeys. METHODS: The authors used retrograde labeling to identify CP and IP L3PNs in the DLPFC of prepubertal, postpubertal, and adult macaque monkeys, and used laser microdissection to capture these neurons for RNA sequencing. RESULTS: At all three ages, CP and IP L3PNs had distinct transcriptomes, with the number of genes differentially expressed between neuronal subtypes increasing with age. For IP L3PNs, age-related shifts in gene expression were most prominent between prepubertal and postpubertal animals, whereas for CP L3PNs such shifts were most prominent between postpubertal and adult animals. CONCLUSIONS: These findings demonstrate the presence of cell type-specific profiles and developmental trajectories of the transcriptomes of PPC-projecting IP and DLPFC-projecting CP L3PNs in monkey DLPFC. The evidence that IP L3PNs reach a mature transcriptome earlier than CP L3PNs suggests that these two subtypes differentially contribute to the maturation of working memory performance across late postnatal development and that they may be differentially vulnerable to the disease process of schizophrenia at specific stages of postnatal development.

Montelukast deprescribing in outpatient specialty clinics: A single center cross-sectional study.

Objective: To identify and evaluate montelukast deprescribing in outpatient specialty clinics. Methods: This was a single-center, retrospective, cross-sectional study conducted at an academic health system in the southern US including 21 specialty clinics. Subjects included adults ≥18 years with an active prescription for montelukast who attended at least one appointment in pulmonology, otolaryngology, or neurology outpatient specialty clinics between January 1, 2021 to December 31, 2022. Patients <18 years and those with diagnoses of uncontrolled asthma or allergic rhinitis were excluded. Outcomes assessed included the frequency and period prevalence of montelukast deprescribing, defined by a documented montelukast discontinuation within the medical record, and evaluation of reasoning for discontinuation mentioned in visit notes. Results: There were 1152 patients who met inclusion criteria. Of these, 43 (3.7 %) experienced a montelukast deprescribing event: 18 (41.9 %) in neurology, 13 (30.2 %) in otolaryngology, and 12 (27.9 %) in pulmonology. Documented reasons for deprescribing were only available for 11 patients (25.6 %); reasons for deprescribing included patient-provider shared decision-making regarding the Black Box Warning [n = 5 (11.6 %)], inadequate treatment response [n = 3 (7.0 %)], suicidal thought development [n = 1 (2.3 %)], adverse drug event [n = 1 (2.3 %)], and pregnancy planning [n = 1 (2.3 %)]. Conclusion: Montelukast deprescribing rates were less than 5 % in outpatient specialty clinics. Factors associated with montelukast deprescribing beget further investigation.

Enhancing transjugular intrahepatic portosystemic shunt procedure efficiency with digital subtraction angiography image overlay technology in esophagogastric variceal bleeding.

BACKGROUND: Transjugular intrahepatic portosystemic shunt (TIPS) is a pivotal intervention for managing esophagogastric variceal bleeding in patients with chronic hepatic schistosomiasis. AIM: To evaluate the efficacy of digital subtraction angiography image overlay technology (DIT) in guiding the TIPS procedure. METHODS: We conducted a retrospective analysis of patients who underwent TIPS at our hospital, comparing outcomes between an ultrasound-guided group and a DIT-guided group. Our analysis focused on the duration of the portosystemic shunt puncture, the number of punctures needed, the total surgical time, and various clinical indicators related to the surgery. RESULTS: The study included 52 patients with esophagogastric varices due to chronic hepatic schistosomiasis. Results demonstrated that the DIT-guided group experienced significantly shorter puncture times (P < 0.001) and surgical durations (P = 0.022) compared to the ultrasound-guided group. Additionally, postoperative assessments showed significant reductions in aspartate aminotransferase, B-type natriuretic peptide, and portal vein pressure in both groups. Notably, the DIT-guided group also showed significant reductions in total bilirubin (P = 0.001) and alanine aminotransferase (P = 0.023). CONCLUSION: The use of DIT for guiding TIPS procedures highlights its potential to enhance procedural efficiency and reduce surgical times in the treatment of esophagogastric variceal bleeding in patients with chronic hepatic schistosomiasis.

Common SYNE2 Genetic Variant Associated With Atrial Fibrillation Lowers Expression of Nesprin-2α1 With Downstream Effects on Nuclear and Electrophysiological Traits.

BACKGROUND: Atrial fibrillation GWAS (genome-wide association studies) identified significant associations for rs1152591 and linked variants in the SYNE2 gene encoding Nesprin-2, which connects the nuclear membrane with the cytoskeleton. METHODS: Reporter gene vector transfection and CRISPR-Cas9 editing were used to identify the causal variant regulating the expression of SYNE2α1. After SYNE2 knockdown or SYNE2α1 overexpression in human stem cell-derived cardiomyocytes, nuclear phenotypes were assessed by imaging and atomic force microscopy. Gene expression was assessed by RNAseq and gene set enrichment analysis. Fura-2 AM staining assessed calcium transients. Optical mapping assessed action potential duration and conduction velocity. RESULTS: The risk allele of rs1152591 had lower promoter and enhancer activity and was significantly associated with lower expression of the short SYNE2α1 isoform in human stem cell-derived cardiomyocytes, without an effect on the expression of the full-length SYNE2 mRNA. SYNE2α1 overexpression had dominant negative effects on the nucleus with its overexpression or SYNE2 knockdown leading to increased nuclear area and decreased nuclear stiffness. Gene expression results from SYNE2α1 overexpression demonstrated both concordant and nonconcordant effects with SYNE2 knockdown. SYNE2α1 overexpression had a gain of function on electrophysiology, leading to significantly faster calcium reuptake and decreased assessed action potential duration, while SYNE2 knockdown showed both shortened assessed action potential duration and decreased conduction velocity. CONCLUSIONS: rs1152591 was identified as a causal atrial fibrillation variant, with the risk allele decreasing SYNE2α1 expression. Downstream effects of SYNE2α1 overexpression include changes in nuclear stiffness and electrophysiology, which may contribute to the mechanism for the risk allele's association with AF.

Perfusion-weighted MRI patterns in neuropsychiatric systemic lupus erythematosus: a systematic review and meta-analysis.

BACKGROUND: Neuropsychiatric Systemic Lupus Erythematosus (NPSLE) is a complex manifestation of Systemic Lupus Erythematosus (SLE) characterized by a wide range of neurological and psychiatric symptoms. This study aims to elucidate the patterns of Perfusion-Weighted MRI (PWI) in NPSLE patients compared to SLE patients without neuropsychiatric manifestations (non-NPSLE) and healthy controls (HCs). MATERIAL AND METHODS: A systematic search was conducted in PubMed/Medline, Embase, Web of Science, and Scopus for studies utilizing PWI in NPSLE patients published through April 14, 2024. Cerebral blood flow (CBF) data from NPSLE, non-NPSLE patients, and HCs were extracted for meta-analysis, using standardized mean difference (SMD) as an estimate measure. For studies lacking sufficient data for inclusion, CBF, cerebral blood volume (CBV), and mean transit time (MTT) were reviewed qualitatively. RESULTS: Our review included eight observational studies employing PWI techniques, including dynamic susceptibility contrast (DSC) and arterial spin labeling (ASL). The meta-analysis of NPSLE compared to non-NPSLE incorporated four studies, encompassing 104 NPSLE patients and 90 non-NPSLE patients. The results revealed an SMD of -1.42 (95% CI: -2.85-0.00, I2: 94%) for CBF in NPSLE compared to non-NPSLE. CONCLUSION: PWI reveals informative patterns of cerebral perfusion, showing a significant reduction in mean CBF in NPSLE patients compared to non-NPSLE patients. Our qualitative synthesis highlights these changes, particularly in the frontal and temporal lobes. However, the existing data exhibits considerable heterogeneity and limitations.

Isotope-Labeled Chemoselective Probes for Labeling, Separation, and Comprehensive Quantitative Analysis of Sub-Metabolome.

The significance of small molecule metabolites as biomarkers for disease diagnosis and prognosis is growing increasingly evident, necessitating the development of highly sensitive qualitative and quantitative methods. Herein, multi-chemoselective probes are synthesized and applied for profiling metabolites, including carboxyl, phosphate, hydroxyl, amino, thiol, and carbonyl compounds. This approach seamlessly integrates magnetic solid-phase materials, orthogonal cleavage sites, isotopic tags, and selective coupling sites, minimizes matrix interference, and enhances quantitative accuracy. Meanwhile, a homemade program, High-Resolution Isotope-Assisted Identification and Quantitative (HRIAIQuant) is developed to process the data, which adeptly filters through 33,874 ion pairs present in human serum, leading to the identification of 701 known metabolites and a remarkable 1,062 potential novel ones. This method is successfully applied to analyze metabolites in multiple brain regions of SAMP8 and SAMR1 models, offering a novel tool for Alzheimer's disease research.

Spatial omics advances for in situ RNA biology.

Spatial regulation of RNA plays a critical role in gene expression regulation and cellular function. Understanding spatially resolved RNA dynamics and translation is vital for bringing new insights into biological processes such as embryonic development, neurobiology, and disease pathology. This review explores past studies in subcellular, cellular, and tissue-level spatial RNA biology driven by diverse methodologies, ranging from cell fractionation, in situ and proximity labeling, imaging, spatially indexed next-generation sequencing (NGS) approaches, and spatially informed computational modeling. Particularly, recent advances have been made for near-genome-scale profiling of RNA and multimodal biomolecules at high spatial resolution. These methods enabled new discoveries into RNA's spatiotemporal kinetics, RNA processing, translation status, and RNA-protein interactions in cells and tissues. The evolving landscape of experimental and computational strategies reveals the complexity and heterogeneity of spatial RNA biology with subcellular resolution, heralding new avenues for RNA biology research.

Synthesis of a 13C/2H Labeled Building Block to Probe the Phosphotyrosine Interactome Using Biomolecular NMR Spectroscopy.

Phosphotyrosine (pTyr) recognition coordinates the assembly of protein complexes, thus controlling key events of cell cycle, cell development and programmed cell death. Although many aspects of membrane receptor function and intracellular signal transduction have been deciphered in the last decades, the details of how phosphorylation alters protein-protein interaction and creates regulating switches of protein activity and localization often remains unclear. We developed a synthetic route to a protected phophotyrosine building block with isolated 13C-1H spins in the aromatic ring. The compound can be used for solid phase peptide synthesis (SPPS) and readily applied to study affinity, dynamics and interactions on an atomic level using NMR spectroscopy. As a first example, we prepared an isotopologue of a pTyr containing 12mer peptide (pY1021) as part of the platelet-derived growth factor to analyze the binding to the phospholipase C-γ (PLCγ-1) SH2 domain.

Mapping conformational changes on bispecific antigen-binding biotherapeutic by covalent labeling and mass spectrometry.

Biotherapeutic's higher order structure (HOS) is a critical determinant of its functional properties and conformational relevance. Here, we evaluated two covalent labeling methods: diethylpyrocarbonate (DEPC)-labeling and fast photooxidation of proteins (FPOP), in conjunction with mass spectrometry (MS), to investigate structural modifications for the new class of immuno-oncological therapy known as bispecific antigen-binding biotherapeutics (BABB). The evaluated techniques unveiled subtle structural changes occurring at the amino acid residue level within the antigen-binding domain under both native and thermal stress conditions, which cannot be detected by conventional biophysical techniques, e.g., near-ultraviolet circular dichroism (NUV-CD). The determined variations in labeling uptake under native and stress conditions, corroborated by binding assays, shed light on the binding effect, and highlighted the potential of covalent-labeling methods to effectively monitor conformational changes that ultimately influence the product quality. Our study provides a foundation for implementing the developed techniques in elucidating the inherent structural characteristics of novel therapeutics and their conformational stability.

Arterial Spin Labeling (ASL) MRI in Evaluating Pancreatic Blood Perfusion in Subjects With Different Glucose Tolerances.

BACKGROUND: The pancreas plays a central role in type 2 diabetes mellitus (T2DM), and its blood flow is usually associated with insulin release demand. PURPOSE: To noninvasively assess pancreatic blood flow (PBF) changes and modulation in people with different glucose tolerance following a glucose challenge using ASL MRI. STUDY TYPE: Prospective. SUBJECTS: Fourteen prediabetes, 22 T2DM, and 40 normal. FIELD STRENGTH/SEQUENCE: Pseudo-continuous ASL with a turbo gradient spin echo sequence at 3.0 T. ASSESSMENT: All normal and subjects (diagnosed by oral glucose tolerance test) underwent ASL after fasting for at least 6 hours. The normal and prediabetes groups additionally had ASL scans at 5, 10, 15, 20, and 25 minutes following oral glucose (50 mL, 5%). PBF maps were generated from the ASL data and measured at body and tail. The ability of baseline PBF (BL-PBF) of body, tail (BL-PBFtail), and their average to determine abnormal glucose tolerance and stage was assessed. STATISTICAL TESTS: ANOVA, Mann-Whitney U test, Kruskal-Wallis H test, paired sample t-test, intra-class correlation coefficient, repeated measures ANOVA, correlation analysis, receiver operating characteristic analysis, and logistic regression analysis. A P value <0.05 was considered significant. RESULTS: There were significant differences in BL-PBF among the three groups. The prediabetes group exhibited significantly lower PBF than the normal group at all time points; Both groups showed similar changing trends in PBF (peaking at the 15th minute and subsequently declining). The BL-PBFtail had the highest diagnostic performance when evaluating abnormal glucose tolerance or stage (area under the curves = 0.800, 0.584, respectively) and was an independent risk factor for glucose tolerance status. DATA CONCLUSION: ASL can noninvasively assess changes in PBF among individuals with varying glucose tolerance and in response to glucose challenge, which could be linked to insulin release demand and might help characterize changes in pancreatic endocrine function. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY: Stage 1.

Development of a bioluminescent immunoassay based on Fc-specific conjugated antibody-nanoluciferase immunoreagents for determining aflatoxin B1.

Aflatoxin B1 (AFB1) is a potent carcinogen, and is among the most hazardous mycotoxins in agricultural products. Therefore, the development of sensitive and convenient detection methods for AFB1 is significant for food safety against mycotoxins. Herein, a bioluminescent enzyme immunoassay (BLEIA) was developed for ultrasensitive detection of AFB1, based on the novel Fc-specific antibody-nanoluciferase (Ab-Nluc) conjugates which were fabricated using an IgG-binding protein-assisted photo-conjugation strategy. In indirect competitive immunoassay format, the proposed BLEIA exhibited the detection limit of 0.0232 ng mL-1, which was 37.4-fold lower than that obtained using the classical enzyme-linked immunosorbent assay (ELISA) based on Ab-horseradish peroxidase (Ab-HRP) chemical conjugates (0.868 ng mL-1). Meanwhile, the BLEIA exhibited high accuracy and precision. Thus, the proposed Fc-specific Ab-Nluc conjugates-based BLEIA provides an ultrasensitive and reliable method for detecting toxins and has potential for use in food safety monitoring.

DeepKINET: a deep generative model for estimating single-cell RNA splicing and degradation rates.

Messenger RNA splicing and degradation are critical for gene expression regulation, the abnormality of which leads to diseases. Previous methods for estimating kinetic rates have limitations, assuming uniform rates across cells. DeepKINET is a deep generative model that estimates splicing and degradation rates at single-cell resolution from scRNA-seq data. DeepKINET outperforms existing methods on simulated and metabolic labeling datasets. Applied to forebrain and breast cancer data, it identifies RNA-binding proteins responsible for kinetic rate diversity. DeepKINET also analyzes the effects of splicing factor mutations on target genes in erythroid lineage cells. DeepKINET effectively reveals cellular heterogeneity in post-transcriptional regulation.

Constructing a highly efficient multifunctional carbon quantum dot platform for the treatment of infectious wounds.

Antibiotic resistance poses a huge threat to public health, which has increased the difficulty and transmission of disease treatment, as well as the burden and cost of medical institutions. In response to the current problems and challenges in inflammation control and treatment of bacterial infected wounds, inspired by antibacterial mechanisms based on active elements such as N, S, Cu and tannic acid (TA), a highly efficient multifunctional carbon quantum dot platform was proposed in this study and constructed through their special assembly in a solvothermal reaction system for the treatment of infected wounds. By introducing active elements such as N, S and Cu, this carbon quantum dot platform is endowed with antibacterial properties, while also achieving good angiogenesis promoting performance through the use of ion Cu. Meanwhile, the good antioxidant activity of TA (one of the precursors used) enables this platform to have better immunomodulatory performance in vivo. The research results on the treatment of bacterial infection models indicate that the multifunctional carbon quantum dots obtained can accelerate the healing of infected wounds by inhibiting bacterial infection, regulating immunoreaction, accelerating collagen deposition and promoting angiogenesis. This multifunctional carbon quantum dot platform shows good clinical application prospects in treating bacterial infected wounds. Additionally, the fluorescence characteristics of such carbon dots can be expected to realize visual therapy in the future.

Development and Preclinical Evaluation of 18F-Labeled PEGylated Sansalvamide A Decapeptide for Noninvasive Evaluation of Hsp90 Status in Pancreas Cancer.

Heat shock protein 90 (Hsp90) is a promising target for cancer therapy and imaging. Accurate detection of Hsp90 levels in tumors via noninvasive PET imaging might be beneficial for management. To achieve this, the precursor compound Dimer-Sansalvamide A (Dimer-San A) was PEGylated and modified by conjugating it with the bifunctional chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). The 18F-labeled PEGylated Dimer-SanA decapeptide (18F-PEGylated San A) was completed within 30 min using a two-step process. In vitro stability and specificity were assessed, including competition studies with the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). MicroPET imaging was performed on PL45 tumor-bearing mice to evaluate probe accumulation and tumor-to-muscle ratios. Biodistribution studies determined the route of excretion. The probe resulted in a radiochemical yield of 23.11% with a purity exceeding 95%. In vitro, 18F-PEGylated San A exhibited high stability and selectively accumulated in Hsp90-positive PL45 cells, with binding effectively blocked by the Hsp90 inhibitor 17AAG, confirming its specificity. MicroPET imaging of PL45 tumor-bearing mice showed significant probe accumulation in tumor tissues at 1 and 2 h postinjection (4.06 ± 0.30 and 3.72 ± 0.61%ID/g, respectively), with optimal tumor-to-muscle ratios observed at 2 h postinjection (6.09 ± 1.92). While 18F-PEGylated San A demonstrates enhanced water solubility, as indicated by increased kidney uptake relative to liver accumulation. The study successfully incorporated PEG units to create the novel probe 18F-PEGylated San A targeting to Hsp90 without affecting its targeting capability, aimed at improving the pharmacokinetics and PET imaging of Hsp90 expression noninvasively.

Effect of Cations on ATP Binding to the N-domain of Na+, K+-ATPase.

The nucleotide-binding domain (N-domain) of the Na+, K+-ATPase (NKA) is physicochemically characterized by a high content of Glu and Asp residues, resulting in a low isoelectric point (pI = 5.0). Acidic proteins are known to interact with cations. The analysis in silico revealed potential cation interaction sites in the NKA N-domain structure. The interaction with cations was tested in vitro by using a recombinant NKA N-domain. The N-domain contains two Trp residues at the protein surface, as determined by acrylamide-mediated fluorescence quenching, that are useful for structural studies through fluorescence changes. Intrinsic fluorescence of the N-domain was decreased by the presence of cations (Na+, K+, Ca2+) indicating an effect on the protein structure. ATP binding also decreased the N-domain intrinsic fluorescence, which allowed nucleotide affinity determination. In the presence of cations, the N-domain affinity for ATP was increased. Molecular docking of fluorescein isothiocyanate (FITC) with the N-domain showed two binding modes with the isothiocyanate group located 5-6 Å close to Lys485 and Lys506 in the nucleotide-binding site. The presence of ATP prevented the FITC covalent labeling of the N-domain demonstrating the competitive behavior for the binding site. It is proposed that cations interact with the N-domain structure and thereby modulate nucleotide (ATP) affinity and possibly affecting NKA catalysis.