Binding Assays Research Articles

MyACR: A Point-of-Care Medical Device for Determination of Albumin–Creatinine Ratio (uACR) in Random Urine Samples as a Marker of Nephropathy

Chronic kidney disease (CKD) is a progressive condition that affects more than 10% of the world’s population. Monitoring urine albumin-to-creatinine ratio (uACR) has become the gold standard for nephropathy diagnosis and control. The objective of the present study was to develop a simple, accurate, sensitive, and rapid point-of-care test (PoCT) device, MyACR, for uACR measurement, intended for use in community healthcare to screen for the risk and monitor the progress of CKD. Albumin and creatinine concentrations in urine samples were determined using spectrophotometric dye (tetrabromophenol blue)-binding and colorimetric Jaffe assay, respectively. Urine samples were diluted with distilled water (1:80) and mixed separately with albumin and creatinine reaction mixture. The creatinine reaction was incubated at room temperature (25 °C) for 30 min before analysis. Optical density (OD) was measured at the wavelengths of 625 nm (albumin) and 515 nm (creatinine). All calibration curves (0–60 mg/L and 0–2 mg/dL for albumin and creatinine) yielded linear relationships with correlation coefficients (R2) of >0.997. Good accuracy (% deviation of mean value (DMV) ≤ 5.42%) and precision (% coefficients of variation (CV) ≤ 12.69%) were observed from both the intra- and inter-day assays for the determination of albumin and creatinine using MyACR. The limit of quantification (LOQ) of albumin and creatinine in urine samples determined using MyACR and a laboratory spectrophotometer were 5 mg/L and 0.25 mg/dL, respectively, using 37.5 μL urine spiked samples (n = 5). The device was well-applied with clinical samples from 20 CKD patients. The median (range) of %DMV of the central (hospital) laboratory method (immune-based assay) was 3.48 (−17.05 to 21.64)%, with a high correlation coefficient (R2 > 0.98). In conclusion, MyACR showed satisfactory test performance in terms of accuracy, reproducibility, and sensitivity. Cost-effectiveness and improvement in clinical decision making need to be proven in future multisite community and home studies.

Structural Characterization of Human Bufavirus 1: Receptor Binding and Endosomal pH-Induced Changes

Bufaviruses (BuV) are members of the Parvoviridae of the Protoparvovirus genus. They are non-enveloped, T = 1 icosahedral ssDNA viruses isolated from patients exhibiting acute diarrhea. The lack of treatment options and a limited understanding of their disease mechanisms require studying these viruses on a molecular and structural level. In the present study, we utilize glycan arrays and cell binding assays to demonstrate that BuV1 capsid binds terminal sialic acid (SIA) glycans. Furthermore, using cryo-electron microscopy (cryo-EM), SIA is shown to bind on the 2/5-fold wall of the capsid surface. Interestingly, the capsid residues stabilizing SIA binding are conserved in all human BuVs identified to date. Additionally, biophysical assays illustrate BuV1 capsid stabilization during endo–lysosomal (pH 7.4–pH 4) trafficking and capsid destabilization at pH 3 and less, which correspond to the pH of the stomach. Hence, we determined the cryo-EM structures of BuV1 capsids at pH 7.4, 4.0, and 2.6 to 2.8 Å, 3.2 Å, and 2.7 Å, respectively. These structures reveal capsid structural rearrangements during endo–lysosomal escape and provide a potential mechanism for this process. The structural insights gained from this study will add to the general knowledge of human pathogenic parvoviruses. Furthermore, the identification of the conserved SIA receptor binding site among BuVs provides a possible targetable surface-accessible pocket for the design of small molecules to be developed as anti-virals for these viruses.

PET imaging of colon cancer CD73 expression using cysteine site-specific 89Zr-labeled anti-CD73 antibody

CD73 is a cell-surface ectoenzyme that hydrolyzes the conversion of extracellular adenosine monophosphate to adenosine, which in turn can promote resistance to immune checkpoint blockade therapy. Immune response may therefore be improved by targeting tumor CD73, and this possibility underlines the need to non-invasively assess tumor CD73 level. In this study, we developed a cysteine site-specific 89Zr-labeled anti-CD73 (89Zr-CD73) IgG immuno-PET technique that can image tumor CD73 expression in living bodies. Anti-CD73 IgG was reduced with tris(2-carboxyethyl)phosphine, underwent sulfohydryl moiety–specific conjugation with deferoxamine–maleimide, and was radiolabeled with 89Zr. CT26 mouse colon cancer cells, CT26/CD73 cells engineered to constitutively overexpress CD73, and 4T1.2 mouse breast cancer cells underwent cell binding assays and western blotting. Balb/c nude mice bearing tumors underwent 89Zr-CD73 IgG PET imaging and biodistribution studies. 89Zr-CD73 IgG showed 20-fold higher binding to overexpressing CT26/CD73 cells compared to low-expressing CT26 cells, and moderate expressing 4T1.2 cells showed uptake that was 38.9 ± 1.51% of CT26/CD73 cells. Uptake was dramatically suppressed by excess unlabeled antibody. CD73 content proportionately increased in CT26 and CT26/CD73 cell mixtures was associated with linear increases in 89Zr-CD73 IgG uptake. 89Zr-CD73 IgG PET/CT displayed clear accumulation in CT26/CD73 tumors with greater uptake compared to CT26 tumors (3.13 ± 1.70%ID/g vs. 1.27 ± 0.31%ID/g at 8 days; P = 0.04). Specificity was further supported by low CT26/CD73 tumor-to-blood ratio of 89Zr-isotype-IgG compared to 89Zr-CD73 IgG (0.48 ± 0.08 vs. 2.68 ± 0.52 at 4 days and 0.53 ± 0.07 vs. 4.81 ± 1.02 at 8 days; both P < 0.001). Immunoblotting and immunohistochemistry confirmed strong CD73 expression in CT26/CD73 tumors and low expression in CT26 tumors. 4T1.2 tumor mice also showed clear 89Zr-CD73 IgG accumulation at 8 days (3.75 ± 0.70%ID/g) with high tumor-to-blood ratio compared to 89Zr-isotype-IgG (4.91 ± 1.74 vs. 1.20 ± 0.28; P < 0.005). 89Zr-CD73 IgG specifically targeted CD73 on high expressing cancer cells in vitro and tumors in vivo. Thus, 89Zr-CD73 IgG immuno-PET may be useful for the non-invasive monitoring of CD73 expression in tumors of living subjects.

Interleukin-1β Signaling Contributes to Cell Cycle Arrest and Apoptotic Cell Death by Leptin via Modulation of AKT and p38MAPK in Hepatocytes.

Leptin, an adipose tissue-derived hormone, has exhibited the potent hepatotoxic effects. However, the underlying molecular mechanisms are not fully understood. In this study, we have elucidated the mechanisms by which leptin exerts cytotoxic effects in hepatocytes, particularly focusing on the role of interleukin-1β (IL-1β) signaling. Leptin significantly induced maturation and secretion of IL-1β in cultured rat hepatocytes. Interestingly, inhibition of IL-1β signaling by pretreatment with an IL-1 receptor antagonist (IL-1Ra) or gene silencing of type I IL-1 receptor (IL-1R1) markedly abrogated leptin-induced cell cycle arrest. The critical role of IL-1β signaling in leptin-induced cell cycle arrest is mediated via upregulation of p16, which acts as an inhibitor of cyclin-dependent kinase. In addition, leptin-induced apoptotic cell death was relieved by inhibition of IL-1β signaling, as determined by annexin V/7-AAD binding assay. Mechanistically, IL-1β signaling contributes to apoptotic cell death and cell cycle arrest by suppressing AKT and activation of p38 mitogen-activated protein kinase (p38MAPK) signaling pathways. Involvement of IL-1β signaling in cytotoxic effect of leptin was further confirmed in vivo using hepatocyte specific IL-1R1 knock out (IL-1R1 KO) mice. Essentially similar results were obtained in vivo, where leptin administration caused the upregulation of apoptotic markers, dephosphorylation of AKT, and p38MAPK activation were observed in wild type mice liver without significant effects in the livers of IL-1R1 KO mice. Taken together, these results demonstrate that IL-1β signaling critically contributes to leptin-induced cell cycle arrest and apoptosis, at least in part, by modulating p38MAPK and AKT signaling pathways.

Synthesis of 64Cu-, 55Co-, and 68Ga-Labeled Radiopharmaceuticals Targeting Neurotensin Receptor-1 for Theranostics: Adjusting InVivo Distribution Using Multiamine Macrocycles.

The development of theranostic radiotracers relies on their binding to specific molecular markers of a particular disease and the use of corresponding radiopharmaceutical pairs thereafter. This study reports the use of multiamine macrocyclic moieties (MAs), as linkers or chelators, in tracers targeting the neurotensin receptor-1 (NTSR-1). The goal is to achieve elevated tumor uptake, minimal background interference, and prolonged tumor retention in NTSR-1-positive tumors. Methods: We synthesized a series of neurotensin antagonists bearing MA linkers and metal chelators. The MA unit is hypothesized to establish a strong interaction with the cell membrane, and the addition of a second chelator may enhance water solubility, consequently reducing liver uptake. Small-animal PET/CT imaging of [64Cu]Cu-DOTA-SR-3MA, [64Cu]Cu-NT-CB-NOTA, [68Ga]Ga-NT-CB-NOTA, [64Cu]Cu-NT-CB-DOTA, and [64Cu]Cu-NT-Sarcage was acquired at 1, 4, 24, and 48 h after injection using H1299 tumor models. [55Co]Co-NT-CB-NOTA was also tested in HT29 (high NTSR-1 expression) and Caco2 (low NTSR-1 expression) colorectal adenocarcinoma tumor models. Saturation binding assay and internalization of [55Co]Co-NT-CB-NOTA were used to test tracer specificity and internalization in HT29 cells. Results: In vivo PET imaging with [64Cu]Cu-NT-CB-NOTA, [68Ga]Ga-NT-CB-NOTA, and [55Co]Co-NT-CB-NOTA revealed high tumor uptake, high tumor-to-background contrast, and sustained tumor retention (≤48 h after injection) in NTSR-1-positive tumors. Tumor uptake of [64Cu]Cu-NT-CB-NOTA remained at 76.9% at 48 h after injection compared with uptake 1 h after injection in H1299 tumor models, and [55Co]Co-NT-CB-NOTA was retained at 60.2% at 24 h compared with uptake 1 h after injection in HT29 tumor models. [64Cu]Cu-NT-Sarcage also showed high tumor uptake with low background and high tumor retention 48 h after injection Conclusion: Tumor uptake and pharmacokinetic properties of NTSR-1-targeting radiopharmaceuticals were greatly improved when attached with different nitrogen-containing macrocyclic moieties. The study results suggest that NT-CB-NOTA labeled with either 64Cu/67Cu, 55Co/58mCo, or 68Ga (effect of 177Lu in tumor to be determined in future studies) and NT-Sarcage labeled with 64Cu/67Cu or 55Co/58mCo may be excellent diagnostic and therapeutic radiopharmaceuticals targeting NTSR-1-positive cancers. Also, the introduction of MA units to other ligands is warranted in future studies to test the generality of this approach.

Synergistic effects of novel penicillin-binding protein 1A amino acid substitutions contribute to high-level amoxicillin resistance of Helicobacter pylori.

The growing resistance to amoxicillin (AMX)-one of the main antibiotics used in Helicobacter pylori eradication therapy-is an increasing health concern. Several mutations of penicillin-binding protein 1A (PBP1A) are suspected of causing AMX resistance; however, only a limited set of these mutations have been experimentally explored. This study aimed to investigate four PBP1A mutations (i.e., T558S, N562H, T593A, and G595S) carried by strain KIN76, a high-level AMX-resistant clinical H. pylori isolate with an AMX minimal inhibition concentration (MIC) of 2 µg/mL. We transformed a recipient strain 26695 with the DNA containing one to four mutation allele combinations of the pbp1 gene from strain KIN76. Transformants were subjected to genomic exploration and antimicrobial susceptibility testing. The resistance was transformable, and the presence of two to four PBP1A mutations (T558S and N562H, or T593A and G595S), rather than separate single mutations, was necessary to synergistically increase the AMX MIC up to 16-fold compared with the wild-type (WT) strain 26695. An AMX binding assay of PBP1A was performed using these strains, and binding was visualized by chasing Bocillin, a fluorescent penicillin analog. This revealed that all four-mutation allele-transformed strains exhibited decreased affinity to AMX on PBP1A than the WT. Protein structure modeling indicated that functional modifications occur as a result of these amino acid substitutions. This study highlights a new synergistic AMX resistance mechanism and establishes new markers of AMX resistance in H. pylori.IMPORTANCEThe development of resistance to antibiotics, including amoxicillin, is hampering the eradication of Helicobacter pylori infection. The identification of mechanisms driving this resistance is crucial for the development of new therapeutic strategies. We have demonstrated in vitro the synergistic role of novel mutations in the pbp1 gene of H. pylori that is suspected to drive amoxicillin resistance. Also deepening our understanding of amoxicillin resistance mechanisms, this study establishes new molecular markers of amoxicillin resistance that may be useful in molecular-based antibiotic susceptibility testing approaches for clinical practice or epidemiologic investigations.

Interaction of Cephalosporins with Human Serum Albumin: A Structural Study.

Modification of the R1 and R2 side chain structures has been used as the main strategy to expand the spectrum of cephalosporins and impart resistance to hydrolysis by β-lactamases. These structural modifications also result in a wide range of plasma protein binding, especially with human serum albumin (HSA). Here, we determined the crystal structures of the HSA complexes with two clinically important cephalosporins, ceftriaxone and cefazolin, and evaluated the binding of cephalosporin to HSA by susceptibility testing and competitive binding assay. Ceftriaxone and cefazolin bind to subdomain IB of HSA, and their cephem core structures are recognized by Arg117 of HSA. Tyr161 of HSA changes its rotamer depending on the cephalosporin, resulting in alterations of the cavity shape occupied by the R2 side chain of cephalosporins. These findings provide structural insight into the mechanisms underlying the HSA binding of cephalosporins.

OBP83b and OBP49a Involved in the Perception of Female-Derived Pheromones in Bactrocera dorsalis (Hendel).

In Bactrocera dorsalis, both males and females release chemical signals to attract mates. In our previous study, we identified ethyl laurate, ethyl myristate, and ethyl palmitate as potent female-derived pheromones that contribute to mate attraction. However, the mechanisms underlying the olfactory recognition remain unclear. In this study, we observed strong antennal and behavioral responses in male B. dorsalis to these female-derived pheromones, and further investigation revealed significant upregulation of OBP49a and OBP83b following exposure to these compounds. Through fluorescence competitive binding assays and RNA interference techniques, we demonstrated the crucial roles of OBP49a and OBP83b in detecting female-derived pheromones. Finally, molecular docking analysis identified key residues, including His134 in OBP83b and a lysine residue in OBP49a, which formed hydrogen bonds with female-derived pheromones, facilitating their binding. These findings not only advance our understanding of olfactory recognition of pheromones in B. dorsalis but also offer potential targets for developing olfaction-interfering techniques for pest control.

Development of Autoimmune Diseases Among Children With Pediatric Acute-Onset Neuropsychiatric Syndrome

Epidemiologic studies indicate a high rate of autoimmune conditions among patients with obsessive-complusive disorder and other psychiatric conditions. Furthering the understanding of the inflammatory diatheses of psychiatric conditions may open doors to new treatment paradigms for psychiatric disorders. To evaluate whether pediatric acute-onset neuropsychiatric syndrome (PANS) is associated with an inflammatory diathesis by assessing signs of immune activation and vasculopathy during a psychiatric symptom exacerbation (flare), estimating the risk of developing arthritis and other autoimmune diseases, and characterizing subtypes of arthritis. This retrospective cohort study used longitudinal clinical data on 193 consecutive patients with PANS followed up within the Stanford Immune Behavioral Health Clinic from September 1, 2012, to December 31, 2021. Medical records were reviewed, and a predefined set of immune markers that were measured during a flare and the features and imaging findings of arthritis and other autoimmune diseases were collected. Immune activation markers included (1) autoimmunity signs (antinuclear antibody, antihistone antibody, antithyroglobulin antibody, C1q binding assay, and complement levels [C3 and C4]); (2) immune dysregulation or inflammation signs (leukopenia, thrombocytosis, C-reactive protein, and erythrocyte sedimentation rate); and (3) vasculopathy signs (livedo reticularis, periungual redness and swelling, abnormally prominent onychodermal band, palatal petechiae, high von Willebrand factor antigen, and high d-dimer). Last, the cumulative risk of developing arthritis and autoimmune diseases was estimated using product limit (Kaplan-Meier) survival probability. The study included data from 193 children (112 boys [58.0%]) who had PANS at a mean (SD) age of 7.5 (3.5) years. They were followed up for a mean (SD) of 4.0 (2.1) years. Among those tested for immune activation markers, 54.2% (97 of 179) had nonspecific markers of autoimmunity, 12.0% (22 of 184) had nonspecific signs of immune dysregulation or inflammation, and 35.8% (69 of 193) had signs of vasculopathy. By 14 years of age, the estimated cumulative incidence of arthritis was 28.3% (95% CI, 20.8%-36.3%), and the estimated cumulative incidence of another autoimmune disease was 7.5% (95% CI, 4.0%-12.4%). Novel findings in the subgroup with arthritis include joint capsule thickening (55.0% [22 of 40]), distal interphalangeal joint tenderness (81.8% [45 of 55]), and spinous process tenderness (80.0% [44 of 55]). Among the 55 patients with arthritis, the most common subtypes of arthritis included enthesitis-related arthritis (37 [67.3%]), spondyloarthritis (27 [49.1%]), and psoriatic arthritis (10 [18.2%]). This study found that patients with PANS show signs of immune activation and vasculopathy during psychiatric symptom flares and have an increased risk of developing arthritis and other autoimmune diseases compared with the general pediatric population. The most common arthritis subtype was enthesitis-related arthritis. These findings suggest that PANS may be part of a multisystem inflammatory condition rather than an isolated psychiatric or neuroinflammatory disorder.

Unraveling the structure and function of a novel SegC protein interacting with the SegAB chromosome segregation complex in Archaea.

Genome segregation is a fundamental process that preserves the genetic integrity of all organisms, but the mechanisms driving genome segregation in archaea remain enigmatic. This study delved into the unknown function of SegC (SSO0033), a novel protein thought to be involved in chromosome segregation in archaea. Using fluorescence polarization DNA binding assays, we discovered the ability of SegC to bind DNA without any sequence preference. Furthermore, we determined the crystal structure of SegC at 2.8 Å resolution, revealing the multimeric configuration and forming a large positively charged surface that can bind DNA. SegC has a tertiary structure folding similar to those of the ThDP-binding fold superfamily, but SegC shares only 5-15% sequence identity with those proteins. Unexpectedly, we found that SegC has nucleotide triphosphatase (NTPase) activity. We also determined the SegC-ADP complex structure, identifying the NTP binding pocket and relative SegC residues involved in the interaction. Interestingly, images from negative-stain electron microscopy revealed that SegC forms filamentous structures in the presence of DNA and NTPs. Further, more uniform and larger SegC-filaments are observed, when SegA-ATP was added. Notably, the introduction of SegB disrupts these oligomers, with ATP being essential for regulating filament formation. These findings provide insights into the functional and structural role of SegC in archaeal chromosome segregation.

TPCA-1 compound, inhibiting testis-specific serine/threonine protein kinase 3 for potential male sterile in Bombyx mori.

Protein kinases are a type of transferase enzyme that catalyze the phosphorylation of protein substrates, including receptor proteins. Testis-specific serine/threonine kinases (TSSKs) are a highly conserved group of protein kinases found in various organisms. They play an essential role in male reproduction by influencing sperm development and function. In this study, we report on the characterization of BmTSSK3, a TSSK from the silkworm, Bombyx mori. We found that BmTSSK3 is specifically expressed in the testis and localized to the sperm flagella, particularly in the sperm tail cyst. Furthermore, we developed BmTSSK3 inhibitors through molecular docking and binding assays. Small molecules 5-(4-Fluorophenyl)-2-ureidothiophene-3-carboxamide (TPCA-1)and Imidurea were identified to bind to BmTSSK3. Using site-specific mutation technology, we identified amino acid residues R134 and S184 as crucial binding sites for small molecules. RNA interference assay and Western blot analysis showed that knockdown of BmTSSK3 significantly decreased histone 3 phosphorylation. To confirm the inhibitory effect of these small molecules, we treated silkworm testes with TPCA-1 and observed a strong inhibitory effect. TPCA-1 is an inhibitor of BmTSSK3, which raises its potential as a future candidate for male sterility of the silkworm. Thus, this study may offer a novel strategy for sterile silkworms as well as insects. © 2024 Society of Chemical Industry.

Clinacanthus nutans (Burm. f.) Lindau Extract Inhibits Dengue Virus Infection and Inflammation in the Huh7 Hepatoma Cell Line

Dengue virus (DENV) infection has emerged as a global health problem, with no specific treatment available presently. Clinacanthus nutans (Burm. f.) Lindau extract has been used in traditional medicine for its anti-inflammatory and antiviral properties. We thus hypothesized C. nutans had a broad-ranged activity to inhibit DENV and the liver inflammation caused by DENV infection. The study showed that treatment using C. nutans extract during DENV infection (co-infection step) showed the highest efficiency in lowering the viral antigen concentration to 22.87 ± 6.49% at 31.25 μg/mL. In addition, the virus–host cell binding assay demonstrated that C. nutans treatment greatly inhibited the virus after its binding to Huh7 cells. Moreover, it could remarkably lower the expression of cytokine and chemokine genes, including TNF-α, CXCL10, IL-6, and IL-8, in addition to inflammatory mediator COX-2 genes. Interestingly, the activation of the NF-κB signaling cascade after C. nutans extract treatment was dramatically decreased, which could be the underlying mechanism of its anti-inflammatory activity. The HPLC profile showed that gallic acid was the bioactive compound of C. nutans extract and might be responsible for the antiviral properties of C. nutans. Taken together, our results revealed the potential of C. nutans extract to inhibit DENV infection and lower inflammation in infected cells.

Pulsatillic acid as a potential inhibitor of protein kinase C-alpha in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a global health concern with a significant impact on morbidity and mortality. Small molecule inhibitors targeting genetic mutations like EGFR and ALK have shown promise in NSCLC treatment. This study focuses on Protein Kinase C-alpha (PKCα), implicated in NSCLC pathogenesis. Overexpression of PKCα correlates with advanced disease stages. Preclinical studies suggest its inhibition can suppress NSCLC cell growth. The research employs molecular docking to identify Pulsatillic acid (PA) as a potential PKCα inhibitor. ADMET predictions support PA's candidacy and PASS analysis and Swiss Target Prediction reveal its biological properties. Fluorescence-based binding assays demonstrate PA's inhibitory potency on PKCα, aligning with molecular docking findings. Cytotoxicity assays show PA's minimal impact on HEK-293 cell viability, with an IC50 of 21.03 μM in A549 cells. mRNA expression analysis in A549 cells indicates PA's potential inhibitory effect on PKCα. In conclusion, this study highlights that PA may emerge as a promising therapeutic candidate for NSCLC, emphasizing the need for further research, validation, and exploration of its translational potential. The study contributes valuable insights into NSCLC treatment strategies, emphasizing the significance of targeting PKCα.

RG-I-containing sugar domains from Centella Asiatica bind strongly to galectin-3 to inhibit cell–cell interactions

BackgroundCentella Asiatica has been shown to have beneficial value for the treatment of tumors. However, its active ingredients and molecular mechanisms of action have not been fully elucidated. Pectic polysaccharides are the primary active components from medicinal plants. Moreover, these polysaccharides are regarded as potential inhibitors of galectins, such as galectin-1, -3, -7, that generally promote tumor growth. Nevertheless, detailed structural analysis of pectic polysaccharides from Centella Asiatica is sorely lacking, as is knowledge of their interactions with galectins.MethodsWater-soluble pectic polysaccharides (WCAP) isolated from Centella Asiatica were purified into two homogeneous fractions (WCAP-A2b and WCAP-A5b) by a combination of anion-exchange and gel-permeation chromatography. Monosaccharide composition, FT-IR, NMR and enzymatic analyses were used to characterize their structural features. Furthermore, the interactions between galectin-1, -3, -7 and a series of these polysaccharides, including two pectin fractions and their structural domains produced by enzymatic hydrolysis, were evaluated by using hemagglutination and biolayer interferometry.ResultsWCAP-A2b and WCAP-A5b have weight averaged molecular weights of 30.0 kDa and 34.0 kDa, respectively, and both polysaccharides consist of rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II) and homogalacturonan (HG) domains, with mass ratios of 1.3: 1.0: 1.4 and 1.1: 1.0: 2.4, respectively. Their RG-I domains contain arabinan, galactan, and/or arabinogalactan, along with neutral sugar side chains that are more prevalent in WCAP-A2b than in WCAP-A5b. Hemagglutination and biolayer interferometry binding assays indicate that galectin-3 vis-à-vis galectin-1 and -7, binds strongly to the RG-I domain (likely via its neutral side chains) in WCAP-A5b, thereby inhibiting galectin-3-mediated cell–cell interactions.ConclusionsOur study provides structural information on pectin polysaccharides from Centella Asiatica. Results suggest that RG-I domains from WCAP-A5b and WCAP-A2b may be developed as potential inhibitors of galectin-3-mediated cell–cell adhesion and tumor growth.Graphical

Postprandial fatty acid-binding protein4 is associated with muscle insulin resistance.

Fatty acid-binding protein4 (FABP4) has been reported to act as a hepatic insulin resistance factor. We previously reported that fasting FABP4 was correlated with insulin resistance measurements derived from the glucose clamp, and another study reported that postprandial FABP4 levels were decreased in healthy volunteers but were not reported (or known) in participants with type 2 diabetes. We have limited knowledge about the direct effect of FABP4 on muscle cells. We investigated the postprandial FABP4 levels in participants with type 2 diabetes, and the basic mechanism of muscle insulin resistance and FABP4. We performed a meal tolerance test and hyperinsulinaemic-euglycaemic clamp in 22 participants with type 2 diabetes and 26 participants without diabetes. We measured fasting and postprandial serum FABP4. We cultured mouse C2C12 muscle cells, and investigated the effect of FABP4 on glucose uptake. We analysed insulin signalling by western blot and insulin binding assay. The postprandial FABP4 level in participants with type 2 diabetes was higher than that in participants without diabetes. Participants without diabetes had lower postprandial FABP4 than fasting except for one participant, whereas one-third of participants with type 2 diabetes had higher postprandial FABP4 than fasting. Postprandial FABP4 was correlated with the muscle insulin resistance M/I value from a glucose clamp in participants without diabetes (r=-0.42, p<0.05). The increase in FABP4 after a meal correlated with the muscle insulin resistance M/I value (r=-0.44, p<0.05) and the difference between fasting and postprandial glucagon in participants with type 2 diabetes (r=0.36, p<0.05). FABP4 alone appears to increase glucose uptake, and the combination of FABP4 and insulin decreases glucose uptake when compared with insulin alone. FABP4 inhibits insulin signalling of muscle cells through decreases in phosphorylation of insulin receptor substrate1 and Akt. The physiological concentration of FABP4 did not inhibit insulin binding to muscle cells. These results suggested that the postprandial FABP4 level is associated with insulin resistance, and FABP4 may suppress insulin signals.

Highly Selective Drug-Derived Fluorescent Probes for the Cannabinoid Receptor Type 1 (CB1R).

The cannabinoid receptor type 1 (CB1R) is pivotal within the endocannabinoid system regulating various signaling cascades with effects in appetite regulation, pain perception, memory formation, and thermoregulation. Still, understanding of CB1R's cellular signaling, distribution, and expression dynamics is very fragmentary. Real-time visualization of CB1R is crucial for addressing these questions. Selective drug-like CB1R ligands with a defined pharmacological profile were investigated for the construction of CB1R fluorescent probes using a reverse design-approach. A modular design concept with a diethyl glycine-based building block as the centerpiece allowed for the straightforward synthesis of novel probe candidates. Validated by computational docking studies, radioligand binding, and cAMP assay, this systematic approach allowed for the identification of novel pyrrole-based CB1R fluorescent probes. Application in fluorescence-based target-engagement studies and live cell imaging exemplify the great versatility of the tailored CB1R probes for investigating CB1R localization, trafficking, pharmacology, and its pathological implications.

Recommendations for Development and Validation of a Fit-For-Purpose Biomarker Assays Using Western Blotting; An-AAPS Sponsored Initiative to Harmonize Industry Practices.

Western blot (WB) assays are routinely used for detection and quantification of biomarkers. Although assay validation to measure biomarkers in complex matrices has become a mainstay process for ligand binding assays (LBA) and mass spectrometry (MS), no guidelines exist yet validate biomarker methods using WB techniques. In this cross-industry white paper, we outlined in detail the key steps for development and for validation of WB assays for protein biomarkers under different contexts of use (COU). In addition, we described how to determine the level of assay validation needed for biomarker assays using Western blotting. For simplicity, we described two paths of WB assay validation. The first path (Path 1) is for biomarkers being analyzed for exploratory research or for internal go- or no/go- decision making. The second path (Path 2) is for clinical decision making such as dose determination or drug response that need to be run in a regulated environment. This work is supported through AAPS Biomarkers and Precision Medicine subteam and represents AAPS members opinion.

Novel Gd-DTPA-peptide for targeted breast tumor magnetic resonance imaging

The prevalence of breast cancer underscores the imperative for early diagnosis in guiding treatment decisions. This study introduces a novel contrast agent, Gd-DTPA-VGB3, derived from the peptide VGB3 targeting vascular endothelial growth factor receptor-1 (VEGFR1) and VEGFR2, to enhance the contrast of conventional drug Magnevist in breast tumor MRI. The MRI contrast agent was synthesized on rink amide resin via Fmoc strategy, incorporating amino acids, and coupling to diethylenetriaminepentaacetic acid (DTPA). Gadolinium (Gd)-DTPA-VGB3 displayed specific binding to VEGFR1/2 in a displacement binding assay. Gd-DTPA-VGB3 exhibited minimal cytotoxicity to normal MCF-10 cells while inhibiting 4T1 mammary carcinoma cell proliferation. Compared to Magnevist, Gd-DTPA-VGB3 demonstrated a 2.8-fold increase in contrast-to-noise ratio (CNR) (355 vs. 125). Gd-DTPA-VGB3 exhibited enhanced accumulation in 4T1 tumor-bearing mice, resulting in significant signal intensity improvement. The findings highlight Gd-DTPA-VGB3's specific binding to VEGFRs, substantiating its potential as a candidate for enhancing MRI contrast in breast cancer diagnostics.

ImmunoPET imaging of LAG-3 expression in tumor microenvironment with 68Ga-labelled cyclic peptides tracers: from bench to bedside

BackgroundLymphocyte activation gene 3 (LAG-3) has been considered as the next generation of immune checkpoint and a promising prognostic biomarker of immunotherapy. As with programmed cell death protein-1/programmed death-ligand 1...

Quercetin-Loaded Nanocarriers as Effective Inhibitors for Copper Metal Ion-Induced γD-Crystallin Aggregation.

Cataract is one of the leading causes of blindness worldwide. Till date, the only solution for cataracts is surgery, which is a resource-intensive solution. A much simpler solution is to find a potential drug that could inhibit aggregation. It is well established that nonamyloid aggregates of eye lens protein result in cataract. γD-Crystallin, a thermodynamically stable protein, is one of the most abundant proteins in the core of the eye lens and is found to aggregate under stress conditions, leading to the cataract. It has also been found that in cataractous lens, the concentration of metals like copper is elevated significantly as compared to healthy eye lens, suggesting their role in inducing aggregation. In our present study, aggregation of γD-Crystallin was carried out in the presence of Cu (II). Using techniques like turbidity assay, CD spectroscopy, ANS binding assay, and microscopic studies like TEM, it could be confirmed that protein aggregates in the presence of Cu (II) and the nature of aggregates is amorphous. Various polyphenols were tested to suppress aggregation of the protein. Quercetin was observed to be the most efficient. To overcome the problems associated with the delivery of polyphenols, such as solubility and bioavailability, quercetin was encapsulated in two types of nanocarriers. Their characterization was done using TEM, DLS, and other techniques. The potency of quercetin-loaded CS-TPP/CS-PLGA NPs as inhibitors of γD-Crystallin aggregation was confirmed by various experiments.