Competitive Assay Research Articles

Receptor interactions of protoxin and activated Vip3Aa structural conformations in Spodoptera exigua.

The Vip3Aa insecticidal protein, produced by Bacillus thuringiensis, has been effectively used in commercial Bt-crops to manage lepidopteran pests. Upon ingestion by larvae, the protoxin is processed by midgut proteases into the activated protein and binds specifically to its receptors in the midgut, leading to insect mortality. Cryo-EM resolution of the trypsin-processed Vip3Aa protein unveiled structural remodelling of the N-terminal region during the transition from protoxin to activated protein. This conformational change has been demonstrated to be crucial for toxicity against Spodoptera exigua larvae, a major global lepidopteran pest. In this study, we investigated the relevance of the structural remodelling for the specific binding to midgut receptors. We conducted in vitro binding assays with radiolabelled proteins and brush border membrane vesicles (BBMV) from S. exigua, employing structural mutants that lock the protein in either its protoxin or its activated conformation. Our results indicate that both structural stages of the protein share binding sites in the midgut epithelium. Moreover, in vivo competition assays revealed that Vip3Aa is able to bind to functional receptors in S. exigua larvae both as protoxin and as activated protein. Altogether, our findings point to both structural conformations contributing to receptor binding. In vivo, either spontaneous structural shift upon proteolytic cleavage or receptor-mediated remodelling could be occurring. However, the timing and context in which the conformational change occurs could influence membrane insertion and toxicity. Our results show the complex interplay between proteolytic processing, protein structure and receptor interactions in Vip3Aa's toxicity. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Seroprevalence and associated risk factors of peste des petits ruminants in sheep and goats in three districts of the Central Oromia Region, Ethiopia

BackgroundPeste des petits ruminants (PPR) is a viral disease that affects domestic and wild small ruminants and camels in Africa, the Middle East, and Asia. Following the successful eradication of rinderpest, the World Organization for Animal Health (WOAH) and the Food and Agriculture Organization (FAO) have undertaken to eradicate PPR by 2030. Regular surveillance and monitoring of the disease in various regions of Ethiopia are crucial to achieving this goal. This study aimed to estimate the prevalence of PPR, assess community awareness of PPR infection, and examine the associated risk factors of the disease in selected districts of the Central Oromia Region, Ethiopia.MethodThe study collected 384 serum samples from 73 flocks containing 217 sheep and 167 goats using a multi-stage sampling technique. A competitive enzyme-linked immunosorbent assay (c-ELISA) was used to detect antibodies against the PPR virus. Additionally, a pre-tested questionnaire was used to gather information on community awareness and potential risk factors for PPRV infection in the study area.ResultsThe study found that the overall prevalence of PPR in flocks was 71.2% [95% confidence interval (CI): 59.4%−81.2%]. The prevalence of PPR at the animal level was 50% (95% CI: 44.9%−55.1%), with sheep having a prevalence of 54.4% (95% CI: 47.0%−60.6%) and goats having a prevalence of 44.3% (95% CI: 36.6%−52.2%). The study also found that districts, flock size, and agroecology were independent predictors of PPRV seropositivity in sheep, whereas districts, origin, and mixed species were independent predictors of PPRV seropositivity in goats.ConclusionThe study revealed a high prevalence of PPR in sheep and goats in the study area. To prevent the spread of the disease, the study suggests quarantining animals before introducing them to districts, regular PPR vaccination, and isolation and molecular characterization of the PPR virus circulating in the study area.

Computational Model-Assisted Development of a Nonenzymatic Fluorescent Glucose-Sensing Assay.

Deep-red fluorescence was implemented in this fully injectable, nonenzymatic glucose biosensor design to allow for better light penetration through the skin, particularly for darker skin tones. In this work, a novel method was developed to synthesize Cy5.5 labeled mannose conjugates (Cy5.5-mannobiose, Cy5.5-mannotriose, and Cy5.5-mannotetraose) to act as the fluorescent competing ligand in a competitive binding assay with the protein Concanavalin A acting as the recognition molecule. Using fluorescence anisotropy (FA) data, a computational model was developed to determine optimal concentration ratios of the assay components to allow for sensitive glucose measurements within the physiological range. The model was experimentally validated by measuring the glucose response via FA of the three Cy5.5-labeled mannose conjugates synthesized with Cy5.5-mannotetraose demonstrating the most sensitive response to glucose across the physiological range. The developed method may be broadly applied to a vast range of commercially available fluorescent dyes and opens up opportunities for glucose measurements using nonenzymatic assays.

Development and Characterization of Sodium Bicarbonate-Based Gel for Cytolytic Vaginosis

Background/Objectives: Cytolytic vaginosis or, classically, Doderlein’s cytolysis is characterized by significant growth of species of the Lactobacillus genus, which leads to high amounts of lactic acid in the vaginal environment. Lactobacillus crispatus has been proposed as a key pathogen in this clinical condition. The symptomatology of cytolytic vaginosis is commonly confused with that of vulvovaginal candidosis, leading to inadequate and ineffective azole therapies. Nevertheless, historically, the use of sodium bicarbonate intimate baths was an effective way to reduce the symptoms of cytolytic vaginosis. Methods: In this study, four HPMC gel prototypes were developed, containing sodium bicarbonate concentrations ranging from 4% to 7% (w/w). These gels were evaluated for their physicochemical properties, antimicrobial activity, interference with lactobacilli adhering to cells, and cellular and tissue biocompatibility. Results: The gels presented pH values of around 9.0, and osmolality between 706 mOsm/kg (F4) and 1065 mOsm/kg (F7). The viscosity upon heating to physiologic temperature and dilution with simulated vaginal fluid was highly affected by the concentration of sodium bicarbonate. Gels with higher sodium bicarbonate concentrations (F6 and F7) were not shown to be stable in these conditions. All formulations exhibited effective antimicrobial activity against seven L. crispatus strains, with MIC values ranging from 6.25% to 25% (v/v) in terms of dilution. Additionally, the 4% (w/w) gel significantly interfered with the adhesion of L. crispatus to epithelial cells in competition and exclusion assays, reducing adhesion by more than 90% in relation to the control. Cytotoxicity tests on the Hec-1A, HeLa, and VK2/E6E7 cell lines indicated that the F4 and F5 gels demonstrated lower cytotoxicity levels compared to those with higher concentrations. Furthermore, ex vivo assays using porcine vaginal tissue confirmed that the 4% gel was non-toxic at a 25% (v/v) dilution. Conclusions: Based on these results, the 4% (w/w) sodium bicarbonate gel (F4) emerges as a promising therapeutic option for cytolytic vaginosis, offering effective bacterial interference, favourable physicochemical properties, and biocompatibility suitable for vaginal application.

DDDR-13. OPTIMIZING BRAIN CANCER THERAPY: BALANCING TUMOR ERADICATION AND NORMAL TISSUE PRESERVATION WITH BPM31510

Abstract The challenges translating glioblastoma therapies lie in safeguarding normal neuronal and glial tissue. Considering that the central nervous system has the lowest regenerative capacity, the exclusive focus on tumor eradication while neglecting normal tissue response, impedes proper healing. This oversight leads to toxicities ultimately culminating in patient demise with marginal gain in survival. BPM31510 is a nanodispersion encapsulating highly hydrophobic oxidized Coenzyme Q10 (CoQ10), allowing for delivery of supraphysiological CoQ10 concentrations into cancer cells. Initial investigations demonstrated differential responsiveness between cancer and non-cancer cells in vitro, an effect associated with high superoxide levels exclusively in cancer cells. To evaluate this divergence between cancer and non-cancer cells, a competitive co-culture cell-based assay was developed with labeled pairs of glioblastoma and non-cancer cells incubated up to three weeks with various concentrations of BPM31510 to ascertain optimal concentration leading to preferential growth of non-cancer cells. Human astrocyte (HA), mouse fibroblast (NIH 3T3), and rat astrocyte (TNC1) cells were paired with U251 human glioma, mouse GL261 and rat C6 glioma, respectively. Cell growth and spatial extent of the plate containing non-cancer or tumor cells were monitored every other day. Using doxorubicin as control, no dosage was discerned in which non-cancer cells exhibited advantage over tumor cells (i.e., either cultures became all tumor cells or both cell lines were eradicated). Using various concentrations of BPM31510, we identified an optimal dose in which cancer cells diminished while non-cancer cells predominated, reflected by markedly elevated mitochondrial superoxide levels in cancer cells prior to cell death. A co-culture cell-based assay was developed to identify an optimal dose of BPM31510 with selective preservation of non-cancer cells alongside tumor cell death. This approach elucidates developing a “fine tuning” strategy, wherein BPM31510 is titrated over time, thereby promoting normal tissue and healing, and ultimately leading to superior and prolonged functional outcomes.

TMIC-63. DUAL IMMUNE CHECKPOINT BLOCKADE IN A NOVEL EGFR-DRIVEN MURINE GLIOMA MODEL INCREASES PERIPHERAL T CELL PROLIFERATION BUT FAILS TO PROLONG SURVIVAL

Abstract Patients with glioblastoma (GBM) survive only 15-18 months following standard-of-care surgical resection and radio-chemotherapy. Immune checkpoint blockade (ICB), effective in extracranial cancers, failed to improve survival in patients with recurrent GBM in a multi-center phase III clinical trial. As such, we are interested in the molecular mechanisms that drive ICB resistance. Oncogenic EGFR activation is observed in ~50% of GBMs and is associated with immunosuppression; however, whether EGFR signaling impairs T cell function in GBM resulting in limited ICB efficacy remains unclear. Therefore, we leveraged the novel MADR-mEGFRvIII glioma model, which expresses murine EGFRvIII under a tetracycline-off system, to investigate the relationship between EGFR activation and responsiveness to dual anti-PD-1 and anti-CTLA-4 (10mg/kg) in vivo. Mice were bled prior to enrollment and weekly thereafter, which confirmed anti-PD-1-PD-1 binding via competitive flow cytometric assay. Following two weeks on treatment, animals were euthanized for immunophenotypic analyses of tumors and tumor-draining lymph nodes. These analyses revealed a significantly increased prevalence of peripheral CD11b+ myeloid cells and enhanced peripheral T cell proliferation, indicated by Ki67 positivity, after treatment with dual ICB compared to isotype control. However, these shifts did not translate to effective ICB-mediated recruitment of peripheral T cells to the tumor microenvironment and no significant survival benefit was observed (p=0.68, n=15/treatment arm). Given our previous findings that EGFR ablation via doxycycline enhanced intratumoral T cell infiltration, we hypothesize the rational combination of EGFR-targeted therapy and ICB will have a synergistic effect; thus, future directions include a study combining the two treatment regimens consisting of a lead-in with doxycycline to increase intratumoral T cell trafficking, followed by treatment with dual ICB. This work will provide insight into the interplay between oncogenic signaling and immunosuppression, contributing to the development of novel combination therapies in treating this lethal disease.

Establishment of Broad-Specificity Monoclonal Antibody-Based Immunoassay for Rapid Detection of Indole-Type and Indazole-Type Synthetic Cannabinoids and Metabolites.

Synthetic cannabinoids (SCs) have emerged as one of the most severely abused categories of new psychoactive substances (NPS), exacerbating the global drug problem and posing significant threats to public health. Presently, a class of new amide-type SCs featuring an indazole or indole core has been identified in numerous cases of illegal drug use, but there is still a lack of comprehensive analysis methods of SC detection. Herein, monoclonal antibodies (mAbs) 2E4 and AE6 targeting 36 indole-type and indazole-type SCs and their metabolites with IC50 ranging from 0.14 to 85.28 ng/mL were prepared and the molecular mechanism of antibody recognition was elaborated. We established the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and gold immunochromatography assay (GICA) based on mAbs 2E4 and AE6 to detect indazole-type and indole-type SCs in urine and hair samples. Under optimal conditions, the proposed method detected ADB-BUTINACA (an indazole-type SC) with limits of detection (LODs) of 0.11 ng/mL for urine and 0.024 ng/mg for hair by ic-ELISA, and 1.02 ng/mL for urine and 0.046 ng/mg for hair by GICA; the LODs of 4F-MDMB-BUTICA (an indole-type SC) detection was 0.036 ng/mL for urine and 0.012 ng/mg for hair by ic-ELISA, and 0.54 ng/mL for urine and 0.03 ng/mg for hair by GICA. Collectively, our study provides a comprehensive foundation for the rapid screening and quantitation of SC derivatives in biological samples.

Characterization of two glutathione S-transferase genes involved in clothianidin resistance in Bradysia odoriphaga.

Glutathione S-transferase (GST) is a key phase II detoxification enzyme involved in xenobiotics metabolism, and plays a pivotal role in the evolution of resistance to various types of insecticides. However, the specific functions of GST genes in clothianidin resistance remain obscure in Bradysia odoriphaga. Here, a specific GST inhibitor, diethyl maleate (DEM), significantly increased the mortality of Bradysia odoriphaga larvae following exposure to clothianidin, and the activity of GST enzyme in clothianidin-resistant (CL-R) strain of Bradysia odoriphaga was markedly greater than that in the SS strain. Two sigma BoGSTs (BoGSTs1 and BoGSTs2) were markedly overexpressed in the CL-R strain and exhibited a higher abundance in the Malpighian tubules or midgut. Exposure to clothianidin resulted in a significant increased expression of BoGSTs1 and BoGSTs2. The knockdown of BoGSTs1 and BoGSTs2 increased sensitivity of larvae to clothianidin in the resistant strain. Furthermore, overexpression of BoGSTs1 and BoGSTs2 led to a significant increase in Escherichia coli cells tolerance to clothianidin. In vitro metabolic assays indicate that these two GSTs cannot directly metabolize clothianidin and its secondary metabolite desmethyl-clothianidin. Disk diffusion assays and fluorescence competitive binding assays indicated that BoGSTs1 and BoGSTs2 play a critical role in clothianidin resistance by antioxidant activity and non-catalytic binding activity. The docking results showed that BoGSTs1 and BoGSTs2 have strong binding affinity toward clothianidin. Collectively, these findings pinpoint the potential role of BoGSTs1 and BoGSTs2 in conferring insecticide resistance in Bradysia odoriphaga and contribute to our understanding of the underlying mechanisms of insecticide resistance. © 2024 Society of Chemical Industry.

Association between thyroid hormones and cognitive functioning in euthyroid elderly adults: a cross-sectional preliminary study from the NHANES 2011–2012 survey

ObjectiveChanges in serum thyroid hormone levels may affect cognitive functioning in euthyroid individuals. This study used representative data from the National Health and Nutrition Examination Survey (NHANES) to comprehensively examine the association of thyroid hormones with different tests of cognitive functioning among US elderly people aged ≥60 years.MethodsThis study was a cross-sectional preliminary study with a total of 734 participants from the NHANES 2011-2012 survey. Thyroid function was measured using competitive binding immune-enzymatic assays, while cognitive functioning was measured using a series of assessments, including the Consortium to Establish a Registry for Alzheimer’s Disease-Word Learning (CERAD W-L), Animal Fluency Test, and Digit Symbol Substitution Test (DSST). Weighted multiple linear regression models and binary logistic regression analyses were used to examine the association between thyroid hormone levels and cognitive functioning. All statistical analyses were performed using SPSS version 20.0, and R software.ResultsWeighted multivariable linear regression showed that FT3 was negatively associated with the Animal Fluency Test and DSST (β=-0.113, 95% CI: -3.279, -0.803, P=0.001; β=-0.062, 95% CI: -6.565, -0.470, P=0.024, respectively) after adjustment for potential covariates. subgroup analysis stratified by sex revealed a negative association between FT3 levels and the Animal Fluency Test in men (β=-0.163, 95% CI: -4.643, -1.153, P=0.001). For female participants, FT3 was negatively associated with not only the Animal Fluency Test but also DSST (β=-0.099, 95% CI: -3.543, -0.093, P=0.039; β=-0.093, 95% CI: -10.288, -1.326, P=0.011). Binary logistic regression showed that the significantly increased adjusted odds ratios (aORs) (95% CI) between the risk of impaired cognitive functioning and FT3 across Q3 and Q4 compared with Q1 were 2.025 (1.092, 3.753) and 2.365 (1.261, 4.433), respectively, for DSST in overall participants. Furthermore, there were significant differences between participants with and without impaired cognitive functioning for serum FT3 levels in overall participants based on DSST score (P=0.020).ConclusionsThere was a significant inverse relationship between FT3 levels within the normal range and cognitive functioning after adjusting for potential covariates. Future longitudinal cohort studies should be conducted to determine the causal relationship between thyroid hormone levels and cognitive functioning.

Beta cell extracellular vesicle PD-L1 as a novel regulator of CD8+ T cell activity and biomarker during the evolution of type 1 diabetes.

Surviving beta cells in type 1 diabetes respond to inflammation by upregulating programmed death-ligand 1 (PD-L1) to engage immune cell programmed death protein 1 (PD-1) and limit destruction by self-reactive immune cells. Extracellular vesicles (EVs) and their cargo can serve as biomarkers of beta cell health and contribute to islet intercellular communication. We hypothesised that the inflammatory milieu of type 1 diabetes increases PD-L1 in beta cell EV cargo and that EV PD-L1 may protect beta cells against immune-mediated cell death. Beta cell lines and human islets were treated with proinflammatory cytokines to model the proinflammatory type 1 diabetes microenvironment. EVs were isolated using ultracentrifugation or size exclusion chromatography and analysed via immunoblot, flow cytometry and ELISA. EV PD-L1 binding to PD-1 was assessed using a competitive binding assay and in vitro functional assays testing the ability of EV PD-L1 to inhibit NOD CD8+ T cells. Plasma EV and soluble PD-L1 were assayed in the plasma of islet autoantibody-positive (Ab+) individuals or individuals with recent-onset type 1 diabetes and compared with levels in non-diabetic control individuals. PD-L1 protein co-localised with tetraspanin-associated proteins intracellularly and was detected on the surface of beta cell EVs. Treatment with IFN-α or IFN-γ for 24 h induced a twofold increase in EV PD-L1 cargo without a corresponding increase in the number of EVs. IFN exposure predominantly increased PD-L1 expression on the surface of beta cell EVs and beta cell EV PD-L1 showed a dose-dependent capacity to bind PD-1. Functional experiments demonstrated specific effects of beta cell EV PD-L1 to suppress proliferation and cytotoxicity of murine CD8+ T cells. Plasma EV PD-L1 levels were increased in Ab+individuals, particularly in those positive for a single autoantibody. Additionally, in Ab+ individuals or those who had type 1 diabetes, but not in control individuals, plasma EV PD-L1 positively correlated with circulating C-peptide, suggesting that higher EV PD-L1 could be protective for residual beta cell function. IFN exposure increases PD-L1 on the beta cell EV surface. Beta cell EV PD-L1 binds PD1 and inhibits CD8+ T cell proliferation and cytotoxicity. Circulating EV PD-L1 is higher in Ab+ individuals than in control individuals. Circulating EV PD-L1 levels correlate with residual C-peptide at different stages in type 1 diabetes progression. These findings suggest that EV PD-L1 could contribute to heterogeneity in type 1 diabetes progression and residual beta cell function and raise the possibility that EV PD-L1 could be exploited as a means to inhibit immune-mediated beta cell death.

Seroprevalence and associated risk factors of bovine herpesvirus 1 in smallholder dairy farms in two districts of Gondar zones, North-West Ethiopia

Bovine herpesvirus 1 (BoHV-1) is the infectious agent that causes infectious bovine rhinotracheitis (IBR), a disease affecting the reproductive and respiratory systems of cattle. Significant economic losses result from infectious bovine rhinotracheitis because of metritis, abortions, placenta retention, recurrent breeding, animal deaths, and losses from trade restrictions. Reports of the diseases have been made in southern, southwestern and in major cities that kept improved breed of dairy cows in Ethiopia with prevalence ranging from 28.5–67 %. However, there is insufficient information available regarding the frequency and spread of IBR in the northwest part of the nation. In northwest Ethiopia, a cross-sectional study was carried out to estimate seroprevalence of BoHV-1 and identify associated risk factors. Dairy farms and farm owners were chosen using a multistage cluster sampling technique, while dairy cattle were chosen using a simple random sample technique. Four hundred and thirty-one dairy cattle from 177 herds in the Debark and Lay-Armachiho districts in the North and Central Gondar zones, respectively, both in Northwestern Ethiopia, were selected to provide serum samples. Owners of dairy animals provided information via questionnaires. Using a competitive enzyme-linked immunosorbent assay (c-ELISA), anti-BoHV-1 antibodies were detected in serum samples. To identify risk factors, univariable and multivariable mixed effect logistic regression models were used. We calculated animal level and herd level seroprevalence of 72 % (95 % CI: 64.9–78.4 %) and 85.7 % (95 % CI: 79.8–90 %), respectively. Parity was associated with seroprevalence of BoHV-1; cows with higher parity had increased seroprevalence of BoHV-1. Bull mating [OR=3.13, (95 % CI: 1.74–5.64)] compared to AI and Debark district [OR=2.73 (95 % CI: 1.63–4.57)] compared to Lay-Armachiho district, were associated with seroprevalence of BoHV-1. The study had shown that BoHV-1 is circulating out of the major cities and also on dairy farms keeping local breeds of dairy cows in Gondar zones, North-West Ethiopia. This suggests need of attention in prevention and control of BoHV-1.

Halogen-bond mediated charge transfer for visual competitive colorimetric detection of fentanyl

Colorimetric assays are inexpensive and attractive tools for the detection of fentanyl (FTN), yet further enhancing their sensitivity remains a major challenge. Herein, we develop a halogen-bond mediated visual competitive colorimetric assay for FTN using Erythrosine B (EB) as a probe. The addition of poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide (F-127) induces the EB aggregation, strongly suppressing the background signal. Upon the introduction of FTN, the subsequent competitive reaction displaces F-127 to generate EB-FTN charge-transfer complexes via N…I halogen bonds between electron-rich amine groups and electron-deficient iodine sites, accompanying a significant absorbance wavelength shift and pink-to-purple color change. The limits of detection of this approach for FTN are 2 mg·L−1 by the naked eye and 0.19 mg·L−1 by UV–vis spectroscopy, which are approximately 3.7-fold to 4 orders of magnitude more sensitive than the reported colorimetric assays. Meanwhile, the present method is well applied for FTN-spiked domestic sewage samples, and an easy-to-use smartphone-based digital image colorimetry is also fabricated. It is expected that such an assay can play a key role in alleviating the worldwide opioid overdose crisis.

A larval expressed chemosensory protein involved in recognition of anthocyanins in Spodoptera litura (Lepidoptera: Noctuidae).

Anthocyanins are secondary metabolites which act as diverse functions during plant growth. Insects can discriminate host plants by their sensitive gustatory systems. It is hypothetical that chemosensory proteins (CSPs) play a crucial role in regulating this behavioral process. However, the underlying molecular mechanisms remain obscure. In the present study, we characterized a CSP SlitCSP8 from the Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae). Quantitative real-time-polymerase chain reaction analysis demonstrated that SlitCSP8 was mainly expressed in the head of the 7th S. litura larvae, especially labrum. Further, recombinant SlitCSP8 was obtained using bacterial expression system. Fluorescence competitive binding assays demonstrated that the purified SlitCSP8 exhibited a strong binding affinity to anthocyanins, a natural compound derived from the host plant. Silencing SlitCSP8 through RNAi significantly reduced the sensitivity of S. litura larvae to anthocyanins-treated leaf disks, the development from larva to pupae was not affected. These data provide insight into the molecular basis that CSP8 can detect anthocyanins in host plants by chemosensory system of insects. It can be further used in designing novel optimal food attractant targeting to the CSPs for pest control.

Development of a monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for screening of diethylstilbestrol in animal-derived foods

Development of a monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for screening of diethylstilbestrol in animal-derived foods

Functional modulation of RAGE activation by multimeric S100B using single-domain antibodies.

S100B is a multifunctional protein primarily found in the brain, where it plays crucial roles in cell proliferation, differentiation, and survival. It has intra- and extracellular functions and, depending on S100B levels, can exhibit both neurotrophic and neurotoxic activities, both mediated by the receptor for advanced glycation end products (RAGE). Here, we report the discovery and characterization of nanobodies (Nbs) targeting dimeric and tetrameric S100B, which are the two most abundant oligomeric functional forms of the protein, aiming to modulate S100B-mediated RAGE activation. Two Nbs were selected for detailed structural and functional studies, and found to bind tetrameric S100B with high affinity, as determined by biolayer interferometry analysis and SEC-stable binary complex formation. Structural and docking analyses revealed preferential contact sites of Nbs with S100B regions implicated in interactions with RAGE, namely residues at the interfacial cleft on dimeric S100B and the at hydrophobic cleft formed by the association of two homodimeric units in the tetramer. In accordance, assays in SH-SY5Y cells showed that Nbs modulate the RAGE-mediated neurotrophic activity of S100B by hindering its functional interactions with the receptor. Biolayer interferometry competition assays between tetrameric S100B and the RAGE-VC1 domain, confirmed that Nbs selectively block S100B-mediated RAGE engagement, in agreement with cell activation experiments. These findings highlight Nbs as powerful tools for elucidating molecular and cellular mechanisms through the modulation of S100B and RAGE functions, inspiring potential therapeutic applications.

Pulse Activation of Retinoic Acid Receptor Enhances Hematopoietic Stem Cell Homing by Controlling CXCR4 Membrane Presentation.

The interplay between metabolic signaling and stem cell biology has gained increasing attention, though the underlying molecular mechanisms remain incompletely elucidated. In this study, we identify and characterize the role of adapalene (ADA), a retinoic acid receptor (RAR) agonist, in modulating the migration behavior of hematopoietic stem cells (HSCs). Our initial findings reveal that ADA treatment suppresses hematopoietic stem and progenitor cell (HSPC) mobilization induced by AMD3100 and G-CSF. Furthermore, we demonstrate that ADA treatment upregulates the surface expression of CXCR4 on HSPCs, resulting in enhanced chemotaxis towards CXCL12. Mechanistically, our study suggests that ADA enhances CXCR4 surface presentation without increasing CXCR4 mRNA levels, pointing towards a non-canonical role of RAR signaling in regulating intracellular trafficking of CXCR4. In vivo experiments show that ADA administration significantly enhances HSC homing efficiency. Additionally, competitive transplantation assays indicate a marked increase in donor chimerism following ADA treatment. These findings highlight the critical role of retinoic acid signaling in regulating HSC homing and suggest its potential for advancing novel HSC-based therapeutic strategies.

A hapten design strategy to enhance the selectivity of monoclonal antibodies against malachite green

Malachite green (MG), a triphenylmethane dye, is used in the aquaculture industry as a disinfectant and insect repellent due to its potent bactericidal and pesticidal properties. However, its use poses potential environmental and health risks. This study analyzed and designed two haptens using computer simulation. Serum data confirmed the feasibility of introducing an arm at the dimethylamine group. Subsequently, a highly selective monoclonal antibody strain was successfully prepared based on the hapten. After optimizing the working conditions of indirect competitive enzyme-linked immunosorbent assay (IC-ELISA), the IC50 value was 0.83 ng/mL, with a detection limit (IC10) of 0.08 ng/mL and a linear range of 0.19–3.52 ng/mL. The developed monoclonal antibody exhibited a crossover rate of less than 0.1% with other similar structures and can be used to establish an immunoassay.

Structure-function relationship of dynorphin B variants using naturally occurring amino acid substitutions.

Dynorphins (Dyn) represent the subset of endogenous opioid peptides with the highest binding affinity to kappa opioid receptors (KOPrs). Activation of the G-protein-coupled pathway of KOPrs has strong anticonvulsant effects. Dyn also bind to mu (MOPrs) and delta opioid receptors (DOPrs) with lower affinity and can activate the β-arrestin pathway. To fully exploit the therapeutic potential of dynorphins and reduce potential unwanted effects, increased selectivity for KOPrs combined with reduced activation of the mTOR complex would be favorable. Therefore, we investigated a series of dynorphin B (DynB) variants, substituted in one or two positions with naturally occurring amino acids for differential opioid receptor activation, applying competitive radio binding assays, GTPγS assays, PRESTO-Tango, and Western blotting on single-opioid receptor-expressing cells. Seven DynB derivatives displayed at least 10-fold increased selectivity for KOPrs over either MOPrs or DOPrs. The highest selectivity for KOPrs over MOPrs was obtained with DynB_G3M/Q8H, and the highest selectivity for KOPrs over DOPrs was obtained with DynB_L5S. Increased selectivity for KOPr over MOPr and DOPr was based on a loss of affinity or potency at MOPr and DOPr rather than a higher affinity or potency at KOPr. This suggests that the investigated amino acid exchanges in positions 3, 5, and 8 are of higher importance for binding and activation of MOPr or DOPr than of KOPr. In tests for signal transduction using the GTPγS assay, none of the DynB derivatives displayed increased potency. The three tested variants with substitutions of glycine to methionine in position 3 displayed reduced efficacy and are, therefore, considered partial agonists. The two most promising activating candidates were further investigated for functional selectivity between the G-protein and the β-arrestin pathway, as well as for activation of mTOR. No difference was detected in the respective read-outs, compared to wild-type DynB. Our data indicate that the assessment of affinity to KOPr alone is not sufficient to predict either potency or efficacy of peptidergic agonists on KOPr. Further assessment of downstream pathways is required to allow more reliable predictions of in vivo effects.

Development of an Indirect Competitive ELISA Based on a Stable Epitope of β-Lactoglobulin for Its Detection in Hydrolyzed Formula Milk Powder.

The target of traditional immunological detection methods for milk allergens is usually the whole β-lactoglobulin molecule. However, thermal processes and hydrolysis can destroy the epitope of β-lactoglobulin and interfere with its accurate detection and labeling in prepackaged foods, posing a health risk to milk-allergic patients. There currently remains a need to excavate and locate recognition sites for β-lactoglobulin in thermally processed and hydrolyzed products. Therefore, a stable epitope of β-lactoglobulin (CAQKKIIAEKTKIPAVFKIDA) was selected as the ideal recognition site, and an indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed using an antibody against this stable β-lactoglobulin epitope in order to improve the detection of β-lactoglobulin in thermally processed and hydrolyzed foods in this study. The stable epitope of β-lactoglobulin was selected using a molecular dynamics simulation, and the binding ability of anti-stable epitope antibodies was characterized using indirect ELISA and indirect competitive ELISA. The limit of detection (LOD) and limit of quantitation (LOQ) of the established ELISA were 0.25 and 1.07 mg·kg-1, respectively. Furthermore, the developed ELISA only showed cross-reactivity to goat milk among 23 common foods, therefore exhibiting high specificity to bovine β-lactoglobulin. In addition, the developed ELISA was able to effectively detect β-lactoglobulin residue in processed commercial foods and hydrolyzed formula milk powder. Our findings provide a novel strategy for accurately detecting milk allergens based on stable epitope recognition in thermally processed and hydrolyzed foods.

Glycocalyx Interactions Modulate the Cellular Uptake of Albumin-Coated Nanoparticles.

Albumin-based nanoparticles (ABNPs) represent promising drug carriers in nanomedicine due to their versatility and biocompatibility, but optimizing their effectiveness in drug delivery requires understanding their interactions with and uptake by cells. Notably, albumin interacts with the cellular glycocalyx, a phenomenon particularly studied in endothelial cells. This observation suggests that the glycocalyx could modulate ABNP uptake and therapeutic efficacy, although this possibility remains unrecognized. In this study, we elucidate the critical role of the glycocalyx in the cellular uptake of a model ABNP system consisting of silica nanoparticles (NPs) coated with native, cationic, and anionic albumin variants (BSA, BSA+, and BSA-). Using various methodologies-including fluorescence anisotropy, dynamic light scattering, microscale thermophoresis, surface plasmon resonance spectroscopy, and computer simulations─we found that both BSA and BSA+, but not BSA-, interact with heparin, a model glycosaminoglycan (GAG). To explore the influence of albumin-GAG interactions on NP uptake, we performed comparative uptake studies in wild-type and GAG-mutated Chinese hamster ovary cells (CHO), along with complementary approaches such as enzymatic GAG cleavage in wild-type cells, chemical inhibition, and competition assays with exogenous heparin. We found that the glycocalyx enhances the cell uptake of NPs coated with BSA and BSA+, while serving as a barrier to the uptake of NPs coated with BSA-. Furthermore, we showed that harnessing albumin-GAG interactions increases cancer cell death induced by paclitaxel-loaded albumin-coated NPs. These findings underscore the importance of albumin-glycocalyx interactions in the rational design and optimization of albumin-based drug delivery systems.