Visualizing the cellular internalization of therapeutic antibodies via pH-sensitive release of AIEgen.

Among the fastest-growing bio-pharmaceuticals, therapeutic antibodies have achieved unprecedented success in treating various diseases. Though powerful, issues such as inefficacy or acquired resistance are waiting to be addressed to benefit more patients with improved therapeutic outcomes. In addition to in vivo distribution, the cellular spatiotemporal information including the antibody-antigen interaction and subsequent internalization is found to be important for the therapeutic effects. To better understand the cellular fate of therapeutic antibodies, especially the cellular internalization process, we employed a pH-sensitive linker to attach a red-emissive AIEgen onto the antibody. The resulting antibody conjugate will undergo AIEgen release to liberate brilliant fluorescence inside acidic endo/lysosomes, allowing wash-free visualization of the internalization process and facilitating the evaluation of antibody-drug efficacy.

Gliotoxin elicits immunotoxicity in the early innate immune system of ducks.

Gliotoxin (GT) belongs to the epipolythiodioxopiperazine (ETP) family, which is considered a crucial virulence determinant among the secondary metabolites produced by Aspergillus fumigatus. The metabolites are commonly found in food and feed, contributing to the invasion and immune escape of Aspergillus fumigatus, thereby posing a significant threat to the health of livestock, poultry, and humans. Heterophil extracellular traps (HETs), a novel form of innate immune defense, have been documented in the chicken's innate immune systems for capturing and eliminating invading microbes. However, the effects and mechanisms of GT on the production of duck HETs in vitro remain unknown. In this study, we first confirmed the presence of HETs in duck innate immune systems and further investigated the molecular mechanism underlying GT-induced HETs release. Our results demonstrate that GT can trigger typical release of HETs in duck. The structures of GT-induced HETs structures were characterized by DNA decoration, citrullinated histones 3, and elastase. Furthermore, NADPH oxidase, glycolysis, ERK1/2 and p38 signaling pathway were found to regulate GT-induced HETs. In summary, our findings reveal that gliotoxin activates HETs release in the early innate immune system of duck while providing new insights into the immunotoxicity of GT towards ducks.

Nanomaterial-based aptamer biosensors for ochratoxin A detection: a review.

Ochratoxin A (OTA) is a widely distributed mycotoxin that often contaminates food, grains and animal feed. It poses a serious threat to human health because of its high toxicity and persistence. Therefore, the development of an inexpensive, highly sensitive, accurate and rapid method for OTA detection is imperative. In recent years, various nanomaterials used in the establishment of aptasensors have attracted great attention due to their large surface-to-volume ratio, good stability and facile preparation. This review summarizes the development of nanomaterial-based aptasensors for OTA determination and sample treatment over the past 5 years. The nanomaterials used in OTA aptasensors include metal, carbon, luminescent, magnetic and other nanomaterials. Finally, the limitations and future challenges in the development of nanomaterial-based OTA aptasensors are reviewed and discussed.

Pharmacokinetics and brain distribution studies of 6-hydroxykynurenic acid and its structural modified compounds.

OBJECTIVES: 6-Hydroxykynurenic acid (6-HKA) is an organic acid component in extracts of Ginkgo biloba leaves and acts as a major contributor to neurorestorative effects, while its oral bioavailability was low. Therefore, using prodrug method to improve the bioavailability and brain content of 6-HKA is significant. METHODS: Three structural modified compounds of 6-HKA were synthesized, and ultra performance liquid chromatography-tandem mass spectrometry methods for quantification of these structural modified compounds in rat plasma and rat brain homogenate were established and comprehensively validated. The methods were effectively applied to investigate the effects of structural modification on apparent permeability coefficients in cells, the pharmacokinetics and the brain distribution in rats. KEY FINDINGS: The results illustrated that esterification can greatly improve the apparent permeability coefficient and bioavailability of 6-HKA. Comparing with direct oral administration of 6-HKA, the bioavailability of isopropyl ester was greatly improved (from 3.96 ± 1.45% to 41.8 ± 15.3%), and the contents of 6-HKA in rat brains (49.7 ± 9.2 ng/g brain) were significantly higher after oral administration. CONCLUSIONS: The bioavailability and the brain content of 6-HKA can be improved by the prodrug method. Among three structural modified compounds, isopropyl-esterified 6-HKA was the most promising treatment.

Real-time imaging of cell-surface proteins with antibody-based fluorogenic probes.

Cell-surface proteins, working as key agents in various diseases, are the targets for around 66% of approved human drugs. A general strategy to selectively detect these proteins in a real-time manner is expected to facilitate the development of new drugs and medical diagnoses. Although brilliant successes were attained using small-molecule probes, they could cover a narrow range of targets due to the lack of suitable ligands and some of them suffer from selectivity issues. We report herein an antibody-based fluorogenic probe prepared via a two-step chemical modification under physiological conditions, to fulfill the selective recognition and wash-free imaging of membrane proteins, establishing a modular strategy with broad implications for biochemical research and for therapeutics.

Advances in Nanoparticle Drug Delivery Systems for Anti-Hepatitis B Virus Therapy: A Narrative Review.

Chronic hepatitis B (CHB) is an infectious viral disease that is prevalent worldwide. Traditional nucleoside analogues, as well as the novel drug targets against hepatitis B virus (HBV), are associated with certain critical factors that influence the curative effect, such as biological stability and safety, effective drug delivery, and controlled release. Nanoparticle drug delivery systems have significant advantages and have provided a basis for the development of anti-HBV strategies. In this review, we aim to review the advances in nanoparticle drug delivery systems for anti-hepatitis B virus therapy by summarizing the relevant literature. First, we focus on the characteristics of nanoparticle drug delivery systems for anti-HBV therapy. Second, we discuss the nanoparticle delivery systems for anti-HBV nucleoside drugs, gene-based drugs, and vaccines. Lastly, we provide an overview of the prospects for nanoparticle-based anti-HBV agents.

The characterisation of the in vitro metabolism and transport of 6-hydroxykynurenic acid, an important constituent of Ginkgo biloba extracts.

6-Hydroxykynurenic acid (6-HKA) is a nitrogen-containing phenolic acid compound in Ginkgo biloba leaves. The pharmacological activities of 6-HKA have been reported and shown that 6-HKA has the potential to become a therapeutic drug and may play an important role in the treatment of nervous system diseases. However, there are few studies on the drug metabolism and transport of 6-HKA. The aim of this study is to investigate the in vitro metabolism of 6-HKA and its interaction with multiple important drug transporters.The in vitro metabolism experiments in the present study demonstrate that 6-HKA might not undergo phase-I or phase-II metabolism in hepatic microsomes/S9 of rats. In addition, some drug transporters, including OAT1/3, OCT2, MDR1, OATP1B1, MATE1/2K and OCTN2, were investigated. The cellular uptake assays indicate that 6-HKA exhibits inhibition to the transport of classical substrates mediated by OAT3, OCT2, MATE2K and OCTN2 but has no significant effect on the transport of substrates mediated by MDR1, OAT1, OATP1B1 or MATE1. Further investigation of cellular accumulation assays shows that 6-HKA might be the substrate of OAT3, but not OCT2 or OCTN2. The bidirectional transport study suggests that 6-HKA is not a substrate of MDR1.The information about the in vitro metabolism of 6-HKA and the interaction between 6-HKA and some transporters will help us to better understand the pharmacokinetic properties of 6-HKA and provide reference for its pharmacodynamics, DDIs and drug-food interactions studies.

Recent progress in the molecular imaging of therapeutic monoclonal antibodies.

Therapeutic monoclonal antibodies have become one of the central components of the healthcare system and continuous efforts are made to bring innovative antibody therapeutics to patients in need. It is equally critical to acquire sufficient knowledge of their molecular structure and biological functions to ensure the efficacy and safety by incorporating new detection approaches since new challenges like individual differences and resistance are presented. Conventional techniques for determining antibody disposition including plasma drug concentration measurements using LC-MS or ELISA, and tissue distribution using immunohistochemistry and immunofluorescence are now complemented with molecular imaging modalities like positron emission tomography and near-infrared fluorescence imaging to obtain more dynamic information, while methods for characterization of antibody's interaction with the target antigen as well as visualization of its cellular and intercellular behavior are still under development. Recent progress in detecting therapeutic antibodies, in particular, the development of methods suitable for illustrating the molecular dynamics, is described here.

The role of attention in retaining the binding of integral features in working memory.

Previous studies have suggested that retaining bindings in working memory (WM) requires more object-based attention than retaining constituent features. However, we still need to address the object-based attention hypothesis to determine both the generality (Does the object-based attention hypothesis of binding apply to feature bindings other than those tested?) and the reality (Was the observed effect in previous studies an artifact of the testing process?). We addressed these two issues by focusing on the binding of integral features, which was ignored in previous studies. Integral features can be manipulated independently but cannot be attended to or processed independently of each other, and they are primarily perceived in a more unitary fashion. Consequently, integral-feature bindings should be processed as integrated units without the help of extra object-based attention. We examined whether or not the object-based attention hypothesis applied to integral-feature bindings (generality), and these results enabled us to check the reality of the hypothesis. In line with our prediction, we found that a secondary task consuming object-based attention did not selectively impair the binding performance (Experiments 1, 2, 3, 5, and 7). The absence of selective binding impairment was not attributable to the use of an invalid secondary task (Experiment 4), failure to memorize the binding between length and width (Experiment 6), tapping the incorrect type of attention (Experiment 6), the feasibility of feature categorization (Experiment 7), or poor task performance (Experiment 7). Overall, these results suggest that the object-based attention hypothesis does not fit for the integral-feature bindings, and that the pivotal role of object-based attention reported by previous studies was reliable.

Object-based attention in retaining binding in working memory: Influence of activation states of working memory.

It has been suggested that retaining bindings in working memory (WM) requires more object-based attention than retaining constituent features. Recent studies have found that when memorized stimuli are presented sequentially, the most recent stimulus is in a highly accessible privileged state such that it is retained in a relatively automatic and resource-free manner, whereas the other stimuli are in a non-privileged state. The current study investigated whether the activation states of WM modulate the role of object-based attention in retaining bindings in WM. To address this question, we presented three colored shapes sequentially and added a transparent-motion task (Experiment 1) or a mental rotation task (Experiment 2) into the WM maintenance phase to consume object-based attention. We consistently found that consuming object-based attention led to a larger impairment to bindings relative to constituent features, which is independent of the WM activation states, suggesting that object-based attention is critical in retaining bindings in WM across activation states of WM.

Excretion stereoselectivity of triticonazole in rat urine and faeces.

The purpose of this study was to study the excretion stereoselectivity of triticonazole enantiomers in rat urine and faeces. Six male Sprague-Dawley (SD) rats were administrated 50 mg/kg rac-triticonazole. Rats urine and faeces were separately and quantitatively collected at the following intervals: 0-3, 3-6, 6-9, 9-12, 12-24, 24-36 and 36-48 h. The faeces samples were homogenized in an aqueous solution containing 0.2% DMSO at the ratio of 1 g: 40 mL. An aliquot of 100 µL rats urine or faeces homogenate was spiked and mixed with 6.0 µL of 1.00 µg/mL flusilazole as an internal standard. The triticonazole enantiomers in urine and faeces were determined by using an HPLC/MS-MS after samples preparation. The excreted amounts of enantiomers in the urine showed a significant difference (P < 0.05) except for 3-6 h. The cumulative excretion rate (Xu0→24) in urine was 26.43 ± 0.08% and 37.58 ± 0.11% for R-(-)- and S-(+)-triticonazole, respectively, indicating high enantioselectivity (P < 0.001). The cumulative excretion rate (Xu0→72) in faeces was 6.93 ± 0.03% and 6.77 ± 0.03% for R-(-)- and S-(+)-triticonazole, respectively, without a difference. The results showed that the total cumulative percentage of triticonazole enantiomers accounted for in urine and faeces was 64.00 ± 0.13% and 13.70 ± 0.32%, the urinary excretion of R-(-)- and S-(+)-triticonazole were significantly different and S-(+)-triticonazole was preferentially excreted. However, the faecal excretion of the enantiomers showed no difference.

Development and validation of a sensitive LC-MS/MS method for the determination of 6-hydroxykynurenic acid in rat plasma and its application to pharmacokinetics study.

A sensitive and specific bioanalytical method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantification of 6-hydroxykynurenic acid (6-HKA) in a small quantity of rat plasma has been developed and comprehensively validated. Tolbutamide (Tol) was used as the internal standard (IS). An aliquot of 50 µL rat plasma sample was deproteinized by 150 µL methanol, and then 100 µL of its supernatant was mixed with 100 µL water after centrifugation. Chromatographic separation was performed on a ZORBAX Eclipse Plus C18 Rapid Resolution HD column (2.1 mm × 100 mm, 1.8 µm) with a gradient mobile phase consisting of water containing 2 mM ammonium formate and methanol at a flow rate of 0.2 mL/min over a total run time of 5.0 min. The mass spectrometer was operated under multiple reactions monitoring (MRM) mode with the transitions m/z 206.1 → 160.0 for 6-HKA and m/z 269.0 → 170.0 for Tol. The linear range was 2.5-250 ng/mL with the square regression coefficient (r2) of 0.997. The lower limit of quantification (LLOQ) was 2.5 ng/mL (Relative Error, RE +1.6% and RSD 4.8%, n = 5). The intra- and inter-day precision and accuracy was <13.3%. The mean recovery of 6-HKA in rat plasma was between the range of 99.3-103.4%. This method was successfully applied to study the pharmacokinetics of 6-HKA in rats after oral administration at a single dose of 20.0 mg/kg and after tail intravenous injection at a single dose of 2.0 mg/kg. Pharmacokinetic parameters bioavailability, Cmax, oral, Tmax, oral, AUC0-24h, oral, AUC0-∞, oral, AUC0-24h, iv and AUC0-∞, iv were 3.96%, 152.0 ±â€¯85.5 ng/mL, 0.4 ±â€¯0.1 h, 340.0 ±â€¯136.3 ng/mL ∗ h, 369.5 ±â€¯135.0 ng/mL ∗ h, 906.6 ±â€¯324.1 ng/mL*h and 932.9 ±â€¯336.5 ng/mL ∗ h, respectively.

Development of a novel LC-MS/MS method for quantitation of triticonazole enantiomers in rat plasma and tissues and application to study on toxicokinetics and tissue distribution.

Triticonazole with an asymmetrical carbon atom has two enantiomers and is a broad-spectrum systemic fungicide, but its disposition in animals is unclear. In this study, a chiral analytical method of LC-MS/MS was developed and validated for the assay of triticonazole enantiomers in rat plasma and tissues. There were no endogenous interferences in the blank plasma and tissues of rats. R-(-)- and S-(+)-triticonazole peaks were separated entirely. The calibration curves were linear from 25 to 2500 ng/mL of each enantiomer. The accuracy, precision, and stability met the requirements of bioanalysis. Therefore, this method is enantioselective, accurate, precise, sensitive and reliable, and has been successfully applied to analyze R-(-)- and S-(+)-triticonazole in rat plasma and to study the toxicokinetics of triticonazole enantiomers in rats. After single oral administration of 50 mg/kg racemate triticonazole to rats, the AUC (0-∞) and Cmax of R-(-)-triticonazole were 3.5 and 3.6 times higher than that of S-(+)-triticonazole, respectively. The content of S-(+)-triticonazole was higher in the kidney whilst R-(-)-triticonazole was higher in the brain and small intestine. The results showed that the toxicokinetics and tissues distribution of triticonazole enantiomers in rats have stereoselective differences.

Bindings in working memory: The role of object-based attention.

Over the past decade, it has been debated whether retaining bindings in working memory (WM) requires more attention than retaining constituent features, focusing on domain-general attention and space-based attention. Recently, we proposed that retaining bindings in WM needs more object-based attention than retaining constituent features (Shen, Huang, & Gao, 2015, Journal of Experimental Psychology: Human Perception and Performance, doi: 10.1037/xhp0000018 ). However, only unitized visual bindings were examined; to establish the role of object-based attention in retaining bindings in WM, more emperical evidence is required. We tested 4 new bindings that had been suggested requiring no more attention than the constituent features in the WM maintenance phase: The two constituent features of binding were stored in different WM modules (cross-module binding, Experiment 1), from auditory and visual modalities (cross-modal binding, Experiment 2), or temporally (cross-time binding, Experiments 3) or spatially (cross-space binding, Experiments 4-6) separated. In the critical condition, we added a secondary object feature-report task during the delay interval of the change-detection task, such that the secondary task competed for object-based attention with the to-be-memorized stimuli. If more object-based attention is required for retaining bindings than for retaining constituent features, the secondary task should impair the binding performance to a larger degree relative to the performance of constituent features. Indeed, Experiments 1-6 consistently revealed a significantly larger impairment for bindings than for the constituent features, suggesting that object-based attention plays a pivotal role in retaining bindings in WM.

Neural mechanisms underlying conflict monitoring over risky decision alternatives: evidence from ERP in a Go/Nogo task.

This study assessed conflict monitoring during presentation of risky decision alternatives, as indexed by the Nogo-N2, Nogo-P3, N2d and P3d event-related potentials (ERP). Decision-makers were tested on a Go/Nogo gambling task in which gain/loss outcomes as well as stimulus type (Go/Nogo) were equiprobable. Frontal-central Nogo-N2 and Nogo-P3 did not significantly differ across risky decision alternatives, whereas N2d and P3d amplitudes were more sensitive to the nature of risky decision alternatives. Frontal-central N2d was moderated by the magnitude of alternatives, with N2d amplitude greater for large than small alternatives, a result that suggests a greater degree of conflict monitoring for the former. Central P3d was associated with alternative valence, such that P3d amplitude was greater for loss than gain valences, again suggestive of more conflict monitoring for the former. The N2d and P3d potentials in risky decision alternatives are discussed in terms of the functional significance of the N2/P3 complex.