Mechanisms of Antioxidant Dipeptides Enhancing Ethanol-Oxidation Cross-Stress Tolerance in Lager Yeast: Roles of the Cell Wall and Membrane.

High concentrations of ethanol could cause intracellular oxidative stress in yeast, which can lead to ethanol-oxidation cross-stress. Antioxidant dipeptides are effective in maintaining cell viability and stress tolerance under ethanol-oxidation cross-stress. In this study, we sought to elucidate how antioxidant dipeptides affect the yeast cell wall and membrane defense systems to enhance stress tolerance. Results showed that antioxidant dipeptide supplementation reduced cell leakage of nucleic acids and proteins by changing cell wall components under ethanol-oxidation cross-stress. Antioxidant dipeptides positively modulated the cell wall integrity pathway and up-regulated the expression of key genes. Antioxidant dipeptides also improved the cell membrane integrity by increasing the proportion of unsaturated fatty acids and regulating the expression of key fatty acid synthesis genes. Moreover, the addition of antioxidant dipeptides significantly (p < 0.05) increased the content of ergosterol. Ala-His (AH) supplementation caused the highest content of ergosterol, with an increase of 23.68 ± 0.01% compared to the control, followed by Phe-Cys (FC) and Thr-Tyr (TY). These results revealed that the improvement of the cell wall and membrane functions of antioxidant dipeptides was responsible for enhancing the ethanol-oxidation cross-stress tolerance of yeast.

Development and validation of a potent and specific inhibitor for the CLC-2 chloride channel.

CLC-2 is a voltage-gated chloride channel that is widely expressed in mammalian tissues. In the central nervous system, CLC-2 appears in neurons and glia. Studies to define how this channel contributes to normal and pathophysiological function in the central nervous system raise questions that remain unresolved, in part due to the absence of precise pharmacological tools for modulating CLC-2 activity. Herein, we describe the development and optimization of AK-42, a specific small-molecule inhibitor of CLC-2 with nanomolar potency (IC50 = 17 ± 1 nM). AK-42 displays unprecedented selectivity (>1,000-fold) over CLC-1, the closest CLC-2 homolog, and exhibits no off-target engagement against a panel of 61 common channels, receptors, and transporters expressed in brain tissue. Computational docking, validated by mutagenesis and kinetic studies, indicates that AK-42 binds to an extracellular vestibule above the channel pore. In electrophysiological recordings of mouse CA1 hippocampal pyramidal neurons, AK-42 acutely and reversibly inhibits CLC-2 currents; no effect on current is observed on brain slices taken from CLC-2 knockout mice. These results establish AK-42 as a powerful tool for investigating CLC-2 neurophysiology.

A general procedure to select calibration drugs for lab-specific validation and calibration of proarrhythmia risk prediction models: An illustrative example using the CiPA model.

INTRODUCTION: In response to the ongoing shift of the regulatory cardiac safety paradigm, a recent White Paper proposed general principles for developing and implementing proarrhythmia risk prediction models. These principles included development strategies to validate models, and implementation strategies to ensure a model developed by one lab can be used by other labs in a consistent manner in the presence of lab-to-lab experimental variability. While the development strategies were illustrated through the validation of the model under the Comprehensive In vitro Proarrhythmia Assay (CiPA), the implementation strategies have not been adopted yet. METHODS: The proposed implementation strategies were applied to the CiPA model by performing a sensitivity analysis to identify a subset of calibration drugs that were most critical in determining the classification thresholds for proarrhythmia risk prediction. RESULTS: The selected calibration drugs were able to recapitulate classification thresholds close to those calculated from the full list of CiPA drugs. Using an illustrative dataset it was shown that a new lab could use these calibration drugs to establish its own classification thresholds (lab-specific calibration), and verify that the model prediction accuracy in the new lab is comparable to that in the original lab where the model was developed (lab-specific validation). DISCUSSION: This work used the CiPA model as an example to illustrate how to adopt the proposed model implementation strategies to select calibration drugs and perform lab-specific calibration and lab-specific validation. Generic in nature, these strategies could be generally applied to different proarrhythmia risk prediction models using various experimental systems under the new paradigm.

Ictal Source Locations and Cortico-Thalamic Connectivity in Childhood Absence Epilepsy: Associations with Treatment Response.

Childhood absence epilepsy (CAE), the most common pediatric epilepsy syndrome, is usually treated with valproic acid (VPA) and lamotrigine (LTG) in China. This study aimed to investigate the ictal source locations and functional connectivity (FC) networks between the cortices and thalamus that are related to treatment response. Magnetoencephalography (MEG) data from 25 patients with CAE were recorded at 300 Hz and analyzed in 1-30 Hz frequency bands. Neuromagnetic sources were volumetrically scanned with accumulated source imaging. The FC networks between the cortices and thalamus were evaluated at the source level through a connectivity analysis. Treatment outcome was assessed after 36-66 months following MEG recording. The children with CAE were divided into LTG responder, LTG non-responder, VPA responder and VPA non-responder groups. The ictal source locations and cortico-thalamic FC networks were compared to the treatment response. The ictal source locations in the post-dorsal medial frontal cortex (post-DMFC, including the medial primary motor cortex and the supplementary sensorimotor area) were observed in all LTG non-responders but in all LTG responders. At 1-7 Hz, patients with fronto-thalamo-parietal/occipital (F-T-P/O) networks were older than those with fronto-thalamic (F-T) networks or other cortico-thalamic networks (p = 0.000). The duration of seizures in patients with F-T-P/O networks at 1-7 Hz was longer than that in patients with F-T networks or other cortico-thalamic networks (p = 0.001). The ictal post-DMFC source localizations suggest that children with CAE might experience initial LTG monotherapy failure. Moreover, the cortico-thalamo-cortical network is associated with age. Finally, the cortico-thalamo-cortical network consists of anterior and posterior cortices and might contribute to the maintenance of discharges.

Assessment of an In Silico Mechanistic Model for Proarrhythmia Risk Prediction Under the CiPA Initiative.

The International Council on Harmonization (ICH) S7B and E14 regulatory guidelines are sensitive but not specific for predicting which drugs are pro-arrhythmic. In response, the Comprehensive In Vitro Proarrhythmia Assay (CiPA) was proposed that integrates multi-ion channel pharmacology data in vitro into a human cardiomyocyte model in silico for proarrhythmia risk assessment. Previously, we reported the model optimization and proarrhythmia metric selection based on CiPA training drugs. In this study, we report the application of the prespecified model and metric to independent CiPA validation drugs. Over two validation datasets, the CiPA model performance meets all pre-specified measures for ranking and classifying validation drugs, and outperforms alternatives, despite some in vitro data differences between the two datasets due to different experimental conditions and quality control procedures. This suggests that the current CiPA model/metric may be fit for regulatory use, and standardization of experimental protocols and quality control criteria could increase the model prediction accuracy even further.