Identification of the Acid-Sensitive Site Critical for Chloral Hydrate (CH) Activation of the Proton-Activated Chloride Channel.

The transmembrane protein TMEM206 was recently identified as the molecular basis of the extracellular proton-activated Cl- channel (PAC), which plays an essential role in neuronal death in ischemia-reperfusion. The PAC channel is activated by extracellular acid, but the proton-sensitive mechanism remains unclear, although different acid-sensitive pockets have been suggested based on the cryo-EM structure of the human PAC (hPAC) channel. In the present study, we firstly identified two acidic amino acid residues that removed the pH-dependent activation of the hPAC channel by neutralization all the conservative negative charged residues located in the extracellular domain of the hPAC channel and some positively charged residues at the hotspot combined with two-electrode voltage-clamp (TEVC) recording in the Xenopus oocytes system. Double-mutant cycle analysis and double cysteine mutant of these two residues proved that these two residues cooperatively form a proton-sensitive site. In addition, we found that chloral hydrate activates the hPAC channel depending on the normal pH sensitivity of the hPAC channel. Furthermore, the PAC channel knock-out (KO) male mice (C57BL/6J) resist chloral hydrate-induced sedation and hypnosis. Our study provides a molecular basis for understanding the proton-dependent activation mechanism of the hPAC channel and a novel drug target of chloral hydrate.SIGNIFICANCE STATEMENT Proton-activated Cl- channel (PAC) channels are widely distributed in the nervous system and play a vital pathophysiological role in ischemia and endosomal acidification. The main discovery of this paper is that we identified the proton activation mechanism of the human proton-activated chloride channel (hPAC). Intriguingly, we also found that anesthetic chloral hydrate can activate the hPAC channel in a pH-dependent manner. We found that the chloral hydrate activates the hPAC channel and needs the integrity of the pH-sensitive site. In addition, the PAC channel knock-out (KO) mice are resistant to chloral hydrate-induced anesthesia. The study on PAC channels' pH activation mechanism enables us to better understand PAC's biophysical mechanism and provides a novel target of chloral hydrate.

Construction of a highly error-prone DNA polymerase for developing organelle mutation systems.

A novel family of DNA polymerases replicates organelle genomes in a wide distribution of taxa encompassing plants and protozoans. Making error-prone mutator versions of gamma DNA polymerases revolutionised our understanding of animal mitochondrial genomes but similar advances have not been made for the organelle DNA polymerases present in plant mitochondria and chloroplasts. We tested the fidelities of error prone tobacco organelle DNA polymerases using a novel positive selection method involving replication of the phage lambda cI repressor gene. Unlike gamma DNA polymerases, ablation of 3'-5' exonuclease function resulted in a modest 5-8-fold error rate increase. Combining exonuclease deficiency with a polymerisation domain substitution raised the organelle DNA polymerase error rate by 140-fold relative to the wild type enzyme. This high error rate compares favourably with error-rates of mutator versions of animal gamma DNA polymerases. The error prone organelle DNA polymerase introduced mutations at multiple locations ranging from two to seven sites in half of the mutant cI genes studied. Single base substitutions predominated including frequent A:A (template: dNMP) mispairings. High error rate and semi-dominance to the wild type enzyme in vitro make the error prone organelle DNA polymerase suitable for elevating mutation rates in chloroplasts and mitochondria.

Emodin, a natural anthraquinone, suppresses liver cancer in vitro and in vivo by regulating VEGFR2 and miR-34a.

The pharmacokinetic (PK) and potential effects of Emodin on liver cancer were systematically evaluated in this study. Both the intragastric administration (i.g.) and hypodermic injection (i.h.) of Emodin exhibited a strong absorption (absorption rate < 1 h) and elimination capacity (t1/2 ≈ 2 h). The tissue distribution of Emodin after i.h. was rapid and wide. The stability of Emodin in three species of liver microsomes wasrat >human> beagle dog. These PK data provided the basis for the subsequent animal experiments. In liver cancer patient tissues, the expression of vascular endothelial growth factor (VEGF)-induced signaling pathways, including phosphorylated VEGF receptor 2 (VEGFR2), AKT, and ERK1/2,were simultaneously elevated, but miR-34a expression was reduced and negatively correlated with SMAD2 and SMAD4. Emodin inhibited the expression of SMAD2/4 in HepG2 cells by inducing the miR-34a level. Subsequently, BALB/c nude mice received a daily subcutaneous injection of HepG2 cells with or without Emodin treatment (1 mg/kg or 10 mg/kg), and Emodin inhibited tumorigenesis and reduced the mortality rate in a dose-dependent manner. In vivo experiments showed that cell proliferation, migration, and invasion were promoted by VEGF or miR-34a signal treatment but were inhibited when combined with Emodin treatment. All these results demonstrated that Emodin inhibited tumorigenesis in liver cancer by simultaneously inhibiting the VEGFR2-AKT-ERK1/2signaling pathway and promoting a miR-34a-mediated signaling pathway.

What is behind the meta-learning initialization of adaptive filter? - A naive method for accelerating convergence of adaptive multichannel active noise control.

Active noise control (ANC) is a typical signal-processing technique that has recently been utilized extensively to combat the urban noise problem. Although numerous advanced adaptive algorithms have been devised to enhance noise reduction performance, few of them have been implemented in actual ANC products due to their high computational complexity and slow convergence. With the rapid development of deep learning technology, Meta-learning-based initialization appears to become an efficient and cost-effective method for accelerating the convergence of adaptive algorithms. However, few dedicated Meta-learning algorithms exist for adaptive signal processing applications, particularly multichannel active noise control (MCANC). Hence, we proposed a modified Model-Agnostic Meta-Learning (MAML) initialization for the MCANC system.1 Additional theatrical research reveals that the nature of MAML, when applied to signal processing, is the expectation of a weight-sum gradient. Based on this discovery, we devised the Monte-Carlo Gradient Meta-learning (MCGM) algorithm, which employed a more straightforward procedure to accomplish the same performance as the Modified MAML algorithm. Furthermore, the numerical simulation of ANC using raw noise samples on measured paths validates the efficacy of the proposed methods in accelerating the convergence of the multichannel-filtered reference least mean square algorithm (McFxLMS).

µ opioid receptor carboxyl terminal-derived peptide alleviates morphine tolerance by inhibiting ß-arrestin2.

The interaction between the µ opioid receptor (MOR) and ß-arrestin2 serves as a model for addressing morphine tolerance. A peptide was designed to alleviate morphine tolerance through interfering with the interaction of MOR and ß-arrestin2. We developed a peptide derived from MOR. The MOR-TAT-pep peptide was expressed in E. coli Bl21(DE3) and purified. The effects of MOR-TAT-pep in alleviating morphine tolerance was examined through behavior tests. The potential mechanism was detected by Western blotting, Mammalian Two-Hybrid and other techniques. The pretreatment with MOR-TAT-pep prior to morphine usage led to an enhanced analgesic effectiveness of morphine and a significant reduction in the development of morphine tolerance. The peptide directly interacted with ß-arrestin2 during morphine treatment and deceased the membrane recruitment of ß-arrestin2. MOR-TAT-pep effectively suppressed the increase of ß-arrestin2 induced by morphine. The MOR-TAT-pep could alleviate morphine tolerance through inhibition of ß-arrestin2.

Downregulating miR-184 relieves calcium oxalate crystal-mediated renal cell damage via activating the Rap1 signaling pathway.

BACKGROUND: Renal calculi are a very prevalent disease with a high incidence. Calcium oxalate (CaOx) is a primary constituent of kidney stones. Our paper probes the regulatory function and mechanism of miR-184 in CaOx-mediated renal cell damage. METHODS: CaOx was used to treat HK2 cells and human podocytes (HPCs) to simulate kidney cell damage. The qRT-PCR technique checked the profiles of miR-184 and IGF1R. The examination of cell proliferation was conducted employing CCK8. TUNEL staining was used to monitor cell apoptosis. Western blot analysis was used to determine the protein profiles of apoptosis-concerned related proteins (including Mcl1, Bcl-XL, and Caspase-3), the NF-κB, Nrf2/HO-1, and Rap1 signaling pathways. ELISA confirmed the levels of the inflammatory factors IL-6, TNF-α, MCP1, and ICAM1. The targeting relationship between miR-184 and IGF1R was validated by dual luciferase assay and RNA immunoprecipitation assay. RESULTS: Glyoxylate-induced rat kidney stones model and HK2 and HPC cells treated with CaOx demonstrated an increase in the miR-184 profile. Inhibiting miR-184 relieved CaOx-mediated renal cell inflammation, apoptosis and oxidative stress and activated the Rap1 pathway. IGF1R was targeted by miR-184. IGF1R activation by IGF1 attenuated the effects of miR-184 on renal cell damage, and Hippo pathway suppression reversed the inhibitory effect of miR-184 knockdown on renal cell impairment. CONCLUSIONS: miR-184 downregulation activates the Rap1 signaling pathway to ameliorate renal cell damage mediated by CaOx.

The effect of obstructive jaundice on the sensitivity of intravenous anesthetic of remimazolam: study protocol for a controlled multicenter trial.

BACKGROUND: It is well known that obstructive jaundice could affect the pharmacodynamics of some anesthetics, and the sensitivity of some anesthetics would increase among icteric patients. Remimazolam is a new ultra-short-acting intravenous benzodiazepine sedative/anesthetic, which is a high-selective and affinity ligand for the benzodiazepine site on the GABAA receptor. However, no study has reported the pharmacodynamics of remimazolam in patients with obstructive jaundice. We hypothesize that obstructive jaundice affects the pharmacodynamics of remimazolam, and the sensitivity of remimazolam increases among icteric patients. METHODS/DESIGN: The study will be performed as a prospective, controlled, multicenter trial. The study design is a comparison of remimazolam requirements to reach a bispectral index of 50 in patients with obstructive jaundice versus non-jaundiced patients with chronic cholecystitisor intrahepatic bile duct stones. Remimazolam was infused at 6 mg/kg/h until this endpoint was reached. DISCUSSION: Remimazolam could be suitable for anesthesia of patients with obstructive jaundice, because remimazolam is not biotransformed in the liver. Hyperbilirubinemia has been well-described to have toxic effects on the brain, which causes the increasing of sensitivity to some anesthetics, such as desflurane, isoflurane, and etomidate. Furthermore, remimazolam and etomidate have the same mechanism of action when exerting an anesthetic effect. We aim to demonstrate that obstructive jaundice affects the pharmacodynamics of remimazolam, and the dose of remimazolam when administered to patients with obstructive jaundice should be modified. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR2100043585 . Registered on 23 February 2021.

[Changes of brain oxidative stress induced by nano-alumina in ICR mice].

OBJECTIVE: To investigate the brain oxidative stress injury induced by nano-alumina particles in ICR mice. METHODS: Sixty male ICR mice were randomly divided into 6 groups: control group, solvent control group, 100 mg/kg micro-alumina particles group, 3 groups exposed to nano-alumina particles at the doses of 50, 100 and 200 mg/kg. The mice were exposed by nasal drip for 30 days. Then levels of malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-PX) in brain tissues of mice were detected. RESULTS: There was no difference of SOD activity in mouse brain between control group [(17.32 +/- 6.23)U/gHb] and 50 mg/kg nano-alumina particles group [(17.89 +/- 1.82) U/gHb]. The SOD activity [(4.93 +/- 2.30)U/gHb] in 200 mg/kg nano-alumina particles group was significantly lower than that in control group (P < 0.05). The MDA levels in 3 nano-alumina particles groups were (0.76 +/- 0.13), (1.00 +/- 0.30) and (1.16 +/- 0.39)nmol/ml, respectively, which were significantly higher than that [( 0.24 +/- 0.09)nmol/ml] in control group (P < 0.05). The GSH levels in 3 nano-alumina particles groups were (0.72 +/- 0.08), (0.55 +/- 0.19) and (0.61 +/- 0.20)mg/gpro, respectively, which were significantly lower than that [(1.55 +/- 0.34)mg/gpro]] in control group (P < 0.05). The CAT activity in 50 and 100 mg/kg nano-alumina particles groups were (10.40 +/- 3.84) and (10.40 +/- 2.00)U/mgpro, respectively, which were significantly higher than that [(5.79 +/- 0.96) U/mgpro] in control group (P < 0.05). The CAT activity [(3.25 +/- 1.04)U/mgpro] in 200 mg/kg nano-alumina particles group was significantly lower than that in control group (P < 0.05 ). CONCLUSION: Nano-alumina particles can induce the oxidative stress damage in brain tissues of mice.

C/EBPα Regulates FOXC1 to Modulate Tumor Growth by Interacting with PPARγ in Hepatocellular Carcinoma.

BACKGROUND: Forkhead box C1 (FOXC1) is an important cancer-associated gene in tumor. PPAR-γ and C/EBPα are both transcriptional regulators involved in tumor development. OBJECTIVE: We aimed to clarify the function of PPAR-γ, C/EBPα in hepatocellular carcinoma (HCC) and the relationship of PPAR-γ, C/EBPα and FOXC1 in HCC. METHODS: Western blotting, immunofluorescent staining, and immunohistochemistry were used to evaluate protein expression. qRT-PCR was used to assess mRNA expression. Co-IP was performed to detect the protein interaction. And ChIP and fluorescent reporter detection were used to determine the binding between protein and FOXC1 promoter. RESULTS: C/EBPα could bind to FOXC1 promoter and PPAR-γ could strengthen C/EBPα's function. Expressions of C/EBPα and PPAR-γ were both negatively related to FOXC1 in human HCC tissue. Confocal displayed that C/EBPα was co-located with FOXC1 in HepG2 cells. C/EBPα could bind to FOXC1 promoter by ChIP. Luciferase activity detection exhibited that C/EBPα could inhibit FOXC1 promoter activity, especially FOXC1 promoter from -600 to -300 was the critical binding site. Only PPAR-γ could not influence luciferase activity but strengthen inhibited effect of C/EBPα. Further, the Co-IP displayed that PPAR-γ could bind to C/EBPα. When C/EBPα and PPAR-γ were both high expressed, cell proliferation, migration, invasion, and colony information were inhibited enormously. C/EBPα plasmid combined with or without PPAR-γ agonist MDG548 treatment exhibited a strong tumor inhibition and FOXC1 suppression in mice. CONCLUSION: Our data establish C/EBPα targeting FOXC1 as a potential determinant in the HCC, which supplies a new pathway to treat HCC. However, PPAR-γ has no effect on FOXC1 expression.