Low-intensity noise exposure takes an essential part in the mechanism of late-onset hereditary hearing loss caused by Abcc1 mutation.

With the development of the social economy, we are exposed to increasing noise in our daily lives. Our previous work found an ABCC1(NM_004996.3:c.A1769G, NP_004987.2:p.N590S) variant which cosegregated with the patients in an autosomal dominant non-syndromic hearing loss family. At present, the specific mechanism of deafness caused by ABCC1 mutation is still not clear. Using the knock-in mouse model simulating human ABCC1 mutation, we found that the occurrence of family-related phenotypes was likely attributed to the combination of the mouse genotype and low-intensity noise. GSH and GSSG are important physiological substrates of ABCC1. The destruction of GSH-GSSG balance in the cochleae of both Abcc1N591S/+ mice and Abcc1N591S/N591S mice during low-intensity noise exposure may result in irreversible damage to the hair cells of the cochleae, consequently leading to hearing loss in mice. The findings offered a potential novel idea for the prevention and management of hereditary hearing loss within this family.

ABCC1 deficiency potentiated noise-induced hearing loss in mice by impairing cochlear antioxidant capacity.

The ABCC1 gene belongs to the ATP-binding cassette membrane transporter superfamily, which plays a crucial role in the efflux of various endogenous and exogenous substances. Mutations in ABCC1 can result in autosomal dominant hearing loss. However, the specific roles of ABCC1 in auditory function are not fully understood. Through immunofluorescence, we found that ABCC1 was expressed in microvascular endothelial cells (ECs) of the stria vascularis (StV) in the murine cochlea. Then, an Abcc1 knockout mouse model was established by using CRISPR/Cas9 technology to elucidate the role of ABCC1 in the inner ear. The ABR threshold did not significantly differ between WT and Abcc1-/- mice at any age studied. After noise exposure, the ABR thresholds of the WT and Abcc1-/- mice were significantly elevated. Interestingly, after 14 days of noise exposure, ABR thresholds largely returned to pre-exposure levels in WT mice but not in Abcc1-/- mice. Our subsequent experiments showed that microvascular integrity in the StV was compromised and that the number of outer hair cells and the number of ribbons were significantly decreased in the cochleae of Abcc1-/- mice post-exposure. Besides, the production of ROS and the accumulation of 4-HNE significantly increased. Furthermore, StV microvascular ECs were cultured to elucidate the role of ABCC1 in these cells under glucose oxidase challenge. Notably, 30 U/L glucose oxidase (GO) induced severe oxidative stress damage in Abcc1-/- cells. Compared with WT cells, the ROS and 4-HNE levels and the apoptotic rate were significantly elevated in Abcc1-/- cells. In addition, the reduced GSH/GSSG ratio was significantly decreased in Abcc1-/- cells after GO treatment. Taken together, Abcc1-/- mice are more susceptible to noise-induced hearing loss, possibly because ABCC1 knockdown compromises the GSH antioxidant system of StV ECs. The exogenous antioxidant N-acetylcysteine (NAC) may protect against oxidative damage in Abcc1-/- murine cochleae and ECs.

Susceptibility of immature spiral ganglion neurons to aminoglycoside-induced ototoxicity is mediated by the TRPV1 channel in mice.

Aminoglycoside antibiotics are among the most common agents that can cause sensorineural hearing loss. From clinical experience, premature babies, whose inner ear is still developing, are more susceptible to aminoglycoside-induced ototoxicity, which is echoed by our previous study carried out in organotypic cultures. This study aimed to investigate whether a nonselective cation channel, TRPV1, contributes to the susceptibility of immature spiral ganglion neurons (SGNs) to the damage caused by aminoglycosides. Through western blotting and immunofluorescence, we found that the TRPV1 expression levels were much higher in immature SGNs than in their mature counterparts. In postnatal day 7 cochlear organotypic cultures, AMG-517 reduced reactive oxygen species generation and inhibited SGN apoptosis under aminoglycoside challenge. However, in adult mice, AMG-517 did not ameliorate the ABR threshold increase at high frequencies (16 kHz and 32 kHz) after aminoglycoside administration, and the SGNs within the cochleae had no morphological changes. By further regulating the function of TRPV1 in primary cultured SGNs with its inhibitor AMG-517 and agonist capsaicin, we demonstrated that TRPV1 is a major channel for aminoglycoside uptake: AMG-517 can significantly reduce, while capsaicin can significantly increase, the uptake of GTTR. In addition, TRPV1 knockdown in SGNs can also significantly reduce the uptake of GTTR. Taken together, our results demonstrated that aminoglycosides can directly enter immature SGNs through the TRPV1 channel. High expression of TRPV1 contributes to the susceptibility of immature SGNs to aminoglycoside-induced damage. The TRPV1 inhibitor AMG-517 has the potential to be a therapeutic agent for preventing aminoglycoside-induced ototoxicity in immature SGNs.

A Novel EYA1 Mutation Causing Alternative RNA Splicing in a Chinese Family With Branchio-Oto Syndrome: Implications for Molecular Diagnosis and Clinical Application.

OBJECTIVES: Branchio-oto syndrome (BOS) primarily manifests as hearing loss, preauricular pits, and branchial defects. EYA1 is the most common pathogenic gene, and splicing mutations account for a substantial proportion of cases. However, few studies have addressed the structural changes in the protein caused by splicing mutations and potential pathogenic factors, and several studies have shown that middle-ear surgery has limited effectiveness in improving hearing in these patients. BOS has also been relatively infrequently reported in the Chinese population. This study explored the genetic etiology in the family of a proband with BOS and provided clinical treatment to improve the patient's hearing. METHODS: We collected detailed clinical features and peripheral blood samples from the patients and unaffected individuals within the family. Pathogenic mutations were identified by whole-exome sequencing and cosegregation analysis and classified according to the American College of Medical Genetics and Genomics guidelines. Alternative splicing was verified through a minigene assay. The predicted three-dimensional protein structure and biochemical experiments were used to investigate the pathogenicity of the mutation. The proband underwent middle-ear surgery and was followed up at 1 month and 6 months postoperatively to monitor auditory improvement. RESULTS: A novel heterozygous EYA1 splicing variant (c.1050+4 A>C) was identified and classified as pathogenic (PVS1(RNA), PM2, PP1). Skipping of exon 11 of the EYA1 pre-mRNA was confirmed using a minigene assay. This mutation may impair EYA1-SIX1 interactions, as shown by an immunoprecipitation assay. The EYA1-Mut protein exhibited cellular mislocalization and decreased protein expression in cytological experiments. Middle-ear surgery significantly improved hearing loss caused by bone-conduction abnormalities in the proband. CONCLUSION: We reported a novel splicing variant of EYA1 in a Chinese family with BOS and revealed the potential molecular pathogenic mechanism. The significant hearing improvement observed in the proband after middle-ear surgery provides a reference for auditory rehabilitation in similar patients.

Antifungal Exploration of Quinoline Derivatives against Phytopathogenic Fungi Inspired by Quinine Alkaloids.

Enlightened from our previous work of structural simplification of quinine and innovative application of natural products against phytopathogenic fungi, lead structure 2,8-bis(trifluoromethyl)-4-quinolinol (3) was selected to be a candidate and its diversified design, synthesis, and antifungal evaluation were carried out. All of the synthesized compounds Aa1-Db1 were evaluated for their antifungal activity against four agriculturally important fungi, Botrytis cinerea, Fusarium graminearum, Rhizoctonia solani, and Sclerotinia sclerotiorum. Results showed that compounds Ac3, Ac4, Ac7, Ac9, Ac12, Bb1, Bb10, Bb11, Bb13, Cb1. and Cb3 exhibited a good antifungal effect, especially Ac12 had the most potent activity with EC50 values of 0.52 and 0.50 µg/mL against S. sclerotiorum and B. cinerea, respectively, which were more potent than those of the lead compound 3 (1.72 and 1.89 µg/mL) and commercial fungicides azoxystrobin (both >30 µg/mL) and 8-hydroxyquinoline (2.12 and 5.28 µg/mL). Moreover, compound Ac12 displayed excellent in vivo antifungal activity, which was comparable in activity to the commercial fungicide boscalid. The preliminary mechanism revealed that compound Ac12 might cause an abnormal morphology of cell membranes, an increase in membrane permeability, and release of cellular contents. These results indicated that compound Ac12 displayed superior in vitro and in vivo fungicidal activities and could be a potential fungicidal candidate against plant fungal diseases.

Design, Synthesis, and Structure-Activity Relationship Studies of Magnolol Derivatives as Antifungal Agents.

Plant pathogenic fungi seriously affect agricultural production and are difficult to control. The discovery of new leads based on natural products is an important way to innovate fungicides. In this study, 30 natural-product-based magnolol derivatives were synthesized and characterized on the basis of NMR and mass spectroscopy. Bioactivity tests on phytopathogenic fungi (Rhizoctonia solani, Fusarium graminearum, Botrytis cinerea, and Sclerotinia sclerotiorum) in vitro of these compounds were performed systematically. The results showed that 11 compounds were active against four kinds of phytopathogenic fungi with EC50 values in the range of 1.40-20.00 µg/mL, especially compound L5 that exhibited excellent antifungal properties against B. cinerea with an EC50 value of 2.86 µg/mL, approximately 2.8-fold more potent than magnolol (EC50 = 8.13 µg/mL). Moreover, compound L6 showed the highest antifungal activity against F. graminearum and Rhophitulus solani with EC50 values of 4.39 and 1.40 µg/mL, respectively, and compound L7 showed good antifungal activity against S. sclerotiorum. Then, an in vivo experiment of compound L5 against B. cinerea was further investigated in vivo using infected tomatoes (curative effect, 50/200 and 36%/100 µg/mL). The physiological and biochemical studies illustrated that the primary action mechanism of compound L5 on B. cinerea might change the mycelium morphology, increase cell membrane permeability, and destroy the function of mitochondria. Furthermore, structure-activity relationship (SAR) studies revealed that hydroxyl groups play a key role in antifungal activity. To sum up, this study provides a reference for understanding the application of magnolol-based antifungal agents in crop protection.

Design, Synthesis, and Antifungal Evaluation of Cryptolepine Derivatives against Phytopathogenic Fungi.

Inspired by the widely antiphytopathogenic application of diversified derivatives from natural sources, cryptolepine and its derivatives were subsequently designed, synthesized, and evaluated for their antifungal activities against four agriculturally important fungi Rhizoctonia solani, Botrytis cinerea, Fusarium graminearum, and Sclerotinia sclerotiorum. The results obtained from in vitro assay indicated that compounds a1-a24 showed great fungicidal property against B. cinerea (EC50 < 4 µg/mL); especially, a3 presented significantly prominent inhibitory activity with an EC50 of 0.027 µg/mL. In the pursuit of further expanding the antifungal spectrum of cryptolepine, ring-opened compound f1 produced better activity with an EC50 of 3.632 µg/mL against R. solani and an EC50 of 5.599 µg/mL against F. graminearum. Furthermore, a3 was selected to be a candidate to investigate its preliminary antifungal mechanism to B. cinerea, revealing that not only spore germination was effectively inhibited and the normal physiological structure of mycelium was severely undermined but also detrimental reactive oxygen was obviously accumulated and the normal function of the nucleus was fairly disordered. Besides, in vivo curative experiment against B. cinerea found that the therapeutic action of a3 was comparable to that of the positive control azoxystrobin. These results suggested that compound a3 could be regarded as a novel and promising agent against B. cinerea for its valuable potency.

Solid-phase microextraction integrated nanobiosensors for the serial detection of cytoplasmic dopamine in a single living cell.

Dopamine participates in many physiological and pathological processes. Dynamic monitoring of dopamine levels in the cytoplasm of a single living cell reflects not only the functional state of dopamine synthesis factors but also the processes of related neurodegenerative diseases. Due to the low content of cytoplasmic dopamine and the difficulty to keep cells alive during the operating process, the detection of cytoplasmic dopamine is still challenging. Herein, a solid-phase microextraction (SPME) technique integrated nanobiosensor was employed to trace and quantify dopamine concentration fluctuations in the cytoplasm of a single living cell. We designed a polypyrrole modified carbon fiber nanoprobe as a bifunctional nanoprobe that can extract cytoplasmic dopamine and then perform electrochemical detection. This bifunctional nanoprobe can detect 10 pmol/L extracted dopamine and detected a 60% decrease of the cytoplasmic dopamine concentration in a single living cell by K+ stimulation. This study allowed for the first time serially detecting cytoplasmic dopamine while keeping the target cell alive, which might yield a new method for research on dopamine neurotoxicity and the related drug action mechanisms for neurodegenerative disease.

Design, Synthesis, and Antifungal Evaluation of 8-Hydroxyquinoline Metal Complexes against Phytopathogenic Fungi.

Phytopathogenic fungal infections have become a major threat to agricultural production, food security, and human health globally, and novel antifungal agents with simple chemical scaffolds and high efficiency are needed. In this study, we designed and synthesized 38 8-hydroxyquinoline metal complexes and evaluated their antifungal activities. The results showed that most of the tested compounds possessed remarkable in vitro antifungal activity. Especially, compound 1e exhibited the highest antifungal potency among all target compounds, with EC50 values of 0.0940, 0.125, 2.95, and 5.96 µg/mL, respectively, against Sclerotinia sclerotiorum, Botrytis cinerea, Fusarium graminearum, and Magnaporthe oryzae. Preliminary mechanistic studies had shown that compound 1e might cause mycelial abnormalities of S. sclerotiorum, cell membrane permeability changes, leakage of cell contents, and inhibition of sclerotia formation and germination. Moreover, the results of in vivo antifungal activity of compound 1e against S. sclerotiorum showed that 1e possessed higher curative effects than that of the positive control azoxystrobin. Therefore, compound 1e is expected to be a novel leading structure for the development of new antifungal agents.

A carbon-supported BiSn nanoparticles based novel sensor for sensitive electrochemical determination of Cd (II) ions.

A novel sensor based on carbon supported BiSn alloy nanoparticles (BiSn@C) was prepared for the sensitive detection of Cd2+. The BiSn@C and Nafion modified glassy carbon electrode (GCE) exhibited improved electrochemical performance in Cd2+ detection, because of its large specific surface area, abundance of active sites, good electrical conductivity, and strong cation exchange ability. Under the optimum conditions, the fabricated sensor showed good linearity of its response from 0.01 µmol/L to 30 µmol/L for the detection of Cd2+ and a limit of detection (LOD) of 3 nmol/L, which is considerably lower than the limit specified for safe drinking water as guided by the World Health Organization. The Nafion/BiSn@C/GCE was successfully applied for determination for trace Cd2+ in river samples with satisfying recoveries using the standard addition method.