PIEZO2 mediates ultrasonic hearing via cochlear outer hair cells in mice.

Ultrasonic hearing and vocalization are the physiological mechanisms controlling echolocation used in hunting and navigation by microbats and bottleneck dolphins and for social communication by mice and rats. The molecular and cellular basis for ultrasonic hearing is as yet unknown. Here, we show that knockout of the mechanosensitive ion channel PIEZO2 in cochlea disrupts ultrasonic- but not low-frequency hearing in mice, as shown by audiometry and acoustically associative freezing behavior. Deletion of Piezo2 in outer hair cells (OHCs) specifically abolishes associative learning in mice during hearing exposure at ultrasonic frequencies. Ex vivo cochlear Ca2+ imaging has revealed that ultrasonic transduction requires both PIEZO2 and the hair-cell mechanotransduction channel. The present study demonstrates that OHCs serve as effector cells, combining with PIEZO2 as an essential molecule for ultrasonic hearing in mice.

Mechanisms in cochlear hair cell mechano-electrical transduction for acquisition of sound frequency and intensity.

Sound signals are acquired and digitized in the cochlea by the hair cells that further transmit the coded information to the central auditory pathways. Any defect in hair cell function may induce problems in the auditory system and hearing-based brain function. In the past 2 decades, our understanding of auditory transduction has been substantially deepened because of advances in molecular, structural, and functional studies. Results from these experiments can be perfectly embedded in the previously established profile from anatomical, histological, genetic, and biophysical research. This review aims to summarize the progress on the molecular and cellular mechanisms of the mechano-electrical transduction (MET) channel in the cochlear hair cells, which is involved in the acquisition of sound frequency and intensity-the two major parameters of an acoustic cue. We also discuss recent studies on TMC1, the molecule likely to form the MET channel pore.

Lack of PDZD7 long isoform disrupts ankle-link complex and causes hearing loss in mice.

Usher syndrome (USH) is the most frequent form of combined hereditary deafness-blindness, characterized by hearing loss and retinitis pigmentosa, with or without vestibular dysfunction. PDZD7 is a PDZ domain-containing scaffold protein that was suggested to be a USH modifier and a contributor to digenic USH. In the inner ear hair cells, PDZD7 localizes at the ankle region of the stereocilia and constitutes the so-called ankle-link complex together with three other USH proteins Usherin, WHRN, and ADGRV1. PDZD7 gene is subjected to alternative splicing, which gives rise to two types of PDZD7 isoforms, namely the long and short isoforms. At present, little is known which specific isoform is involved in ankle-link formation and stereocilia development. In this work, we showed that PDZD7 long isoform, but not short isoforms, localizes at the ankle region of the stereocilia. Moreover, we established Pdzd7 mutant mice by introducing deletions into exon 14 of the Pdzd7 gene, which causes potential premature translational stop in the long isoform but leaves short isoforms unaffected. We found that lack of PDZD7 long isoform affects the localization of other ankle-link complex components in the stereocilia. Consequently, Pdzd7 mutant mice showed stereocilia development deficits and hearing loss as well as reduced mechanotransduction (MET) currents, suggesting that PDZD7 long isoform is indispensable for hair cells. Furthermore, by performing yeast two-hybrid screening, we identified a PDZD7 long isoform-specific binding partner PIP5K1C, which has been shown to play important roles in hearing and might participate in the function and/or transportation of PDZD7.

Redox regulation of cancer metastasis: molecular signaling and therapeutic opportunities.

Cancer metastasis is the major cause of cancer-related mortality. Accumulated evidence has shown that high-metastasis potential cancer cells have more reactive oxygen species (ROS) accumulation compared with low-metastasis potential cancer cells. ROS can function as second messengers to regulate multiple cancer metastasis-related signaling pathways via reversible oxidative posttranslational modifications of cysteine in key redox-sensitive proteins, which leads to the structural and functional change of these proteins. Because ROS can promote cancer metastasis, therapeutic strategies aiming at inducing/reducing cellular ROS level or targeting redox sensors involved in metastasis hold great potential in developing new efficient approaches for anticancer therapy. In this review, we summarize recent findings on regulation of tumor metastasis by key redox sensors and describe the potential of targeting redox signaling pathways for cancer therapy.

Improving the performance of coupled solid carbon source biofilm carriers through pore-forming methods.

Coupled solid carbon source biofilm carriers (CCBs) was usually utilized to enhance the treatment efficiency of low carbon/nitrogen (C/N) wastewater. However, current CCBs have low carbon release capacity because of its small inner mass transfer coefficient. Therefore, this study innovatively applied pore-forming methods to modify CCBs. After orthogonal selections, two porous CCBs, which were respectively prepared through circulating freezing pore-forming method (CCB2) and ammonium bicarbonate pore-forming method (CCB3), were proposed and further applied in sequencing batch moving bed biofilm reactors (SBMBBRs). The results indicated that circulating freezing pore-forming method could improve the mechanical strength and carbon source release rate of CCBs. In addition, CCB2 could significantly enhance the total nitrogen (TN) removal efficiency of SBMBBRs, when compared with the non-porous CCBs (i.e., CCB1). Further biofilm and simultaneous nitrification and denitrification (SND) rate calculation attributed this enhancement to the higher biofilm amount (i.e., 0.06 g g-1 CCB) and the higher SND rate (i.e., 33.60%). Microbial community analysis reiterated these observations that CCB2 and CCB3 could accumulate Proteobacteria, Actinobacteriota and Nitrospirota, and also stimulate nitrification and denitrification associated pathways. More importantly, the cost calculation indicated CCB2 cost only 47.37% of CCB1 and 31.34% of CCB3, showing highly economic applicability. Overall, our results collectively proved that CCBs manufactured by circulating freezing pore-forming method could provide more carbon releasing points and microorganisms attaching positions, exhibiting effectively nitrogen removal when treating low C/N wastewater.

Selection and synthesization of multi-carbon source composites to enhance simultaneous nitrification-denitrification in treating low C/N wastewater.

Low carbon/nitrogen ratio (C/N) wastewater is widespread and difficult to treat. To find a resolution to this issue, this study systematically evaluated the constituents of composite solid carbon (i.e., skeletons, carbon sources and crosslinking agents), and proposed a new multi-carbon source composite S1 (MCSC.S1). The effects on nitrogen removal were further determined through a sequencing batch moving bed biofilm reactor (SBMBBR). The results showed that MCSC.S1, which was composed of polyvinyl alcohol-sodium alginate (PVA-SA), corncob + poly (R-ß-hydroxybutyrate) (CC + PHB), and H3BO3-4% CaCl2+Na2SO4 had high stability and absorption. With MCSC.S1, total nitrification removal was enhanced by more than 48.56% through releasing carbon and absorbing the attached denitrifying bacteria. In addition, it was found that MCSC.S1 can simulate the simultaneous nitrification and denitrification (SND) process and contribute to 29.85% of the total nitrogen removal. 16S gene-based analysis attributed this supplementary nitrogen removal to the enrichment of nitrification (i.e., Proteobacteria, Actinobacteria and Chloroflexi), denitrification of associated bacteria (i.e., Nitrospirota) in MCSC.S1 added reactor, and the increase in nitrogen recycling associated genes. These findings collectively demonstrate that the new MCSC.S1 could effectively enhance nitrogen removal efficiency in low C/N ratio wastewater.

The potential of electrotrophic denitrification coupled with sulfur recycle in MFC and its responses to COD/SO42- ratios.

Electrotrophic denitrification is a promising novel nitrogen removal technique. In this study, the performance and the mechanism of electrotrophic denitrification coupled with sulfate-sulfide cycle were investigated under different anodic influent COD/SO42- ratios. The results showed that electrotrophic denitrification contributed to more than 22% total nitrogen removal in cathode chamber. Higher COD/SO42- ratios would deteriorate the sulfate reduction but enhance methane production. Further mass balance indicated that the electron flow utilized by methanogenic archaea (MA) increased while that utilized by sulfate-reducing bacteria (SRB) decreased as the COD/SO42- ratio increased from 0.44 to 1.11. However, higher COD/SO42- ratios would produce more electrons to strengthen electrotrophic denitrification. Microbial community analysis showed that the biocathode was predominantly covered by Thiobacillus that encoded with narG gene. These findings collectively suggest that electrotrophic denitrification could be a sustainable approach to simultaneously remove COD and nitrogen under suitable COD/SO42- ratio based on sulfur cycle in wastewater.

Template-independent genome editing in the Pcdh15av-3j mouse, a model of human DFNB23 nonsyndromic deafness.

Although frameshift mutations lead to 22% of inherited Mendelian disorders in humans, there is no efficient in vivo gene therapy strategy available to date, particularly in nondividing cells. Here, we show that nonhomologous end-joining (NHEJ)-mediated nonrandom editing profiles compensate the frameshift mutation in the Pcdh15 gene and restore the lost mechanotransduction function in postmitotic hair cells of Pcdh15av-3J mice, an animal model of human nonsyndromic deafness DFNB23. Identified by an ex vivo evaluation system in cultured cochlear explants, the selected guide RNA restores reading frame in approximately 50% of indel products and recovers mechanotransduction in more than 70% of targeted hair cells. In vivo treatment shows that half of the animals gain improvements in auditory responses, and balance function is restored in the majority of injected mutant mice. These results demonstrate that NHEJ-mediated reading-frame restoration is a simple and efficient strategy in postmitotic systems.

Potential applications of endogenous sulfide for enhanced denitrification of low C/N domestic wastewater in anodic mixotrophic denitrification microbial fuel cell: The mechanism of electrons transfer and microbial community.

Anodic mixotrophic denitrification microbial fuel cell (MFC) was developed for pollutants removal and electricity generation in treatment of low C/N domestic wastewater. The experimental results show that the MFC achieved up to 100% of acetate, 100% of sulfide, and more than 91% of nitrate removal efficiency in all the MFCs. Particularly, thiosulfate was generated as the main intermediate of sulfide oxidation, and the sulfate generation ratio ranged from 66.93% to 73.76%. Those electrons produced during the acetate and sulfide oxidation were mainly used for denitrification and electricity generation. The microbial community analysis revealed that heterotrophic denitrifying bacteria (HDB) and sulfide-based autotrophic denitrifying bacteria (SADB) were the dominant bacteria for pollutants removal, and those facultative autotrophic bacterium (FAB) were key functional genera for high sulfate generation under both low and high sulfide concentrations. Meanwhile, the microbial functional prediction revealed that sulfide oxidation gene of Sqr and Sox were highly expressed. Moreover, a preliminary sulfide-based autotrophic denitrification (SAD) potential estimation indicated that the sulfide generated in the WWTPs had great potential for denitrification.

TMC1 is an essential component of a leak channel that modulates tonotopy and excitability of auditory hair cells in mice.

Hearing sensation relies on the mechano-electrical transducer (MET) channel of cochlear hair cells, in which transmembrane channel-like 1 (TMC1) and transmembrane channel-like 2 (TMC2) have been proposed to be the pore-forming subunits in mammals. TMCs were also found to regulate biological processes other than MET in invertebrates, ranging from sensations to motor function. However, whether TMCs have a non-MET role remains elusive in mammals. Here, we report that in mouse hair cells, TMC1, but not TMC2, provides a background leak conductance, with properties distinct from those of the MET channels. By cysteine substitutions in TMC1, we characterized four amino acids that are required for the leak conductance. The leak conductance is graded in a frequency-dependent manner along the length of the cochlea and is indispensable for action potential firing. Taken together, our results show that TMC1 confers a background leak conductance in cochlear hair cells, which may be critical for the acquisition of sound-frequency and -intensity.

Improved calcium sensor GCaMP-X overcomes the calcium channel perturbations induced by the calmodulin in GCaMP.

GCaMP, one popular type of genetically-encoded Ca2+ indicator, has been associated with various side-effects. Here we unveil the intrinsic problem prevailing over different versions and applications, showing that GCaMP containing CaM (calmodulin) interferes with both gating and signaling of L-type calcium channels (CaV1). GCaMP acts as an impaired apoCaM and Ca2+/CaM, both critical to CaV1, which disrupts Ca2+ dynamics and gene expression. We then design and implement GCaMP-X, by incorporating an extra apoCaM-binding motif, effectively protecting CaV1-dependent excitation-transcription coupling from perturbations. GCaMP-X resolves the problems of detrimental nuclear accumulation, acute and chronic Ca2+ dysregulation, and aberrant transcription signaling and cell morphogenesis, while still demonstrating excellent Ca2+-sensing characteristics partly inherited from GCaMP. In summary, CaM/CaV1 gating and signaling mechanisms are elucidated for GCaMP side-effects, while allowing the development of GCaMP-X to appropriately monitor cytosolic, submembrane or nuclear Ca2+, which is also expected to guide the future design of CaM-based molecular tools.

Itraconazole suppresses the growth of glioblastoma through induction of autophagy: involvement of abnormal cholesterol trafficking.

Glioblastoma is one of the most aggressive human cancers with poor prognosis, and therefore a critical need exists for novel therapeutic strategies for management of glioblastoma patients. Itraconazole, a traditional antifungal drug, has been identified as a novel potential anticancer agent due to its inhibitory effects on cell proliferation and tumor angiogenesis; however, the molecular mechanisms involved are still unclear. Here, we show that itraconazole inhibits the proliferation of glioblastoma cells both in vitro and in vivo. Notably, we demonstrate that treatment with itraconazole induces autophagic progression in glioblastoma cells, while blockage of autophagy markedly reverses the antiproliferative activities of itraconazole, suggesting an antitumor effect of autophagy in response to itraconazole treatment. Functional studies revealed that itraconazole retarded the trafficking of cholesterol from late endosomes and lysosomes to the plasma membrane by reducing the levels of SCP2, resulting in repression of AKT1-MTOR signaling, induction of autophagy, and finally inhibition of cell proliferation. Together, our studies provide new insights into the molecular mechanisms regarding the antitumor activities of itraconazole, and may further assist both the pharmacological investigation and rational use of itraconazole in potential clinical applications.