Efficient expansion and CRISPR-Cas9-mediated gene correction of patient-derived hepatocytes for treatment of inherited liver diseases.

Cell-based ex vivo gene therapy in solid organs, especially the liver, has proven technically challenging. Here, we report a feasible strategy for the clinical application of hepatocyte therapy. We first generated high-quality autologous hepatocytes through the large-scale expansion of patient-derived hepatocytes. Moreover, the proliferating patient-derived hepatocytes, together with the AAV2.7m8 variant identified through screening, enabled CRISPR-Cas9-mediated targeted integration efficiently, achieving functional correction of pathogenic mutations in FAH or OTC. Importantly, these edited hepatocytes repopulated the injured mouse liver at high repopulation levels and underwent maturation, successfully treating mice with tyrosinemia following transplantation. Our study combines ex vivo large-scale cell expansion and gene editing in patient-derived transplantable hepatocytes, which holds potential for treating human liver diseases.

Laser Shock Peening: Fundamentals and Mechanisms of Metallic Material Wear Resistance Improvement.

With the rapid development of the advanced manufacturing industry, equipment requirements are becoming increasingly stringent. Since metallic materials often present failure problems resulting from wear due to extreme service conditions, researchers have developed various methods to improve their properties. Laser shock peening (LSP) is a highly efficacious mechanical surface modification technique utilized to enhance the microstructure of the near-surface layer of metallic materials, which improves mechanical properties such as wear resistance and solves failure problems. In this work, we summarize the fundamental principles of LSP and laser-induced plasma shock waves, along with the development of this technique. In addition, exemplary cases of LSP treatment used for wear resistance improvement in metallic materials of various nature, including conventional metallic materials, laser additively manufactured parts, and laser cladding coatings, are outlined in detail. We further discuss the mechanism by which the microhardness enhancement, grain refinement, and beneficial residual stress are imparted to metallic materials by using LSP treatment, resulting in a significant improvement in wear resistance. This work serves as an important reference for researchers to further explore the fundamentals and the metallic material wear resistance enhancement mechanism of LSP.

Critical role of TPRN rings in the stereocilia for hearing.

Inner ear hair cells detect sound vibration through the deflection of mechanosensory stereocilia. Cytoplasmic protein TPRN has been shown to localize at the taper region of the stereocilia, and mutations in TPRN cause hereditary hearing loss through an unknown mechanism. Here, using biochemistry and dual stimulated emission depletion microscopy imaging, we show that the TPRN, together with its binding proteins CLIC5 and PTPRQ, forms concentric rings in the taper region of stereocilia. The disruption of TPRN rings, triggered by the competitive inhibition of the interaction of TPRN and CLIC5 or exogenous TPRN overexpression, leads to stereocilia degeneration and severe hearing loss. Most importantly, restoration of the TPRN rings can rescue the damaged auditory function of Tprn knockout mice by exogenously expressing TPRN at an appropriate level in HCs via promoter recombinant adeno-associated virus (AAV). In summary, our results reveal highly structured TPRN rings near the taper region of stereocilia that are crucial for stereocilia function and hearing. Also, TPRN ring restoration in stereocilia by AAV-Tprn effectively repairs damaged hearing, which lays the foundation for the clinical application of AAV-mediated gene therapy in patients with TPRN mutation.

Precise regulation of the multicolor spectrum of carbon dots based on the bionic leaf vein ultrasonic microreactor.

Carbon dots (CDs) are a fascinating new type of fluorescent carbon nanomaterial with excellent photoelectric properties. However, preparing long-wavelength and multicolor-emitting CDs has been challenging, limiting their large-scale applications. Fortunately, a new efficient method has been proposed to co-regulate CDs' multicolor spectra using an ultrasonic microreactor. Inspired by plant leaves, a bionic vein microchannel was designed with good fluidity and high energy transfer efficiency. The optimal microchannel structural parameters were determined after investigating the effects of fractal angle, depth-to-width ratio, and inlet angle on the flow uniformity of the microchannel using numerical simulations. The efficiency of ultrasonic energy transfer was improved by directly coupling the microreactor and the sandwich transducer to fabricate the ultrasonic microreactor. Simulation results showed that the ultrasonic microreactor's vibration resonated along the longitudinal direction, and the ultrasonic intensity of the microreactor was maximal and uniform. A high-efficiency and controllable ultrasonic microreactor system was built to synthesize the CDs in situ. The influence of the ultrasound field intensity on CDs' preparation in a microreactor was simultaneously investigated to verify the ultrasound enhancement, and the PLQY of the high-performance CDs was found to be 83.1%. The CDs' multicolor spectra from the blue to the red region can be precisely tuned by adjusting key reaction parameters such as reaction temperature, flow rate, and precursor concentration. This new method shows promising applications in lighting, display, and other fields, making CDs a versatile and exciting new material to explore.

Highly Stable and Photoluminescent CsPbBr3/Cs4PbBr6 Composites for White-Light-Emitting Diodes and Visible Light Communication.

Inorganic lead halide perovskite is one of the most excellent fluorescent materials, and it plays an essential role in high-definition display and visible light communication (VLC). Its photochromic properties and stability determine the final performance of light-emitting devices. However, efficiently synthesizing perovskite with high quality and stability remains a significant challenge. Here, we develop a facile and environmentally friendly method for preparing high-stability and strong-emission CsPbBr3/Cs4PbBr6 composites using ultrasonication and liquid paraffin. Tuning the contents of liquid paraffin, bright-emission CsPbBr3/Cs4PbBr6 composite powders with a maximum PLQY of 74% were achieved. Thanks to the protection of the Cs4PbBr6 matrix and liquid paraffin, the photostability, thermostability, and polar solvent stability of CsPbBr3/Cs4PbBr6-LP are significantly improved compared to CsPbBr3 quantum dots and CsPbBr3/Cs4PbBr6 composites that were prepared without liquid paraffin. Moreover, the fabricated CsPbBr3/Cs4PbBr6-LP-based WLEDs show excellent luminescent performance with a power efficiency of 129.5 lm/W and a wide color gamut, with 121% of the NTSC and 94% of the Rec. 2020, demonstrating a promising candidate for displays. In addition, the CsPbBr3/Cs4PbBr6-LP-based WLEDs were also demonstrated in a VLC system. The results suggested the great potential of these high-performance WLEDs as an excitation light source to achieve VLC.

Highly emissive green CsPbBr3/Cs4PbBr6 composites: formation kinetics, excellent heat, light, and polar solvent resistance, and flexible light-emitting application.

Benefit from their near-unity photoluminescence quantum yield (PL QY), narrow emission band, and widely tunable bandgap, metal halide perovskites have shown promising in light-emitting applications. Despite such promise, how to facile, environmentally-friendly, and large-scale prepare solid metal halide perovskite with high emission and stability remains a challenging. Herein, we demonstrate a convenient and environmentally-friendly method for the mass synthesis of solid CsPbBr3/Cs4PbBr6 composites using high-power ultrasonication. Adjusting key experimental parameters, bright emitting CsPbBr3/Cs4PbBr6 solids with a maximum PL QY of 71% were obtained within 30 min. XRD, SEM, TEM, Abs/PL, XPS, and lifetime characterizations provide solid evidence for forming CsPbBr3/Cs4PbBr6 composites. Taking advantage of these composites, the photostability, thermostability, and polar solvent stability of CsPbBr3/Cs4PbBr6 are much improved compared to CsPbBr3. We further demonstrated CsPbBr3/Cs4PbBr6 use in flexible/stretchable film and high-power WLEDs. After being subjected to bending, folding, and twisting, the film retains its bright emission and exhibits good resistance to mechanical deformation. Additionally, our WLEDs display a superior, durable high-power-driving capability, operating currents up to 300 mA and maintaining high luminous intensity for 50 hours. Such highly emissive and stable metal halide perovskites make them promising for solid-state lighting, lasing, and flexible/stretchable display device applications.

Multicolor Luminescent Carbon Dots: Tunable Photoluminescence, Excellent Stability, and Their Application in Light-Emitting Diodes.

Carbon dots (CDs) are attracting much interest due to their excellent photoelectric properties and wide range of potential applications. However, it is still a challenge to regulate their bandgap emissions to achieve full-color CDs with high emissions. Herein, we propose an approach for producing full-color emissive CDs by employing a solvent engineering strategy. By only tuning the volume ratio of water and dimethylformamide (H2O/DMF), the photoluminescence (PL) emission wavelengths of the CDs can be changed from 451 to 654 nm. Different fluorescence features of multicolor CDs were systematically investigated. XRD, SEM, TEM, Abs/PL/PLE, XPS, and PL decay lifetime characterizations provided conclusive evidence supporting the extent to which the solvent controlled the dehydration and carbonization processes of the precursors, leading to a variation in their emission color from red to blue. The as-prepared CDs exhibited excellent and stable fluorescence performance even after being heated at 80 °C for 48 h and with UV light continuously irradiated for 15 h. Based on their excellent fluorescent properties and photothermal stability, bright multicolor light-emitting diodes with a high CRI of up to 91 were obtained. We anticipate that these full-color emissive CDs are beneficial for applications in lighting, display, and other fields.

AAV-ie-K558R mediated cochlear gene therapy and hair cell regeneration.

The cochlea consists of multiple types of cells, including hair cells, supporting cells and spiral ganglion neurons, and is responsible for converting mechanical forces into electric signals that enable hearing. Genetic and environmental factors can result in dysfunctions of cochlear and auditory systems. In recent years, gene therapy has emerged as a promising treatment in animal deafness models. One major challenge of the gene therapy for deafness is to effectively deliver genes to specific cells of cochleae. Here, we screened and identified an AAV-ie mutant, AAV-ie-K558R, that transduces hair cells and supporting cells in the cochleae of neonatal mice with high efficiency. AAV-ie-K558R is a safe vector with no obvious deficits in the hearing system. We found that AAV-ie-K558R can partially restore the hearing loss in Prestin KO mice and, importantly, deliver Atoh1 into cochlear supporting cells to generate hair cell-like cells. Our results demonstrate the clinical potential of AAV-ie-K558R for treating the hearing loss caused by hair cell death.

Ubiquitin ligase MARCH5 localizes to peroxisomes to regulate pexophagy.

Mitochondria and peroxisomes are independent but functionally closely related organelles. A few proteins have been characterized as dual-organelle locating proteins with distinct or similar roles on mitochondria and peroxisomes. MARCH5 is a mitochondria-associated ubiquitin ligase best known for its regulatory role in mitochondria quality control, fission, and fusion. Here, we used a proximity tagging system, PUP-IT, and identified new interacting proteins of MARCH5. Our data uncover that MARCH5 is a dual-organelle locating protein that interacts with several peroxisomal proteins. PEX19 binds the transmembrane region on MARCH5 and targets it to peroxisomes. On peroxisomes, MARCH5 binds and mediates the ubiquitination of PMP70. Furthermore, we find PMP70 ubiquitination and pexophagy induced by mTOR inhibition are blocked in the absence of MARCH5. Our study suggests novel roles of MARCH5 on peroxisomes.

Structure-Based Design of Dual-Acting Compounds Targeting Adenosine A2A Receptor and Histone Deacetylase as Novel Tumor Immunotherapeutic Agents.

Adenosine is an immunosuppressive factor in the tumor microenvironment mainly through activation of the A2A adenosine receptor (A2AR), which is a mechanism hijacked by tumors to escape immune surveillance. Small-molecule A2AR antagonists are being evaluated in clinical trials as immunotherapeutic agents, but their efficacy is limited as standalone therapies. To enhance the antitumor effects of A2AR antagonists, dual-acting compounds incorporating A2AR antagonism and histone deacetylase (HDAC) inhibitory actions were designed and synthesized, based on co-crystal structures of A2AR. Compound 24e (IHCH-3064) exhibited potent binding to A2AR (Ki = 2.2 nM) and selective inhibition of HDAC1 (IC50 = 80.2 nM), with good antiproliferative activity against tumor cell lines in vitro. Intraperitoneal administration of 24e (60 mg/kg, bid) inhibited mouse MC38 tumor growth with a tumor growth inhibition rate of 95.3%. These results showed that dual-acting compounds targeting A2AR and HDAC are potentially immunotherapeutic agents that are worth further exploring.

Elucidation of Distinct Modular Assemblies of Smoothened Receptor by Bitopic Ligand Measurement.

Class F G protein-coupled receptors are characterized by a large extracellular domain (ECD) in addition to the common transmembrane domain (TMD) with seven α-helixes. For smoothened receptor (SMO), structural studies revealed dissected ECD and TMD, and their integrated assemblies. However, distinct assemblies were reported under different circumstances. Using an unbiased approach based on four series of cross-conjugated bitopic ligands, we explore the relationship between the active status and receptor assembly. Different activity dependency on the linker length for these bitopic ligands corroborates the various occurrences of SMO assembly. These results reveal a rigid "near" assembly for active SMO, which is in contrast to previous results. Conversely, inactive SMO adopts a free ECD, which would be remotely captured at "far" assembly by cholesterol. Altogether, we propose a mechanism of cholesterol flow-caused SMO activation involving an erection of ECD from far to near assembly.

Rational Remodeling of Atypical Scaffolds for the Design of Photoswitchable Cannabinoid Receptor Tools.

Azobenzene-embedded photoswitchable ligands are the widely used chemical tools in photopharmacological studies. Current approaches to azobenzene introduction rely mainly on the isosteric replacement of typical azologable groups. However, atypical scaffolds may offer more opportunities for photoswitch remodeling, which are chemically in an overwhelming majority. Herein, we investigate the rational remodeling of atypical scaffolds for azobenzene introduction, as exemplified in the development of photoswitchable ligands for the cannabinoid receptor 2 (CB2). Based on the analysis of residue-type clusters surrounding the binding pocket, we conclude that among the three representative atypical arms of the CB2 antagonist, AM10257, the adamantyl arm is the most appropriate for azobenzene remodeling. The optimizing spacer length and attachment position revealed AzoLig 9 with excellent thermal bistability, decent photopharmacological switchability between its two configurations, and high subtype selectivity. This structure-guided approach gave new impetus in the extension of new chemical spaces for tool customization for increasingly diversified photo-pharmacological studies and beyond.

Multiregional profiling of the brain transmembrane proteome uncovers novel regulators of depression.

Transmembrane proteins play vital roles in mediating synaptic transmission, plasticity, and homeostasis in the brain. However, these proteins, especially the G protein-coupled receptors (GPCRs), are underrepresented in most large-scale proteomic surveys. Here, we present a new proteomic approach aided by deep learning models for comprehensive profiling of transmembrane protein families in multiple mouse brain regions. Our multiregional proteome profiling highlights the considerable discrepancy between messenger RNA and protein distribution, especially for region-enriched GPCRs, and predicts an endogenous GPCR interaction network in the brain. Furthermore, our new approach reveals the transmembrane proteome remodeling landscape in the brain of a mouse depression model, which led to the identification of two previously unknown GPCR regulators of depressive-like behaviors. Our study provides an enabling technology and rich data resource to expand the understanding of transmembrane proteome organization and dynamics in the brain and accelerate the discovery of potential therapeutic targets for depression treatment.

The zinc finger protein Miz1 suppresses liver tumorigenesis by restricting hepatocyte-driven macrophage activation and inflammation.

Chronic inflammation plays a central role in hepatocellular carcinoma (HCC), but the contribution of hepatocytes to tumor-associated inflammation is not clear. Here, we report that the zinc finger transcription factor Miz1 restricted hepatocyte-driven inflammation to suppress HCC, independently of its transcriptional activity. Miz1 was downregulated in HCC mouse models and a substantial fraction of HCC patients. Hepatocyte-specific Miz1 deletion in mice generated a distinct sub-group of hepatocytes that produced pro-inflammatory cytokines and chemokines, which skewed the polarization of the tumor-infiltrating macrophages toward pro-inflammatory phenotypes to promote HCC. Mechanistically, Miz1 sequestrated the oncoprotein metadherin (MTDH), preventing MTDH from promoting transcription factor nuclear factor κB (NF-κB) activation. A distinct sub-group of pro-inflammatory cytokine-producing hepatocytes was also seen in a subset of HCC patients. In addition, Miz1 expression inversely correated with disease recurrence and poor prognosis in HCC patients. Our findings identify Miz1 as a tumor suppressor that prevents hepatocytes from driving inflammation in HCC.

Structural basis for CD97 recognition of the decay-accelerating factor CD55 suggests mechanosensitive activation of adhesion GPCRs.

The adhesion G protein-coupled receptor CD97 and its ligand complement decay-accelerating factor CD55 are important binding partners in the human immune system. Dysfunction in this binding has been linked to immune disorders such as multiple sclerosis and rheumatoid arthritis, as well as various cancers. Previous literatures have indicated that the CD97 includes 3 to 5 epidermal growth factor (EGF) domains at its N terminus and these EGF domains can bind to the N-terminal short consensus repeat (SCR) domains of CD55. However, the details of this interaction remain elusive, especially why the CD55 binds with the highest affinity to the shortest isoform of CD97 (EGF1,2,5). Herein, we designed a chimeric expression construct with the EGF1,2,5 domains of CD97 and the SCR1-4 domains of CD55 connected by a flexible linker and determined the complex structure by crystallography. Our data reveal that the two proteins adopt an overall antiparallel binding mode involving the SCR1-3 domains of CD55 and all three EGF domains of CD97. Mutagenesis data confirmed the importance of EGF5 in the interaction and explained the binding specificity between CD55 and CD97. The architecture of CD55-CD97 binding mode together with kinetics suggests a force-resisting shearing stretch geometry when forces applied to the C termini of both proteins in the circulating environment. The potential of the CD55-CD97 complex to withstand tensile force may provide a basis for the mechanosensing mechanism for activation of adhesion G protein-coupled receptors.

Organized cannabinoid receptor distribution in neurons revealed by super-resolution fluorescence imaging.

G-protein-coupled receptors (GPCRs) play important roles in cellular functions. However, their intracellular organization is largely unknown. Through investigation of the cannabinoid receptor 1 (CB1), we discovered periodically repeating clusters of CB1 hotspots within the axons of neurons. We observed these CB1 hotspots interact with the membrane-associated periodic skeleton (MPS) forming a complex crucial in the regulation of CB1 signaling. Furthermore, we found that CB1 hotspot periodicity increased upon CB1 agonist application, and these activated CB1 displayed less dynamic movement compared to non-activated CB1. Our results suggest that CB1 forms periodic hotspots organized by the MPS as a mechanism to increase signaling efficacy upon activation.

Correction: Recent development of AAV-based gene therapies for inner ear disorders.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.