AAV-mediated Gene Cocktails Enhance Supporting Cell Reprogramming and Hair Cell Regeneration.

Mammalian cochlear hair cells (HCs) are essential for hearing, and damage to HCs results in severe hearing impairment. Damaged HCs can be regenerated by neighboring supporting cells (SCs), thus the functional regeneration of HCs is the main goal for the restoration of auditory function in vivo. Here, cochlear SC trans-differentiation into outer and inner HC by the induced expression of the key transcription factors Atoh1 and its co-regulators Gfi1, Pou4f3, and Six1 (GPAS), which are necessary for SCs that are destined for HC development and maturation via the AAV-ie targeting the inner ear stem cells are successfully achieved. Single-cell nuclear sequencing and lineaging tracing results showed that the majority of new Atoh1-derived HCs are in a state of initiating differentiation, while GP (Gfi1, Pou4f3) and GPS (Gfi1, Pou4f3, and Six1) enhanced the Atoh1-induced new HCs into inner and outer HCs. Moreover, the patch-clamp analysis indicated that newborn inner HCs induced by GPAS forced expression have similar electrophysiological characteristics to those of native inner HCs. Also, GPAS can induce HC regeneration in the HC-damaged mice model. In summary, the study demonstrates that AAV-mediated co-regulation of multiple genes, such as GPAS, is an effective means to achieve functional HC regeneration in the mouse cochlea.

Eggshell membrane powder reinforces adhesive polysaccharide hydrogels for wound repair.

Multifunctional polysaccharide hydrogels with strong tissue adhesion, and antimicrobial and hemostatic properties are attractive wound healing materials. In this study, a chitosan-based hydrogel (HCS) was designed, and its properties were enhanced by incorporating oxidized eggshell membrane (OEM). Hydrogel characterization and testing results showed that the hydrogel had excellent antimicrobial properties, cytocompatibility, satisfactory adhesion properties on common substrates, and wet-state adhesion capacity. A rat liver injury model confirmed the significant hemostatic effect of the hydrogel. Finally, the ability of the hydrogel to promote wound healing was verified using rat skin wound repair experiments. Our findings indicate that HCS/OEM hydrogels with added eggshell membrane fibers have better self-healing properties, mechanical strength, adhesion, hemostatic properties, and biocompatibility than HCS hydrogels, in addition to having superior repair performance in wound repair experiments. Overall, the multifunctional polysaccharide hydrogels fabricated in this study are ideal for wound repair.

Pcolce2 overexpression promotes supporting cell reprogramming in the neonatal mouse cochlea.

Hair cell (HC) damage is a leading cause of sensorineural hearing loss, and in mammals supporting cells (SCs) are unable to divide and regenerate HCs after birth spontaneously. Procollagen C-endopeptidase enhancer 2 (Pcolce2), which encodes a glycoprotein that acts as a functional procollagen C protease enhancer, was screened as a candidate regulator of SC plasticity in our previous study. In the current study, we used adeno-associated virus (AAV)-ie (a newly developed adeno-associated virus that targets SCs) to overexpress Pcolce2 in SCs. AAV-Pcolce2 facilitated SC re-entry into the cell cycle both in cultured cochlear organoids and in the postnatal cochlea. In the neomycin-damaged model, regenerated HCs were detected after overexpression of Pcolce2, and these were derived from SCs that had re-entered the cell cycle. These findings reveal that Pcolce2 may serve as a therapeutic target for the regeneration of HCs to treat hearing loss.

AAV-mediated Gpm6b expression supports hair cell reprogramming.

Irreversible damage to hair cells (HCs) in the cochlea leads to hearing loss. Cochlear supporting cells (SCs) in the murine cochlea have the potential to differentiate into HCs. Neuron membrane glycoprotein M6B (Gpm6b) as a four-transmembrane protein is a potential regulator of HC regeneration according to our previous research. In this study, we found that AAV-ie-mediated Gpm6b overexpression promoted SC-derived organoid expansion. Enhanced Gpm6b prevented the normal decrease in SC plasticity as the cochlea develops by supporting cells re-entry cell cycle and facilitating the SC-to-HC transformation. Also, overexpression of Gpm6b in the organ of Corti through the round window membrane injection facilitated the trans-differentiation of Lgr5+ SCs into HCs. In conclusion, our results suggest that Gpm6b overexpression promotes HC regeneration and highlights a promising target for hearing repair using the inner ear stem cells combined with AAV.

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.

Dual-bioactive molecules loaded aligned core-shell microfibers for tendon tissue engineering.

Development of a controlled delivery ultrafine fibrous system with two bioactive molecules is required to stimulate tendon healing in different phase. In this study, we used emulsion stable jet electrospinning to fabricate aligned poly(L-lactic acid) (PLLA) based ultrafine fibers with two small bioactive molecules of L-Arginine (Arg) and low molecular weight hyaluronic acid (HA). The results demonstrated that the aligned Arg/HA/PLLA microfibrous scaffold showed core-shell structure and allowed sequential release of Arg and HA due to their different electric charge. The scaffold also showed enhanced hydrophilicity, cell migration, spread and proliferation. Using an Achilles tendon repair model in rats, we demonstrated that this novel fibrous scaffold can prevent adhesion and promote tendon regeneration. Additionally, two p53 and ER-α-mediated signalling pathways were described as the probable main path of synergistic effects of the novel scaffold on tendon generation. Thus, this study may provide an important strategy for developing biofunctional and biomimetic tendon scaffolds.

Rps14 upregulation promotes inner ear progenitor proliferation and hair cell regeneration in the neonatal mouse cochlea.

Sensorineural hearing loss a result from hair cell damage, which is irreversible in mammals owing to the lack of hair cell regeneration, but recent researches have shown that Lgr5+ supporting cells are progenitors capable of regenerating hair cells. RPS14 (ribosomal protein S14) is a 40S ribosomal subunit component and is associated with erythrocyte differentiation, and in this study, we used a novel adeno-associated virus-inner ear system to upregulate Rps14 expression in cultured hair cell progenitors and observed an enhancement on their ability to proliferate and differentiate into hair cells. Similarly, Rps14 overexpression in the mice cochlea could promote supporting cells proliferation by activating the Wnt signalling pathway. In addition, over-expressing Rps14 induced hair cells regeneration in the organ of Corti, and lineage tracing showed that the new hair cells had transformed from Lgr5+ progenitors. In conclusion, our analysis reveals the potential role of Rps14 in driving hair cell regeneration in mammalian.

AAV-Net1 facilitates the trans-differentiation of supporting cells into hair cells in the murine cochlea.

Mechanosensitive hair cells (HCs) in the cochlear sensory epithelium are critical for sound detection and transduction. Mammalian HCs in the cochlea undergo cytogenesis during embryonic development, and irreversible damage to hair cells postnatally is a major cause of deafness. During the development of the organ of Corti, HCs and supporting cells (SCs) originate from the same precursors. In the neonatal cochlea, damage to HCs activates adjacent SCs to act as HC precursors and to differentiate into new HCs. However, the plasticity of SCs to produce new HCs is gradually lost with cochlear development. Here, we delineate an essential role for the guanine nucleotide exchange factor Net1 in SC trans-differentiation into HCs. Net1 overexpression mediated by AAV-ie in SCs promoted cochlear organoid formation and HC differentiation under two and three-dimensional culture conditions. Also, AAV-Net1 enhanced SC proliferation in Lgr5-EGFPCreERT2 mice and HC generation as indicated by lineage tracing of HCs in the cochleae of Lgr5-EGFPCreERT2/Rosa26-tdTomatoloxp/loxp mice. We further found that the up-regulation of Wnt/ß-catenin and Notch signaling in AAV-Net1-transduced cochleae might be responsible for the SC proliferation and HC differentiation. Also, Net1 overexpression in SCs enhanced SC proliferation and HC regeneration and survival after HC damage by neomycin. Taken together, our study suggests that Net1 might serve as a potential target for HC regeneration and that AAV-mediated gene regulation may be a promising approach in stem cell-based therapy in hearing restoration.

Natural glycosidic antioxidants from Cynanchum atratum roots.

Two new rhamnosides, 18-O-α-l-rhamnopyranosylabietic acid (1) and (E)-3,5-dimethoxystilben-4'-O-α-l-rhamnopyranoside (2), five known glucosides (3-7) along with three others were isolated from Cynanchum atratum roots. The structures of new compounds were elucidated by physical data analyses such as NMR, UV, IR, HR-ESI-MS, as well as acid hydrolysis. All of them were assessed for their antioxidant activities through 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical ion (ABTS•+), 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH•) and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical (PTIO•) assay, with l-ascorbic acid used as the positive control. As a result, compounds 3-5 exhibited obvious antioxidant activities. These bioactive components could be promising antioxidants.

Hierarchical Network Enabled Flexible Textile Pressure Sensor with Ultrabroad Response Range and High-Temperature Resistance.

Thin, lightweight, and flexible textile pressure sensors with the ability to detect the full range of faint pressure (<100 Pa), low pressure (≈KPa) and high pressure (≈MPa) are in significant demand to meet the requirements for applications in daily activities and more meaningfully in some harsh environments, such as high temperature and high pressure. However, it is still a significant challenge to fulfill these requirements simultaneously in a single pressure sensor. Herein, a high-performance pressure sensor enabled by polyimide fiber fabric with functionalized carbon-nanotube (PI/FCNT) is obtained via a facile electrophoretic deposition (EPD) approach. High-density FCNT is evenly wrapped and chemically bonded to the fiber surface during the EPD process, forming a conductive hierarchical fiber/FCNT matrix. Benefiting from the large compressible region of PI fiber fabric, abundant yet firm contacting points and high elastic modulus of both PI and CNT, the proposed pressure sensor can be customized and modulated to achieve both an ultra-broad sensing range, long-term stability and high-temperature resistance. Thanks to these merits, the proposed pressure sensor could monitor the human physiological information, detect tiny and extremely high pressure, can be integrated into an intelligent mechanical hand to detect the contact force under high-temperature.

Isolation and characterization of antioxidative monoterpenes from Cynanchum atratum roots.

One novel monoterpene rhamnoside (1) and 7 known monoterpenes (2-8) were isolated from the ethanol extract of Cynanchum atratum for the first time. Their structures were identified by comprehensive spectroscopic data analysis such as nuclear magnetic resonance, high-resolution electrospray ionization mass spectra, optical rotatory dispersion, and acid hydrolysis. In the subsequent antioxidant assay, compound 8 exhibited obvious 2,2-diphenyl-2-picrylhydrazyl hydrate radical scavenging activity.

Two new iridoid glycosides from Gardeniae Fructus.

Two new iridoid glycosides, genipin 1,10-di-O-α-l-rhamnoside (1) and genipin 1,10-di-O-ß-d-xylopyranoside (2), along with thirteen known compounds (3-15) were isolated from Gardeniae Fructus. Their structures were elucidated by physical data analyses such as NMR, UV, IR, HR-ESI-MS, as well as chemical hydrolysis. All compounds were tested for their tyrosinase inhibitory and antioxidant activities. At a concentration of 25 µM, compound 13 showed obvious mushroom tyrosinase inhibition activity with % inhibition value of 36.52 ± 1.98%, with kojic acid used as the positive control (46.09 ± 1.29%). At a concentration of 1 mM, compounds 8 and 9 exhibited considerable DPPH radical scavenging activities, with radical scavenging rates of 48.54 ± 0.47%, 58.59 ± 0.39%, respectively, with l-ascorbic acid used as the positive control (59.02 ± 0.77%).

High-performance x-ray source based on graphene oxide-coated Cu2S nanowires grown on copper film.

Full static x-ray computed tomography (CT) technology has enabled higher precision and resolution imaging and has been applied in many applications such as diagnostic medical imaging, industrial inspection and security screening. In this technique, the x-ray source section is mainly composed of a thermionic cathode and electron beam scanning system. However, they have several shortcomings such as limited scanning angle, long response time and large volume. Distributed and programmable cold cathode (i.e. carbon nanotubes, ZnO nanowires (NWs)) field-emission x-ray sources are expected to solve these problems. However, there have been several long-standing challenges to the application of such cold field emitters for x-ray sources, such as the short lifetime and rigorous fabrication process, which have fundamentally prevented their widespread use. Here, we propose and demonstrate a cold field-emission x-ray source based on a graphene oxide (GO)-coated cuprous sulfide nanowire (Cu2S NW/GO) cathode. The proposed Cu2S NW/GO x-ray source provides stable emission (>18 h at a direct voltage of 2600 V) and has a low threshold (4.5 MV m-1 for obtaining a current density of 1 µA cm-2), benefiting from the demonstrated key features such as in situ epitaxy growth of Cu2S NWs on Cu, nanometer-scale sharp protrusions within GO and charge transfer between the Cu2S NWs and GO layer. Our research provides a simple and robust method to obtain a high-performance cold field emitter, leading to great potential for the next generation of x-ray source and CT.

Highly Sensitive and Wide Linear-Response Pressure Sensors Featuring Zero Standby Power Consumption under Bending Conditions.

The ability of a flexible pressure sensor to possess zero power consumption in standby mode, high sensitivity, and wide linear-response range is critical in real flexible matrix-based scenes. However, when the conventional flexible pressure sensors are attached on a curved surface, a pseudosignal response is generated because of the normal stress, resulting in a short linear-response range. Here, a flexible piezoresistive pressure sensor with high performance, zero standby power consumption is demonstrated. The flexible pressure sensor is fabricated from polydimethylsiloxane (PDMS)/carbon black (CB), patterned polyimide (PI) spacer layer, and laser-induced graphene (LIG) interdigital electrodes. Benefiting from the hierarchical structure and sufficient roughness of PDMS/CB and LIG interdigital electrodes, the proposed pressure sensors (PDMS/CB/PI/LIG) exhibit high sensitivity (43 kPa-1), large linear-response range (0.4-13.6 kPa), fast response (<40 ms), and long-term cycle stability (>1800 cycles). The resulting pressure sensor also features zero standby power consumption merit under certain bending conditions (bending angle: 0-5o). Furthermore, the effect of the hole diameter of the PI spacer layer on the performance of the pressure sensors is experimentally and theoretically investigated. As a proof of concept, a bioinspired artificial haptic neuron system has been successfully equipped to modulate the number of lit LED lights. The proposed high-performance pressure sensor has promising potential to be used in flexible and wearable electronics, especially for the applications in actual flexible matrix-based scenes.