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Purpose: Pueraria lobata (P. lobata), a dual-purpose food and medicine, displays limited efficacy in alcohol detoxification and liver protection, with previous research primarily focused on puerarin in its dried roots. In this study, we investigated the potential effects and mechanisms of fresh P. lobata root-derived exosome-like nanovesicles (P-ELNs) for mitigating alcoholic intoxication, promoting alcohol metabolism effects and protecting the liver in C57BL/6J mice. Methods: We isolated P-ELNs from fresh P. lobata root using differential centrifugation and characterized them via transmission electron microscopy, nanoscale particle sizing, ζ potential analysis, and biochemical assays. In Acute Alcoholism (AAI) mice pre-treated with P-ELNs, we evaluated their effects on the timing and duration of the loss of the righting reflex (LORR), liver alcohol metabolism enzymes activity, liver and serum alcohol content, and ferroptosis-related markers. Results: P-ELNs, enriched in proteins, lipids, and small RNAs, exhibited an ideal size (150.7 ± 82.8 nm) and negative surface charge (-31 mV). Pre-treatment with 10 mg/(kg.bw) P-ELNs in both male and female mice significantly prolonged ebriety time, shortened sobriety time, enhanced acetaldehyde dehydrogenase (ALDH) activity while concurrently inhibited alcohol dehydrogenase (ADH) activity, and reduced alcohol content in the liver and serum. Notably, P-ELNs demonstrated more efficacy compared to P-ELNs supernatant fluid (abundant puerarin content), suggesting alternative active components beyond puerarin. Additionally, P-ELNs prevented ferroptosis by inhibiting the reduction of glutathione peroxidase 4 (GPX4) and reduced glutathione (GSH), and suppressing acyl-CoA synthetase long-chain family member 4 (ACSL4) elevation, thereby mitigating pathological liver lipid accumulation. Conclusion: P-ELNs exhibit distinct exosomal characteristics and effectively alleviate alcoholic intoxication, improve alcohol metabolism, suppress ferroptosis, and protect the liver from alcoholic injury. Consequently, P-ELNs hold promise as a therapeutic agent for detoxification, sobriety promotion, and prevention of alcoholic liver injury.
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Intoxicación Alcohólica , Exosomas , Hígado , Ratones Endogámicos C57BL , Raíces de Plantas , Pueraria , Animales , Pueraria/química , Exosomas/metabolismo , Exosomas/efectos de los fármacos , Exosomas/química , Ratones , Masculino , Intoxicación Alcohólica/tratamiento farmacológico , Raíces de Plantas/química , Hígado/efectos de los fármacos , Hígado/metabolismo , Etanol/química , Etanol/farmacología , Extractos Vegetales/farmacología , Extractos Vegetales/química , Alcoholismo/tratamiento farmacológico , IsoflavonasRESUMEN
Magnetic proximity interaction provides a promising route to manipulate the spin and valley degrees of freedom in van der Waals heterostructures. Here, we report a control of valley pseudospin in the WS2/MoSe2 heterostructure by utilizing the magnetic proximity effect of few-layered CrBr3 and, for the first time, observe a substantial difference in valley polarization of intra/interlayer excitons under different circularly polarized laser excitations, referred to as chirality-dependent valley polarization. Theoretical and experimental results reveal that the spin-selective charge transfer between MoSe2 and CrBr3, as well as between MoSe2 and WS2, is mostly responsible for the chiral feature of valley polarization in comparison with the proximity exchange field. This means that a long-distance manipulation of exciton behaviors in multilayer heterostructures can be achieved through spin-selective charge transfer. This work marks a significant advancement in the control of spin and valley pseudospin in multilayer structures.
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Proximate-induced magnetic interactions present a promising strategy for precise manipulation of valley degrees of freedom. Taking advantage of the splendid valleytronic platform of transition metal dichalcogenides, magnetic two-dimensional VSe2 with different phases are introduced to intervene in the spin of electrons and modulate their valleytronic properties. When constructing the heterostructures, 1T-VSe2/WX2 (X = S and Se) showcases significant improvement in the valley polarizations at room temperature, while 2H-VSe2/WX2 exhibits superior performance at low temperatures and demonstrates heightened sensitivity to the external magnetic field. Simultaneously, considerable valley splitting with a large geff factor up to -29.0 is observed in 2H-VSe2/WS2, while it is negligible in 1T-VSe2/WX2. First-principles calculations reveal a phase-dependent magnetic proximity mechanism on the valleytronic modulations, which is dominated by interfacial charge transfer in 1T-VSe2/WX2 and the proximity exchange field in 2H-VSe2/WX2 heterostructures. The effective control over valley degrees of freedom will bridge the valleytronic physics and devices, rendering enormous potential in the field of valley quantum applications.
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Parkinson's disease (PD) is the second most frequently diagnosed neurodegenerative disease, and it is characterized by the intracellular and extracellular accumulation of α-synuclein (α-syn) and Tau, which are major components of cytosolic protein inclusions called Lewy bodies, in the brain. Currently, there is a lack of effective methods that preventing PD progression. It has been suggested that the plasminogen activation system, which is a major extracellular proteolysis system, is involved in PD pathogenesis. We investigated the functional roles of plasminogen in vitro in an okadaic acid-induced Tau hyperphosphorylation NSC34 cell model, ex vivo using brains from normal controls and methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, and in vivo in a widely used MPTP-induced PD mouse model and an α-syn overexpression mouse model. The in vitro, ex vivo and in vivo results showed that the administered plasminogen crossed the bloodâbrain barrier (BBB), entered cells, and migrated to the nucleus, increased plasmin activity intracellularly, bound to α-syn through lysine binding sites, significantly promoted α-syn, Tau and TDP-43 clearance intracellularly and even intranuclearly in the brain, decreased dopaminergic neurodegeneration and increased the tyrosine hydroxylase levels in the substantia nigra and striatum, and improved motor function in PD mouse models. These findings indicate that plasminogen plays a wide range of pivotal protective roles in PD and therefore may be a promising drug candidate for PD treatment.
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Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Plasminógeno , Animales , Ratones , alfa-Sinucleína , Modelos Animales de Enfermedad , Proteínas de Unión al ADN/metabolismo , Dopamina , Enfermedades Neurodegenerativas/metabolismo , Enfermedad de Parkinson/metabolismo , Plasminógeno/metabolismo , Serina Proteasas , Proteínas tau/metabolismo , Neuronas Dopaminérgicas/patologíaRESUMEN
Hepatocellular carcinoma (HCC) has high incidence and mortality rates, and it is characterized by invasiveness, poor prognosis, and limited treatment opportunities. The objective of our research was to assess the role of circ_0016142 in HCC. The ferroptosis inducer RSL3 and the iron chelator deferoxamine were used to treat cells to induce or inhibit ferroptosis, respectively, and cell viability and proliferation were assessed in Hep3B and HA22T cells by CCK8 and EdU assays, respectively. ROS, MDA, GSH, and Fe2+ levels were determined using commercial kits. RT-qPCR and western blotting were performed to determine the relative expression levels of entities of interest. Dual-luciferase reporter and RNA pull-down assays were performed to assess the relationship between circ_0016142/GPX4 and miR-188-3p. The results showed that circ_0016142/GPX4 was overexpressed, whereas miR-188-3p was downregulated in HCC. Circ_0016142 silencing reduced cell proliferation and GSH levels and increased ROS, MDA, and Fe2+ levels in HCC cells, and this was reversed by the miR-188-3p inhibitor. GPX4-overexpression abolished the effect of miR-188-3p mimic in HCC cells. In conclusion, circ_0016142 silencing suppressed HCC cell proliferation by inducing ferroptosis via the miR-188-3p/GPX4 axis.
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Carcinoma Hepatocelular , Ferroptosis , Neoplasias Hepáticas , MicroARNs , Humanos , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Proliferación Celular , Ferroptosis/genética , Neoplasias Hepáticas/genética , MicroARNs/genética , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Especies Reactivas de Oxígeno , ARN Circular/genéticaRESUMEN
Memristors with nonvolatile storage performance and simulated synaptic functions are regarded as one of the critical devices to overcome the bottleneck in traditional von Neumann computer architecture. 2D van der Waals heterostructures have paved a new way for the development of advanced memristors by integrating the intriguing features of different materials and offering additional controllability over their optoelectronic properties. Herein, planar memristors with both electrical and optical tunability based on ReS2 /WS2 van der Waals heterostructure are demonstrated. The devices show unique unipolar nonvolatile behavior with high Roff /Ron ratio of up to 106 , desirable endurance, and retention, which are superior to pure ReS2 and WS2 devices. When decreasing the channel length, the set voltage can be notably reduced while the high Roff /Ron ratios are retained. By introducing electrostatic doping through the gate control, the set voltage can be tailored in a wide range from 4.50 to 0.40 V. Furthermore, biological synaptic functions and plasticity, including spike rate-dependent plasticity and paired-pulse facilitation, are successfully realized. By employing optical illumination, resistive switching can also be modulated, which is dependent on the illumination energy and power. A mechanism related to the interlayer charge transfer controlled by optical excitation is revealed.
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An optimization design of the bending-vibration resistance of magnetorheological elastomer carbon fibre reinforced polymer sandwich sheets (MECFRPSSs) was studied in this paper. Initially, by adopting the classical laminate theory, the Reddy's high-order shear deformation theory, the Rayleigh-Ritz method, etc., an analytical model of the MECFRPSSs was established to predict both bending and vibration parameters, with the three-point bending forces and a pulse load being considered separately. After the validation of the model was completed, the optimization design work of the MECFRPSSs was conducted based on an optimization model developed, in which the thickness, modulus, and density ratios of magnetorheological elastomer core to carbon fibre reinforced polymer were taken as design variables, and static bending stiffness, the averaged damping, and dynamic stiffness parameters were chosen as objective functions. Subsequently, an artificial bee colony algorithm was adopted to execute single-objective, dual-objective, and multi-objective optimizations to obtain the optimal design parameters of such structures, with the convergence effectiveness being examined in a validation example. It was found that it was hard to improve the bending, damping, and dynamic stiffness behaviours of the structure simultaneously as the values of design variables increased. Some compromised results of design parameters need to be determined, which are based on Pareto-optimal solutions. In further engineering application of the MECFRPSSs, it is suggested to use the corresponding design parameters related to a turning point to better exert their bending-vibration resistance.
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Intra-abdominal infections (IAI) is common surgical emergencies and have been reported as major contributors to non-trauma deaths in hospitals worldwide. The principles of IAI management included early diagnosis, adequate source control, appropriate antimicrobial therapy, and prompt physiologic stabilization using critical care resources, combined with an optimal surgical approach. In order to facilitate clinical management, establish a global standard and provide guidance for clinicians, the World Society of Emergency Surgery (WSES), the Global Alliance for Infection in Surgery (GAIS), the Surgical Infection Society-Europe (SIS-E), the World Surgical Infection Society (WSIS), and the American Association for the Surgery of Trauma (AAST) worked together to complete an international multi-society document, which provided the evidence-based clinical pathways. Herein, we made a comprehensive interpretation for the clinical pathways combined with the latest domestic and international research developments, aiming to provide evidence for domestic doctors on the diagnosis and treatment of IAI, and ultimately benefit patients.
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Antiinfecciosos , Infecciones Intraabdominales , Médicos , Humanos , Vías Clínicas , Antiinfecciosos/uso terapéutico , Europa (Continente)RESUMEN
Alzheimer's disease (AD) is the most common neurodegenerative disorder in the world. The aggregation of both amyloid beta (Aß) peptides extracellularly and Tau proteins intracellularly plays key roles in the pathological consequences of AD, which lead to cholinergic neurodegeneration and eventually death. Currently, there are no effective methods to stop the progression of AD. Using ex vivo, in vivo and clinical approaches, we investigated the functional effects of plasminogen on the widely used FAD, Aß42 oligomer or Tau intracranial injection-induced AD mouse model and explored its therapeutic effects on patients with AD. The results show that intravenously injected plasminogen rapidly crosses the bloodâbrain barrier (BBB); increases plasmin activity in the brain; colocalizes with and effectively promotes the clearance of Aß42 peptide and Tau protein deposits ex vivo and in vivo; increases the choline acetyltransferase (ChAT) level and decreases the acetylcholinesterase (AChE) activity; and improves the memory functions. Clinically, when GMP-level plasminogen was administered to 6 AD patients for 1-2 weeks, their average scores on the Minimum Mental State Examination (MMSE), which is a standard scoring system used to measure the memory loss and cognitive deficits, were extremely significantly improved by 4.2 ± 2.23 points, e.g., an average increase from 15.5 ± 8.22 before treatment to 19.7 ± 7.09 after treatment. The preclinical study and pilot clinical study suggest that plasminogen is effective in treating AD and may be a promising drug candidate.
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Enfermedad de Alzheimer , Humanos , Ratones , Animales , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Proteínas tau/metabolismo , Plasminógeno , Acetilcolinesterasa , Fragmentos de Péptidos/metabolismoRESUMEN
Aminoglycosides (AGs) are widely used to treat severe infections. However, systemically administered AGs preferentially kill cochlear hair cells, resulting in irreversible hearing loss. Recently, we found that AGs bind to RIPOR2 and trigger its rapid translocation in cochlear hair cells. Reducing RIPOR2 expression entirely prevents AG-induced hair cell death and subsequent hearing loss in mice. Next using yeast two-hybrid screening, we found that RIPOR2 interacts with GABARAP, a key macroautophagy/autophagy pathway protein. Following AG treatment, RIPOR2 colocalizes with GABARAP and regulates the activation of autophagy. Remarkably, reducing the expression of GABARAP, or another key autophagy protein MAP1LC3B/LC3B, entirely prevents AG-induced hair cell death and subsequent hearing loss in mice. Furthermore, we found that AGs activate the autophagy pathway specific to mitochondria. Reducing the expression of PINK1 or PRKN/parkin, two key mitophagy proteins, protects hair cells against AG toxicity. Thus, our findings demonstrated that RIPOR2-mediated autophagic dysfunction is essential for AG-induced hearing loss and provided potential therapeutic strategies for preventing AG toxicity.
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Sordera , Pérdida Auditiva , Ratones , Animales , Aminoglicósidos/toxicidad , Aminoglicósidos/metabolismo , Autofagia , Antibacterianos/farmacología , Pérdida Auditiva/inducido químicamente , Pérdida Auditiva/metabolismo , Células Ciliadas AuditivasRESUMEN
In order to explore the clinical value of large bone flap craniotomy, the effects of standard large bone flap craniotomy on cerebral hemodynamic indexes, incidence of postoperative intracranial infection, and neurological function in patients with severe craniocerebral trauma are investigated. 89 patients with severe craniocerebral trauma admitted from January 2020 to June 2021 are analyzed retrospectively. All patients are divided into a large craniotomy group (n = 45) and control group (n = 44) according to different surgical methods. The large craniotomy group is treated with large craniotomy decompression, and the control group is treated with traditional craniotomy decompression. The incidence of intracranial infection in each group is recorded, and NIHSS is applied to observe the neurological function recovery of 2 groups before and 1 month after operation. Besides, the patients are followed up after surgery and the Kaplan-Meier survival curve is obtained to compare the survival rate of patients in the two groups. It is clearly evident that the two surgical methods have certain clinical efficacy in the treatment of patients with severe craniocerebral trauma. Comparatively, the large craniotomy can further improve brain blood supply and improve neurological function recovery. Also, it can obtain low incidence of postoperative adverse reactions and intracranial infection.
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Traumatismos Craneocerebrales , Craneotomía , Traumatismos Craneocerebrales/cirugía , Craneotomía/métodos , Hemodinámica , Humanos , Estudios Retrospectivos , Colgajos QuirúrgicosRESUMEN
Aminoglycosides (AGs) are potent antibiotics that are capable of treating a wide variety of life-threatening infections; however, they are ototoxic and cause irreversible damage to cochlear hair cells. Despite substantial progress, little is known about the molecular pathways critical for hair cell function and survival that are affected by AG exposure. We demonstrate here that gentamicin, a representative AG antibiotic, binds to and within minutes triggers translocation of RIPOR2 in murine hair cells from stereocilia to the pericuticular area. Then, by interacting with a central autophagy component, GABARAP, RIPOR2 affects autophagy activation. Reducing the expression of RIPOR2 or GABARAP completely prevents AG-induced hair cell death and subsequent hearing loss in mice. Additionally, abolishing the expression of PINK1 or Parkin, two key mitochondrial autophagy proteins, prevents hair cell death and subsequent hearing loss caused by AG. In summary, our study demonstrates that RIPOR2-mediated autophagic dysfunction is essential for AG-induced hearing loss.
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Aminoglicósidos , Autofagia , Moléculas de Adhesión Celular , Pérdida Auditiva , Aminoglicósidos/toxicidad , Animales , Antibacterianos/toxicidad , Moléculas de Adhesión Celular/genética , Gentamicinas/toxicidad , Pérdida Auditiva/inducido químicamente , Ratones , Proteínas Quinasas , Ubiquitina-Proteína LigasasRESUMEN
Several degenerative disorders of the central nervous system, including Parkinson's disease (PD), are related to the pathological aggregation of proteins. Antibodies against toxic disease proteins, such as α-synuclein (SNCA), are therefore being developed as possible therapeutics. In this work, one peptide (YVGSKTKEGVVHGVA) from SNCA was used as the epitope to construct magnetic molecularly imprinted composite nanoparticles (MMIPs). These composite nanoparticles were characterized by dynamic light scattering (DLS), high-performance liquid chromatography (HPLC), isothermal titration calorimetry (ITC), Brunauer-Emmett-Teller (BET) analysis, and superconducting quantum interference device (SQUID) analysis. Finally, the viability of brain endothelial cells that were treated with MMIPs was measured, and the extraction of SNCA from CRISPR/dCas9a-activated HEK293T cells from the in vitro model system was demonstrated for the therapeutic application of MMIPs.
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Impresión Molecular , Nanopartículas , Células Endoteliales/metabolismo , Epítopos , Células HEK293 , Humanos , Impresión Molecular/métodos , alfa-Sinucleína/metabolismoRESUMEN
Cisplatin is one of the most widely used chemotherapeutic drugs across the world. However, the serious ototoxic effects, leading to permanent hair cell death and hearing loss, significantly limit the utility of cisplatin. In zebrafish, the functional mechanotransduction channel is required for cisplatin ototoxicity. However, it is still unclear the extent to which the mechanotransduction channel is involved in cisplatin uptake and ototoxicity in mammalian hair cells. Herein, we show that genetically disrupting mechanotransduction in mouse partially protects hair cells from cisplatin-induced hair cell death. Using a fluorescent-dye conjugated cisplatin, we monitored cisplatin uptake in cochlear explants and found that functional mechanotransduction is required for the uptake of cisplatin in murine hair cells. In addition, cimetidine, an inhibitor of the organic cation transporter, also partially protects hair cells from cisplatin ototoxicity. Notably, the otoprotective effects of cimetidine do not require mechanotransduction. These findings suggest that both the mechanotransduction channel and the organic cation transporter are critical for cisplatin ototoxicity in murine hair cells.
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Collapsin response mediator protein 1 (CRMP1), also known as dihydropyrimidinase-related protein 1, participates in cytoskeleton remodeling during axonal guidance and neuronal migration. In cochlear hair cells, the assembly and maintenance of the cytoskeleton is of great interest because it is crucial for the morphogenesis and maintenance of hair cells. Previous RNA sequencing analysis found that Crmp1 is highly expressed in cochlear hair cells. However, the expression profile and functions of CRMP1 in the inner ear remain unknown. In this study, the expression and localization of CRMP1 in hair cells was investigated using immunostaining, and was shown to be highly expressed in both outer and inner hair cells. Next, the stereocilia morphology of Crmp1-deficient mice was characterized. Abolishing CRMP1 did not affect the morphogenesis of hair cells. Interestingly, scanning electron microscopy detected hair cell loss at the basal cochlear region, an area responsible for high-frequency auditory perception, in Crmp1-deficient mice. Correspondingly, an auditory brainstem response test showed that mice lacking CRMP1 had progressive hearing loss at high frequencies. In summary, these data suggest that CRMP1 is required for high-frequency auditory perception.
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Proteínas del Tejido Nervioso/metabolismo , Semaforina-3A , Estereocilios , Animales , Citoesqueleto/metabolismo , Audición , Ratones , Neurogénesis , Semaforina-3A/metabolismo , Estereocilios/metabolismoRESUMEN
Oyster shells are rich in calcium, and thus, the potential use of waste shells is in the production of calcium phosphate (CaP) minerals for osteopathic biomedical applications, such as scaffolds for bone regeneration. Implanted scaffolds should stimulate the differentiation of induced pluripotent stem cells (iPSCs) into osteoblasts. In this study, oyster shells were used to produce nano-grade hydroxyapatite (HA) powder by the liquid-phase precipitation. Then, biphasic CaP (BCP) bioceramics with two different phase ratios were obtained by the foaming of HA nanopowders and sintering by two different two-stage heat treatment processes. The different sintering conditions yielded differences in structure and morphology of the BCPs, as determined by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Brunauer-Emmett-Teller (BET) surface area analysis. We then set out to determine which of these materials were most biocompatible, by co-culturing with iPSCs and examining the gene expression in molecular pathways involved in self-renewal and differentiation of iPSCs. We found that sintering for a shorter time at higher temperatures gave higher expression levels of markers for proliferation and (early) differentiation of the osteoblast. The differences in biocompatibility may be related to a more hierarchical pore structure (micropores within macropores) obtained with briefer, high-temperature sintering.
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Exoesqueleto/química , Hidroxiapatitas/química , Células Madre Pluripotentes Inducidas/metabolismo , Exoesqueleto/metabolismo , Animales , Materiales Biocompatibles/química , Regeneración Ósea/fisiología , Fosfatos de Calcio/química , Adhesión Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Cerámica/química , Humanos , Hidroxiapatitas/síntesis química , Hidroxiapatitas/metabolismo , Hidroxiapatitas/farmacología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Osteoblastos/citología , Osteogénesis/efectos de los fármacos , Osteogénesis/fisiología , Ostreidae/metabolismo , Porosidad/efectos de los fármacos , Ingeniería de Tejidos/métodos , Andamios del Tejido/químicaRESUMEN
Quantum-dot color conversion (QDCC) is a promising technique for next-generation full-color displays, such as QD converted organic light-emitting diodes and micro light-emitting diodes. Although present QDCC research has made some progress on the experimental aspect, the optical model and corresponding mathematical expression that can lay an indispensable foundation for QDCC have not been reported yet. In this paper, we present a theoretical model for precisely describing the complete optical behavior of QDCC, including optical transmission, scattering, absorption, and conversion process. A key parameter of QDCC, called dosage factor (DoF), is defined to quantitatively express the total consumption of QDs that can be calculated as the product of film thickness and QD concentration. Theoretical relations are established between DoF and three key performance indicators of QDCC, namely the light conversion efficiency (LCE), blue light transmittance (BLT), and optical density (OD). The maximum LCE value can be predicted based on this theoretical model, as well as the relationship between the slope of the OD curve and the molar absorption coefficient of blue light. This theoretical model is verified by both simulation and experiment. Results show that the simulation and experimental data highly match the theoretical model, and the goodness of fit reaches higher than 96% for LCE, BLT, and OD. Based on this, the optimal interval of DoF is recommended that provides key guiding significance to the QDCC related experiment.
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Programmed death-ligand 1 protein (PD-L1) has been posited to have a major role in suppressing the immune system during pregnancy, tissue allografts, autoimmune disease and other diseases, such as hepatitis. Photodynamic therapy uses light and a photosensitizer to generate singlet oxygen, which causes cell death (phototoxicity). In this work, photosensitizers (such as merocyanine) were immobilized on the surface of magnetic nanoparticles. One peptide sequence from PD-L1 was used as the template and imprinted onto poly(ethylene-co-vinyl alcohol) to generate magnetic composite nanoparticles for the targeting of PD-L1 on tumor cells. These nanoparticles were characterized using dynamic light scattering, high-performance liquid chromatography, Brunauer-Emmett-Teller analysis and superconducting quantum interference magnetometry. Natural killer-92 cells were added to these composite nanoparticles, which were then incubated with human hepatoma (HepG2) cells and illuminated with visible light for various periods. The viability and apoptosis pathway of HepG2 were examined using a cell counting kit-8 and quantitative real-time polymerase chain reaction. Finally, treatment with composite nanoparticles and irradiation of light was performed using an animal xenograft model.
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Stereocilia of cochlear hair cells are specialized mechanosensing organelles that convert sound-induced vibration to electrical signals. Glutaredoxin domain-containing cysteine-rich protein 2 (GRXCR2) is localized at the base of stereocilia and is necessary for stereocilia morphogenesis and auditory perception. However, the detailed functions of GRXCR2 in hair cells are still largely unknown. Here, we report that GRXCR2 interacts with chloride intracellular channel protein 5 (CLIC5) which is also localized at the base of stereocilia and required for normal hearing in human and mouse. Immunolocalization analyses suggest that GRXCR2 is not required for the localization of CLIC5 to the stereociliary base during development, or vice versa. Using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we deleted 60 amino acids near the N-terminus of GRXCR2 essential for its interaction with CLIC5. Interestingly, mice harboring this in-frame deletion in Grxcr2 exhibit moderate hearing loss at lower frequencies and severe hearing loss at higher frequencies although the morphogenesis of stereocilia is minimally affected. Thus, our findings reveal that the interaction between GRXCR2 and CLIC5 is crucial for normal hearing.
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Induced pluripotent stem cells are usually derived by reprogramming transcription factors (OSKM), such as octamer-binding transcription factor 4 (OCT4), (sex determining region Y)-box 2 (SOX2), Krüppel-like factor 4 (KLF4), and cellular proto-oncogene (c-Myc). However, the genomic integration of transcription factors risks the insertion of mutations into the genome of the target cells. Recently, the clustered regularly interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9) system has been used to edit genomes. In this work, dCas9-VPR (dCas9 with a gene activator, VP64-p65-Rta (VPR), fused to its c-terminus) and guide RNA (gRNA) combined to form ribonucleoproteins, which were immobilized on magnetic peptide-imprinted chitosan nanoparticles. These were then used to activate OSKM genes in human embryonic kidney (HEK) 293T cells. Four pairs of gRNAs were used for the binding site recognition to activate the OSKM genes. Transfected HEK293T cells were then prescreened for the high expression of OSKM proteins by immunohistochemistry images. The optimal gRNAs for OSKM expression were identified using quantitative real-time polymerase chain reaction and the staining of OSKM proteins. Finally, we found that the activated expression of one of the OSKM genes is up to three-fold higher than that of the other genes, enabling precise control of the cell differentiation.