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Introduction: Optimizing the dynamics of daylily (Hemerocallis citrina Baroni) growth under various planting patterns is critical for enhancing production efficiency. This study presents a comprehensive model to simulate daylily growth and optimize planting patterns to maximize bud yield while minimizing land resource utilization. Methods: The model incorporates source-sink relationship specific to daylilies into physiological process modeling, considering environmental factors such as micro-light and temperature climate, and CO2 concentration. Spatial factors, including planting pattern, row spacing, plant spacing, and plant density were examined for their impact on light interception, photosynthesis, and resource efficiency. Employing partial least square path modeling (PLS-PM), we analyzed the interrelations and causal relationships between planting configurations and physiological traits of daylily canopy leaves and buds. Through in situ simulations of 36 planting scenarios, we identified an optimal configuration (Scenario ID5) with a density of 83,000 plants·ha-1, row spacing of 0.8 m, and equidistant planting with a plant spacing of 0.15 m. Results and discussion: Our research findings indicate that increased Wide+Narrow row spacing can enhance yield to a certain extent. Although planting patterns influence daylily yield, their overall impact is relatively minor, and there is no clear pattern regarding the impact of plant spacing on individual plant yield. This modeling approach provides valuable insights into daylily plant growth dynamics and planting patterns optimization, offering practical guidance for both farmers and policymakers to enhance daylily productivity while minimizing land use.
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Lung cancer (LC) is the leading cause of cancer-related mortality worldwide, underscoring an urgent need for strategies that enable early detection and phenotypic classification. Here, we conducted a label-free surface-enhanced Raman spectroscopic (SERS) analysis of serum exosomes from 643 participants to elucidate the biochemical deregulation associated with LC progression and the unique phenotypes of different LC subtypes. Iodide-modified silver nanofilms were prepared to rapidly acquire SERS spectra with a high signal-to-noise ratio using 0.5 µL of patient exosomes. We performed interpretable and automated machine learning (ML) analysis of differential SERS features of serum exosomes to build LC diagnostic models, which achieved accuracies of 100% and 81% for stage I lung adenocarcinoma and its preneoplasia, respectively. In addition, the ML-derived exosomal SERS models effectively recognized different LC subtypes and disease stages to guide precision treatment. Our findings demonstrate that spectral fingerprinting of circulating exosomes holds promise for decoding the clinical status of LC, thus aiding in improving the clinical management of patients.
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Medical device research and development are characterized by high costs, extended timelines, inherent risks, and the necessity for interdisciplinary knowledge and skills. It is significantly influenced by policies, making the understanding of medical device innovation both important and challenging. This paper takes a dual approach to analyze medical device innovation. We reviewed representative clinical product of bougie and stylet and summarized the common characteristics and trend of these product. Innovations in these products often involve adding depth markings, replacing material and design structure, enhancing visualization, deciding between reusable or disposable designs, and integrating multi-functional features. This underscores the delicate balance between technological advancements and medical costs for widespread clinical applicability. We explored the guiding role of policy in medical device innovation, emphasizing its impact through an analysis of medical device regulations and policies in China. By offering insights from the perspectives of medical device companies and regulators, this paper aims to elucidate the critical aspects of medical device innovation, assisting researchers in mitigating risks during product development.
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Scalable fiber lithium-ion batteries (FLIBs) have garnered significant attention due to huge potential applications in wearable technology. However, their widespread applications have been limited by inadequate cycle and calendar life, primarily due to the high permeability of the encapsulation layer to water vapor in ambient air. To address this challenge, an ultra-high barrier composite tube is developed by blending polytrifluorochloroethylene (PCTFE) with organically modified montmorillonite (OMMT) for the continuous packaging of FLIBs. Due to the high crystallinity (≈40.21%) and small free volume (103.443 Å3), the PCTFE tube exhibited a low water vapor transmission rate (WVTR) of 0.123 mg day-1 pkg-1. Furthermore, through the melt extrusion, OMMT with its plate-like morphology are fully exfoliated and dispersed within the PCTFE matrix. This created more complex pathways for water, increasing the diffusion path length and thereby reducing WVTR to 0.006 mg day-1 pkg-1. This innovation enabled an ultra-long calendar life of 200 days and cycle life of 870 cycles for FLIBs, with over 80% capacity retention in ambient air. Additionally, 2%OMMT-PCTFE-FLIBs exhibited excellent flexibility, retaining an impressive 85.31% capacity after 10 000 bending cycles. This research presents a simple yet effective approach to enhance the lifetime and practicality of FLIBs through building a high-performance polymer-based encapsulation layer.
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Background: Osteoporosis is a prevalent global health condition, primarily affecting the aging population, and several therapies for osteoporosis have been widely used. However, available drugs for osteoporosis are far from satisfactory because they cannot alleviate disease progression. This study aimed to explore potential drug targets for osteoporosis through Mendelian randomization analysis. Methods: Using cis-expression quantitative trait loci (cis-eQTL) data of druggable genes and two genome-wide association studies (GWAS) datasets related to osteoporosis (UK Biobank and FinnGen cohorts), we employed mendelian randomization (MR) analysis to identify the druggable genes with causal relationships with osteoporosis. Subsequently, a series of follow-up analyses were conducted, such as colocalization analysis, cell-type specificity analysis, and correlation analysis with risk factors. The association between potential drug targets and osteoporosis was validated by qRT-PCR. Results: Six druggable genes with causal relationships with osteoporosis were identified and successfully replicated, including ACPP, DNASE1L3, IL32, PPOX, ST6GAL1, and TGM3. Cell-type specificity analysis revealed that PPOX and ST6GAL1 were expressed in all cell types in the bone samples, while IL32, ACPP, DNASE1L3, and TGM3 were expressed in specific cell types. The GWAS data showed there were seven risk factors for osteoporosis, including vitamin D deficiency, COPD, physical activity, BMI, MMP-9, ALP and PTH. Furthermore, ACPP was associated with vitamin D deficiency and COPD; DNASE1L3 was linked to physical activity; IL32 correlated with BMI and MMP-9; and ST6GAL1 was related to ALP, physical activity, and MMP-9. Among these risk factors, only MMP-9 had a high genetic correlation with osteoporosis. The results of qRT-PCR demonstrated that IL32 was upregulated while ST6GAL1 was downregulated in peripheral blood of osteoporosis patients. Conclusion: Our findings suggested that those six druggable genes offer potential drug targets for osteoporosis and require further clinical investigation, especially IL32 and ST6GAL1.
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To improve the informatization and intelligence level of high-speed railway (HSR) bridge construction, a parametric modeling method for continuous beam bridges based on Building Information Modeling (BIM) is proposed in this study. By this method, the parametric families of continuous beam components and key construction machinery are established, and the rapid modeling of overall continuous beam bridge and the simulation of critical construction process are realized as well. Taking the Caoxian-Shangqiu bridge of Xiong'an-Shangqiu HSR as a case study, the parametric modeling method is applied to conduct the engineering application on the prestressed duct layout and rebar clash detection. The results indicate that the modeling efficiencies of HSR continuous beam bridge and construction machinery are significantly increased by the established parametric modeling method. Based on the BIM model of continuous beam bridge, the improvement in the precision of prestressed duct layout and the elimination of rebar clash points can be achieved. The research achievement can guide the visualization of construction disclosure, enhance construction efficiency, and provide reference and technical support for the construction management and control of HSR continuous beam bridges.
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Modelos Teóricos , Ferrovias , Engenharia/métodosRESUMO
Long-term inflammation can cause chronic pain and trigger patients' anxiety by sensitizing the central nervous system. However, effective drugs with few side effects for treating chronic pain-induced anxiety are still lacking. The anxiolytic and anti-inflammatory effects of ruscogenin (RUS), an important active compound in Ophiopogon japonicus, were evaluated in a mouse model of chronic inflammatory pain and N9 cells. RUS (5, 10, or 20 mg/kg/day, i.g.) was administered once daily for 7 days after CFA injection; pain- and anxiety-like behaviors were assessed in mice. Anti-inflammatory effect of RUS (0.1, 1, 10 µM) on N9 microglia after LPS treatment was evaluated. Inflammatory markers (TNF-α, IL-1ß, IL-6, CD86, IL-4, ARG-1, and CD206) were measured using qPCR. The levels of IBA1, ROS, NF-κB, TLR4, P-IKK, P-IκBα, and P65, MAPKs (ERK, JNK, and P38), NLRP3 (caspase-1, ASC, and NLRP3) were detected by Western blotting or immunofluorescence staining. The potential target of RUS was validated by molecular docking and adeno-associated virus injection. Mice in CFA group exhibited allodynia and anxiety-like behaviors. LPS induced neuroinflammation in N9 cells. Both CFA and LPS increased the levels of IBA1, ROS, and inflammatory markers. RUS (10 mg/kg in vivo and 1 µM in vitro) alleviated these alterations through NF-κB/MAPKs/NLRP3 signaling pathways but had no effect on pain hypersensitivity. TLR4 strongly interacted with RUS, and TLR4 overexpression abolished the effects of RUS on anxiety and neuroinflammation. RUS exerts anti-inflammatory and anxiolytic effects via TLR4-mediated NF-κB/MAPKs/NLRP3 signaling pathways, which provides a basis for the treatment of chronic pain-induced anxiety.
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In this study, sixteen compounds were isolated from the branches of Mitragyna diversifolia, including twelve triterpenes (1-12), a phenolic compound (13), and three flavonoids (14-16). Among them, compounds 1-7, and 10-16 were reported for the first time from this plant. Compounds 7, 14, and 15 exhibited significant inhibitory activities against α-glucosidase, with IC50 values of 18.48 ± 2.74, 12.14 ± 1.58 and 35.77 ± 4.52⯵M, respectively. Furthermore, the inhibitory kinetics of α-glucosidase revealed that all fractions, active compounds 7, 14, and 15 belong to the mix inhibition type. In molecular docking, the analysis showed that compounds 13, 14, 15, and 16 possessed superior binding capacities with α-glucosidase (-8.3, -9.6, -9.9, and -9.2â¯kcal/mol, respectively). The results of the glucose uptake experiment indicated that only compound 14 showed a significant promotion effect on the glucose uptake rate of 3T3-L1 adipocytes (P < 0.05). Meanwhile, compounds 13, 14, 15, and 16 possessed potent antioxidant abilities with DPPH, ABTS, and FRAP. In DNA and protein oxidative damage assays, compound 15 had a stronger effect than the positive control Vc. The network-based pharmacological analysis platform was used to predict the diabetes-related target proteins of active compounds 7, 13, 14, 15, and 16, and two candidate targets (ALB and PPARG) related to their therapeutic effects on diabetes were identified.
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BACKGROUND: Hospital water systems represent critical environments for the transmission of pathogens, including multidrug-resistant strains like mucoid Pseudomonas aeruginosa (M-PA). Conventional disinfection methods often struggle to eradicate these pathogens effectively, highlighting the need for innovative approaches. OBJECTIVE: This study aimed to develop an enhanced photodynamic disinfection strategy targeting M-PA from hospital water systems, using curcumin-mediated photodynamic inactivation (PDI) with specific spectral range. METHODS: An M-PA strain isolated from hospital water was subjected to photodynamic treatment using curcumin as the photosensitizer. The efficacy of different wavelengths of light and varying concentrations of curcumin, with and without Tris-EDTA adjuvants, was evaluated through bacterial enumeration, ROS level measurements, transcriptome analysis, and assessment of virulence factors and biofilm formation. In vivo experiments utilizing a DSS-induced colitis mouse model assessed the protective effects of the photodynamic treatment against M-PA infection. RESULTS: Our findings demonstrated that the combination of curcumin-mediated PDI with specific spectral range effectively reduced M-PA counts in water, particularly when supplemented with Tris-EDTA. Transcriptome analysis revealed significant downregulation of virulence-related genes under sublethal photodynamic conditions. Furthermore, photodynamic treatment inhibited pyocyanin production and biofilm formation in M-PA, highlighting its potential to disrupt pathogenicity mechanisms. In vivo experiments showed that PDI attenuated M-PA-induced colitis in mice, indicating its protective efficacy. CONCLUSION: This study presents a promising photodynamic disinfection strategy for combating M-PA from hospital water. By optimizing curcumin-mediated PDI with specific spectral range and adjuvants, our approach demonstrates substantial efficacy in reducing bacterial counts, inhibiting virulence factors, and preventing M-PA-associated colitis.
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Vaccinations are essential for preventing and treating disease, especially cancer nanovaccines, which have gained considerable interest recently for their strong anti-tumor immune capabilities. Vaccines can prompt the immune system to generate antibodies and activate various immune cells, leading to a response against tumor tissues and reducing the negative effects and recurrence risks of traditional chemotherapy and surgery. To enhance the flexibility and targeting of vaccines, nanovaccines utilize nanotechnology to encapsulate or carry antigens at the nanoscale level, enabling more controlled and precise drug delivery to enhance immune responses. Cancer nanovaccines function by encapsulating tumor-specific antigens or tumor-associated antigens within nanomaterials. The small size of these nanomaterials allows for precise targeting of T cells, dendritic cells, or cancer cells, thereby eliciting a more potent anti-tumor response. In this paper, we focus on the classification of carriers for cancer nanovaccines, the roles of different target cells, and clinically tested cancer nanovaccines, discussing strategies for effectively inducing cytotoxic T lymphocytes responses and optimizing antigen presentation, while also looking ahead to the translational challenges of moving from animal experiments to clinical trials.
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Atherosclerosis is a persistent inflammatory condition of the blood vessels associated with abnormalities in lipid metabolism. Development of biomimetic nanoplatforms provides an effective strategy. Herein, inspired by the peptide CLIKKPF spontaneously coupling to phosphatidylserine (PS) on the inner leaflet of cell membranes specifically, MM@NPs were constructed by macrophage membrane spontaneous encapsulation of cyclodextrin-based nanoparticles modified with the peptide CLIKKPF and loaded with the hydrophobic compound resveratrol. MM@NPs could be specifically phagocytized by the activated endothelium with the overexpressed VCAM-1 for enhancing target delivery into the pathological lesion. Additionally, for the ApoE-/- mice, MM@NPs provide comprehensive treatment efficiency in reducing oxidant stress, alleviating the inherent inflammation, and decreasing cholesterol deposition, subsequently resulting in the atherosclerotic plaque regression. Therefore, MM@NPs could be one possible candidate for improving lipid metabolism and inflammation in atherosclerosis.
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Aterosclerose , Ciclodextrinas , Inflamação , Metabolismo dos Lipídeos , Macrófagos , Nanopartículas , Animais , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Aterosclerose/patologia , Camundongos , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Ciclodextrinas/química , Ciclodextrinas/farmacologia , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Nanopartículas/química , Células RAW 264.7 , Resveratrol/química , Resveratrol/farmacologia , Nanomedicina , Membrana Celular/metabolismo , Membrana Celular/efeitos dos fármacos , HumanosRESUMO
Abnormal biosynthesis of spliceosomes and ribosomes can lead to their dysfunction, which in turn disrupts protein synthesis and results in various diseases. While genetic factors have been extensively studied, our understanding of how environmental compounds interfere with spliceosome and ribosome biosynthesis remains limited. In the present study, we employed a Reduced Transcriptome Analysis (RTA) approach, integrating large-scale transcriptome data sets of zebrafish and compiling a specific zebrafish gene panel focusing on the spliceosome and ribosome, to elucidate the potential disruptors targeting their biosynthesis. Transcriptomic data sets for 118 environmental substances and 1400 related gene expression profiles were integrated resulting in 513 exposure signatures. Among these substances, several categories including PCB126, transition metals Lanthanum (La) and praseodymium (Pr), heavy metals Cd2+ and AgNO3 and atrazine were highlighted for inducing the significant transcriptional alterations. Furthermore, we found that the transcriptional patterns were distinct between categories, yet overlapping patterns were generally observed within each group. For instance, over 82 % differentially expressed ribosomal genes were shared between La and Pr within the equivalent concentration range. Additionally, transcriptional complexities were also evident across various organs and developmental stages of zebrafish, with notable differences in the inhibition of the transcription of various spliceosome subunits. Overall, our results provide novel insights into the understanding of the adverse effects of environmental compounds, thereby contributing to their environmental risk assessments.
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Ribossomos , Spliceossomos , Transcriptoma , Peixe-Zebra , Peixe-Zebra/genética , Animais , Spliceossomos/metabolismo , Spliceossomos/efeitos dos fármacos , Ribossomos/metabolismo , Perfilação da Expressão Gênica , Poluentes Químicos da Água/toxicidadeRESUMO
Metformin has been widely detected in aquatic ecosystems, yet the knowledge of its impact on aquatic organisms, particularly at environmentally relevant concentrations, remains limited. In the present study, we characterized the developmental toxicity of metformin in zebrafish, utilizing a transcriptome-guided toxicological assessment framework. Transcriptomic analysis conducted at metformin concentrations within the µg/L range revealed significant disruptions in biological processes associated with nucleotide, hydrocarbon, and amino acid metabolism, suggesting a significant disturbance in energy homeostasis. This observation was corroborated by energy-targeted metabolomic analysis, wherein a considerable number of metabolites involved in purine metabolism, pyrimidine metabolism, and the citrate cycle displayed significant alterations. Notably, most intermediates in the citrate cycle such as acetyl-CoA exhibited remarkable decreases. Additionally, our study identified significant impediments in zebrafish embryonic development, including decreased yolk extension progress, spontaneous contraction and body length, and increased yolk sac area and yolk/while body lipid content ratio, at metformin concentrations as low as 0.12 µg/L. Furthermore, the disruption of energy homeostasis by metformin was observed to persist into adulthood even after a prolonged recovery period. The present findings highlighted the disruptive effects of metformin on energy homeostasis and embryonic development in teleost at environmentally relevant concentrations, thereby prompting a reevaluation of its environmental risk to nontarget aquatic organisms.
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BACKGROUND: Suicide is a global public health crisis, and little evidence has focused on associations between suicide attempts and childhood maltreatment (CM) in Chinese middle school students. OBJECTIVE: This study aimed to explore the relationship between childhood maltreatment and suicide attempts and the sex difference in Chinese middle school students. PARTICIPANTS AND SETTING: In this prospective cohort study, students in grades 7 and 8 filled out the questionnaire at baseline and 6-month follow-up. METHODS: Demographic data, childhood maltreatment, and suicide attempts were surveyed. Depression, anxiety, and stress were measured at baseline as covariates. Logistic regression was employed to measure the impact of childhood maltreatment and its sex differences on suicide attempts. RESULTS: Among 782 students, 39.6 % suffered from childhood maltreatment, and the incidence rate of suicide attempts in 6 months was 4.60 % (36/782). After controlling for covariates, childhood maltreatment (adjusted odds ratio, OR = 2.899, 95%CI = 1.349-6.227) and its subtypes, physical abuse (adjusted OR = 4.077, 95%CI = 1.593-10.505) and emotional neglect (adjusted OR = 2.179, 95%CI = 1.059-4.481) were independent risk factors of suicide attempts. The association remained significant in females but not males, while no interactions between sex and childhood maltreatment were found. CONCLUSIONS: Childhood maltreatment was prevalent in Chinese middle school students and closely associated with the incidence of suicide attempts, which provided valuable evidence for suicide prevention.
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Wound infections caused by Staphylococcus aureus often result in localized suppurative lesions that severely impede the healing process, so it is urgent to develop a dress with efficient antimicrobial and pro-healing functions. In this study, the bifunctional injectable hydrogel lactoferrin (Lf)/NZ2114/lithium magnesium silicate hydrogel (LMSH) was first successfully prepared through the electrostatic interaction method. The physical, biological, and efficacy properties are systematically analyzed with good shear-thinning capacity and biocompatibility. More importantly, it inhibits infection and promotes wound healing in a mouse wound infection model after 14 d treatment, and the bactericidal rate and healing rate were over 99.92% and nearly 100%, respectively. Meanwhile, the massive reduction of inflammatory cells, restoration of tissue structure, and angiogenesis in mice showed the anti-inflammatory and pro-healing properties of the hydrogel. The healed wounds showed thickening with more hair follicles and glands, suggesting that the hydrogel Lf/NZ2114/LMSH (Three in One) could be a better dressing candidate for the treatment of S. aureus-induced wound infections.
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Selective activation of light alkanes is an essential reaction in the petrochemical industry for producing commodity chemicals, such as light olefins and aromatics. Because of the much higher intrinsic activities of noble metals in comparison to non-noble metals, it is desirable to employ solid catalysts with low noble metal loadings to reduce the cost of catalysts. Herein, we report the introduction of a tiny amount of Pt (at levels of hundreds of ppm) as a promoter of the Ga2O3 clusters encapsulated in ZSM-5 zeolite, which leads to â¼20-fold improvement in the activity for ethane dehydrogenation reaction. A combination of experimental and theoretical studies shows that the isolated Pt atoms stabilized by small Ga2O3 clusters are the active sites for activating the inert C-H bonds in ethane. The synergy of atomically dispersed Pt and Ga2O3 clusters confined in the 10MR channels of ZSM-5 can serve as a bifunctional catalyst for the direct ethane-benzene coupling reaction for the production of ethylbenzene, surpassing the performances of the counterpart catalysts made with PtGa nanoclusters and nanoparticles.
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Background: Parkinson's disease psychosis (PDP) is a common and distressing complication of Parkinson's disease (PD), characterized by hallucinations and delusions. This research aimed to assess the pharmacokinetics and safety of NH130, a selective serotonin 5-HT2A inverse agonist, as a potential PDP treatment in healthy individuals. Methods: We conducted clinical pharmacokinetic studies and safety evaluations for NH130, employing a physiologically based pharmacokinetic (PBPK) model to predict its behavior in human body. Results: In a single-dose escalation study, healthy volunteers received NH130 at varying doses (2 mg, 6 mg, 12 mg, 24 mg, 40 mg, 60 mg, and 90 mg) or a placebo. The drug demonstrated favorable pharmacokinetics, with no serious adverse events (AEs) reported. Clinical plasma concentrations correlated well with PBPK model predictions, validating the model's utility for guiding future clinical development. Conclusion: NH130 showed promising pharmacokinetic characteristics and safety profile, supporting its progression to multi-dose trials and suggesting its potential as a therapeutic agent for PDP. Clinical Trial Registration: http://www.chinadrugtrials.org.cn/index.html, Identifier CTR20230409.
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BACKGROUND: Intervertebral disc degeneration (IDD) is associated with back pain; back pain is a world-wide contributor to poor quality of life, while necroptosis has the characteristics of necroptosis and apoptosis, however, its role in IDD is still unclear. Therefore, the aim of this study was to identify biomarkers associated with necroptosis in IDD. PURPOSE: To explore biomarkers associated with necroptosis in IDD, reveal the pathogenesis of IDD, as well as provide new directions for the diagnosis and treatment of this disease. STUDY DESIGN/SETTINGS: Retrospective cohort study. Our study employs scRNA-seq coupled with MR analysis to investigate the causal relationship between necroptosis and IDD, laying a foundational groundwork for unveiling the intricate pathogenic mechanisms of this condition. METHODS: Data quality control and normalisation was executed in single-cell dataset, GSE205535. Then, different cell types were obtained by cell annotation through marker genes. Subsequently, chi-square test was employed to assess the distribution difference of different cell types between IDD and control to screen key cells. AUCell was applied to calculate necroptosis-related genes (NRGs) scores of all cell types, further key cells were divided into high and low NRGs groups according to the median AUC scores of different cell types. Afterwards, the differentially expressed genes (DEGs) within the two score groups were screened. Then, the genes that had causal relationship with IDD were selected as biomarkers by univariate and multivariate Mendelian randomization (MR) analysis. Finally, the expression of biomarkers in different cell types and pseudo-time analysis was analyzed separately. RESULTS: In GSE205535, 16 different cell populations identified by UMAP cluster analysis were further annotated to 8 cell types using maker genes. Afterwards, 53 DEGs were screened between the high and low NRGs groups. In addition, 9 genes with causal relationship with IDD were obtained by univariate MR analysis, further multivariate MR analysis proved that NT5E and TMEM158 had a direct causal relationship with IDD, which were used as biomarkers in this study. This study not only found that the expression levels of NT5E and TMEM158 were higher in IDD group, but also found that fibrochondrocytes and inflammatory chondrocytes were the key cells of NT5E and TMEM158, respectively. In the end, the biomarkers had the same expression trend in the quasi-time series, and both of them from high to low and then increased. CONCLUSION: NT5E and TMEM158, as biomarkers of necroptotic apoptotic IDD, were causally associated with IDD. CLINICAL SIGNIFICANCE: The understanding of chondrocytes as key cells provides new perspectives for deeper elucidation of the pathogenesis of IDD, improved diagnostic methods, and the development of more effective treatments. These findings are expected to provide a more accurate and personalised approach to clinical diagnosis and treatment, thereby improving the prognosis and quality of life of patients with IDD.
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The editing efficiencies of prime editing (PE) using ribonucleoprotein (RNP) and RNA delivery are not optimal due to the challenges in solid-phase synthesis of long PE guide RNA (pegRNA) (>125 nt). Here, we develop an efficient, rapid and cost-effective method for generating chemically modified pegRNA (125-145 nt) and engineered pegRNA (epegRNA) (170-190 nt). We use an optimized splint ligation approach and achieve approximately 90% production efficiency for these RNAs, referred to as L-pegRNA and L-epegRNA. L-epegRNA demonstrates enhanced editing efficiencies across various cell lines and human primary cells with improvements of up to more than tenfold when using RNP delivery and several hundredfold with RNA delivery of PE, compared to epegRNA produced by in vitro transcription. L-epegRNA-mediated RNP delivery also outperforms plasmid-encoded PE in most comparisons. Our study provides a solution to obtaining high-quality pegRNA and epegRNA with desired chemical modifications, paving the way for the use of PE in therapeutics and various other fields.
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BACKGROUND: Physical symptoms and aversion induced by opioid withdrawal strongly affect the management of opioid addiction. YTH N6-methyladenosine (m6A) RNA binding protein 1 (YTHDF1), an m6A-binding protein, from the periaqueductal gray (PAG) reportedly contributes to morphine tolerance and hyperalgesia. However, the role of YTHDF1 in morphine withdrawal remains unclear. METHODS: A naloxone-precipitated morphine withdrawal model was established in C57/BL6 mice or transgenic mice. YTHDF1 was knocked down via adeno-associated virus transfection. Combined with the results of the single-cell RNA sequencing analysis, the changes in morphine withdrawal somatic signs and conditioned place aversion (CPA) scores were compared when YTHDF1 originating from different neurons in the ventrolateral periaqueductal gray (vlPAG) was knocked down. We further explored the role of inflammatory factors and transcription factors related to inflammatory response in morphine withdrawal. RESULTS: Our results revealed that YTHDF1 expression was upregulated in the vlPAG of mice with morphine withdrawal and that the knockdown of vlPAG YTHDF1 attenuated morphine withdrawal-related somatic signs and aversion. The levels of NF-κB and p-NF-κB were reduced after the inhibition of YTHDF1 in the vlPAG. YTHDF1 from vlPAG inhibitory neurons, rather than excitatory neurons, facilitated morphine withdrawal responses. The inhibition of YTHDF1 in vlPAG somatostatin (Sst)-expressing neurons relieved somatic signs of morphine withdrawal and aversion, whereas the knockdown of YTHDF1 in cholecystokinin (Cck)-expressing or parvalbumin (PV)-expressing neurons did not change morphine withdrawal-induced responses. The activity of c-fos + neurons, the intensity of the calcium signal, the density of dendritic spines, and the frequency of mIPSCs in the vlPAG, which were increased in mice with morphine withdrawal, were decreased with the inhibition of YTHDF1 from vlPAG inhibitory neurons or Sst-expressing neurons. Knockdown of NF-κB in Sst-expressing neurons also alleviated morphine withdrawal-induced responses. CONCLUSIONS: YTHDF1 originating from Sst-expressing neurons in the vlPAG is crucial for the modulation of morphine withdrawal responses, and the underlying mechanism might be related to the regulation of the expression and phosphorylation of NF-κB.