Polyglycerol-Functionalized ß-Cyclodextrins as Crosslinkers in Thermoresponsive Nanogels for the Enhanced Dermal Penetration of Hydrophobic Drugs.

Thermoresponsive nanogels (tNGs) are promising candidates for dermal drug delivery. However, poor incorporation of hydrophobic drugs into hydrophilic tNGs limits the therapeutic efficiency. To address this challenge, ß-cyclodextrins (ß-CD) are functionalized by hyperbranched polyglycerol serving as crosslinkers (hPG-ßCD) to fabricate ßCD-tNGs. This novel construct exhibits augmented encapsulation of hydrophobic drugs, shows the appropriate thermal response to dermal administration, and enhances the dermal penetration of payloads. The structural influences on the encapsulation capacity of ßCD-tNGs for hydrophobic drugs are analyzed, while concurrently retaining their efficacy as skin penetration enhancers. Various synthetic parameters are considered, encompassing the acrylation degree and molecular weight of hPG-ßCD, as well as the monomer composition of ßCD-tNGs. The outcome reveals that ßCD-tNGs substantially enhance the aqueous solubility of Nile Red elevating to 120 µg mL-1 and augmenting its dermal penetration up to 3.33 µg cm-2. Notably, the acrylation degree of hPG-ßCD plays a significant role in dermal drug penetration, primarily attributed to the impact on the rigidity and hydrophilicity of ßCD-tNGs. Taken together, the introduction of the functionalized ß-CD as the crosslinker in tNGs presents a novel avenue to enhance the efficacy of hydrophobic drugs in dermatological applications, thereby offering promising opportunities for boosted therapeutic outcomes.

Cellular Localization of FOXO3 Determines Its Role in Cataractogenesis.

The transcription factor forkhead box protein (FOX)-O3 is a core regulator of cellular homeostasis, stress response, and longevity. The cellular localization of FOXO3 is closely related to its function. Herein, the role of FOXO3 in cataract formation was explored. FOXO3 showed nuclear translocation in lens epithelial cells (LECs) arranged in a single layer on lens capsule tissues from both human cataract and N-methyl-N-nitrosourea (MNU)-induced rat cataract, also in MNU-injured human (H)-LEC lines. FOXO3 knockdown inhibited the MNU-induced increase in expression of genes related to cell cycle arrest (GADD45A and CCNG2) and apoptosis (BAK and TP53). H2 is highly effective in reducing oxidative impairments in nuclear DNA and mitochondria. When H2 was applied to MNU-injured HLECs, FOXO3 underwent cleavage by MAPK1 and translocated into mitochondria, thereby increasing the transcription of oxidative phosphorylation-related genes (MTCO1, MTCO2, MTND1, and MTND6) in HLECs. Furthermore, H2 mediated the translocation of FOXO3 from the nucleus to the mitochondria within the LECs of cataract capsule tissues of rats exposed to MNU. This intervention ameliorated MNU-induced cataracts in the rat model. In conclusion, there was a correlation between the localization of FOXO3 and its function in cataract formation. It was also determined that H2 protects HLECs from injury by leading FOXO3 mitochondrial translocation via MAPK1 activation. Mitochondrial FOXO3 can increase mtDNA transcription and stabilize mitochondrial function in HLECs.

Glycometabolic reprogramming in cementoblasts: A vital target for enhancing cell mineralization.

Cementum, a constituent part of periodontal tissues, has important adaptive and reparative functions. It serves to attach the tooth to alveolar bone and acts as a barrier delimit epithelial growth and bacteria evasion. A dynamic and highly responsive cementum is essential for maintaining occlusal relationships and the integrity of the root surface. It is a thin layer of mineralized tissue mainly produced by cementoblasts. Cementoblasts are osteoblast-like cells essential for the restoration of periodontal tissues. In recent years, glucose metabolism has been found to be critical in bone remodeling and osteoblast differentiation. However, the glucose metabolism of cementoblasts remains incompletely understood. First, immunohistochemistry staining and in vivo tracing with 18 F-fluorodeoxyglucose (18 F-FDG) revealed significantly higher glucose metabolism in cementum formation. To test the bioenergetic pathways of cementoblast differentiation, we compared the bioenergetic profiles of mineralized and unmineralized cementoblasts. As a result, we observed a significant increase in the consumption of glucose and production of lactate, coupled with the higher expression of glycolysis-related genes. However, the expression of oxidative phosphorylation-related genes was downregulated. The verified results were consistent with the RNA sequencing results. Likewise, targeted energy metabolomics shows that the levels of glycolytic metabolites were significantly higher in the mineralized cementoblasts. Seahorse assays identified an increase in glycolytic flux and reduced oxygen consumption during cementoblast mineralization. Apart from that, we also found that lactate dehydrogenase A (LDHA), a key glycolysis enzyme, positively regulates the mineralization of cementoblasts. In summary, cementoblasts mainly utilized glycolysis rather than oxidative phosphorylation during the mineralization process.

CXXC5 mitigates P. gingivalis-inhibited cementogenesis by influencing mitochondrial biogenesis.

BACKGROUND: Cementoblasts on the tooth-root surface are responsible for cementum formation (cementogenesis) and sensitive to Porphyromonas gingivalis stimulation. We have previously proved transcription factor CXXC-type zinc finger protein 5 (CXXC5) participates in cementogenesis. Here, we aimed to elucidate the mechanism in which CXXC5 regulates P. gingivalis-inhibited cementogenesis from the perspective of mitochondrial biogenesis. METHODS: In vivo, periapical lesions were induced in mouse mandibular first molars by pulp exposure, and P. gingivalis was applied into the root canals. In vitro, a cementoblast cell line (OCCM-30) was induced cementogenesis and submitted for RNA sequencing. These cells were co-cultured with P. gingivalis and examined for osteogenic ability and mitochondrial biogenesis. Cells with stable CXXC5 overexpression were constructed by lentivirus transduction, and PGC-1α (central inducer of mitochondrial biogenesis) was down-regulated by siRNA transfection. RESULTS: Periapical lesions were enlarged, and PGC-1α expression was reduced by P. gingivalis treatment. Upon apical inflammation, Cxxc5 expression decreased with Il-6 upregulation. RNA sequencing showed enhanced expression of osteogenic markers, Cxxc5, and mitochondrial biogenesis markers during cementogenesis. P. gingivalis suppressed osteogenic capacities, mitochondrial biogenesis markers, mitochondrial (mt)DNA copy number, and cellular ATP content of cementoblasts, whereas CXXC5 overexpression rescued these effects. PGC-1α knockdown dramatically impaired cementoblast differentiation, confirming the role of mitochondrial biogenesis on cementogenesis. CONCLUSIONS: CXXC5 is a P. gingivalis-sensitive transcription factor that positively regulates cementogenesis by influencing PGC-1α-dependent mitochondrial biogenesis. Video Abstract.

A MnO2 nanosheet-mediated CRISPR/Cas12a system for the detection of organophosphorus pesticides in environmental water.

Nowadays, easy, convenient, and sensitive sensing strategies are still critical for organophosphorus pesticides in environmental water samples. Herein, a novel organophosphorus pesticide (OP) assay based on acetylcholinesterase (AChE) and a MnO2 nanosheet-mediated CRISPR/Cas12a reaction is reported. The single-strand DNA (ssDNA) activator of CRISPR/Cas12a was simply adsorbed on the MnO2 nanosheets as the nanoswitches of the assay. In the absence of target OPs, AChE hydrolyzed acetylcholine (ATCh) to thiocholine (TCh), which reduced the MnO2 nanosheets to Mn2+, resulting in the release of the activator followed by activation of the CRISPR/Cas12a system. The activated Cas12a thereafter nonspecifically cleaved the FAM/BHQ1-labeled ssDNA (FQ-reporter), producing a fluorescence signal. Upon the addition of target OPs, the hydrolysis of ATCh by AChE was inhibited owing to OPs combining with AChE, and thus effective quantification of OPs could be achieved by measuring the fluorescence changes of the system. As a proof of concept, dichlorvos (DDVP) was chosen as a model OP analyte to address the feasibility of the proposed method. Attributed to the excellent trans-cleavage activity of Cas12a, the fluorescent biosensor exhibits a satisfactory limit of detection (LOD) for DDVP at 0.135 ng mL-1. In addition, the excellent recoveries for the detection of DDVP in environmental water samples demonstrate the applicability of the proposed assay in real sample research.

Impacts of a new diagnosis-related group point payment system on children's medical services in China: Length of stay and costs.

BACKGROUND: Paediatric healthcare is always highlighted in medical and health care system reform in China. Zhejiang Province established a new diagnosis-related group (DRG) point payment reform in 2020 to regulate provider behaviours and control medical costs. We conducted this study to evaluate impacts of the DRG point payment policy on provider behaviours and resource usage in children's medical services. METHODS: Data from patients' discharge records from July 2019 to December 2020 in Children's Hospital, Zhejiang University School of Medicine were collected for analysis. We employed the interrupted time series approach to reveal the trend before and after the DRG point payment reform and the difference-in-differences analysis to estimate the independent outcome changes attributed to the reform. RESULTS: We found that the upward trend of length of stay slightly slowed, and the total costs began to decrease at the post-policy stage. Although independent effects of the reform were not presented among the whole sample, the length of stay and hospitalisation costs of moderate-hospital-stay paediatric patients, non-surgical patients, and infant patients were found to decrease rapidly after the reform. CONCLUSION: DRG point payments can changed the provider behaviours and eventually reduce healthcare resource usage in children's medical services.

Spatio-temporal transcriptome profiling and subgenome analysis in Brassica napus.

Brassica napus is an important oil crop and an allotetraploid species. However, the detailed analysis of gene function and homoeologous gene expression in all tissues at different developmental stages was not explored. In this study, we performed a global transcriptome analysis of 24 vegetative and reproductive tissues at six developmental stages (totally 111 tissues). These samples were clustered into eight groups. The gene functions of silique pericarp were similar to roots, stems and leaves. In particular, glucosinolate metabolic process was associated with root and silique pericarp. Genes involved in protein phosphorylation were often associated with stamen, anther and the early developmental stage of seeds. Transcription factor (TF) genes were more specific than structural genes. A total of 17 100 genes that were preferentially expressed in one tissue (tissue-preferred genes, TPGs), including 889 TFs (5.2%), were identified in the 24 tissues. Some TPGs were identified as hub genes in the co-expression network analysis, and some TPGs in different tissues were involved in different hormone pathways. About 67.0% of the homoeologs showed balanced expression, whereas biased expression of homoeologs was associated with structural divergence. In addition, the spatiotemporal expression of homoeologs was related to the presence of transposable elements (TEs) and regulatory elements (REs); more TEs and fewer REs in the promoters resulted in divergent expression in different tissues. This study provides a valuable transcriptional map for understanding the growth and development of B. napus, for identifying important genes for future crop improvement, and for exploring gene expression patterns in the B. napus.

Histone demethylase LSD1 promotes RIG-I poly-ubiquitination and anti-viral gene expression.

Under RNA virus infection, retinoic acid-inducible gene I (RIG-I) in host cells recognizes viral RNA and activates the expression of type I IFN. To investigate the roles of protein methyltransferases and demethylases in RIG-I antiviral signaling pathway, we screened all the known related enzymes with a siRNA library and identified LSD1 as a positive regulator for RIG-I signaling. Exogenous expression of LSD1 enhances RIG-I signaling activated by virus stimulation, whereas its deficiency restricts it. LSD1 interacts with RIG-I, promotes its K63-linked polyubiquitination and interaction with VISA/MAVS. Interestingly, LSD1 exerts its function in antiviral response not dependent on its demethylase activity but through enhancing the interaction between RIG-I with E3 ligases, especially TRIM25. Furthermore, we provide in vivo evidence that LSD1 increases antiviral gene expression and inhibits viral replication. Taken together, our findings demonstrate that LSD1 is a positive regulator of signaling pathway triggered by RNA-virus through mediating RIG-I polyubiquitination.

An adaptive test based on principal components for detecting multiple phenotype associations using GWAS summary data.

Extensive evidence from genome-wide association studies (GWAS) has shown that jointly analyzing multiple phenotypes can improve the power of the association test compared to the traditional single variant versus single trait approach. Here we propose an adaptive test based on principal components (ATPC) that is powerful and efficient for discovering the association between a single variant and multiple traits. Our method only needs GWAS summary statistics that are often available. We first estimate the trait correlation matrix by LD score regression. Then, based on the correlation matrix, we construct a series of test statistics that contain different numbers of principal components. The ultimate test statistic combines the P values of these principal component-based statistics by using the aggregated Cauchy association test. The analytical P-value of the test statistic can be computed quickly without the permutation process, which is the notable feature of our proposed method. The extensive simulation studies demonstrate that ATPC can control the type I error rates and have powerful and robust performance compared to several existing tests in a wide range of simulation settings. The analysis of the lipids GWAS summary data from the Global Lipids Genetics Consortium shows that ATPC identifies 230 new SNPs that are missed by the original single trait association analysis. By searching the GWAS Catalog, some SNPs and mapped genes identified by ATPC are reported to be associated with lipid traits. Through further analysis for GWAS results, we also find some Gene Ontology terms and biological pathways related to lipids.

The Applications and Potentials of Extracellular Vesicles from Different Cell Sources in Periodontal Regeneration.

Periodontitis is a chronic infectious disease worldwide that can cause damage to periodontal supporting tissues including gingiva, bone, cementum and periodontal ligament (PDL). The principle for the treatment of periodontitis is to control the inflammatory process. Achieving structural and functional regeneration of periodontal tissues is also essential and remains a major challenge. Though many technologies, products, and ingredients were applied in periodontal regeneration, most of the strategies have limited outcomes. Extracellular vesicles (EVs) are membranous particles with a lipid structure secreted by cells, containing a large number of biomolecules for the communication between cells. Numerous studies have demonstrated the beneficial effects of stem cell-derived EVs (SCEVs) and immune cell-derived EVs (ICEVs) on periodontal regeneration, which may be an alternative strategy for cell-based periodontal regeneration. The production of EVs is highly conserved among humans, bacteria and plants. In addition to eukaryocyte-derived EVs (CEVs), a growing body of literature suggests that bacterial/plant-derived EVs (BEVs/PEVs) also play an important role in periodontal homeostasis and regeneration. The purpose of this review is to introduce and summarize the potential therapeutic values of BEVs, CEVs and PEVs in periodontal regeneration, and discuss the current challenges and prospects for EV-based periodontal regeneration.

Improved thermostability of D-allulose 3-epimerase from Clostridium bolteae ATCC BAA-613 by proline residue substitution.

d-allulose, a rare sugar that is scarce in nature, exerts several beneficial effects and has commercial potential. d-allulose 3-epimerase (DAEase) plays a vital role in catalyzing the isomerization from d-fructose to d-allulose. However, the industrial application of DAEase for d-allulose production is hindered by its poor long-term thermostability. In the present research, we introduced a proline residue (i) to restrict its spatial conformation and (ii) to reduce the entropy of the unfolded state of DAEase. The t1/2 value of the double-site Clostridium bolteae DAEase mutant Cb-51P/89P was prolonged to 58 min at 55 °C, a 2.32-fold increase compared with wild-type DAEase. The manipulation did not cause obvious changes in the enzymatic properties, including optimum pH, optimal temperature, optimum metal ion, and enzymatic activity. As the accumulation of multiple small effects through proline substitution could dramatically improve the thermostability of the mutant protein, our method to improve the thermostability while roughly retaining the original enzymatic properties is promising.

Nanocarriers for Skin Applications: Where Do We Stand?

Skin penetration of active molecules for treatment of diverse diseases is a major field of research owing to the advantages associated with the skin like easy accessibility, reduced systemic-derived side effects, and increased therapeutic efficacy. Despite these advantages, dermal drug delivery is generally challenging due to the low skin permeability of therapeutics. Although various methods have been developed to improve skin penetration and permeation of therapeutics, they are usually aggressive and could lead to irreversible damage to the stratum corneum. Nanosized carrier systems represent an alternative approach for current technologies, with minimal damage to the natural barrier function of skin. In this Review, the use of nanoparticles to deliver drug molecules, genetic material, and vaccines into the skin is discussed. In addition, nanotoxicology studies and the recent clinical development of nanoparticles are highlighted to shed light on their potential to undergo market translation.

Histone demethylase KDM3A is required for enhancer activation of hippo target genes in colorectal cancer.

Hippo pathway is involved in tumorigenesis, and its regulation in cytosol has been extensively studied, but its regulatory mechanisms in the nuclear are not clear. In the current study, using a FBS-inducing model following serum starvation, we identified KDM3A, a demethylase of histone H3K9me1/2, as a positive regulator for hippo target genes. KDM3A promotes gene expression through two mechanisms, one is to upregulate YAP1 expression, and the other is to facilitate H3K27ac on the enhancers of hippo target genes. H3K27ac upregulation is more relevant with gene activation, but not H3K4me3; and KDM3A depletion caused H3K9me2 upregulation mainly on TEAD1-binding enhancers rather than gene bodies, further resulting in H3K27ac decrease, less TEAD1 binding on enhancers and impaired transcription. Moreover, KDM3A is associated with p300 and required for p300 recruitment to enhancers. KDM3A deficiency delayed cancer cell growth and migration, which was rescued by YAP1 expression. KDM3A expression is correlated with YAP1 and hippo target genes in colorectal cancer patient tissues, and may serve as a potential prognosis mark. Taken together, our study reveals novel mechanisms for hippo signaling and enhancer activation, which is critical for tumorigenesis of colorectal cancer.

Enhanced Thermostability of D-Psicose 3-Epimerase from Clostridium bolteae through Rational Design and Engineering of New Disulfide Bridges.

D-psicose 3-epimerase (DPEase) catalyzes the isomerization of D-fructose to D-psicose (aka D-allulose, a low-calorie sweetener), but its industrial application has been restricted by the poor thermostability of the naturally available enzymes. Computational rational design of disulfide bridges was used to select potential sites in the protein structure of DPEase from Clostridium bolteae to engineer new disulfide bridges. Three mutants were engineered successfully with new disulfide bridges in different locations, increasing their optimum catalytic temperature from 55 to 65 °C, greatly improving their thermal stability and extending their half-lives (t1/2) at 55 °C from 0.37 h to 4-4.5 h, thereby greatly enhancing their potential for industrial application. Molecular dynamics simulation and spatial configuration analysis revealed that introduction of a disulfide bridge modified the protein hydrogen-bond network, rigidified both the local and overall structures of the mutants and decreased the entropy of unfolded protein, thereby enhancing the thermostability of DPEase.

Large Scale Production of Continuous Hydrogel Fibers with Anisotropic Swelling Behavior by Dynamic-Crosslinking-Spinning.

Hydrogel microfibers have been considered as a potential biomaterial to spatiotemporally biomimic 1D native tissues such as nerves and muscles which are always assembled hierarchically and have anisotropic response to external stimuli. To produce facile hydrogel microfibers in a mathematical manner, a novel dynamic-crosslinking-spinning (DCS) method is demonstrated for direct fabrication of size-controllable fibers from poly(ethylene glycol diacrylate) oligomer in large scale, without microfluidic template and in a biofriendly environment. The diameter of fibers can be precisely controlled by adjusting the spinning parameters. Anisotropic swelling property is also dependent on inhomogeneous structure generated in spinning process. Comparing with bulk hydrogels, the resulting fibers exhibit superior rapid water adsorption property, which can be attributed to the large surface area/volume ratio of fiber. This novel DCS method is one-step technology suitable for large-scale production of anisotropic hydrogel fibers which has a promising application in the area such as biomaterials.

A case report of percutaneous intramyocardial septal radiofrequency ablation in an adult with re-obstruction after Morrow procedure.

Background: Some patients with hypertrophic cardiomyopathy (HCM) re-occur with drug-refractory symptoms but are not eligible for re-operation after the Morrow procedure. Traditional treatment options are limited. We present the first case of the use of ultrasound-guided percutaneous intramyocardial septal radiofrequency ablation (PIMSRA) for the treatment of a patient with HCM combined with congenital anatomically corrected malposition of the great arteries (MGA) after Morrow procedure. Case summary: A 61-year-old male patient with congenital MGA, who had been treated with the Morrow procedure for HCM, had worsening symptoms in recent years that were difficult to control medically. He was diagnosed with occult obstructive HCM by stress echocardiography. After multi-disciplinary discussion, this patient was treated with PIMSRA. The post-operative clinical outcome was remarkable, with a significant decrease in septal thickness and disappearance of the left anterior branch conduction block. Conclusion: Percutaneous intramyocardial septal radiofrequency ablation is feasible and can be one of the options for the treatment of patients with HCM, especially those who cannot choose Morrow procedure. However, it still needs a large sample of clinical trials to validate its clinical effectiveness.

Simultaneous Enhancement of the Thermostability and Catalytic Activity of D-Allulose 3-Epimerase from Clostridium bolteae ATTC BAA-613 Based on the "Back to Consensus Mutations" Hypothesis.

D-Allulose is a high value rare sugar with multiple physiological functions and commercial potential that can be enzymatically synthesized from D-fructose by D-allulose 3-epimerase (DAEase). Poor catalytic activity and thermostability of DAEase prevent the industrial production of D-allulose. In this work, rational design was applied to a previously identified DAEase from Clostridium bolteae ATCC BAA-613 based on the "back to consensus mutations" hypothesis, and the catalytic activity of the Cb-I265 V variant was enhanced 2.5-fold. Furthermore, the Cb-I265 V/E268D double-site variant displayed 2.0-fold higher specific catalytic activity and 1.4-fold higher thermostability than the wild-type enzyme. Molecular docking and kinetic simulation results indicated increased hydrogen bonds between the active pocket and substrate, possibly contributing to the improved thermal stability and catalytic activity of the double-site mutant. The findings outlined a feasible approach for the rational design of multiple preset functions of target enzymes simultaneously.

Exploring the Performance of ChatGPT-4 in the Taiwan Audiologist Qualification Examination: Preliminary Observational Study Highlighting the Potential of AI Chatbots in Hearing Care.

Background: Artificial intelligence (AI) chatbots, such as ChatGPT-4, have shown immense potential for application across various aspects of medicine, including medical education, clinical practice, and research. Objective: This study aimed to evaluate the performance of ChatGPT-4 in the 2023 Taiwan Audiologist Qualification Examination, thereby preliminarily exploring the potential utility of AI chatbots in the fields of audiology and hearing care services. Methods: ChatGPT-4 was tasked to provide answers and reasoning for the 2023 Taiwan Audiologist Qualification Examination. The examination encompassed six subjects: (1) basic auditory science, (2) behavioral audiology, (3) electrophysiological audiology, (4) principles and practice of hearing devices, (5) health and rehabilitation of the auditory and balance systems, and (6) auditory and speech communication disorders (including professional ethics). Each subject included 50 multiple-choice questions, with the exception of behavioral audiology, which had 49 questions, amounting to a total of 299 questions. Results: The correct answer rates across the 6 subjects were as follows: 88% for basic auditory science, 63% for behavioral audiology, 58% for electrophysiological audiology, 72% for principles and practice of hearing devices, 80% for health and rehabilitation of the auditory and balance systems, and 86% for auditory and speech communication disorders (including professional ethics). The overall accuracy rate for the 299 questions was 75%, which surpasses the examination's passing criteria of an average 60% accuracy rate across all subjects. A comprehensive review of ChatGPT-4's responses indicated that incorrect answers were predominantly due to information errors. Conclusions: ChatGPT-4 demonstrated a robust performance in the Taiwan Audiologist Qualification Examination, showcasing effective logical reasoning skills. Our results suggest that with enhanced information accuracy, ChatGPT-4's performance could be further improved. This study indicates significant potential for the application of AI chatbots in audiology and hearing care services.

Label/immobilization-free Cas12a-based electrochemiluminescence biosensor for sensitive DNA detection.

Electrochemiluminescence (ECL) is one of the most sensitive techniques in the field of diagnostics. However, they typically require luminescent labeling and electrode surface biological modification, which is a time-consuming and laborious process involving multiple steps and may also lead to low reaction efficiency. Fabricating label/modification-free biosensors has become one of the most attractive parts for simplifying the ECL assays. In this work, the ECL luminophores carbon dots (CDs) were encapsulated in DNA hydrogel in situ by a simple rolling circle amplification (RCA) reaction. Upon binding of the target DNA, active Cas12a induces a collateral cleavage of the hydrogel's ssDNA backbone, resulting in a programmable degradation of the hydrogel and the release of CDs. By directly measuring the released CDs ECL, a simple and rapid label/modification-free detection of the target HPV-16 was realized. It is noted that this method allowed for 0.63 pM HPV-16 DNA detection without any amplification step, and it could take only ∼60 min for a fast test of a human serum sample. These results showed that our label/modification-free ECL biosensor has great potential for use in simple, rapid, and sensitive point-of-care (POC) detection.