Molecular-level periodic arrays of long-chain poly(3-hexylthiophene-2,5-diyl) driven by an electric field.

Two-dimensional (2D) periodic arrays of conductive polymers represent attractive platforms for wiring functional molecules into the integrated circuits of molecular electronics. However, the large-scale assembly of polymer periodic arrays at the molecular level faces challenges such as curling, twisting, and aggregation. Here, we assembled the periodic arrays of long-chain poly(3-hexylthiophene-2,5-diyl) (P3HT, Mw = 65 k) at the solid-liquid interface by applying an electric field, within which the charged chain segments were aligned. Atomic force microscopy (AFM) imaging revealed that individual P3HT chains assemble into monolayers featuring face-on orientation, extended chain conformation and isolated packing, which is thermodynamically more stable than folded chains in 2D polycrystals. The assembly process is initiated with the formation of disordered clusters and progresses through voltage-dependent nucleation and growth of extended-chain arrays, wherein continuous conformational adjustments along the nucleation pathway exhibit dependence on the cluster size.

An atomic force microscopy and total internal reflection fluorescence microscopy correlated system (AFM-TIRF) for fluorescence imaging and spectroscopy of a single particle.

Combining atomic force microscopy (AFM) with other optical microscopic techniques is pivotal in nanoscale investigations, particularly leveraging the surface-sensitive properties of total internal reflection fluorescence microscopy (TIRF). A novel design that integrates AFM with a multi-wavelength TIRF is displayed, providing simultaneous fluorescence imaging and spectral acquisition capabilities. We elaborate on the considerations in the instrument design process and demonstrate the performance and potential applications of the instrument through fluorescence imaging and spectroscopy testing of individual nanoparticles. This AFM and TIRF correlated system (AFM-TIRF) emerges as a promising option for single-molecule fluorescence studies, enabling simultaneous manipulation and detection of fluorescence from individual molecules.

Rare variations within the serine/arginine-rich splicing factor PtoRSZ21 modulate stomatal size to determine drought tolerance in Populus.

Rare variants contribute significantly to the 'missing heritability' of quantitative traits. The genome-wide characteristics of rare variants and their roles in environmental adaptation of woody plants remain unexplored. Utilizing genome-wide rare variant association study (RVAS), expression quantitative trait loci (eQTL) mapping, genetic transformation, and molecular experiments, we explored the impact of rare variants on stomatal morphology and drought adaptation in Populus. Through comparative analysis of five world-wide Populus species, we observed the influence of mutational bias and adaptive selection on the distribution of rare variants. RVAS identified 75 candidate genes correlated with stomatal size (SS)/stomatal density (SD), and a rare haplotype in the promoter of serine/arginine-rich splicing factor PtoRSZ21 emerged as the foremost association signal governing SS. As a positive regulator of drought tolerance, PtoRSZ21 can recruit the core splicing factor PtoU1-70K to regulate alternative splicing (AS) of PtoATG2b (autophagy-related 2). The rare haplotype PtoRSZ21hap2 weakens binding affinity to PtoMYB61, consequently affecting PtoRSZ21 expression and SS, ultimately resulting in differential distribution of Populus accessions in arid and humid climates. This study enhances the understanding of regulatory mechanisms that underlie AS induced by rare variants and might provide targets for drought-tolerant varieties breeding in Populus.

Association of circulating vitamin levels with thyroid diseases: a Mendelian randomization study.

Background: Previous observational studies have shown conflicting results of vitamins supplementation for thyroid diseases. The causal relationships between vitamins and thyroid diseases are unclear. Therefore, we conducted a two-sample bidirectional Mendelian randomization (MR) study to explore association of circulating vitamin levels with thyroid diseases. Methods: We performed a bidirectional MR analysis using genome-wide association study (GWAS) data. Genetic tool variables for circulating vitamin levels include vitamins A, B9, B12, C, D, and E, Genetic tool variables of thyroid diseases include autoimmune hyperthyroidism, autoimmune hypothyroidism, thyroid nodules (TNs), and Thyroid cancer (TC). Inverse-variance weighted multiplicative random effects (IVW-RE) was mainly used for MR Analysis, weighted median (WM) and MR Egger were used as supplementary methods to evaluate the relationships between circulating vitamin levels and thyroid diseases. Sensitivity and pluripotency were evaluated by Cochran's Q test, MR-PRESSO, Radial MR, MR-Egger regression and leave-one-out analysis. Results: Positive MR evidence suggested that circulating vitamin C level is a protective factor in autoimmune hypothyroidism (ORIVW-RE=0.69, 95%CI: 0.58-0.83, p = 1.05E-04). Reverse MR Evidence showed that genetic susceptibility to autoimmune hyperthyroidism is associated with reduced level of circulating vitamin A(ORIVW-RE = 0.97, 95% CI: 0.95-1.00, p = 4.38E-02), genetic susceptibility of TNs was associated with an increased level of circulating vitamin D (ORIVW-RE = 1.02, 95% CI: 1.00-1.03, p = 6.86E-03). No causal and reverse causal relationship was detected between other circulating vitamin levels and thyroid diseases. Conclusion: Our findings provide genetic evidence supporting a bi-directional causal relationship between circulating vitamin levels and thyroid diseases. These findings provide information for the clinical application of vitamins prevention and treatment of thyroid diseases.

Efficacy and safety of ultrasound-assisted wound debridement in the treatment of diabetic foot ulcers: a systematic review and meta-analysis of 11 randomized controlled trials.

Objective: Research data suggests that ultrasound-assisted wound debridement (UAWD) can effectively promote the healing of diabetic foot ulcers (DFU). However, existing research is not consistent with this viewpoint. Therefore, we conducted this study to investigate the effect of UAWD on the healing of diabetic foot ulcers. Methods: From the establishment of the database to January 2024, we searched 8 databases to study the effectiveness and safety of UAWD in the treatment of DFU. Two authors independently screened the qualifications of the articles, while two authors extracted relevant data. Statistical analysis was conducted using Review Manager 5.4 and STATA 18.0 software. Results: A total of 11 randomized controlled studies were included, with 6 countries and 696 participants participating. Our findings showed that UAWD was associated with a significant benefit in healing rate (OR = 2.60, 95% CI: [1.67, 4.03], P < 0.0001, I2 = 25%), wound healing time (MD = -11.94, 95% CI: [-23.65, -0.23], P = 0.05, I2 = 99%), percentage reduction in wound size (MD = 14.2, 95% CI: [10.8, 17.6], P = 0.47, I2 = 32%), effectiveness of treatment (OR = 10.3, 95% CI: [4.68, 22.66], P < 0.00001, I2 = 0%). Moreover, UAWD did not cause any significant adverse reactions. However, there was no obvious difference in wound blood perfusion (MD = 0.25, 95% CI: [-0.01, 0.52], P = 0.06, I2 = 90%), transcutaneous oxygen partial pressure (MD = 14.34, 95% CI: [-10.03, 38.71], P = 0.25, I2 = 98%). Conclusion: UAWD can significantly improve wound healing rate, shorten wound healing time, accelerate wound area reduction, and improve clinical treatment effectiveness without significant adverse reactions. Although there is no significant difference in transcutaneous oxygen pressure and wound blood flow perfusion between UAWD and SWC. So we look forward to more scientifically blinded, placebo-controlled, high-quality studies in the future, to enable researchers to obtain more complete and accurate analytical data, in order to improve the scientific and credibility of the evidence. Systematic review registration: https://www.crd.york.ac.uk/prospero/, identifier CRD42024501198.

Suppression of circular RNA serum and glucocorticoid-induced kinase 1 elevates antioxidant molecules and angiogenesis in trophoblast cells to attenuate preeclampsia via microRNA-508-3p to target and restrain PUM homolog 1.

AIM: Preeclampsia (PE) is a pregnancy-specific syndrome characterized by hypertension and proteinuria. Recently, multiple circular RNAs (circRNAs) were considered latent clinical diagnostic markers or therapeutic targets. This study was to explore the impact of circRNA serum and glucocorticoid-induced kinase 1 (SGK1) on PE via influencing the microRNA (miR)-508-3p/PUM homolog 1 (PUM1) axis. METHODS: Placental tissues of 34 pregnant women with PE and 34 normal pregnant women were collected to measure circRNA SGK1 (circSGK1), miR-508-3p, and PUM1. Human placental trophoblasts HTR-8/SVneo were transfected with plasmids, thereafter to observe proliferation, migration, invasion, and apoptosis, analyze antioxidant molecules Troxerutin (TXN), Glutamate-cysteine ligase catalytic subunit (GCLC), NAD (P) H-quinone oxidoreductase 1 (NQO1), and determine angiogenesis. After the construction of the PE rat model, antioxidant molecules TXN, GCLC, and NQO1, vascular-associated factor vascular endothelial growth factor A (VEGF-A), and histopathological conditions were tested. Examination of the binding of circSGK1 and miR-508-3p with PUM1 was performed. RESULTS: Our data showed that circSGK1 expression was elevated in the placenta of patients with PE. Silenced circSGK1 or elevated miR-508-3p promoted the growth and antioxidant molecules and angiogenesis in trophoblast cells; CircSGK1 combined with miR-508-3p, and miR-508-3p targeted PUM1. CONCLUSIONS: In summary, suppression of circSGK1 augments antioxidant molecules and angiogenesis in trophoblast cells to attenuate PE via miR-508-3p to target PUM1.

Increasing carbon to nitrogen ratio promoted anaerobic ammonia-oxidizing bacterial enrichment and advanced nitrogen removal in mainstream anammox system.

The effects of fluctuating organic carbon to nitrogen (C/N) ratios on mainstream simultaneous partial nitrification, anammox, and denitrification (SNAD) process were studied over 376-day period. The nitrogen removal efficiency decreased from 85.0 ± 6.6 % to 75.8 ± 2.8 % as C/N ratio decreased (3.4 â†’ 1.7), but increased to 82.0 ± 1.9 % when C/N ratio raised to 2.9 and to 78.4 ± 3.0 % when C/N ratio decreased again (2.9 â†’ 2.1), indicating that high C/N ratios promoted nitrogen removal. As C/N ratio raised (1.7 â†’ 2.9), anaerobic ammonia-oxidizing bacteria (AnAOB) abundance increased from 1.3 × 109 to 2.0 × 109 copies/L, which explained the improved nitrogen removal. With an elevated C/N ratio, partial nitrification and endogenous partial denitrification reactions were enhanced, providing more nitrite for AnAOB. Additionally, the aerobic_chemoheterotrophy function and particle sizes increased, forming more stable anoxic microenvironment for AnAOB. Overall, increasing C/N ratio promoted the stability of mainstream SNAD.

Results of scoliosis screening among primary and middle school students in Chuzhou City / 预防医学

Objective@#To investigate the screening results of adolescent scoliosis in Chuzhou City, Anhui Province, and analyze the influencing factors for scoliosis, so as to provide insights into the prevention and control of scoliosis among adolescents.@*Methods@#Students were selected from six primary and middle schools in Chuzhou City using the stratified random cluster sampling method from April to June 2023. Demographic information, daily behaviors and postures, and exercise status were collected through questionnaire surveys. Scoliosis was screened and diagnosed according to the Screening for Abnormal Spinal Curvature in Children and Adolescents. Influencing factors for scoliosis among primary and middle school students were identified using a multivariable logistic regression model.@*Results@#A total of 1 823 questionnaires were allocated, and 1 768 effective questionnaires were recovered, with an effective response rate of 96.98%. There were 537 primary school students, 1 000 junior high school students and 231 senior high school students, with an average age of (13.40±1.92) years. There were 948 male students (53.62%) and 820 female students (46.38%). A total of 131 cases of scoliosis were screened positive, with a positive rate of 7.41%. The results of multivariable logistic regression analysis showed that gender (female, OR=1.759, 95%CI: 1.135-2.727), body mass index (OR=0.593, 95%CI: 0.538-0.654), sleeping position (side lying, OR=0.598, 95%CI: 0.377-0.951; prone lying, OR=2.336, 95%CI: 1.201-4.545), frequency of reading in bed (often, OR=2.099, 95%CI: 1.201-3.670), way of carrying backpack (shoulders, OR=0.580, 95%CI: 0.370-0.908), and exercise level (OR=0.428, 95%CI: 0.296-0.618) were influencing factors of scoliosis among primary and middle school students.@*Conclusion@#The positive rate of scoliosis screening among primary and middle school students in Chuzhou City was 7.41%, which was influenced by gender, age, body mass index, sleeping posture, reading in bed, way of carrying backpack and exercise level.

One-step, reagent-free construction of nano-enzyme as visual and reusable biosensor for oxidase substrates.

The substrates of oxidase are biologically essential substances that are closely associated with human physiological health. However, current biosensing methods suffer from tough recyclability and undesired denaturation of enzyme due to impurity interference. Herein, we have developed a visual and reusable biosensor for detecting substrate using glucose oxidase (GOx) as a model oxidase. GOx was immobilized onto gold nanoparticles (AuNPs) at -20 °C in one step without additional reagents. The resulting nano-enzyme generated coloimetric signals by coupling with horseradish peroxidase (HRP) using TMB as the substrate. Our results demonstrated that the immobilized GOx exhibited satisfactory sensitivity (0.68 µM) for glucose detection and higher inherent stability than free GOx under harsh conditions, enabling reliable detection of glucose in complex fluids (colored beverages and saliva). Furthermore, the nano-enzyme retained 80 % activity even after four cycles of catalytic oxidation. This strategy constructs a universal biosensor for substrates with nano-enzyme which rely only on intrinsic cysteine within the oxidase while avoiding functional handle modification.

Privately vertically mining of sequential patterns based on differential privacy with high efficiency and utility.

Sequential pattern mining is one of the fundamental tools for many important data analysis tasks, such as web browsing behavior analysis. Based on frequent patterns, decision-makers can obtain both economic gains and social values. Sequential data, on the other hand, frequently contain sensitive information, and directly analyzing these data will raise user concerns from a privacy perspective. Differential privacy (DP), as the most popular privacy model, has been employed to address this privacy concern. Most existing DP-Solutions are designed to combine horizontal sequence pattern mining algorithms with differential privacy. Due to the inefficiency of horizontal algorithms, their DP-Solutions cannot ensure high efficiency and accuracy while offering a high privacy guarantee. Therefore, we proposed privVertical, a new private sequence pattern mining scheme combining the vertical mining algorithm with differential privacy to achieve the above objective. Unlike DP-solutions based on horizontal algorithms, privVertical can promote efficiency by avoiding performing costly database scans or costly projection database constructions. Moreover, to promote accuracy, a differentially private hash MapList (called privHashMap) is designed to record frequent concurrency items and their noisy support based on the Sparse Vector Technique. PrivHashMap is used to pre-pruning excessive infrequent candidate sequences in private mining, and Sparse Vector Technique is used to promote the accuracy of PrivHashMap. After pruning these invalid candidate sequences, less noise is required to achieve the same level of privacy, increasing the accuracy of private mining. Theoretical privacy analysis proves privVertical satisfies [Formula: see text]-differential privacy. Experiments show that privVertical achieves higher accuracy and efficiency while achieving the same privacy level.

Research progress on MOFs and their derivatives as promising and efficient electrode materials for electrocatalytic hydrogen production from water.

Hydrogen energy is considered to be the most potential "ultimate energy source" due to its high combustion calorific value, cleanliness, and pollution-free characteristics. Furthermore, the production of hydrogen via the electrolysis of water has the advantages of simplicity, high efficiency, environmentally safe, and high-purity hydrogen. However, it is also associated with issues such as high-power consumption for the reaction and limited large-scale application of noble metal catalysts. Metal-organic frameworks (MOFs) are porous composite materials composed of metal ions and organic functional groups through orderly coordination with large specific surface areas and large porosity. Herein, we focus on the research status of MOFs and their transition metal derivatives for electrocatalytic water splitting to produce hydrogen and briefly describe the reaction mechanism and evaluation parameters of the electrocatalytic hydrogen evolution and oxygen evolution reactions. Furthermore, the relationship between the catalytic behavior and catalytic activity of different MOF-based catalysts and their morphology, elemental composition, and synthetic strategy is analyzed and discussed. The reasons for the excellent activity and poor stability of the original MOF materials for the electrolysis of water reaction are shown through analysis, and using various means to improve the catalytic activity by changing the electronic structure, active sites, and charge transfer rate, MOF-based catalysts were obtained. Finally, we present perspectives on the future development of MOFs for the electrocatalytic decomposition of water.

Increasing specific biomass nitrogen load facilitated nitrite accumulation in mainstream wastewater treatment.

By regulating influent nitrogen loading rate (NLR) and solids retention time (SRT), the effect of specific biomass nitrogen load (BNL) on the start-up of mainstream partial nitrification (PN) was investigated in five parallel sequencing batch reactors inoculated with ordinary nitrification sludge. The results showed that increasing BNL by both methods could achieve nitrite accumulation. Moreover, a high initial activity of ammonium oxidizing bacteria (AOB) accelerated nitrite accumulation. Increasing influent NLR (ammonium: 55-70 mg N/L) achieved only 30%-40% of nitrite accumulation ratio (NAR) and gradually decreased with reactor operation. By increasing BNL via controlling SRT (30 days), desirable PN with an average NAR of 81.7 ± 4.4% (effluent nitrite: ∼10 mg N/L) was obtained. Nitrite oxidizing bacteria (NOB) were effectively inhibited, and the AOB to NOB activity ratio increased from 1.5 to 7.8, promoting efficient nitrite accumulation. Overall, increasing BNL by regulating SRT was a potential method for start-up of mainstream PN.

Comprehensive Assessment of 16S rRNA Gene Amplicon Sequencing for Microbiome Profiling across Multiple Habitats.

The 16S rRNA gene works as a rapid and effective marker for the identification of microorganisms in complex communities; hence, a huge number of microbiomes have been surveyed by 16S amplicon-based sequencing. The resolution of the 16S rRNA gene is always considered only at the genus level; however, it has not been verified on a wide range of microbes yet. To fully explore the ability and potential of the 16S rRNA gene in microbial profiling, here, we propose Qscore, a comprehensive method to evaluate the performance of amplicons by integrating the amplification rate, multitier taxonomic annotation, sequence type, and length. Our in silico assessment by a "global view" of 35,889 microbe species across multiple reference databases summarizes the optimal sequencing strategy for 16S short reads. On the other hand, since microbes are unevenly distributed according to their habitats, we also provide the recommended configuration for 16 typical ecosystems based on the Qscores of 157,390 microbiomes in the Microbiome Search Engine (MSE). Detailed data simulation further proves that the 16S amplicons produced with Qscore-suggested parameters exhibit high precision in microbiome profiling, which is close to that of shotgun metagenomes under CAMI metrics. Therefore, by reconsidering the precision of 16S-based microbiome profiling, our work not only enables the high-quality reusability of massive sequence legacy that has already been produced but is also significant for guiding microbiome studies in the future. We have implemented the Qscore as an online service at http://qscore.single-cell.cn to parse the recommended sequencing strategy for specific habitats or expected microbial structures. IMPORTANCE 16S rRNA has long been used as a biomarker to identify distinct microbes from complex communities. However, due to the influence of the amplification region, sequencing type, sequence processing, and reference database, the accuracy of 16S rRNA has not been fully verified on a global range. More importantly, the microbial composition of different habitats varies greatly, and it is necessary to adopt different strategies according to the corresponding target microbes to achieve optimal analytical performance. Here, we developed Qscore, which evaluates the comprehensive performance of 16S amplicons from multiple perspectives, thus providing the best sequencing strategies for common ecological environments by using big data.

Flex Meta-Storms elucidates the microbiome local beta-diversity under specific phenotypes.

MOTIVATION: Beta-diversity quantitatively measures the difference among microbial communities thus enlightening the association between microbiome composition and environment properties or host phenotypes. The beta-diversity analysis mainly relies on distances among microbiomes that are calculated by all microbial features. However, in some cases, only a small fraction of members in a community plays crucial roles. Such a tiny proportion is insufficient to alter the overall distance, which is always missed by end-to-end comparison. On the other hand, beta-diversity pattern can also be interfered due to the data sparsity when only focusing on nonabundant microbes. RESULTS: Here, we develop Flex Meta-Storms (FMS) distance algorithm that implements the "local alignment" of microbiomes for the first time. Using a flexible extraction that considers the weighted phylogenetic and functional relations of microbes, FMS produces a normalized phylogenetic distance among members of interest for microbiome pairs. We demonstrated the advantage of FMS in detecting the subtle variations of microbiomes among different states using artificial and real datasets, which were neglected by regular distance metrics. Therefore, FMS effectively discriminates microbiomes with higher sensitivity and flexibility, thus contributing to in-depth comprehension of microbe-host interactions, as well as promoting the utilization of microbiome data such as disease screening and prediction. AVAILABILITY AND IMPLEMENTATION: FMS is implemented in C++, and the source code is released at https://github.com/qdu-bioinfo/flex-meta-storms.

Increasing the Mechanical Stability of Polymer-Gold Interfacial Connection: A Parallel Covalent Strategy.

Thiol-gold (S-Au) chemistry has been widely used in coating and functionalizing gold surfaces because it is robust and highly efficient. However, recent studies have shown that the S-Au-based self-assembled monolayers can lead to significant instability under external mechanical loading (e.g., in a swelled polymer film). Such instability limits further applications of S-Au chemistry-based functional materials. Here, we report a surface-modifying procedure based on a parallel covalent strategy. By employing dendritic macromolecules as a "middle layer" between the gold surface and polymer, the interfacial connecting strength increased by at least 350% as revealed by atomic force microscopy-based single molecule force spectroscopy (AFM-SMFS). The ultimate cleavage structure is confirmed to be an amide bond by control SMFS experiments, fluorescent microscopy, and dynamic force spectroscopy. This study/concept paves the way to prepare stable stimuli-responsive polymer brushes on solid surfaces and study mechanophores with high force stability.

Supramolecular Exosome Array for Efficient Capture and In Situ Detection of Protein Biomarkers.

Exosomes are an emerging source for disease biomarker discovery due to the high stability of proteins protected by phospholipid bilayers. However, liquid biopsy with exosomes remains challenging due to the extreme complexity of biological samples. Herein, we introduced an amphiphile-dendrimer supramolecular probe (ADSP) for the efficient capture and high-throughput analysis of exosomes, enabling the array-based assay for marker proteins. Amphiphilic amphotericin B was functionalized onto highly branched globular dendrimers, which can then insert into the exosome membrane efficiently, forming a supramolecular complex through multivalent interactions between the probe and the bilayer of exosomes. The ADSP can be easily coated onto magnetic beads or the nitrocellulose membrane, facilitating the capture of exosomes from a minimum amount of clinical samples. The captured exosomes can be detected with target protein antibodies via Western blotting or in a high-throughput array-based dot blotting format. This new strategy exhibited excellent extraction capability from trace body fluids with superior sensitivity (less than 1 µL plasma), good quantitation ability (R2 > 0.99), and high throughput (96 samples in one batch) using clinical plasma samples. The combination of proteomics and ADSP will provide a platform for the discovery and validation of protein biomarkers for cancer diagnosis and prognosis.

The influence of verapamil on the pharmacokinetics of the pan-HER tyrosine kinase inhibitor neratinib in rats: the role of P-glycoprotein-mediated efflux.

Neratinib, an irreversible pan-HER tyrosine kinase inhibitor, has been approved for the treatment of HER2-positive (HER2+) early-stage and brain metastatic breast cancer. Thus far, the pharmacology effects and pharmacodynamics of neratinib have been well studied. However, the disposition of neratinib and its influencing factors in vivo remain unclear. P-glycoprotein (P-gp), one of the most extensively studied transporters, substantially restricts penetration of drugs into the body or deeper compartments (i.e., blood-brain barrier, BBB), regarding drug resistance and drug-drug interactions. Thereby, the aim of this study was to investigate the influence of verapamil (a P-gp inhibitor) on the pharmacokinetics of neratinib in rats. Here, we have established a high specific, selective and sensitive ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify plasma concentrations of neratinib in rats. Pharmacokinetic results showed that verapamil significantly increased the system exposure of neratinib, as Cmax increased by 2.09-fold and AUC0-t increased by 1.64-fold, respectively. Additionally, the in vitro transport of neratinib was evaluated using Madin-Darby canine kidney II (MDCK II) and human MDR1 gene overexpressed MDCK (MDCK-MDR1) cell line models. As a result, the net flux ratio was over than 2 and decreased over 50% by verapamil, suggesting that neratinib was a substrate of P-gp. Hence, our findings have highlighted the important role of P-gp in the system exposure of neratinib in vivo, and drug-drug interaction should be considered when coadministration of P-gp inhibitors with neratinib. These findings may support the further clinical development and application of neratinib.

Exploring the impact of PEGylation on the cell-nanomicelle interactions by AFM-based single-molecule force spectroscopy and force tracing.

PEGylation has been considered the gold standard method for the modification of various drug delivery systems since the last century. However, the impact of PEGylation on the dynamic interaction between drug carriers and cell membranes has not been quantitatively clarified. Herein, the cellular binding and receptor-mediated endocytosis of a model PEGylated polypeptide nanomicelle were systematically investigated at the single-particle level using AFM-based single-molecule force spectroscopy (SMFS) and force tracing. A self-assembled elastin-like polypeptide (ELP) nanomicelle, which is capable of cross-linking, gastrin-releasing peptide (GRP) modification, and PEGylation was prepared. The cross-linked ELP-based nanomicelles exhibited outstanding stability in a broad temperature range of 4-40 °C, which facilitate the drug loading, as well as our cell-nanomicelle study at the single particle level. The unbinding force between the cross-linked ELP-based nanomicelles and the GRP receptor (GRPR)-containing cell (PC-3) membranes was quantitatively measured by AFM-SMFS. It is found that the PEGylated GRP-displaying nanomicelles exhibit the highest unbinding force, indicating the enhanced specific binding effect of PEGylation. Furthermore, the receptor-mediated endocytosis of the cross-linked ELP-based nanomicelles was monitored with the help of force tracing based on AFM-SMFS. Our results show that PEGylation decreases the endocytic force, duration, and engulfment depth of the PEGylated GRP-displaying nanomicelles, but increases their endocytic velocity, which results from the elimination of non-specific interactions during endocytosis. These observations demonstrate the diverse and complex roles of PEGylation on the interaction of polypeptide nanomicelles to cell membranes and may shed light on the rational design of organic polymer-based drug delivery systems aiming for active and passive targeting strategies. STATEMENT OF SIGNIFICANCE: A self-assembled elastin-like polypeptide (ELP) nanomicelle, which can be easily cross-linked, gastrin-releasing peptide (GRP) modified, and PEGylated, is designed. The AFM-SMFS experiment shows that PEGylation can enhance specific binding of the nanomicelles to the receptors on cell membranes. The force tracing experiment indicates that PEGylation decreases the endocytic force as well as engulfment depth of the nanomicelles through the elimination of non-specific interactions. PEGylation can benefit the drug delivery systems aiming at active targeting, while might not be an ideal modification for drug carriers designed for passive targeting, whose cellular uptake mainly depends on non-specific interactions.

O-Sulfation disposition of curcumin and quercetin in SULT1A3 overexpressing HEK293 cells: the role of arylsulfatase B in cellular O-sulfation regulated by transporters.

Extensive phase II metabolic reactions (i.e., glucuronidation and sulfation) have resulted in low bioavailability and decreased biological effects of curcumin and quercetin. Compared to glucuronidation, information on the sulfation disposition of curcumin and quercetin is limited. In this study, we identified that BCRP and MRP4 played a critical role in the cellular excretion of curcumin-O-sulfate (C-O-S) and quercetin-O-sulfate (Q-O-S) by integrating chemical inhibition with transporter knock-down experiments. Inhibited excretion of sulfate (C-O-S and Q-O-S) caused significant reductions in cellular O-sulfation of curcumin (a maximal 74.4% reduction) and quercetin (a maximal 76.9% reduction), revealing a strong interplay of sulfation with efflux transport. It was further identified that arylsulfatase B (ARSB) played a crucial role in the regulation of cellular O-sulfation by transporters. ARSB overexpression significantly enhanced the reduction effect of MK-571 on the cellular O-sulfation (fmet) of the model compound (38.8% reduction for curcumin and 44.2% reduction for quercetin). On the contrary, ARSB knockdown could reverse the effect of MK-571 on the O-sulfation disposition of the model compound (29.7% increase for curcumin and 47.3% increase for quercetin). Taken together, ARSB has been proven to be involved in cellular O-sulfation, accounting for transporter-dependent O-sulfation of curcumin and quercetin. A better understanding of the interplay beneath metabolism and transport will contribute to the exact prediction of in vivo drug disposition and drug-drug interactions.

Cinobufotalin inhibits the epithelial-mesenchymal transition of hepatocellular carcinoma cells through down-regulate ß-catenin in vitro and in vivo.

Hepatocellular carcinoma (HCC) is one of the malignant tumors with high incidence and mortality. The prognosis of HCC is poor due to the high postoperative recurrence rate and metastasis rate. Epithelial-mesenchymal transition (EMT) plays a key role in the metastasis of HCC, which is closely related to the invasion, intrahepatic metastasis and low survival rate. Here we demonstrated that cinobufotalin can upregulate epithelial markers (E-cadherin) and downregulate mesenchymal markers (N-cadherin, snail, slug and ZEB1) in HepG2, SMMC-7721 and SNU-368 cells. We further found that the mRNA and protein expression of ß-catenin and its target genes (i.e. MMP7 and DKK1), which are related to tumor invasion and metastasis, were decreased after cinobufotalin treatment. Overexpression of ß-catenin promoted EMT of HepG2 and SMMC-7721 cells, and cinobufotalin could antagonize this induction. While Knockdown of ß-catenin could inhibit EMT and cinobufotalin enhanced this inhibition. In addition, cinobufotalin significantly suppressed the tumor EMT, as demonstrated by increased E-cadherin expression and decreased N-cadherin and vimentin expression, and inhibited formation and metastasis of lung metastases in vivo. In conclusion, our study has revealed a novel anticancer mechanism of cinobufotalin, which inhibits EMT progress by downregulating ß-catenin, and then prevents the migration and invasion of HCC. These results provide convincing evidence for the development of cinobufotalin as a potential HCC metastasis inhibitor.