Liquid-Liquid Phase Separation of DDR1 Counteracts the Hippo Pathway to Orchestrate Arterial Stiffening.

BACKGROUND: The Hippo-YAP (yes-associated protein) signaling pathway is modulated in response to various environmental cues. Activation of YAP in vascular smooth muscle cells conveys the extracellular matrix stiffness-induced changes in vascular smooth muscle cells phenotype and behavior. Recent studies have established a mechanoreceptive role of receptor tyrosine kinase DDR1 (discoidin domain receptor 1) in vascular smooth muscle cells. METHODS: We conduced 5/6 nephrectomy in vascular smooth muscle cells-specific Ddr1-knockout mice, accompanied by pharmacological inhibition of the Hippo pathway kinase LATS1 (large tumor suppressor 1), to investigate DDR1 in YAP activation. We utilized polyacrylamide gels of varying stiffness or the DDR1 ligand, type I collagen, to stimulate the cells. We employed multiple molecular biological techniques to explore the role of DDR1 in controlling the Hippo pathway and to determine the mechanistic basis by which DDR1 exerts this effect. RESULTS: We identified the requirement for DDR1 in stiffness/collagen-induced YAP activation. We uncovered that DDR1 underwent stiffness/collagen binding-stimulated liquid-liquid phase separation and co-condensed with LATS1 to inactivate LATS1. Mutagenesis experiments revealed that the transmembrane domain is responsible for DDR1 droplet formation. Purified DDR1 N-terminal and transmembrane domain was sufficient to drive its reversible condensation. Depletion of the DDR1 C-terminus led to failure in co-condensation with LATS1. Interaction between the DDR1 C-terminus and LATS1 competitively inhibited binding of MOB1 (Mps one binder 1) to LATS1 and thus the subsequent phosphorylation of LATS1. Introduction of the single-point mutants, histidine-745-proline and histidine-902-proline, to DDR1 on the C-terminus abolished the co-condensation. In mouse models, YAP activity was positively correlated with collagen I expression and arterial stiffness. LATS1 inhibition reactivated the YAP signaling in Ddr1-deficient vessels and abrogated the arterial softening effect of Ddr1 deficiency. CONCLUSIONS: These findings identify DDR1 as a mediator of YAP activation by mechanical and chemical stimuli and demonstrate that DDR1 regulates LATS1 phosphorylation in an liquid-liquid phase separation-dependent manner.

A dissipative particle dynamics simulation of controlled loading and responsive release of theranostic agents from reversible crosslinked triblock copolymer vesicles.

To control the transport stability and release efficiency of loaded theranostic drugs in triblock copolymer carriers, the reversible crosslinking ability is of great significance. A molecular level exploration of such a function is needed to extend existing stabilizing and responsive dissociation mechanisms of carriers. Here, dissipative particle dynamics simulations were used to first demonstrate the formation of triblock copolymer vesicular carriers. Chemical crosslinking was used to strengthen the structural stability of the vesicle shell to avoid drug leakage. Reversible decrosslinking along with dissociation of the vesicle and release of loaded drugs were then explored. The structural, energetic and dynamical properties of the system were discussed at the molecular level. The regulation mechanism of drug release patterns was revealed by systematically exploring the effect of intra and intermolecular repulsive interactions. The results indicate that the chemical crosslinking of copolymers enhanced the compactness of the vesicle shell with a strengthened microstructure, increased binding energy, and limited chain migration, thus achieving more stable delivery of drugs. In terms of drug release, we clarified how the pairwise interactions of beads in the solution system affect the responsive dissociation of the vesicle and associated release patterns (speed and amount) of drugs. More efficient delivery and smart release of theranostic drugs are achieved using such reversible crosslinked triblock copolymer vesicles.

A Participation Degree-Based Fault Detection Method for Wireless Sensor Networks.

In wireless sensor networks (WSNs), there are many challenges for outlier detection, such as fault detection, fraud detection, intrusion detection, and so on. In this paper, the participation degree of instances in the hierarchical clustering process infers the relationship between instances. However, most of the existing algorithms ignore such information. Thus, we propose a novel fault detection technique based on the participation degree, called fault detection based on participation degree (FDP). Our algorithm has the following advantages. First, it does not need data training in labeled datasets; in fact, it uses the participation degree to measure the differences between fault points and normal points without setting distance or density parameters. Second, FDP can detect global outliers without local cluster influence. Experimental results demonstrate the performance of our approach by applying it to synthetic and real-world datasets and contrasting it with four well-known techniques: isolation forest (IF), local outlier factor (LOF), one-class support vector machine (OCS), and robust covariance (RC).

A Study of the Relationship Between the Severity of Posterior Capsular Opacification Detected by Objective Detection Techniques and Visual Acuity.

PURPOSE: To explore the severity of posterior capsule opacification (PCO) using objective detection techniques and its relationship with visual acuity. SETTING: The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China. DESIGN: Prospective cohort study. METHODS: All patients underwent slit-lamp examination, intraocular pressure measurement (IOP), best-corrected visual acuity (BCVA) before neodymium: yttrium aluminium garnet (Nd:YAG) laser capsulotomy, followed by examination after fully dilated, including IOLMaster 700, optical coherence tomography (OCT), Sirius anterior segment analysis system (Sirius), color fundus photography (CFP). Conducting BCVA and IOP post-treatment again. Recording the thickness and density of posterior capsule, color fundus photography quality (CFPQ) and OCT Signal Strength (OCTSS). Analysis using Spearman correlation analysis, heatmaps, and ROC curves. RESULTS: A total of 83 eyes in 78 patients were included in this study. Spearman correlation analysis revealed correlations between pre-treatment BCVA and IOLMaster 700 PCO thickness (MT), IOLMaster 700 cumulative effect (MCE), Sirius PCO thickness (ST), Sirius maximum density (SMD), Sirius cumulative effect (SCE), OCTSS, and CFPQ (correlation coefficients were 0.500, 0.484, 0.465, -0.256, 0.317, -0.442, -0.412, all P＜0.05). The improvement of Vision Acuity (ImpVA) showed correlations with MT, MCE, ST, SCE, OCTSS, and CFPQ (correlation coefficients were -0.452, -0.471, -0.346, -0.278, 0.320, 0.381, all P＜0.05). For ImpVA, the predictive ability of IOLMaster 700 was superior to Sirius, and the joint model was significantly better than single factors. CONCLUSIONS: Posterior capsule thickness and cumulative effect were reliable indicators for evaluating PCO. Compared to Sirius, IOLMaster 700 demonstrates superior predictive ability and higher correlation.

Endothelial discoidin domain receptor 1 senses flow to modulate YAP activation.

Mechanotransduction in endothelial cells is critical to maintain vascular homeostasis and can contribute to disease development, yet the molecules responsible for sensing flow remain largely unknown. Here, we demonstrate that the discoidin domain receptor 1 (DDR1) tyrosine kinase is a direct mechanosensor and is essential for connecting the force imposed by shear to the endothelial responses. We identify the flow-induced activation of endothelial DDR1 to be atherogenic. Shear force likely causes conformational changes of DDR1 ectodomain by unfolding its DS-like domain to expose the buried cysteine-287, whose exposure facilitates force-induced receptor oligomerization and phase separation. Upon shearing, DDR1 forms liquid-like biomolecular condensates and co-condenses with YWHAE, leading to nuclear translocation of YAP. Our findings establish a previously uncharacterized role of DDR1 in directly sensing flow, propose a conceptual framework for understanding upstream regulation of the YAP signaling, and offer a mechanism by which endothelial activation of DDR1 promotes atherosclerosis.

Echinatin maintains glutathione homeostasis in vascular smooth muscle cells to protect against matrix remodeling and arterial stiffening.

Decreased vascular compliance of the large arteries as indicated by increased pulse wave velocity is shown to be associated with atherosclerosis and the related cardiovascular events. The positive correlation between arterial stiffening and disease progression derives a hypothesis that softening the arterial wall may protect against atherosclerosis, despite that the mechanisms controlling the cellular pathological changes in disease progression remain unknown. Here, we established a mechanical-property-based screening to look for compounds alleviating the arterial wall stiffness through their actions on the interaction between vascular smooth muscle cells (VSMCs) and the wall extracellular matrix (ECM). We found that echinatin, a chalcone preferentially accumulated in roots and rhizomes of licorice (Glycyrrhiza inflata), reduced the stiffness of ECM surrounding cultured VSMCs. We examined the potential beneficial effects of echinatin on mitigating arterial stiffening and atherosclerosis, and explored the mechanistic basis by which the compound exert the effects. Administration of echinatin in mice fed on an adenine diet and in hyperlipidemia mice subjected to 5/6 nephrectomy mitigated arterial stiffening and atherosclerosis. Mechanistic insights were gained from the RNA-sequencing results showing that echinatin upregulated the expression of glutamate cysteine ligases (GCLs), both the catalytic (GCLC) and modulatory (GCLM) subunits. Further study indicated that upregulation of GCLC/GCLM in VSMCs by echinatin maintains the homeostasis of glutathione (GSH) metabolism; adequate availability of GSH is critical for counteracting arterial stiffening. As a consequence of regulating the GSH synthesis, echinatin inhibits ferroptosis and matrix remodeling that being considered two contributors of arterial stiffening and atherosclerosis. These data demonstrate a pivotal role of GSH dysregulation in damaging the proper VSMC-ECM interaction and uncover a beneficial activity of echinatin in preventing vascular diseases.

Amylose-3,5-dimethylphenylcarbamate immobilized on monolithic silica stationary phases for chiral separations in capillary electrochromatography.

Chiral monolithic silica capillary columns were prepared by immobilization of amylose-3,5-dimethylphenylcarbamate (ADMPC) bearing a small fraction of 3-(triethoxysilyl)propyl residues through intermolecular polycondensation of the triethoxysilyl groups. The obtained columns were used for chiral separations in capillary electrochromatography (CEC). The effects of the silica monolith nature and the used chiral selector concentration on the resulting enantiomeric separations were investigated. Fifteen chiral compounds, including acidic, neutral, and basic substances were evaluated and twelve showed partial or baseline separation at some of the different conditions tested. These results demonstrated the promising applicability of ADMPC-immobilized monolithic silica columns in CEC enantioseparations, but also revealed the need for further improvements on the level of baseline separations and efficiencies.

Clinical and mechanism advances of neuronal intranuclear inclusion disease.

Due to the high clinical heterogeneity of neuronal intranuclear inclusion disease (NIID), it is easy to misdiagnose this condition and is considered to be a rare progressive neurodegenerative disease. More evidence demonstrates that NIID involves not only the central nervous system but also multiple systems of the body and shows a variety of symptoms, which makes a clinical diagnosis of NIID more difficult. This review summarizes the clinical symptoms in different systems and demonstrates that NIID is a multiple-system intranuclear inclusion disease. In addition, the core triad symptoms in the central nervous system, such as dementia, parkinsonism, and psychiatric symptoms, are proposed as an important clue for the clinical diagnosis of NIID. Recent studies have demonstrated that expanded GGC repeats in the 5'-untranslated region of the NOTCH2NLC gene are the cause of NIID. The genetic advances and possible underlying mechanisms of NIID (expanded GGC repeat-induced DNA damage, RNA toxicity, and polyglycine-NOTCH2NLC protein toxicity) are briefly summarized in this review. Interestingly, inflammatory cell infiltration and inflammation were observed in the affected tissues of patients with NIID. As a downstream pathological process of NIID, inflammation could be a therapeutic target for NIID.

Recent Progress in Electrospun Nanofiber-Based Degenerated Intervertebral Disc Repair.

Annulus fibrosus fissure and fibrosis of nucleus pulposus are severe morphological characteristics of intervertebral disc degeneration. Currently, surgery or drugs are used to relieve pain in such cases. Tissue engineering is a new multidisciplinary strategy with great potential for use in joint replacement and organ regeneration. Based on the natural anatomy of intervertebral discs, intervertebral disc scaffolds are fabricated by exploiting the special arrangement of extracellular matrix fibers. Electrospun nanofibers possess clear advantages in repairing degenerated intervertebral discs. This article reviews and summarizes recently developed methods for improving and fabricating electrospun nanofiber annulus fibrosus scaffolds in terms of nanofiber alignment, material selection, loading additives, and the progress made in combining other advanced technologies with electrospun nanofibers. In addition, the improvement in mechanical properties and biocompatibility of nucleus pulposus scaffolds by electrospun nanofiber-reinforced hydrogels is discussed. Finally, complete intervertebral disc scaffolds can be fabricated using the disc-like angle-ply structure and other emerging fabrication methods. Taken together, electrospun nanofiber intervertebral disc scaffolds are promising for clinical applications.

Geometric Constraints Regulate Energy Metabolism and Cellular Contractility in Vascular Smooth Muscle Cells by Coordinating Mitochondrial DNA Methylation.

Vascular smooth muscle cells (SMCs) can adapt to changes in cellular geometric cues; however, the underlying mechanisms remain elusive. Using 2D micropatterned substrates to engineer cell geometry, it is found that in comparison with an elongated geometry, a square-shaped geometry causes the nuclear-to-cytoplasmic redistribution of DNA methyltransferase 1 (DNMT1), hypermethylation of mitochondrial DNA (mtDNA), repression of mtDNA gene transcription, and impairment of mitochondrial function. Using irregularly arranged versus circumferentially aligned vascular grafts to control cell geometry in 3D growth, it is demonstrated that cell geometry, mtDNA methylation, and vessel contractility are closely related. DNMT1 redistribution is found to be dependent on the phosphoinositide 3-kinase and protein kinase B (AKT) signaling pathways. Cell elongation activates cytosolic phospholipase A2, a nuclear mechanosensor that, when inhibited, hinders AKT phosphorylation, DNMT1 nuclear accumulation, and energy production. The findings of this study provide insights into the effects of cell geometry on SMC function and its potential implications in the optimization of vascular grafts.

Enantioselectivity of monolithic silica stationary phases immobilized with different concentrations cellulose tris (3,5-dimethylphenylcarbamate), analyzed with different mobile phases in capillary electrochromatography.

The 3,5-dimethylphenylcarbamate derivatives of cellulose bearing 3-(triethoxysilyl)propyl residues were immobilized in a capillary format onto a monolithic silica support by intermolecular polycondensation of the triethoxysilyl groups. The resulting columns were used for chiral separations using capillary electrochromatography. The effects of the synthesizing solvent, the selector coating procedure, the chiral selector concentration onto the silica monolith and the mobile phase pH value, on the separation of enantiomers were studied. The column-to-column reproducibility and stability also were evaluated. A test set of 14 chiral substances, including acidic, neutral, bifunctional and basic compounds, was used to investigate the effects of the factors mentioned above. Twelve pairs of enantiomers showed enantioselectivity at some of the different conditions tested. The column-to-column repeatability was satisfactory, and the prepared columns were stable under the adopted analysis conditions.

Number of and distance between response elements in Kaposi's sarcoma-associated herpesvirus ORF57 promoter influence its activation by replication and transcription activator and its repression by interferon regulatory factor 7.

Kaposi's sarcoma-associated herpesvirus ORF57 expression is highly responsive to replication and transcription activator (RTA) and interferon regulatory factor 7 (IRF-7). Three RTA response elements (RREs) have been identified in the ORF57 promoter. Here, we show evidence of another functional RRE located between nt 82003 and 82081, which can complement the loss of RTA activation resulting from RRE1 deletion. Repeats of a recombination signal-binding protein Jkappa (RBP-Jkappa) site enhanced RTA activation, which could not be suppressed by IRF-7. Alteration of the distance between the RBP-Jkappa site and RRE2 modulated responsiveness to RTA and IRF-7. These results will help to elucidate the precise regulation of viral gene expression.

High-performance liquid chromatographic evaluation of a coated cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase having a small-pore silica support.

A two-step coating/precipitation synthetic procedure has been developed for the preparation of cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase (CSP) having a small-pore silica support. With this synthetic strategy, monodisperse, spherical CSP particles can be produced without the need for a wasteful and time-consuming sieving process. The performance of the synthesized CSP towards a variety of racemates was evaluated in the normal-phase HPLC mode. HPLC separation experiments revealed that the synthesized CSP exhibited a chiral recognition ability fully comparable to the corresponding commercial columns prepared using conventional large-pore silica as the support. Moreover, the synthesized CSP was successfully applied to semipreparative enantioseparation of a new triazole antifungal agent.

In Situ Observation of Light Illumination-Induced Degradation in Organometal Mixed-Halide Perovskite Films.

Organometal mixed-halide perovskite materials hold great promise for next-generation solar cells, light-emitting diodes, lasers, and photodetectors. Except for the rapid progress in the efficiency of perovskite-based devices, the stability issue over prolonged light illumination has severely hindered their practical application. The deterioration mechanism of organometal halide perovskite materials under light illumination has seldom been conducted to date, which is indispensable to the understanding and optimization of photon-harvesting process inside perovskite-based optoelectronic devices. Here, explicit degradation pathways and comprehensive microscopic understandings of white-light-induced degradation have been put forward for two organometal mixed-halide perovskite materials (e.g., MAPbI3-xClx and MAPbBr3-xClx) under high vacuum conditions. In situ compositional analysis and real-time film characterizations reveal that the decomposition of both mixed-halide perovskites starts at the grain boundaries, leading to the formation of hydrocarbons and ammonia gas with the residuals of PbI2(Cl), Pb, or PbClxBr2-x in the films. The degradation has been correlated to the localized trap states that induce strong coupling between photoexcited carriers and the crystal lattice.

Synthesis of chiral stationary phases with radical polymerization reaction of cellulose phenylcarbamate derivatives and vinylized silica gel.

Cellulose phenylcarbamate derivatives having methacrylate groups were synthesized with regioselective and non-regioselective procedures. These derivatives were chemically immobilized onto a vinylized silica gel, respectively, via a radical co-polymerization reaction. The immobilization was efficiently attained using a small amount of AIBN. The chiral recognition abilities of the prepared chiral stationary phases (CSPs) were evaluated by HPLC resolution of test enantiomers. It was observed that most of the enantiomers were completely resolved with markedly high column efficiency of 30,000-40,000 plates per metre for the eluted peaks. The effect of the amount of methacrylolyl chloride used for preparation on resolution was investigated. A direct comparison of the chiral recognition ability was made on the regioselectively and non-regioselectively prepared CSPs. In addition, the chemically bonded-type of CSPs were found to be relatively stable with addition of solvents such as tetrahydrofuran (THF) and chloroform into the mobile phase, which can lead to the dissolution of cellulose derivatives on the coated CSPs. Thus the choice of solvents used as the mobile phase is greatly extended and better resolution of several test enantiomers was observed on the prepared CSPs with THF and chloroform as a composition in the mobile phase. The batch-to-batch and run-to-run reproducibility was also discussed on the newly prepared CSPs.

Synthesis of covalently bonded cellulose derivative chiral stationary phases with a bifunctional reagent of 3-(triethoxysilyl)propyl isocyanate.

A bifunctional reagent of 3-(triethoxysilyl)propyl isocyanate (TEPI) was initially adopted as a spacer reagent to prepare the bonded types of chiral stationary phases (CSPs) with cellulose derivatives. The silica-based CSPs were chemically prepared with non-regioselective and regioselective approaches and their chiral resolving capabilities were evaluated in terms of HPLC resolution of test enantiomers. It was observed that the chiral recognition capabilities of the non-regioselectively prepared CSPs were influenced by the amount of TEPI used. And also, the regioselectively prepared CSP generally showed a slightly higher resolution power than the non-regioselectively prepared CSP, while the non-regioselective procedures were highly advantageous to rapid preparation. In addition, chiral recognition of the prepared CSPs was affected by the properties of the used silica matrices.

Short columns with molecularly imprinted monolithic stationary phases for rapid separation of diastereomers and enantiomers.

Three molecularly imprinted monolithic columns with different length but almost identical column volume had been prepared. It was observed that the separation factors of diastereomers and enantiomers were almost unaffected by column length. However, the short column with dimension of 38 mm x 8 mm i.d. showed much lower resistance to flow rate so that it could be operated at much higher flow rates. By combining stepwise gradient elution with elevated flow rate, the diastereomers of cinchonine and cinchonidine and the enantiomers of Cbz-DL-Trp and Fmoc-DL-Trp were successfully separated within 3 min on the short column with dimension of 38 mm x 8 mm i.d. Based on the above results, a cinchonine imprinted monolithic disk with dimension of 10mm x 16 mm i.d. was further developed. The SEM image and the pore size distribution profile showed that large flow-through pores are present on the prepared monolith, which allowed mobile phase to flow through the disk with very low resistance. Chromatographic performances on the monolithic disk were almost unchanged compared with the long columns. A rapid separation of cinchonine and cinchonidine was achieved in 2.5 min at the flow rate of 9.0 ml/min. Furthermore, it was observed that there was almost no effect of the flow rate on the dynamic binding capacity at high flow rates. In addition, the effect of the loading concentration of analytes on the dynamic binding capacity, namely adsorption isotherm, was also investigated. A non-linear adsorption isotherm of cinchonine was observed on the molecularly imprinted monolith with cinchonine as template, which might be a main reason to result in the peak tailing of template molecule.

Plasma low-density lipoprotein immobilized silica as stationary phase in nano-liquid chromatography.

The feasibility to attach plasma low-density lipoprotein (LDL) particles covalently onto 5 µm silica particles and use them as stationary phase in nano-liquid chromatography (nano-LC) for interaction and oxidation studies was clarified. Before the immobilization, both epoxy silica and aldehyde-activated particles were synthesized, LDL was immobilized to the particles via its protein component and capillary columns were packed with LDL-modified silica materials. The performance of the capillary columns was tested with neutral steroids, and the column with LDL immobilized to aldehyde-activated silica was selected for further studies due to its stronger retention toward steroids. The retention factors of the steroids were used as indicators of the column stability, and the RSDs from 0.8 to 5.7% (n=12) for 168 successive runs within 14 days carried out in the same capillary column and from 0.8 to 3.6% (n=6) in three different capillaries demonstrated that the capillary column was stable and that the capillary column-to-capillary column reproducibility was good. The lifetime of LDL-modified silica stationary phase was around 14 days. The applicability of the column for the separation of steroids and ß-blockers was based mainly on the hydrophobic interactions with lipids of LDL in the stationary phase. The LDL immobilized silica column was successfully exploited also in the copper-mediated in situ oxidation of LDL. The results achieved demonstrated that nano-LC with plasma LDL immobilized silica phase can be exploited as nano-biomimicking tool for interaction studies and as a microreactor for oxidation studies with low consumption of reagents and human materials.

Monodispersed, molecularly imprinted polymers for cinchonidine by precipitation polymerization.

Three monodispersed, molecularly imprinted polymers (MIPs) for cinchonidine (CD) have been synthesized by precipitation polymerization. MIP1 was prepared using methacrylic acid (MAA) as a functional monomer and divinylbenzene (DVB) as a cross-linker and MIP2 was prepared with further addition of 2-hydroxyethyl methacrylate (HEMA) as a co-monomer. For the preparation of MIP3, core-shell type MIP, monodispersed DVB homopolymers, which are prepared by precipitation polymerization, were used as a core and CD-imprinted MAA-DVB copolymer phases were coated onto the core. Three MIPs synthesized gave monodispersed, spherical beads in micrometer sizes. The binding characteristics and molecular recognition properties of MIP1-3 were examined by Scatchard analysis and chromatographic studies. The association constant of CD with MIP1 was the highest among MIPs prepared, while that with MIP3 was the lowest. The template molecule, CD, was more retained than its stereoisomer, cinchonine, on the three MIPs, and the stereoseparation factor of 38 was obtained with MIP3.

Preparation and evaluation of a novel chiral stationary phase based on covalently bonded chitosan for ligand-exchange chromatography.

A novel chiral stationary phase based on chitosan covalently bonded onto silica gels has been prepared and used for the separation of various alpha-amino acid enantiomers as well as alpha-hydroxycarboxylic acid enantiomers by chiral ligand-exchange chromatography with copper(II) as a complexing ion. The methanol content and copper(II) ion concentration in the eluent affected retentivity and enantioselectivity. Furthermore, a plausible chiral recognition mechanism for resolution of alpha-amino acids was proposed.