Fluorescence Recovery After Photobleaching Research Articles

Characterization of a Charged Biomimetic Lipid Membrane for Unique Antifouling Effects against Clinically Relevant Matrices in Biosensing.

Clinically relevant matrices such as human blood and serum can cause substantial interference in biosensing measurements, severely compromising the effectiveness of the sensors. We report the characterization of a positively charged lipid membrane that has demonstrated unique features to suppress the nonspecific signal for antifouling effects by using SPR, fluorescence recovery after photobleaching (FRAP), and MALDI-TOF-MS. The ethylphosphocholine (EPC) lipid membrane proved to be exceptionally effective at reducing irreversible interactions from human serum on a Protein A surface. The membrane formation conditions and their effects on membrane fluidity and mobility were characterized for understanding the antifouling functions when various capture molecules were immobilized. Specifically, EPC lipid membranes on a Protein A substrate appear to exhibit a strong interaction, likely through the electrostatic effect with the negatively charged proteins that resulted in a stable hydration layer. The strong interaction also limited lipid mobility, contributing to a robust, protective interface that remained undamaged in undiluted serum. Tailoring a surface with antifouling lipid membranes allows for a range of biosensing applications in highly complex biological media.

Dissecting SOX9 dynamics reveals its differential regulation in osteoarthritis.

The transcription factor SOX9 is integral to tissue homeostasis and is implicated in skeletal malformation, campomelic dysplasia, and osteoarthritis (OA). Despite extensive research, the complete regulatory landscape of SOX9 transcriptional activity, interconnected with signaling pathways (TGFβ, WNT, BMP, IHH, NFκB, and HIF), remains challenging to decipher. This study focuses on elucidating SOX9 signaling in OA pathology using Fluorescence Recovery After Photobleaching (FRAP) to assess SOX9 activity directly in live human primary chondrocytes (hPCs). Single cell FRAP data revealed two distinct subpopulations with differential SOX9 dynamics, showing varied distribution between healthy and OA hPCs. Moreover, inherently elevated SOX9-DNA binding was observed in healthy hPCs compared to preserved and OA counterparts. Anabolic factors (BMP7 and GREM1) and catabolic inhibitors (DKK1 and FRZb) were found to modulate SOX9 transcriptional activity in OA-hPCs. These findings provide valuable insights into the intricate regulation of SOX9 signaling in OA, suggesting potential therapeutic avenues for modulating SOX9 activity in diseased states.

The Alphabet Soup of Microscopy: An Introduction to Advanced Imaging Techniques. Part II: What the FLIP, FLAP, FRAP, FRET, and FLIM is Going On?

Abstract The use of fluorescence in imaging has been a pivotal factor in advancing our scientific understanding. As microscopy continues to evolve, the terminology used to describe these techniques becomes increasingly complex, often resulting in a bewildering array of acronyms that resemble alphabet soup. Among the most prominent acronyms are those associated with advanced fluorescence microscopy techniques: Fluorescence Loss in Photobleaching (FLIP), Fluorescence Localization after Photobleaching (FLAP), Fluorescence Recovery after Photobleaching (FRAP), Förster Resonance Energy Transfer (FRET), and Fluorescence Lifetime Imaging Microscopy (FLIM). Each of these methods provides invaluable insights into molecular dynamics and interactions within living cells and tissues. This article, Part 2 of the Alphabet Soup of Microscopy series, aims to clarify these widely used fluorescence microscopy techniques and to illuminate their contributions to our understanding of cellular processes.

Mechanosensitive recruitment of Vinculin maintains junction integrity and barrier function at epithelial tricellular junctions.

Apical cell-cell junctions, including adherens junctions and tight junctions, adhere epithelial cells to one another and regulate selective permeability at both bicellular junctions and tricellular junctions (TCJs). Although several specialized proteins are known to localize at TCJs, it remains unclear how actomyosin-mediated tension transmission at TCJs contributes to the maintenance of junction integrity and barrier function at these sites. Here, utilizing the embryonic epithelium of gastrula-stage Xenopus laevis embryos, we define a mechanism by which the mechanosensitive protein Vinculin helps anchor the actomyosin network at TCJs, thus maintaining TCJ integrity and barrier function. Using an optogenetic approach to acutely increase junctional tension, we find that Vinculin is mechanosensitively recruited to apical junctions immediately surrounding TCJs. In Vinculin knockdown (KD) embryos, junctional actomyosin intensity is decreased and becomes disorganized at TCJs. Using fluorescence recovery after photobleaching (FRAP), we show that Vinculin KD reduces actin stability at TCJs and destabilizes Angulin-1, a key tricellular tight junction protein involved in regulating barrier function at TCJs. When Vinculin KD embryos are subjected to increased tension, TCJ integrity is not maintained, filamentous actin (F-actin) morphology at TCJs is disrupted, and breaks in the signal of the tight junction protein ZO-1 signal are detected. Finally, using a live imaging barrier assay, we detect increased barrier leaks at TCJs in Vinculin KD embryos. Together, our findings show that Vinculin-mediated actomyosin organization is required to maintain junction integrity and barrier function at TCJs and reveal new information about the interplay between adhesion and barrier function at TCJs.

Reversible in Situ Control over Monolayer Organization.

Cr2+ and Cr3+ ions are shown to mediate the formation, morphology, and organization of arachidic acid (AA) Langmuir-Blodgett (LB) monolayers. This finding, based on cyclic voltammetry (CV), linear sweep voltammetry (LSV) and fluorescence recovery after photobleaching (FRAP) measurements, show that Langmuir monolayer formation depends on subphase pH and metal ion concentration. Following monolayer deposition on ITO, the LB monolayer organization can be modified reversibly through control of the Cr oxidation state, which has not been shown before by other monolayers formed with other divalent metal ions. The dynamics and the mobility of a chromophore (perylene) incorporated into the monolayer sense changes in Cr oxidation state-dependent organization of the LB monolayer. Demonstrating reversible changes in monolayer organization provides an opportunity to control chemical and electron access to the interface support.

Light at the end of the tunnel: FRAP assays combined with super resolution microscopy confirm the presence of a tubular vacuole network in meristematic plant cells

Abstract Plant vacuoles play key roles in cellular homeostasis, performing catabolic and storage functions, and regulating pH and ion balance. Despite their essential role, there is still no consensus on how vacuoles are established. A model proposing that the endoplasmic reticulum is the main contributor of membrane for growing vacuoles in meristematic cells has been challenged by a study proposing that plant vacuoles are formed de novo by homotypic fusion of multivesicular bodies (MVBs). Here, we use the Arabidopsis thaliana root as a model system to provide a systematic overview of successive vacuole biogenesis stages, starting from the youngest cells proximate to the quiescent center. We combine in vivo high- and super-resolution (STED) microscopy to demonstrate the presence of tubular and connected vacuolar structures in all meristematic cells. Using customized fluorescence recovery after photobleaching (FRAP) assays, we establish different modes of connectivity and demonstrate that thin, tubular vacuoles, as observed in cells near the quiescent center, form an interconnected network. Finally, we argue that a growing body of evidence indicates that vacuolar structures cannot originate from MVBs alone but receive membrane material from different sources simultaneously.

The METHYLTRANSFERASE B-SERRATE interaction mediates the reciprocal regulation of microRNA biogenesis and RNA m6A modification.

In eukaryotes, RNA N6-methyladenosine (m6A) modification and microRNA (miRNA)-mediated RNA silencing represent two critical epigenetic regulatory mechanisms. The m6A methyltransferase complex (MTC) and the microprocessor complex both undergo liquid-liquid phase separation to form nuclear membraneless organelles. Although m6A methyltransferase has been shown to positively regulate miRNA biogenesis, a mechanism of reciprocal regulation between the MTC and the microprocessor complex has remained elusive. Here, we demonstrate that the MTC and the microprocessor complex associate with each other through the METHYLTRANSFERASE B (MTB)-SERRATE (SE) interacting module. Knockdown of MTB impaired miRNA biogenesis by diminishing microprocessor complex binding to primary miRNAs (pri-miRNAs) and their respective MIRNA loci. Additionally, loss of SE function led to disruptions in transcriptome-wide m6A modification. Further biochemical assays and fluorescence recovery after photobleaching (FRAP) assay indicated that SE enhances the liquid-liquid phase separation and solubility of the MTC. Moreover, the MTC exhibited enhanced retention on chromatin and diminished binding to its RNA substrates in the se mutant background. Collectively, our results reveal the substantial regulatory interplay between RNA m6A modification and miRNA biogenesis.

Engineering Planar Gram-Negative Outer Membrane Mimics Using Bacterial Outer Membrane Vesicles.

Antibiotic resistance is a major challenge in modern medicine. The unique double membrane structure of gram-negative bacteria limits the efficacy of many existing antibiotics and adds complexity to antibiotic development by limiting transport of antibiotics to the bacterial cytosol. New methods to mimic this barrier would enable high-throughput studies for antibiotic development. In this study, we introduce an innovative approach to modify outer membrane vesicles (OMVs) from Aggregatibacter actinomycetemcomitans, to generate planar supported lipid bilayer membranes. Our method first involves the incorporation of synthetic lipids into OMVs using a rapid freeze-thaw technique to form outer membrane hybrid vesicles (OM-Hybrids). Subsequently, these OM-Hybrids can spontaneously rupture when in contact with SiO2 surfaces to form a planar outer membrane supported bilayer (OM-SB). We assessed the formation of OM-Hybrids using dynamic light scattering and a fluorescence quenching assay. To analyze the formation of OM-SBs from OM-Hybrids we used quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence recovery after photobleaching (FRAP). Additionally, we conducted assays to detect surface-associated DNA and proteins on OM-SBs. The interaction of an antimicrobial peptide, polymyxin B, with the OM-SBs was also assessed. These findings emphasize the capability of our platform to produce planar surfaces of bacterial outer membranes, which in turn, could function as a valuable tool for streamlining the development of antibiotics.

Quantifying surface tension and viscosity in biomolecular condensates by FRAP-ID

Many proteins with intrinsically disordered regions undergo liquid-liquid phase separation under specific conditions in vitro and in vivo. These complex biopolymers form a metastable phase with distinct mechanical properties defining the timescale of their biological functions. However, determining these properties is nontrivial, even in vitro, and often requires multiple techniques. Here we report the measurement of both viscosity and surface tension of biomolecular condensates via correlative fluorescence microscopy and atomic force microscopy (AFM) in a single experiment (fluorescence recovery after probe-induced dewetting, FRAP-ID). Upon surface tension evaluation via regular AFM-force spectroscopy, controlled AFM indentations induce dry spots in fluorescent condensates on a glass coverslip. The subsequent rewetting exhibits a contact line velocity that is used to quantify the condensed-phase viscosity. Therefore, in contrast with fluorescence recovery after photobleaching (FRAP), where molecular diffusion is observed, in FRAP-ID fluorescence recovery is obtained through fluid rewetting and the subsequent morphological relaxation. We show that the latter can be used to cross-validate viscosity values determined during the rewetting regime. Making use of fluid mechanics, FRAP-ID is a valuable tool to evaluate the mechanical properties that govern the dynamics of biomolecular condensates and determine how these properties impact the temporal aspects of condensate functionality.

Dried Blood Matrix as a New Material for the Detection of DNA Viruses.

The gold standard for diagnosing viruses such as the Hepatitis B Virus has remained largely unchanged, relying on conventional methods involving extraction, purification, and polymerase chain reaction (PCR). This approach is hindered by limited availability, as it is time-consuming and requires highly trained personnel. Moreover, it suffers from low recovery rates of the nucleic acid molecules for samples with low copy numbers. To address the challenges of complex instrumentation and low recovery rate of DNA, a drying process coupled with thermal treatment of whole blood is employed, resulting in the creation of a dried blood matrix characterized by a porous structure with a high surface-to-volume ratio where it also inactivates the amplification inhibitors present in whole blood. Drawing on insights from Brunauer-Emmett-Teller (BET)- Barrett-Joyner-Halenda (BJH) analysis, scanning electron microscopy (SEM), and fluorescence recovery after photobleaching (FRAP), detection assay is devised for HBV, as a demonstration, from whole blood with high recovery of DNA and simplified instrumentation achieving a limit of detection (LOD) of 10 IU mL-1. This assay can be completed in <1.5 h using a simple heater, can be applied to other DNA viruses, and is expected to be suitable for point-of-care, especially in low-resource settings.

Cholinergic stimulation stabilizes TRPM4 in the plasma membrane of cortical pyramidal neurons.

TRPM4 is a calcium activated non-selective cation channel, impermeable to Ca2+, in neurons it has been implicated in the regulation of the excitability and in the persistent firing. Cholinergic stimulation is also implicated in changes in excitability that leads neurons to an increased firing frequency, however it is not clear whether TRPM4 is involved in the cholinergic-induced increase in firing frequency. Here using a combination of patch clamp electrophysiology, Ca2+ imaging, immunofluorescence, fluorescence recovery after photobleaching (FRAP) and pharmacological approach, we demonstrate that carbachol (Cch) increases firing frequency, intracellular Ca2+ and that TRPM4 inhibition using 9-Ph and CBA reduces firing frequency and decreases the peak in intracellular Ca2+ induced by Cch in cortical pyramidal neurons in culture. Moreover, we determined that cholinergic stimulation reduces TRPM4 recycling and stabilizes TRPM4 in the plasma membrane. Together our results indicate that cholinergic stimulation increases firing in a TRPM4 dependent manner, and also increases the TRPM4 stability in the membrane, suggesting that TRPM4 is locked in microdomains in the membrane, possibly signaling or cytoskeleton proteins complexes.

Diffusion coefficients in scaffolds made with temperature controlled cryoprinting and an ink made of sodium alginate and agar

Temperature Controlled Cryoprinting (TCC), is a tissue engineering technique wherein each deposited voxel is frozen with precise control over cooling rates and the direction of freezing. This control allows for the generation of ice crystals with controlled shape and orientation. Recently we found that the macroscale fidelity of the TCC print is substantially improved by using a 3D printing ink composed of a mixture of two compounds: one that solidifies through chemical crosslinking (sodium alginate) and another that solidifies through physical (thermal) effects (agar). In this study we examine the hypothesis that the combination of sodium alginate and agar, affects also the fidelity of the microstructure and thereby the diffusivity of the scaffold. The ability of this technology to generate controlled diffusivity within the tissue scaffold was examined with a directional solidified TCC sample using fluorescence recovery after photobleaching (FRAP) and scanning electron microscope (SEM). We find that the diffusion coefficient in m2/s × 10−10 is: 1.62 ± 1.27 for the unfrozen sample, 2.40 ±1.54 for the rapidly frozen sample and 9.72± 4.50 for the slow frozen sample. This points to two conclusions. One is that the diffusivity is slow frozen samples is higher than that in unfrozen samples and in rapidly frozen sample. A second observation is that a relatively narrow range of diffusivity variance was obtained when using 2%w/v sodium alginate and 2%w/v of agar. However, when the concentration of agar was reduced to 0.5w/v a much wider spread of diffusivities was measure, 4.07±1.65. This suggests that the addition of agar has also an effect on the microscale fidelity, and consequently the diffusivity. The anisotropic diffusion properties of TCC-printed directional solidification samples were also validated through both FRAP and SEM.

A BODIPY-Naphtholimine-BF2 Dyad for Precision Photodynamic Therapy, Targeting, and Dual Imaging of Endoplasmic Reticulum and Lipid Droplets in Cancer.

Currently, effective therapeutic modalities for pancreatic ductal adenocarcinoma (PDAC) are quite limited, leading to gloomy prognosis and ∼6-months median patient survival. Recent advances showed the promise of photodynamic therapy (PDT) for PDAC patients. Next generation photosensitizers (PS) are based on "organelle-targeted-PDT" and provide new paradigm in the field of precision medicines to address the current challenge for treating PDAC. In this investigation, we have constructed a novel PS, named as N b B, for precise and simultaneous targeting of endoplasmic reticulum (ER) and lipid droplets (LDs) in PDAC, based on the fact that malignant PDAC cells are heavily relying on ER for hormone synthesis. Our live cell imaging and fluorescence recovery after photobleaching (FRAP) experiments revealed that N b B is quickly targeted to ER and subsequently to LDs and shows simultaneous dual fluorescence color due to polar and nonpolar milieu of ER and LDs. Interestingly, the same molecule generates triplet state and singlet oxygen efficiently and causes robust ER stress and cellular lipid peroxidation, leading to apoptosis in two different PDAC cells in the presence of light. Together, we present, for the first time, a potential next generation precision medicine for ER-LD organelle specific imaging and PDT of pancreatic cancer.

Smad2/3/4 complex could undergo liquid liquid phase separation and induce apoptosis through TAT in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) represents one of the most significant causes of mortality due to cancer-related deaths. It has been previously reported that the TGF-β signaling pathway may be associated with tumor progression. However, the relationship between TGF-β signaling pathway and HCC remains to be further elucidated. The objective of our research was to investigate the impact of TGF-β signaling pathway on HCC progression as well as the potential regulatory mechanism involved. MethodsWe conducted a series of bioinformatics analyses to screen and filter the most relevant hub genes associated with HCC. E. coli was utilized to express recombinant protein, and the Ni–NTA column was employed for purification of the target protein. Liquid liquid phase separation (LLPS) of protein in vitro, and fluorescent recovery after photobleaching (FRAP) were utilized to verify whether the target proteins had the ability to drive force LLPS. Western blot and quantitative real-time polymerase chain reaction (qPCR) were utilized to assess gene expression levels. Transcription factor binding sites of DNA were identified by chromatin immunoprecipitation (CHIP) qPCR. Flow cytometry was employed to examine cell apoptosis. Knockdown of target genes was achieved through shRNA. Cell Counting Kit-8 (CCK-8), colony formation assays, and nude mice tumor transplantation were utilized to test cell proliferation ability in vitro and in vivo.ResultsWe found that Smad2/3/4 complex could regulate tyrosine aminotransferase (TAT) expression, and this regulation could relate to LLPS. CHIP qPCR results showed that the key targeted DNA binding site of Smad2/3/4 complex in TAT promoter region is −1032 to −1182. In addition. CCK-8, colony formation, and nude mice tumor transplantation assays showed that Smad2/3/4 complex could repress cell proliferation through TAT. Flow cytometry assay results showed that Smad2/3/4 complex could increase the apoptosis of hepatoma cells. Western blot results showed that Smad2/3/4 complex would active caspase-9 through TAT, which uncovered the mechanism of Smad2/3/4 complex inducing hepatoma cell apoptosis.ConclusionThis study proved that Smad2/3/4 complex could undergo LLPS to active TAT transcription, then active caspase-9 to induce hepatoma cell apoptosis in inhibiting HCC progress. The research further elucidate the relationship between TGF-β signaling pathway and HCC, which contributes to discover the mechanism of HCC development.

Supported Biomembrane Systems Incorporating Multiarm Polymers and Bioorthogonal Tethering.

To functionalize interfaces with supported biomembranes and membrane proteins, the challenge is to build stabilized and supported systems that mimic the native lipid microenvironment. Our objective is to control substrate-to-biomembrane spacing and the tethering chemistry so proteoliposomes can be fused and conjugated without perturbation of membrane protein function. Furthermore, the substrates need to exhibit low protein and antibody nonspecific binding to use these systems in assays. We have employed protein orthogonal coupling schemes in concert with multiarm poly(ethylene glycol) (PEG) technology to build supported biomembranes on microspheres. The lipid bilayer structures and tailored substrates of the microsphere-supported biomembranes were analyzed via flow cytometry, confocal fluorescence, and super-resolution imaging microscopy, and the lateral fluidity was quantified using fluorescence recovery after photobleaching (FRAP) techniques. Under these conditions, the 4-arm-PEG20,000-NH2 based configuration gave the most desirable tethering system based on lateral diffusivity and coverage.

Spatially heterogeneous dynamics of droplets formed by arginine-rich dipeptides and poly-A RNA during liquid-liquid phase separation

Arginine-rich dipeptides (proline-arginine (PR)) are highly toxic products encoded by aberrant repeat expansion in the C9ORF72 gene, which is the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These dipeptides have the capability to undergo liquid-liquid phase separation (LLPS) with components inside the membrane-less organelles (MLO) that could have a detrimental effect on fundamental cellular processes, potentially leading to injury or death in motor neurons. In this study, Raman spectroscopy was used to investigate the spatial variation within these liquid droplets in vitro, revealing concentration gradients of RNA in droplets. The findings were compared with fluorescence recovery after photobleaching (FRAP), offering insights into the spatially heterogeneous dynamics within the droplets. Understanding the mechanism is essential for unraveling the pathogenesis of neuronal diseases and may provide future therapeutic strategies.

Ionic Effect on the Microenvironment of Biomolecular Condensates.

Biomolecules such as proteins and RNA could organize to form condensates with distinct microenvironments through liquid-liquid phase separation (LLPS). Recent works have demonstrated that the microenvironment of biomolecular condensates plays a crucial role in mediating biological activities, such as the partition of biomolecules, and the subphase organization of the multiphasic condensates. Ions could influence the phase transition point of LLPS, following the Hofmeister series. However, the ion-specific effect on the microenvironment of biomolecular condensates remains unknown. In this study, we utilized fluorescence lifetime imaging microscopy (FLIM), fluorescence recovery after photobleaching (FRAP), and microrheology techniques to investigate the ion effect on the microenvironment of condensates. We found that ions significantly affect the microenvironment of biomolecular condensates: salting-in ions increase micropolarity and reduce the microviscosity of the condensate, while salting-out ions induce opposing effects. Furthermore, we manipulate the miscibility and multilayering behavior of condensates through ion-specific effects. In summary, our work provides the first quantitative survey of the microenvironment of protein condensates in the presence of ions from the Hofmeister series, demonstrating how ions impact micropolarity, microviscosity, and viscoelasticity of condensates. Our results bear implications on how membrane-less organelles would exhibit varying microenvironments in the presence of continuously changing cellular conditions.

Bottlebrush Block Copolymers at the Interface of Immiscible Liquids: Adsorption and Lateral Packing.

Amphiphilic bottlebrush block copolymers (BBCPs), having a hydrophilic bottlebrush polymer (BP) linked covalently to a hydrophobic BP, were found to segregate to liquid-liquid interfaces to minimize the free energy of the system. The key parameter influencing the outcome of the experiments is the ratio between the degree of polymerization of the backbone (NBB) and that of the side-chain brushes (NSC). Specifically, a spherical, star-like configuration results when NBB < NSC, while a cylindrical, bottlebrush-like shape is preferred when NBB > NSC. Dynamic interfacial tension (γ) and fluorescence recovery after photobleaching (FRAP) measurements show that the BBCP configuration influences the areal density and in-plane diffusion at the fluid interface. The characteristic relaxation times associated with BBCP adsorption (τA) and reorganization (τR) were determined by fitting time-dependent interfacial tension measurements to a sum of two exponential relaxation functions. Both τA and τR initially increased with NBB up to 92 repeat units, due to the larger hydrodynamic radius in solution and slower in-plane diffusivity, attributed to a shorter cross-sectional diameter of the side-chains near the block junction. This trend reversed at NBB = 190, with shorter τA and τR attributed to increased segregation strength and exposure of the bare water/toluene interface due to tilting and/or wiggling of the backbone chains, respectively. The adsorption energy barrier decreased with higher NBB, due to a reduced BBCP packing density at the fluid interface. This study provides fundamental insights into macromolecular assembly at fluid interfaces, as it pertains to unique bottlebrush block architectures.

Abstract 1128: Smooth Muscle Myosin Ii Dynamics And Specificity To Muscle Type

Smooth muscle myosin II (SMII), the main motor protein in vascular smooth muscle cells (VSMC), generates the contractile forces crucial to VSMC function. SMII filaments assemble into bundles that bind and pull on the actin cytoskeleton. VSMCs exist on a functional spectrum between the contractile and synthetic phenotypes. Our previous work established a discernible difference in EGFP-SMII filaments between phenotypes. The synthetic phenotype is highly dynamic with SMII filaments freely assembling into bundles and dissociating into monomers. The contractile phenotype features stabilized SMII bundles leading to organized contractile machinery. While known that VSMC function changes with artery size, differences in SMII dynamics remain understudied. We will investigate these differences in SMII between VSMCs of large conducting, small conducting, and resistive arteries to determine SMII functional properties unique to each VSMC type. We will extend this to looking at smooth muscle cells (SMC) from other tissue types. We aim to identify tissue-specific SMII properties to discover mechanisms that predispose specific tissues to pathologies and potential tissue-specific targets for therapeutics. We recently engineered a novel endogenously-tagged EGFP-SMII mouse, which allowed us to observe changes in VSMC contractile machinery directly and to better understand SMII dynamics. We isolated SMCs from the EGFP-SMII mouse from the vasculature, gastrointestinal, and genitourinary tracts. We measured SMII filament assembly, SMII fluorescent recovery after photobleaching (FRAP), and whole-cell and organoid contractility using traction force microscopy. With the isolation of single cells, we observed a very rapid transition in aortic VSMC phenotype, from contractile to synthetic, with the contractile machinery demonstrating synthetic changes accordingly. The physiologic, native whole-tissue state had stable SMII dynamics, while isolated cells (in a pathologic, cultured state) exhibited rapid filament turnover. We also observed greater stability in aortic tissue ex vivo compared to other vessels and tissues containing SMII. These results suggest aortic SMII has unique properties and pathways specific to the type and state of the tissue.

Network pharmacology of Dracaena sp. in Guangxi and its related species leaf secondary metabolites possess antioxidant properties

In this study, metabolite maps were conducted on leaf samples accessions of Dracaena sp. in Guangxi (C6, C13, CZ4, JX2, RM1, and BM3) and six related species including D. angustifolia (CH), D. elliptica (XZ), D. cochinchinensis (CB), D. cambodiana (HN), D. marginata (HB), and Yucca schidigera (SL). We identified 2,971 compounds including carboxylic acids and their derivatives, organooxygen compounds, prenol lipids, benzenes and their substituted derivatives, and flavonoids. Of those, 73 characteristic secondary metabolites were screened out by weighted correlation network analysis (WGCNA). Pterostilbene, 9-cis-retinal, and dracorubin were unique to the Dracaena sp. in Guangxi, and 3-hydroxytridecanoic acid and chrysin were highly abundant in them. An in vitro antioxidant activity assay disclosed that all 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) scavenging rates and all fluorescence recovery after photobleaching (FRAP) rates were in the ranges of 75–87 % and 3.10–3.25 μmol/mL, respectively, for the Dracaena sp. in Guangxi. Hence, the latter have relatively high and stable foliar antioxidant activity. A network pharmacological analysis of the top 20 metabolites in Dracaena leaf and their 446 antioxidant-related disease targets revealed ten core action targets, including GAPDH, AKT1, MAPK3, VEGFA, CASP3, TNF, MAPK1, SRC, EGFR, and MAPK8. An antioxidant-related compound-target-pathway network was constructed and verified by molecular docking. The results of this study provide a theoretical basis for mining and exploiting the foliar secondary metabolites of Dracaena.