Stabilization of F-actin by Salmonella effector SipA resembles the structural effects of inorganic phosphate and phalloidin.

Entry of Salmonella into host enterocytes relies on its pathogenicity island 1 effector SipA. We found that SipA binds to F-actin in a 1:2 stoichiometry with sub-nanomolar affinity. A cryo-EM reconstruction revealed that SipA's globular core binds at the groove between actin strands, whereas the extended C-terminal arm penetrates deeply into the inter-strand space, stabilizing F-actin from within. The unusually strong binding of SipA is achieved by a combination of fast association via the core and very slow dissociation dictated by the arm. Similar to Pi, BeF3, and phalloidin, SipA potently inhibited actin depolymerization by actin depolymerizing factor (ADF)/cofilin, which correlated with increased filament stiffness, supporting the hypothesis that F-actin's mechanical properties contribute to the recognition of its nucleotide state by protein partners. The remarkably strong binding to F-actin maximizes the toxin's effects at the injection site while minimizing global influence on the cytoskeleton and preventing pathogen detection by the host cell.

[Effects of conditioned medium of alveolar epithelial cells on vascular endothelial cell damage].

OBJECTIVE: To observe the effect of lipopolysaccharide (LPS) induced conditioned medium of alveolar epithelial cells on the inflammatory response and cell damage of vascular endothelial cells, and explore its mechanism. METHODS: The LPS induced type II alveolar epithelial cells (A549) conditioned medium was used as a stimulus to induce human umbilical vein endothelial cells (HUVEC) damage. The cell counting kit-8 (CCK-8) was used to detect the effect of 0% (blank group), 12.5%, 25%, 50%, 75% and 100% A549 cell conditioned medium cultured for 6, 12, 24 and 48 hours on the cell viability of HUVEC. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α)] and vasoactive substances [vascular endothelial growth factor (VEGF), endothelin-1 (ET-1)] in the supernatant. Phalloidin staining was used to observe the effects of A549 cells conditioned medium on cell morphology. The expressions of protein kinase B/nuclear factor-κB (AKT/NF-κB) pathway in HUVEC induced by conditioned medium was detected by Western blotting. RESULTS: Compared with the blank group, A549 cells conditioned medium with concentrations of 12.5%, 25%, and 50% had no significant effects on cell viability of HUVEC after 6, 12, and 24 hours, but the activity of HUVEC decreased significantly after 48 hours. Therefore, 12.5%, 25%, 50% A549 cell conditioned medium stimulated for 24 hours was selected as the induction condition for follow-up experiments. Compared with the blank group, the level of IL-6 was significantly increased in 12.5% and 50% conditioned medium groups (ng/L: 2 438.95±64.89, 3 036.41±96.69 vs. 1 736.75±20.99, both P < 0.05), the level of TNF-α was significantly increased in 12.5% and 25% conditioned medium groups (ng/L: 174.08±11.09, 81.37±8.17 vs. 50.03±0.26, both P < 0.01), the levels of VEGF and ET-1 were significantly increased in 12.5%, 25% and 50% conditioned medium groups [VEGF (ng/L): 173.60±41.44, 192.49±12.38, 318.89±27.90 vs. 66.68±19.65; ET-1 (ng/L): 54.88±1.37, 36.69±0.29, 24.07±0.73 vs. 10.67±0.25, all P < 0.01]. Phalloidin staining showed that HUVEC induced by 25% A549 cells conditioned medium were irregular in shape, uneven in size, disordered in arrangement, widened in gap, dense and unclear in microfilament structure and serrated in cell membrane. Furthermore, the average fluorescence intensity of 25% conditioned medium group significantly increased compared to the blank group (67 205.60±3 430.40 vs. 56 272.67±7 650.95, P < 0.05). Western blotting showed that compared with the blank group, the expression of HUVEC cells phosphonated inhibitor α of NF-κB (p-IκBα) was significantly decreased in the 12.5%, 25%, and 50% conditioned medium groups (p-IκBα/IκBα: 0.38±0.08, 0.67±0.12, 0.31±0.07 vs. 1.00±0.00, all P < 0.01), the expressions of phosphonated-AKT (p-AKT) and VEGF were significantly increased (p-AKT/AKT: 1.50±0.18, 1.42±0.27, 1.61±0.14 vs. 1.00±0.00, VEGF/GAPDH: 1.37±0.10, 1.53±0.22, 1.40±0.12 vs. 1.00±0.00, all P < 0.05), the expression of phosphonated NF-κB p65 (p-P65) was significantly increased in the 25% conditioned medium group (p-P65/P65: 1.45±0.14 vs. 1.00±0.00, P < 0.05). CONCLUSIONS: LPS induced conditional culture medium of alveolar epithelial cells induced endothelial cell damage via activating AKT/NF-κB pathway.

Antiviral Effect of Extracellular Matrix Protein ABI3BP on Vesicular Stomatitis Virus and Its Mechanism: A Preliminary Study In Vitro.

Objective To explore the influence of extracellular matrix protein ABI-interactor 3-binding protein (ABI3BP) on vesicular stomatitis virus (VSV) genome replication and innate immune signaling pathway.Methods The small interfering RNA (siRNA) was transfected to knock down ABI3BP gene in human skin fibroblast BJ-5ta cells. VSV-green fluorescent protein (VSV-GFP)-infected cell model was established. The morphological changes and F-actin stress fiber formation were detected on ABI3BP knockdown cells by phalloidin immunofluorescence staining. The mRNA level of virus replication was detected by RT-qPCR in BJ-5ta cells after VSV-GFP infection; western blotting was performed to detect the changes in interferon regulatory factor 3 (IRF3) and TANK-binding kinase 1 (TBK1) phosphorylation levels.Results The VSV-GFP-infected BJ-5ta cell model was successfully established. Efficient knockdown of ABI3BP in BJ-5ta cells was achieved. Phalloidin immunofluorescence staining revealed structural rearrangement of intracellular F-actin after ABI3BP gene knockdown. Compared with the control group, the gene copy number of VSV-GFP in ABI3BP knockdown cells increased by 2.2 - 3.5 times (P<0.01) and 2.2 - 4.0 times (P<0.01) respectively when infected with VSV of multiplicity of infection 0.1 and 1. The expression of viral protein significantly increased in ABI3BP knockdown cells after virus infection. The activation of type-I interferon pathway, as determined by phosphorylated IRF3 and phosphorylated TBK1, was significantly decreased in ABI3BP knockdown cells after VSV-GFP infection.Conclusions Extracellular matrix protein ABI3BP plays an important role in maintaining the formation and rearrangement of actin structure. ABI3BP gene deletion promotes RNA virus replication, and ABI3BP is an important molecule that maintains the integrity of type I interferon pathway.

Jiawei Buzhong Yiqi decoction ameliorates polycystic ovary syndrome via oocyte-granulosa cell communication.

ETHNOPHARMACOLOGICAL RELEVANCE: Jiawei Buzhong Yiqi Decoction (JWBZYQ), from records of FuqingzhuNvke, is a classical formula for treating obese women related infertility. JWBZYQ has been shown to be effective in treating polycystic ovary syndrome (PCOS) in both clinical studies and practical practice, with the pharmacological mechanism remaining unknown. AIM OF THE STUDY: To explore the potential therapeutic effects and mechanistic insights of JWBZYQ in PCOS. MATERIALS AND METHODS: An overweight PCOS rat model was established via testosterone propionate (TP) injection and 45% high-fat diet (HFD). Then they were categorized into five distinct groups: Control group, Model group, low-dose of JWBZYQ (JWBZYQ1) group, high-dose of JWBZYQ (JWBZYQ2) group, and metformin (Met) group. Body weight, estrous cycle, and sex hormone levels were observed. Hematoxylin-Eosin staining was employed to investigate the histological characteristics of the ovaries. To identify the pathways that changed significantly, transcriptome analysis was performed. The protein and mRNA levels of key molecules in ovarian zona pellucida (ZP) organization, transzonal projections (TZPs) assembly, steroid hormone receptors, and steroidogenesis were assessed using phalloidin staining, immunohistochemistry, Western blot, and polymerase chain reaction. RESULTS: RNA-seq analysis demonstrated that regulation of hormone secretion, cilium assembly, cell projection assembly, and ZP production may all have crucial impact on the etiology of PCOS and therapeutic effect of JWBZYQ. In particular, PCOS rats exhibited elevated expressions of ZP1-3, which can be reversed by JWBZYQ2 particularly. Simultaneously, TZPs assembly was totally disrupted in PCOS rats, evidenced by the phalloidin staining, upregulated calcium-/calmodulin-dependent protein kinase II beta (CaMKIIß), and deficient p-CaMKIIß, myosin X (MYO10), proline-rich tyrosine kinase 2 (PTK2), and Fascin. Nonetheless, JWBZYQ or metformin treatment revived the disturbance, repairing the oocyte-granulosa cell communication, regulating steroidogenesis in PCOS rats. In this way, JWBZYQ and metformin exerted remarkable effects in alleviating altered ovarian morphology and function in PCOS rats, with JWBZYQ2 revealing the best effect. CONCLUSIONS: JWBZYQ restored the altered ovarian morphology and function by regulating the oocyte-granulosa cell communication, which was related with ZP organization and TZPs assembly in the ovary.

Three-dimensional visualization of the lymphatic, vascular and neural network in rat lung by confocal microscopy.

In order to demonstrate the intricate interconnection of pulmonary lymphatic vessels, blood vessels, and nerve fibers, the rat lung was selected as the target and sliced at the thickness of 100 µm for multiply immunofluorescence staining with lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), alpha smooth muscle actin (α-SMA), phalloidin, cluster of differentiation 31 (CD31), and protein gene product 9.5 (PGP9.5) antibodies. Taking the advantages of the thicker tissue section and confocal microscopy, the labeled pulmonary lymphatic vessels, blood vessels, and nerve fibers were demonstrated in rather longer distance, which was more convenient to reconstruct a three-dimensional (3D) view for analyzing their spatial correlation in detail. It was clear that LYVE-1+ lymphatic vessels were widely distributed in pulmonary lobules and closely to the lobar bronchus. Through 3D reconstruction, it was also demonstrated that LYVE-1+ lymphatic vessels ran parallel to or around the α-SMA+ venules, phalloidin+ arterioles and CD31+ capillaries, with PGP9.5+ nerve fibers traversing alongside or wrapping around them, forming a lymphatic, vascular and neural network in the lung. By this study, we provide a detailed histological view to highlight the spatial correlation of pulmonary lymphatic, vascular and neural network, which may help us for insight into the functional role of this network under the physiological and pathological conditions.

Toxicokinetics and tissue distribution of phalloidin in mice.

Phalloidin, a bicyclic heptapeptide found in Amanita mushroom, specifically binds to F-actin in the liver causing cholestatic hepatotoxicity. However, the toxicokinetics and tissue distribution properties of phalloidin as well as their underlying mechanisms have to be studied further. The area under the plasma concentration curve (AUC) of phalloidin increased in proportion to the doses (0.2, 0.4, and 0.8 mg/kg for intravenous injection and 2, 5, and 10 mg/kg for oral administration). Phalloidin exhibited dose-independent low volume of distribution (395.6-456.9 mL/kg) and clearance (21.4-25.5 mL/min/kg) and low oral bioavailability (2.4%-3.3%). This could be supported with its low absorptive permeability (0.23 ± 0.05 × 10-6 cm/s) in Caco-2 cells. The tissue-to-plasma AUC ratios of intravenously injected and orally administered phalloidin were the highest in the liver and intestines, respectively, and also high in the kidneys, suggesting that the liver, kidneys, and intestines could be susceptible to phalloidin exposure and that active transport via the hepatic and renal organic anion transporters (OATP1B1, OATP1B3, and OAT3) may contribute to the higher distribution of phalloidin in the liver and kidneys.

Phalloidin Staining for F-Actin in Hepatic Stellate Cells.

During the development of liver fibrosis, hepatic stellate cells undergo a transition from a quiescent phenotype into a proliferative, fibrogenic, and contractile, α-smooth muscle actin-positive myofibroblast. These cells acquire properties that are strongly associated with the reorganization of the actin cytoskeleton. Actin possesses a unique ability to polymerize into filamentous actin (F-actin) form its monomeric globular state (G-actin). F-actin can form robust actin bundles and cytoskeletal networks by interacting with a number of actin-binding proteins that provide important mechanical and structural support for a multitude of cellular processes including intracellular transport, cell motility, polarity, cell shape, gene regulation, and signal transduction. Therefore, stains with actin-specific antibodies and phalloidin conjugates for actin staining are widely used to visualize actin structures in myofibroblasts. Here we present an optimized protocol for F-actin staining for hepatic stellate cells using a fluorescent phalloidin.

Knockdown of G Protein-coupled Estrogen Receptor 1 (GPER1) Enhances Tumor-supportive Properties in Cervical Carcinoma Cells.

BACKGROUND/AIM: A wide variety of answers can be found regarding the question of whether G-protein-coupled estrogen receptor 1 (GPER1) is tumor supportive or tumor suppressive. In cervical carcinoma (CC), the function of GPER1 is poorly understood. In this work, we aimed to clarify what role GPER1 plays in CC, tumor promoting of tumor suppressive. MATERIALS AND METHODS: Transient GPER1 silencing was conducted using RNAi and approved by RT-qPCR. Clonogenic potential was tested by colony and sphere formation. Expression of SERPINE1/PAI-1 was quantified by RT-qPCR and Western blot. Morphological changes were analyzed using Phalloidin staining. Localization of GPER1 in tumor spheres was examined by immunofluorescence. RESULTS: After GPER1 knockdown, more colonies formed in HeLa and SiHa, and larger colonies formed in C33-A and SiHa CC cells. Size of HeLa and SiHa tumor spheres was also increased. In addition, number of HeLa tumor spheres was elevated, and larger secondary colonies were present. C33-A only formed tumor sphere-like clusters showing no differences in number and size. Phalloidin staining revealed greater cellular length-to-width ratio and increased average filopodia length. Expression of SERPINE1/PAI-1 was increased in HeLa and decreased in C33-A. In SiHa cells, SERPINE1 was slightly decreased, whereas the protein PAI-1 was increased. Strong expression of GPER1 was detectable in peripheral areas and in sprouts of tumor spheres. CONCLUSION: GPER1 appears to be tumor suppressive in CC, as GPER1 knockdown provoked increased stem cell properties and increased migration/invasion. EMT also appears to be enhanced. Of interest is the increase in SERPINE1/PAI-1 expression after GPER1 knockdown.

In situ FRET-based localization of the N terminus of myosin binding protein-C in heart muscle cells.

Cardiac myosin binding protein-C (cMyBP-C) is a thick filament-associated regulatory protein frequently found mutated in patients suffering from hypertrophic cardiomyopathy (HCM). Recent in vitro experiments have highlighted the functional significance of its N-terminal region (NcMyBP-C) for heart muscle contraction, reporting regulatory interactions with both thick and thin filaments. To better understand the interactions of cMyBP-C in its native sarcomere environment, in situ Foerster resonance energy transfer-fluorescence lifetime imaging (FRET-FLIM) assays were developed to determine the spatial relationship between the NcMyBP-C and the thick and thin filaments in isolated neonatal rat cardiomyocytes (NRCs). In vitro studies showed that ligation of genetically encoded fluorophores to NcMyBP-C had no or little effect on its binding to thick and thin filament proteins. Using this assay, FRET between mTFP conjugated to NcMyBP-C and Phalloidin-iFluor 514 labeling the actin filaments in NRCs was detected by time-domain FLIM. The measured FRET efficiencies were intermediate between those observed when the donor was attached to the cardiac myosin regulatory light chain in the thick filaments and troponin T in the thin filaments. These results are consistent with the coexistence of multiple conformations of cMyBP-C, some with their N-terminal domains binding to the thin filament and others binding to the thick filament, supporting the hypothesis that the dynamic interchange between these conformations mediates interfilament signaling in the regulation of contractility. Moreover, stimulation of NRCs with ß-adrenergic agonists reduces FRET between NcMyBP-C and actin-bound Phalloidin, suggesting that cMyBP-C phosphorylation reduces its interaction with the thin filament.

Actin: Static and Dynamic Studies.

The actin cytoskeleton is a highly dynamic network in plant cells, which is precisely regulated by numerous actin-binding proteins. Hence, characterizing the biochemical activities of actin-binding proteins is of great importance. Here we describe methods for determining the binding and bundling of microfilaments as well as methods for visualizing microfilaments using fluorescent phalloidin and single-molecule TIRF imaging.

Optimized Fixation and Phalloidin Staining of Basally Localized F-Actin Networks in Collectively Migrating Follicle Cells.

The follicular epithelial cells of the Drosophila egg chamber have become a premier model to study how cells globally orient their actin-based machinery for collective migration. The basal surface of each follicle cell has lamellipodial and filopodial protrusions that extend from its leading edge and an array of stress fibers that mediate its adhesion to the extracellular matrix; these migratory structures are all globally aligned in the direction of tissue movement. To understand how this global alignment is achieved, one must be able to reliably visualize the underlying F-actin; however, dynamic F-actin networks can be difficult to preserve in fixed tissues. Here, we describe an optimized protocol for the fixation and phalloidin staining of the follicular epithelium. We also provide a brief primer on relevant aspects of the image acquisition process to ensure high quality data are collected.

In vitro and in vivo evaluation of a modified porcine acellular dermal matrix for soft tissue augmentation.

OBJECTIVES: To evaluate the effects of a modified porcine acellular dermal matrix (P-ADM), subepithelial connective tissue graft (SCTG) and other commercial bovine acellular dermal matrix membrane materials (B-ADM) on gingival soft tissue augmentation in the oral esthetic zone. MATERIAL AND METHODS: The characteristics of P-ADM were observed by scanning electron microscope (SEM), Hematoxylin and eosin (H&E) and Masson's trichrome staining (Masson staining). The biocompatibility of P-ADM was verified by CCK8, phalloidin and living/dead cell staining. Beagle dog models were constructed and the thickness of gingiva was analyzed by the intraoral scanner. The morphology was observed by H&E and Masson staining. RESULTS: Scanning electron microscopy, H&E and Masson staining showed that the P-ADM was mainly composed of collagen fibers, with no component of nuclear. The results of CCK8, phalloidin and living/dead cell staining indicated that the P-ADM had good cytocompatibility and no cytotoxicity. Human gingival fibroblasts were able to adhere and stretch on the surface of the material with pseudopodia. The SCTG group outperformed the B-ADM and P-ADM groups in terms of effectiveness, according to the analysis of digital oral scanning data at various time points following incremental soft tissue surgery. Compared with the B-ADM group, the effect of soft tissue increment was better in the P-ADM group. CONCLUSIONS: P-ADM, as a biocompatible biomaterial, can be used as an alternative biomaterial for oral soft tissue thickening. However, the results of this study need to be verified by more clinical trials.

Stage-dependent localization of F-actin and Na+ /K+ -ATPase in zebrafish embryos detected using optimized cryosectioning immunostaining protocol.

The increasing use of the zebrafish model in biomedical and (eco)toxicological studies aimed at understanding the function of various proteins highlight the importance of optimizing existing methods to study gene and protein expression and localization in this model. In this context, zebrafish cryosections are still underutilized compared with whole-mount preparations. In this study, we used zebrafish embryos (24-120 hpf) to determine key factors for the preparation of high-quality zebrafish cryosections and to determine the optimal protocol for (immuno)fluorescence analyses of Na+ /K+ -ATPase and F-actin, across developmental stages from 1 to 5 dpf. The results showed that the highest quality zebrafish cryosections were obtained after the samples were fixed in 4% paraformaldehyde (PFA) for 1 h, incubated in 2.5% bovine gelatin/25% sucrose mixture, embedded in OCT, and then sectioned to 8 µm thickness at -20°C. Fluorescence microscopy analysis of phalloidin-labeled zebrafish skeletal muscle revealed that 1-h-4% PFA-fixed samples allowed optimal binding of phalloidin to F-actin. Further immunofluorescence analyses revealed detailed localization of F-actin and Na+ /K+ -ATPase in various tissues of the zebrafish and a stage-dependent increase in their respective expression in the somitic muscles and pronephros. Finally, staining of zebrafish cryosections and whole-mount samples revealed organ-specific and zone-dependent localizations of the Na+ /K+ -ATPase α1-subunit. RESEARCH HIGHLIGHTS: This study brings optimization of existing protocols for preparation and use of zebrafish embryos cryosections in (immuno)histological analyses. It reveals stage-dependent localization/expression of F-actin and Na+ /K+ -ATPase in zebrafish embryos.

Visualizing Cofilin-Actin Filaments by Immunofluorescence and CryoEM: Essential Steps for Observing Cofilactin in Cells.

Fluorescence microscopy of cytoskeletal proteins in situ using immunolabeling, fluorescent reagents, or expression of tagged proteins has been a common practice for decades but often with too little regard for what might not be visualized. This is especially true for assembled filamentous actin (F-actin), for which binding of fluorescently labeled phalloidin is taken as the gold standard for its quantification even though it is well known that F-actin saturated with cofilin (cofilactin) binds neither fluorescently labeled phalloidin nor genetically encoded F-actin reporters, such as LifeAct. Here, using expressed fluorescent cofilactin reporters, we show that cofilactin is the major component of some actin-containing structures in both normal and stressed neurons and present various fixation, permeabilization, and cryo-preservation methods for optimizing its observation.

The immunolocalization of cluster of differentiation 31, phalloidin and alpha smooth muscle actin on vascular network of normal and ischemic rat brain.

Cluster of differentiation 31 (CD31), phalloidin and alpha smooth muscle actin (α-SMA) have been widely applied to label the cerebral blood vessels in the past years. Although CD31 is mainly used as endothelial marker in determining the cerebral capillaries, it seems likely that its labeling efficiency is closely correlated with the antibodies from the polyclonal or monoclonal one, as well as the conditions of blood vessels. In order to test this phenomenon, we compared the labeling characteristics of goat polyclonal anti-CD31 (gP-CD31) and mouse monoclonal anti-CD31 (mM-CD31) with those of phalloidin and α-SMA on the rat brain in health and ischemia/reperfusion (I/R) with the middle cerebral artery occlusion. By multiple immunofluorescence staining, it was found that gP-CD31 labeling expressed extensively on the cerebral capillaries forming the vascular networks on the normal and ischemic regions, but mM-CD31 labeling mainly presented on the capillaries in the ischemic region. In contrast to the vascular labeling with gP-CD31, phalloidin and α-SMA were mainly expressed on the wall of cortical penetrating arteries, and less on that of capillaries. By three-dimensional reconstruction analysis, it was clearly shown that gP-CD31 labeling was mainly located on the lumen side of vascular wall and was surrounded by phalloidin labeling and α-SMA labeling. These results indicate that gP-CD31 is more sensitive than mM-CD31 for labeling the cerebral vasculature, and is highly compatible with phalloidin and α-SMA for evaluating the cerebral vascular networks under the physiological and pathological conditions.

Neuromuscular organization and haptoral armament of Polyclithrum ponticum (Monogenea: Gyrodactylidae).

Most gyrodactylids have a haptor armed with a pair of hamuli, two connecting bars and 16 marginal hooks. In some gyrodactylids, however, the haptor is disc-shaped and reinforced by additional sclerites. The genus Polyclithrum has arguably the most elaborate haptor in this group. This study aimed to gain better understanding of the anatomy of Polyclithrum by examining neuromusculature and haptoral armament of Polyclithrum ponticum, a species parasitizing Mugil cephalus in the Black Sea, with emphasis on haptoral sclerites and musculature in connection with host-attachment mechanisms. Musculature was stained by phalloidin, the nervous system by anti-serotonin and anti-FMRFamide antibodies, and haptoral sclerites were visualized in reflected light. The study provided new information on sclerites: in addition to previously described supplementary sclerites (A1-6), ear-shaped sclerites (ESSs) and two paired groups of ribs, reflected light revealed a rod-shaped process on the ESSs and a pair of small posterior sclerites. The sclerites were shown to be operated by 16 muscles, the most prominent of which were two transverse muscles connecting the hamular roots, three muscles attached to sclerite A2, the muscle fibres of anterior ribs and a set of extrinsic muscles. The nervous system consists of a pair of cerebral ganglia connected by a commissure and three pairs of nerve cords that unite in the haptor to form a loop between the opposite cords. The arrangement of sclerites and muscles suggests that Polyclithrum initiates the attachment by clamping a host's surface with longitudinally folded haptor and then secures its position with marginal hooks.

Study on Long-Term Tracing of Fibroblasts on Three-Dimensional Tissue Engineering Scaffolds Based on Graphene Quantum Dots.

In order to find a convenient and stable way to trace human skin fibroblasts (HSFs) in three-dimensional tissue engineering scaffolds for a long time, in this experiment, Graphene Oxide Quantum Dots (GOQDs), Amino Graphene Quantum Dots (AGQDs) and Carboxyl Graphene Quantum Dots (CGQDs) were used as the material source for labeling HSFs. Exploring the possibility of using it as a long-term tracer of HSFs in three-dimensional tissue engineering scaffolds, the contents of the experiment are as follows: the HSFs were cultured in a cell-culture medium composed of three kinds of Graphene Quantum Dots for 24 h, respectively; (1) using Cell Counting Kit 8 (CCK8), Transwell migration chamber and Phalloidin-iFlior 488 to detect the effect of Graphene Quantum Dots on the biocompatibility of HSFs; (2) using a living cell workstation to detect the fluorescence labeling results of three kinds of Graphene Quantum Dots on HSFs, and testing the fluorescence attenuation of HSFs for 7 days; (3) the HSFs labeled with Graphene Quantum Dots were inoculated on the three-dimensional chitosan demethylcellulose sodium scaffold, and the living cell workstation was used to detect the spatial distribution of the HSFs on the three-dimensional scaffold through the fluorescence properties of the HSFs.. Experimental results: (1) the results of CCK8, Transwell migration, and FITC-Phalloidin cytoskeleton test showed that the three kinds of Graphene Quantum Dots had no effect on the biological properties of HSFs (p < 0.05); (2) the results of the fluorescence labeling experiment showed that only AGQDs could make HSFs fluorescent, and cells showed orange−red fluorescence; (3) the results of long-range tracing of HSFs which were labeled by with AGQDs showed that the fluorescence life of the HSFs were as long as 7 days; (4) The spatial distribution of HSFs can be detected on the three-dimensional scaffold based on their fluorescence properties, and the detection time can be up to 7 days.

Quantitative Microstructural Analysis of Cellular and Tissue Remodeling in Human Glaucoma Optic Nerve Head.

Purpose: To measure quantitatively changes in lamina cribrosa (LC) cell and connective tissue structure in human glaucoma eyes. Methods: We studied 27 glaucoma and 19 age-matched non-glaucoma postmortem eyes. In 25 eyes, LC cross-sections were examined by confocal and multiphoton microscopy to quantify structures identified by anti-glial fibrillary acidic protein (GFAP), phalloidin-labeled F-actin, nuclear 4',6-diamidino-2-phenylindole (DAPI), and by second harmonic generation imaging of LC beams. Additional light and transmission electron microscopy were performed in 21 eyes to confirm features of LC remodeling, including immunolabeling by anti-SOX9 and anti-collagen IV. All glaucoma eyes had detailed clinical histories of open-angle glaucoma status, and degree of axon loss was quantified in retrolaminar optic nerve cross-sections. Results: Within LC pores, the proportionate area of both GFAP and F-actin processes was significantly lower in glaucoma eyes than in controls (P = 0.01). Nuclei were rounder (lower median aspect ratio) in glaucoma specimens (P = 0.02). In models assessing degree of glaucoma damage, F-actin process width was significantly wider in glaucoma eyes with more damage (P = 0.024), average LC beam width decreased with worse glaucoma damage (P = 0.042), and nuclear count per square millimeter rose with worse damage (P = 0.019). The greater cell count in LC pores represented 92.3% astrocytes by SOX9 labeling. The results are consistent with replacement of axons in LC pores by basement membrane labeled by anti-collagen IV and in-migrating astrocytes. Conclusions: Alteration in LC structure in glaucoma involves migration of astrocytes into axonal bundles, change in astrocyte orientation and processes, production of basement membrane material, and thinning of connective tissue beams.

Eye Movements But Not Vision Drive the Development of Palisade Endings.

Purpose: To test whether visual experience and/or eye movements drive the postnatal development of palisade endings in extraocular muscles. Methods: In three newborn cats, the right eye was covered until 30 days from postnatal (P) day 7 (before opening their eyes), and in three cats both eyes were covered until 45 days, also from P7. To block eye movements, another seven cats received a retrobulbar injection of botulinum neurotoxin A (BoNT-A) into the left orbit at birth and survived for 45 days (three cats) and 95 days (four cats). The distal third of the rectus muscles containing the palisade endings was used for whole-mount preparation and triple-fluorescence labeling with anti-neurofilament along with (1) anti-synaptophysin and phalloidin or (2) anti-growth associated protein 43 (GAP43) and phalloidin. Immunolabeled specimens were analyzed in the confocal laser scanning microscope. Results: After unilateral and bilateral dark rearing, palisade endings were qualitatively and quantitatively equal to those from age-matched controls. After BoNT-A induced eye immobilization for 45 or 95 days, palisade endings were absent in the superior rectus and lateral rectus muscles and only present in the inferior rectus and medial rectus muscle. These BoNT-A-treated palisade endings were rudimentary and reduced in number, and the expression of the neuronal developmental protein GAP43 was significantly reduced. Conclusions: This study demonstrates that eye immobilization, but not visual deprivation, affects palisade ending development. Palisade endings develop in the first month of life, and the present findings indicate that, during this time window, palisade endings are prone to oculomotor perturbations.

Rhizoma Paridis saponins suppresses vasculogenic mimicry formation and metastasis in osteosarcoma through regulating miR-520d-3p/MIG-7 axis.

Osteosarcoma (OS) is a highly metastatic bone cancer that usually affects children. Rhizoma Paridis saponins (RPS) have been identified to show a broad-spectrum anti-tumor activity. Our previous study has identified vasculogenic mimicry (VM) as an indicator of poor prognosis for OS. Rhizoma Paridis ethanol extract exhibits potent anti-OS property. However, the anti-metastatic effect of RPS on OS and the detailed mechanisms remain unknown. RPS was characterized by liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/Q-TOF/MS) analysis. The anti-OS, anti-metastasis and anti-VM activities of RPS were investigated using in vitro biological assays and a xenograft mouse model. Western blot, qRT-PCR, ELISA, Phalloidin staining and immunohistochemistry assays were conducted to investigate the molecular mechanism of RPS. A total of 34 phytochemicals from RPS were identified by LC/Q-TOF/MS. RPS dose-dependently suppressed the OS cell proliferation, metastasis and VM formation in vitro and in vivo. Mechanically, we found that RPS downregulated migration-inducing gene 7 (MIG-7) expression, resulting in inhibition of the PI3K/MMPs/Ln-5γ2 pathway and cell protrusion formation. Additionally, we confirmed that RPS downregulated MIG-7 by upregulating miR-520d-3p expression. Our results suggests that RPS inhibits the VM formation and metastasis of OS by modulating the miR-520d-3p/MIG-7 signaling axis.