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Sustained liver injuries predominantly promote oxidative stress and inflammation that lead to the progression of chronic liver disease (CLD), including fibrosis, cirrhosis, and hepatocellular carcinoma. Boldine, an alkaloid isolated from Peumus boldus, has been shown to have antioxidant and anti-inflammatory effects. Currently, there is no definitive treatment option available for CLD. Therefore, we investigated the hepatoprotective effect of boldine against carbon tetrachloride (CCl 4 )-induced chronic liver injury in rats. CCl 4 (2 mL/kg., b.w., i.p.) was administered twice weekly for 5 weeks to induce chronic liver injury in rats. Separate groups of rats were given boldine (20 mg/kg b.w., and 40 mg/kg b.w.) and silymarin (100 mg/kg b.w.) orally, daily. Serum transaminases, lipid peroxidation, and antioxidant levels were measured, and nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (cox-2), interleukin-1 β (IL-1β), and α-smooth muscle actin (α-SMA) gene and protein expressions were evaluated. CCl 4 administration increased liver marker enzymes of hepatotoxicity in serum and oxidative stress markers, inflammatory genes and α-smooth muscle actin expression in liver tissue. Boldine concurrent treatment suppressed CCl 4 -induced elevation of transaminase levels in serum, restored enzymic and non-enzymic antioxidants, and downregulated NF-κB, TNF-α, Cox-2 and IL-1β expressions, thereby suppressing hepatic inflammation. Boldine administration also repressed α-SMA expression. The results of this study demonstrate the antioxidant, anti-inflammatory, and antifibrotic properties of boldine, and it can be a potential therapeutic candidate in the treatment of CLD., (© 2024 Wiley Periodicals LLC.)

Objective: To investigate the effects of Porphyromonas gingivalis derived outer membrane vesicles (Pg OMV) on osteoclast differentiation of macrophages and its underlying mechanisms. Methods: The morphology and the size distribution of Pg OMV were analyzed by transmission electron microscopy and nanoparticle tracing analysis, respectively. The osteoclast precursors were treated with 1, 3 and 10 mg/L Pg OMV (1, 3 and 10 mg/L OMV treatment group) or phosphate buffer solution (PBS)(control group). The formation of osteoclasts was analyzed by tartrate-resistant acid phosphase (TRAP) staining and F-actin staining and real-time quantitative PCR (RT-qPCR) were used to detect the expression of Fos and matrix metallopeptidase 9 (MMP9). Polymyxin B (PMB) was used to block lipopolysaccharide (LPS) and then Pg OMV was used to treat osteoclast precursor (PMB-OMV treatment group), and OMV treatment group was used as control. TRAP and F-actin staining were used to observe the formation of osteoclasts and actin rings. The effect of Pg OMV on the expression of Toll-like receptor (TLR) 2 and TLR4 in preosteoclasts was detected by Western blotting. The osteoclast precursors were pretreated with 10, 50, 100 and 200 μmol/L C29, an inhibitor of TLR2, and then treated with Pg OMV(OMV+10, 50, 100 and 200 μmol/L C29 treatment group) and OMV treatment group without C29 pretreatment was control. TRAP and F-actin staining were used to observe the formation of osteoclasts and actin rings. The osteoclast precursor cells were treated with OMV (OMV treatment group) and OMV incubated with PMB (PMB-OMV treatment group) and the expression of TLR2 in osteoclast precursor was detected by Western blotting. Results: Pg OMV showed classical vesicular structures, and the average particle size of Pg OMV were 179.2 nm. A large number of actin rings were observed in the 3 and 10 mg/L OMV treatment groups. The percentages of TRAP-positive osteoclast area in 3 mg/L OMV treatment group [(22.6±2.1)%] and 10 mg/L OMV treatment group [(32.0±2.3)%] were significantly increased compared with control group [(4.9±0.5)%] ( P <0.001). Compared with the control group (1.000±0.029), the mRNA relative expression of Fos in 3 mg/L OMV treatment group (1.491±0.114) and 10 mg/L OMV treatment group (1.726±0.254) was significantly increased ( P =0.013, P =0.001). Compared with the control group (1.007±0.148), the mRNA relative expression of MMP9 in the group of 10 mg/L OMV (2.232±0.097) was significantly increased ( P <0.001). Actin ring formation was less in PMB-OMV treatment groups than in OMV treatment groups. The proportion of TRAP-positive osteoclasts area [(14.8±3.8)%] in PMB-OMV treatment group was significantly lower than OMV treatment group [(31.5±6.7) %] ( P =0.004). The relative expression of TLR2 in OMV treatment group (1.359±0.134) was significantly higher than that in the control group (1.000±0.000) ( t =4.62, P =0.044). Compared with the OMV treatment group [(29.4±1.7)%], 50, 100 and 200 μmol/L C29 significantly decreased the formation of osteoclasts [(24.0±1.7)%, (18.5±2.1)%, (9.1±1.3) %] ( P =0.026, P <0.001, P <0.001). TLR2 protein expression in PMB-OMV group (0.780±0.046) was significantly lower than that in OMV group (1.000±0.000)( t =8.32, P =0.001). Conclusions: Pg OMV can promote osteoclast differentiation by carrying LPS, TLR2 plays an important role in Pg OMV mediated osteoclast differentiation.

Exposure to particulate matter ≤ 2.5 µm (PM 2.5 ) is a risk factor for many lung diseases. Although the toxicologic effects of PM 2.5 on airway epithelium are well-described, the effects of PM 2.5 on fibroblasts in the lung are less studied. Here, we sought to examine the effects of PM 2.5 on the differentiation of fibroblasts into myofibroblasts. Although a single treatment of fibroblasts did not result in a change in collagen or the myofibroblast marker α-SMA, exposing fibroblasts to sequential treatments with PM 2.5 at low concentrations caused a robust increase in these proteins. Treatment of fibroblasts with IMD0354, an inhibitor to nuclear factor κB, but not with an antagonist to aryl hydrocarbon receptor, abolished the ability of PM 2.5 to induce myofibroblast differentiation. These data demonstrate that potential impact of PM 2.5 to fibroblast activation and fibrosis and support the importance of utilizing low concentrations and varying exposure protocols to toxicologic studies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: This study investigated the effects of dexamethasone (Dex) on human trabecular meshwork (TM) cells, a model of glucocorticoid-induced glaucoma, and evaluated the impact of ripasudil (Rip) as a co-delivery or sequential dosing strategy., Methods: In vitro experiments were conducted to assess the effects of Dex and Rip on TM cells. Confocal microscopy was used to evaluate the impact of Dex and Rip on F-actin staining signals. Contractility of the TM cells upon Dex and Rip treatment mimicking co-delivery and sequential delivery was quantified using collagen gel contraction assay. Transepithelial electrical resistance (TEER) values and fluorescein isothiocyanate (FITC)-dextran permeability were also measured to assess the impact of Dex and Rip on TM cells., Results: Dex and Rip did not exhibit cytotoxicity at the maximum tested concentration (20 µM). Dex-treated TM cells exhibited higher F-actin staining signals compared to controls, which were reduced when co-treated with Rip. Rip inhibited Dex-induced collagen gel contraction activity in both co-delivery and sequential treatments. Dex resulted in increased TEER values as the dose increased, whereas TEER values were maintained when co-treated with Rip., Conclusions: Co-delivery of Rip has the potential to prevent glaucoma symptoms when patients are treated with Dex. This study highlights the importance of identifying strategies to reduce the side effects of prolonged use of glucocorticoids, such as Dex, in the treatment of various diseases., Translational Relevance: This study demonstrates the potential of co-delivering ripasudil with dexamethasone to mitigate glucocorticoid-induced ocular hypertension and a secondary glaucoma that resembles primary open-angle glaucoma, providing insights for the development of novel preventive strategies in clinical care.

The uc.291 transcript controls keratinocytes differentiation by physical interaction with ACTL6A and subsequent induction of transcription of the genes belonging to the epidermal differentiation complex (EDC). Uc.291 is also implicated in the dedifferentiation phenotype seen in poorly differentiated cutaneous squamous cell carcinomas. Here, we would like to investigate the contribution of uc.291 to the unbalanced differentiation state of keratinocytes observed in hyperproliferative skin disorders, e. g., psoriasis. Psoriasis is a multifactorial inflammatory disease, caused by alteration of keratinocytes homeostasis. The imbalanced differentiation state, triggered by the infiltration of immune cells, represents one of the events responsible for this pathology. In the present work, we explore the role of uc.291 and its interactor ACTL6A in psoriasis skin, using quantitative real-time PCR (RT-qPCR), immunohistochemistry and bioinformatic analysis of publicly available datasets. Our data suggest that the expression of the uc.291 and of EDC genes loricrin and filaggrin (LOR, FLG) is reduced in lesional skin compared to nonlesional skin of psoriatic patients; conversely, the mRNA and protein level of ACTL6A are up-regulated. Furthermore, we provide evidence that the expression of uc.291, FLG and LOR is reduced, while ACTL6A mRNA is up-regulated, in an in vitro psoriasis-like model obtained by treating differentiated keratinocytes with interleukin 22 (IL-22). Furthermore, analysis of a publicly available dataset of human epidermal keratinocytes treated with IL-22 (GSE7216) confirmed our in vitro results. Taken together, our data reveal a novel role of uc.291 and its functional axis with ACTL6A in psoriasis disorder and a proof of concept that biological inhibition of this molecular axis could have a potential pharmacological effect against psoriasis and, in general, in skin diseases with a suppressed differentiation programme., (© 2023. The Author(s).)

Cytotoxicity of nanoparticles, typically evaluated by biochemical-based assays, often overlook the cellular biophysical properties such as cell morphology and cytoskeletal actin, which could serve as more sensitive indicators for cytotoxicity. Here, we demonstrate that low-dose albumin-coated gold nanorods (HSA@AuNRs), although being considered noncytotoxic in multiple biochemical assays, can induce intercellular gaps and enhance the paracellular permeability between human aortic endothelial cells (HAECs). The formation of intercellular gaps can be attributed to the changed cell morphology and cytoskeletal actin structures, as validated at the monolayer and single cell levels using fluorescence staining, atomic force microscopy, and super-resolution imaging. Molecular mechanistic study shows the caveolae-mediated endocytosis of HSA@AuNRs induces the calcium influx and activates actomyosin contraction in HAECs. Considering the important roles of endothelial integrity/dysfunction in various physiological/pathological conditions, this work suggests a potential adverse effect of albumin-coated gold nanorods on the cardiovascular system. On the other hand, this work also offers a feasible way to modulate the endothelial permeability, thus promoting drug and nanoparticle delivery across the endothelium., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Pulpotomy is an effective treatment for retaining vital pulp after pulp exposure caused by caries removal and/or trauma. The expression of alpha smooth muscle actin (α-SMA) is increased during the wound-healing process, and α-SMA-positive fibroblasts accelerate tissue repair. However, it remains largely unknown whether α-SMA-positive fibroblasts influence pulpal repair. In this study, we established an experimental rat pulpotomy model and found that the expression of α-SMA was increased in dental pulp after pulpotomy relative to that in normal dental pulp. In vitro results showed that the expression of α-SMA was increased during the induction of odontogenic differentiation in dental pulp stem cells (DPSCs) compared with untreated DPSCs. Moreover, α-SMA overexpression promoted the odontogenic differentiation of DPSCs via increasing mitochondrial function. Mechanistically, α-SMA overexpression activated the mammalian target of rapamycin (mTOR) signaling pathway. Inhibition of the mTOR signaling pathway by rapamycin decreased the mitochondrial function in α-SMA-overexpressing DPSCs and suppressed the odontogenic differentiation of DPSCs. Furthermore, we found that α-SMA overexpression increased the secretion of transforming growth factor beta-1 (TGF-β1). In sum, our present study demonstrates a novel mechanism by which α-SMA promotes odontogenic differentiation of DPSCs by increasing mitochondrial respiratory activity via the mTOR signaling pathway., (© 2023 Scandinavian Division of the International Association for Dental Research. Published by John Wiley & Sons Ltd.)

Cold plasma can be beneficial for promoting skin wound healing and has a high potential of being effectively used in treating various wounds. Our aim was to verify the effect of cold plasma in accelerating wound healing and investigate its underlying mechanism in vitro and in vivo. For the in vivo experiments, 2 full-thickness dermal wounds were created in each mouse (n = 30). While one wound was exposed to 2 daily plasma treatments for 3 min, the other wound served as a control. The wounds were evaluated by imaging and histological analyses at 4, 7, and 11 days post the wound infliction process. Immunohistochemical studies were also performed at the same time points. In vitro proliferation and scratch assay using HaCaT keratinocytes and fibroblasts were performed. The expression levels of wound healing-related genes were analyzed by real-time polymerase chain reaction and western blot analysis. On day 7, the wound healing rates were 53.94% and 63.58% for the control group and the plasma-treated group, respectively. On day 11, these rates were 76.05% and 93.44% for the control and plasma-treated groups, respectively, and the difference between them was significant ( P  = .039). Histological analysis demonstrated that plasma treatment promotes the formation of epidermal keratin and granular layers. Immunohistochemical studies also revealed that collagen 1, collagen 3, and alpha-smooth muscle actin appeared more abundantly in the plasma-treated group than in the control group. In vitro, the proliferation of keratinocytes was promoted by plasma exposure. Scratch assay showed that fibroblast exposure to plasma increased their migration. The expression levels of collagen 1, collagen 3, and alpha-smooth muscle actin were elevated upon plasma treatment. In conclusion, cold plasma can accelerate skin wound healing and is well tolerated.

Objectives: To evaluate the potential effect of EphrinB2 in human thoracic aortic dissection (TAD) and to illustrate the mechanisms governing the role of EphrinB2 in the growth of human aortic smooth muscle cells (HASMC)., Methods: In the study, EphrinB2 expression was investigated by qRT-PCR and immunohistochemistry in 12 pairs of TAD and adjacent human tissues. HASMCs were used for in vitro experiments. Next, EphrinB2 overexpression and depletion in HASMCs were established by EphrinB2-overexpressing vectors and small interfering RNA, respectively. The transfection efficiency was evaluated by qRT-PCR and Western blot. The effects of overexpression and depletion of EphrinB2 on cell proliferation, migration, and invasion were tested in vitro. Cell Counting Kit-8, flow cytometry and transwell migration/invasion, and wound healing assay were used to explore the function of EphrinB2 on HASMC cell lines. The relationship between EphrinB2 and F-actin was assessed by Western blot, immunofluorescence, and Co-IP., Results: We found that EphrinB2 was a prognostic biomarker of TAD patients. Moreover, EphrinB2 expression negatively correlated to aortic dissection tissues, and disease incidence of males, suggesting that EphrinB2 might act as a TAD suppressor by promoting proliferation or decreasing apoptosis in HASMC. Next, over-expression of EphrinB2 in HASMC lines drove cell proliferation, migration, and invasion, and inhibited apoptosis while knockdown EphrinB2 showed the opposite phenomenon, respectively. Furthermore, the level of F-actin in mRNA, protein, and distribution in HASMC cell lines highly matched with the expression of EphrinB2, which indicated that EphrinB2 could mediate the HASMC cytoskeleton via inducing F-actin., Conclusions: In conclusion, our results first provided the pivotal role of EphrinB2 in HASMC proliferation initiated by mediating F-actin and demonstrated a prognostic biomarker and the potential targets for therapy to prevent thoracic aortic dissection.

Mechanosensitivity of cell spread area to substrate stiffness has been established both through experiments and different types of mathematical models of varying complexity including both the mechanics and biochemical reactions in the cell. What has not been addressed in previous mathematical models is the role of cell membrane dynamics on cell spreading, and an investigation of this issue is the goal of this work. We start with a simple mechanical model of cell spreading on a deformable substrate and progressively layer mechanisms to account for the traction dependent growth of focal adhesions, focal adhesion induced actin polymerization, membrane unfolding/exocytosis and contractility. This layering approach is intended to progressively help in understanding the role each mechanism plays in reproducing experimentally observed cell spread areas. To model membrane unfolding we introduce a novel approach based on defining an active rate of membrane deformation that is dependent on membrane tension. Our modeling approach allows us to show that tension-dependent membrane unfolding plays a critical role in achieving the large cell spread areas experimentally observed on stiff substrates. We also demonstrate that coupling between membrane unfolding and focal adhesion induced polymerization works synergistically to further enhance cell spread area sensitivity to substrate stiffness. This enhancement has to do with the fact that the peripheral velocity of spreading cells is associated with contributions from the different mechanisms by either enhancing the polymerization velocity at the leading edge or slowing down of the retrograde flow of actin within the cell. The temporal evolution of this balance in the model corresponds to the three-phase behavior observed experimentally during spreading. In the initial phase membrane unfolding is found to be particularly important.

The aim of this study was to investigate the underlying protective mechanisms of asiaticoside (AS) against liver fibrosis (LF) both in vivo and in vitro. A rat model with carbon tetrachloride (CCl 4 )-induced liver fibrosis is employed to verify the effect and mechanism of AS on the process of liver fibrosis in vivo experiment. Hematoxylin/eosin and sirius red staining was conducted to assess the severity of liver injury and fibrosis. Further, the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), glutamyl transferase (GGT), and total bilirubin (TBil) were measured. In addition, LX2 cells were cultured for vitro experiment to investigate the influence of AS on hepatic stellate cells (HSCs). Overproduction of α-smooth muscle actin and type I collagen is characteristic of LF and HSCs, as determined by immunohistochemical and Western blot analyses. The expression levels of molecules associated with the Notch signaling pathway (i.e., Notch-1, Jagged-1, and Delta-like-4) were assessed by Western blot analysis. The results revealed that AS attenuated LF, as defined by reduced deposition of collagen, expression of α-smooth muscle actin and collagen type 1, and expression of biochemical parameters (alanine aminotransferase, aspartate aminotransferase, and hydroxyproline). Notably, AS suppressed the expression levels of Notch-1, Jagged-1, and Delta-like-4 in activated HSCs and LF. Collectively, these results demonstrate that AS prevented the progression of LF by modulating the Notch signaling pathway, indicating that AS has potential therapeutic effects against LF., (© 2022. The Author(s) under exclusive licence to The Japanese Society of Pharmacognosy.)

Objective: To explore the effect of macrophage-derived exosomes on the activation of hepatic stellate cells and its possible mechanism. Methods: Differential ultracentrifugation was used to extract macrophage exosomes. The exosomes were co-cultured with the mouse hepatic stellate cell line JS1, and a control group was established with phosphate buffered saline (PBS). Cell immunofluorescence was used to observe the expressional conditions of F-actin. Cell counting kit-8 (CCK8) was used to detect the survival rate of JS1 cells in the two groups. The activation indices of JS1 cells [collagen type Ⅰ (Col Ⅰ) and α-smooth muscle actin (α-SMA)] and its key signal pathway activation index expression level [transforming growth factor (TGF)-β1/Smads, platelet-derived growth factor (PDGF)] in the two groups were determined using Western blot and RT-PCR. Data comparison between two groups was performed using an independent sample t-test. Results: The membrane structure of exosomes was clearly observed by transmission electron microscopy. The expression of exosome marker proteins CD63 and CD81 was positive, suggesting that exosomes were successfully extracted. Exosomes were co-cultured with JS1 cells. Compared with the PBS control group, there was no statistically significant difference in the proliferation rate of JS1 cells in the exosomes group ( P ＞0.05). The expression of F-actin was significantly increased in the exosome group. The mRNA and protein expression levels of α-SMA and ColⅠwere significantly increased in exosome group JS1 cells (all P ＜0.05). The mRNA relative expression levels of α-SMA in PBS and exosome group were 0.25±0.07 and 1.43±0.19, respectively, while that of ColⅠ was 1.03±0.04 and 1.57±0.06, respectively. The mRNA and protein expressions of PDGF were significantly increased in exosome group JS1 cells ( P ＜0.05). The mRNA relative expression levels of PDGF in the PBS group and exosome group were 0.27±0.04 and 1.65±0.12, respectively. There were no statistically significant differences in the mRNA and protein expressions of TGF-β1, Smad2 and Smad3 between the two groups ( P ＞0.05). Conclusion: Macrophage-derived exosomes significantly promote the activation of hepatic stellate cells. JS1 cells may be the underlying mechanism for the up-regulation of PDGF expression.

Latently infected cells harboring replication-competent proviruses represent a major barrier to HIV-1 cure. One major effort to purge these cells has focused on developing the "shock and kill" approach for forcing provirus reactivation to induce cell killing by viral cytopathic effects, host immune responses, or both. We conducted kinetic and mechanistic studies of HIV-1 protein expression, virion production, and cell-to-cell virus transmission during provirus reactivation. Provirus-activated ACH-2 cells stimulated with romidepsin (RMD) or PMA produced Nef early, and then Env and Gag in parallel with the appearance of virions. Env on the surface of provirus-activated cells and cellular F-actin were critical in the formation of virological synapses to mediate cell-to-cell transmission of HIV-1 from provirus-activated cells to uninfected cells. This HIV-1 cell-to-cell transmission was substantially more efficient than transmission seen via cell-free virus spread and required F-actin remodeling and CD4, but not chemokine receptors. Resting human primary CD4 + T cells including naïve and memory subpopulations and, especially the memory CD4 + T cells, were highly susceptible to HIV-1 infection via cell-to-cell transmission. Cell-to-cell transmission of HIV-1 from provirus-activated cells was profoundly decreased by protease inhibitors (PIs) and neutralizing antibodies (nAbs) that recognize the CD4-binding site (CD4bs) such as VRC01, but not by reverse transcriptase (RT) inhibitor Emtricitabine (FTC). Therefore, our results suggest that PIs with potent blocking abilities should be used in clinical application of the "shock and kill" approach, most likely in combination with CD4bs nAbs, to prevent new HIV-1 infections., (© 2022 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

The study aimed to assess the effect of simvastatin on gene expression of LDLR, SREBPs, and SCD1 in rat hepatic tissues fed with high-fat diets (HFD) and its association with some biochemical parameters. Thirty-two male Wister albino rats were divided into four equal groups (three test and one control groups). The biochemical parameters were determined by using spectrophotometer techniques and the Elisa method. Low-density lipoprotein receptor, sterol regulatory element-binding proteins, stearoyl-CoA desaturase1, Beta-actin were analysed by real-time quantitative polymerase chain reaction (RT-PCR) method. At the end of study, the livers of the rats were separated and changes of hepatic tissue were determined. LDLR, SREBP2, and SCD1 expression increased significantly when compared G1 versus G4 and G2 versus G4. The expression of LDLR, SREBP2, and SCD1 also increased significantly when compared G2 versus G3, G1versus G3 and G1 versus G3 and G2 versus G3. The serum level of cholesterol, triglyceride, glucose, LDL, and HDL increased significantly when compared G1 versus G3. LDL showed significantly decreased when compared G1 versus G2. Cholesterol, glucose and HDL and triglyceride levels were increased significantly when compared G1 versus G4 and G2. Treatment of rats with HFD and simvastatin 20 mg/kg, triglyceride and LDL were almost the same as a control group and LDLR expression increased 98% in liver tissue. Gene expressions may be up-regulated in liver tissue and they showed different effects on biochemical parameters.

Background: The intermittent administration of parathyroid hormone (PTH) has been prescribed to osteoporotic patients due to its bone anabolic effects. In addition to its actions on bone cells, PTH appears to affect bone-specific blood vessels. These blood vessels are derived from bone marrow sinusoids, which express EphB4, a hallmark of veinous vascular endothelial cells. Given the presence of osteo-vascular interactions, it is important to elucidate the effects of PTH on bone cells and blood vessels in murine models., Highlights: PTH stimulates preosteoblastic proliferation and osteoblastic bone formation. The former appears to be directly affected by PTH, whereas the latter requires osteoclast-mediated coupling. The administration of PTH through high-frequency dosage schemes accelerates bone turnover featuring remodeling-based bone formation, whereas low-frequency schemes cause mainly remodeling-based and partly modeling-based bone formation. Normally, many blood vessels lack alpha smooth muscle actin (αSMA)-immunoreactive vascular muscle cells surrounding basement membranes, indicating them being capillaries. However, PTH administration increases the number of blood vessels surrounded by αSMA-positive cells. These αSMA-positive cells spread out of blood vessels and express alkaline phosphatase and c-kit, suggesting their potential to differentiate into osteogenic and vascular endothelial/perivascular cells. Unlike bone cells, αSMA-positive cells did not appear in the periphery of blood vessels in the kidney and liver, and the thickness of the tunica media did not change regardless of PTH administration., Conclusion: Based on the results of the study and presence of osseous-vascular interactions, PTH appears to influence not only osteoblastic cells, but also blood vessels in bone., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (Copyright © 2022 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.)

Purpose: The etiology of adolescent idiopathic scoliosis (AIS) remains unclear. The chondrogenic differentiation of mesenchymal stem cells (MSCs) is important in AIS, and the Ras homolog gene family member A (RHOA) is associated with chondrogenesis. The purpose of this study was to explore the effect of RHOA on the chondrogenic differentiation of MSCs in AIS., Methods: We isolated MSCs from patients with AIS (AIS MSCs) and individuals without AIS (control MSCs). The inhibitor Y27632 was used to inhibit the function of RHOA/ROCK signaling, and plasmid-based overexpression and siRNA-mediated knockdown were used to manipulate RHOA expression. CCK-8 was used to detect cell viability. The phosphorylation levels of LIMK1, MLC2 and cofilin were detected by Western blotting. The mRNA expression of aggrecan, SOX9, and COL2A1 were confirmed using RT-PCR. Immunofluorescence was used to analyze F-actin and collagen II. Alcian blue staining was performed to assess the secretion of glycosaminoglycans (GAGs)., Results: We found that RHOA was significantly upregulated in AIS MSCs, and the phosphorylation levels of LIMK1, MLC2, and cofilin were increased. The mRNA expressions of aggrecan, SOX9, and COL2A1 were notably reduced in AIS MSCs. However, these effects were abolished by Y27632 treatment and RHOA knockdown in AIS MSCs. In addition, RHOA knockdown in AIS MSCs increased the content of collagen II and GAGs. RHOA overexpression in the control MSCs markedly activated the RHOA/ROCK signaling and decreased the expression of aggrecan, SOX9, and COL2A1, F-actin, and GAGs., Conclusion: RHOA regulates the chondrogenic differentiation ability of MSCs in AIS via the RHOA/ROCK signaling pathway and this regulation may involve SOX9.

Activation and/or modulation of the membrane-associated receptors plays a critical role in brain development. Thyroid hormone (TH) acts on both nuclear receptors (thyroid hormone receptor, TR) and membrane-associated receptors, particularly integrin αvβ3 in neurons and glia. Integrin αvβ3-mediated signal transduction mediates various cellular events during development including morphogenesis, migration, synaptogenesis, and intracellular metabolism. However, the involvement of integrin αvβ3-mediated TH action during brain development remains poorly understood. Thus, we examined the integrin αvβ3-mediated effects of TH (T 3 , T 4 , and rT 3 ) in the neurons and astrocytes using primary cerebellar culture, astrocyte-enriched culture, Neuro-2A clonal cells, and co-culture of neurons and astrocytes. We found that TH augments dendrite arborization of cerebellar Purkinje cells. This augmentation was suppressed by knockdown of integrin αvβ3, as well as TRα and TRβ. A selective integrin αvβ3 antagonist, LM609, was also found to suppress TH-induced arborization. However, whether this effect was a direct action of TH on Purkinje cells or due to indirect actions of other cells subset such as astrocytes was not clarified. To further study neuron-specific molecular mechanisms, we used Neuro-2A clonal cells and found TH also induces neurite growth. TH-induced neurite growth was reduced by co-exposure with LM609 or knockdown of TRα, but not TRβ. Moreover, co-culture of Neuro-2A and astrocytes also increased TH-induced neurite growth, indicating astrocytes may be involved in neuritogenesis. TH increased the localization of synapsin-1 and F-actin in filopodia tips. TH exposure also increased phosphorylation of FAK, Akt, and ERK1/2. Phosphorylation was suppressed by co-exposure with LM609 and TRα knockdown. These results indicate that TRs and integrin αvβ3 play essential roles in TH-induced dendritogenesis and neuritogenesis. Furthermore, astrocytes-neuron communication via TR-dependent and TR-independent signaling through membrane receptors and F-actin are required for TH-induced neuritogenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ariyani, Miyazaki, Amano and Koibuchi.)

Pesticide resistance in pests drives the development of RNA interference (RNAi)-based technology as a novel approach for pest control. To investigate the effects of the positional dependency of double-stranded RNAs (dsRNAs), we newly designed four different 200 bp dsRNAs targeting Colorado potato beetle (CPB) β-Actin gene, termed as dsACT200-1 to dsACT200-4, to compare their insecticidal activity to CPB larvae together with our previously used 200 bp and 700 bp dsRNAs (dsACT200 and dsACT700), respectively (He et al., 2020a). Each of dsRNAs harbors different numbers of expected siRNAs predicted by sequence-based prediction platform, dsACT200 and dsACT200-2 have a relatively higher number of siRNA than other 200 bps dsRNAs. When CPB larvae were fed with in vitro synthesized dsRNA-painted potato leaves, all the tested dsRNAs showed significant effects to protect against CPB larvae. Combined with the survival rate of CPB larvae, β-Actin gene expression level and the surviving CPB larvae weight, various positional dsRNAs from the same allele showed different plant protection activity against CPB larvae and partially correlated with the predicted siRNA numbers and distribution on the target sequence. This study suggests the specific allelic locus for rational dsRNA design triggering RNAi efficiency for target gene silencing is an essential factor in enhancing the insecticidal activity., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objective: The purpose of this study was to investigate the effects of different durations of static progressive stretching (SPS) on posttraumatic knee contracture in rats, including range of motion (ROM), gait analysis, myofibroblast proliferation, and collagen regulation., Methods: The posttraumatic knee contracture model was established, and male Wistar rats were randomly divided into the 20-minute SPS treatment, 30-minute SPS treatment (S30), 40-minute SPS treatment, untreated, immobilization, and control groups. At Week 1, 2, and 4 of treatment intervention, joint ROM and gait were measured and compared. Knee joint samples stained with hematoxylin and eosin and Masson trichrome were used to observe alterations in pathological structures. Collagen density and cell numbers in the posterior joint capsule were used to assess joint capsule fibrosis and inflammation. Immunohistochemistry was used to detect type I collagen and α-smooth muscle actin expression., Results: The S30 group improved the most; ROM, stance, mean intensity, print area, and stride length were 115 (SD = 5) degrees, 0.423 (SD = 0.074) seconds, 156.020 (SD = 7.952), 2.116 (SD = 0.078) cm2, and 11.758 (SD = 0.548) cm, respectively. The numbers of myofibroblasts, fibroblasts, and inflammatory cells decreased, and collagen proliferation was significantly suppressed in the S30 group compared with the other groups., Conclusion: S30 significantly improved posttraumatic knee contracture in rats, with reduced type I collagen and α-smooth muscle actin expression, decreased the numbers of myofibroblasts and inflammatory cells, suppressed fibrotic and inflammatory changes in the joint capsule, and increased joint mobility. This study provided basic evidence for an optimal standard-of-care treatment approach for posttraumatic knee joint contracture in rats, which may have significance for humans., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Physical Therapy Association. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The supplementation of dimethyl alpha-ketoglutarate (DMKG) during the in vitro maturation (IVM) process has been shown to improve the in vitro developmental competences of porcine oocytes. Here, the effects of DMKG supplementation in IVM medium on the development competencies of ovine oocytes were investigated by analyzing the nuclear maturation rate to metaphase II (MII) stage, ATP synthesis, cortical granules (CGs) dynamic, F-actin polymerization, mitochondrial activity, mitochondrial damage, reactive oxygen species (ROS) production, intracellular glutathione (GSH) production, DNA damage, cellular apoptosis, fertilization capacity and blastocyst development potential of ovine oocytes. In addition, the oxidative stress damage model induced by H 2 O 2 treatment was applied to confirm the antioxidative effect of DMKG supplementation on the development of ovine oocytes. The results showed that compared with MII oocytes without DMKG supplementation (Control group), 3 mM DMKG supplementation during IVM significantly (P < 0.05) increased nuclear maturation rate, ATP synthesis, CGs dynamic, F-actin polymerization, mitochondrial activity, GSH production and embryonic developmental competence and decreased ROS production, mitochondrial damage, DNA damage and cellular apoptosis level of ovine MII oocytes. Moreover, the reductions in the developmental competences of ovine MII oocytes caused by H 2 O 2 induced oxidative stress damages were effectively ameliorated by the co-supplementation in IVM of 3 mM DMKG (P < 0.05). Our results demonstrate the promising effect of DMKG supplementation on the in vitro developmental competence of ovine oocytes via the reduction of oxidative stress damages and indicates further research into the clinical applications of DMKG and the development of ovine breeding technologies is warranted., Competing Interests: Declaration of competing interest The authors report no conflicts of interest in this work., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objective: To explore the effect of hypoxia on the chemosensitivity of B-acute lymphoblastic leukemia （B-ALL） cells to Vincristine (VCR) and the mechanisms., Methods: B-ALL cells SUP-B15, Nalm-6 and RS4;11 were selected as the research objects. The cells were divided into the control group and the hypoxia mimic group (CoCl 2 pretreatment). The two groups were treated with VCR at different concentrations for 24 hours, CCK-8 was used to detect cell viability, flow cytometry was used to detect cell apoptosis, and Western bolt method was used to detect hypoxia inducible factor (HIF-1α), BAX, Bcl-2 and β-actin protein expression. Quantitative real-time fluorescent PCR (qRT-PCR) was used to detect BAX and β-actin mRNA levels., Results: CoCl 2 could simulate hypoxic environment to induce the expression of HIF-1α. The cells SUP-B15 and RS4;11 of the hypoxia mimic group were lower sensitivity to VCR as compared with the control group; the apoptosis rate of the hypoxia mimic group was lower than that of the control group after 80 nmol/L VCR treatment. The expression levels of BAX protein and mRNA in the hypoxia mimic group were lower than those of the control group, and there was no significant difference in the expression levels of Bcl-2 protein between two groups., Conclusion: Under hypoxic conditions, HIF-1α may mediate VCR resistance in B-ALL cells by downregulating the pro-apoptotic protein BAX.

Objective: To investigate the protective effect and potential mechanism of mitochondrial coenzyme Q (MitoQ) on mitochondria-dependent apoptosis in type II alveolar epithelial cells induced by lipopolysaccharide (LPS)., Methods: The type II lung epithelial cell line (A549) were cultured with different concentrations of LPS in vitro, a cell model of acute lung injury (ALI) was reproduced, the optimal concentration of LPS was obtained according to the half maximal inhibitory concentration (IC 50 ). The cells were pretreated with different concentrations of MitoQ to determine the best intervention concentration of MitoQ. The cells were divided into four groups: the cells in blank control group were cultured in DMEM; the cells in LPS group were stimulated with 10 mg/L of LPS for 24 hours; the cells in MitoQ+LPS group were pretreated with 1 μmol/L MitoQ for 60 minutes, and then were co-cultured with 10 mg/L of LPS for 24 hours; and the cells in MitoQ+phosphatidylinositol 3-kinase (PI3K) selective inhibitor LY294002+LPS group were pretreated with 1 μmol/L MitoQ and 20 μmol/L LY294002 for 60 minutes, and then were co-cultured with 10 mg/L of LPS for 24 hours. Cell viability was measured using cell counting kit-8 (CCK-8). The cell apoptosis rate was determined by flow cytometry and TdT-mediated dUTP-nick end labeling (TUNEL) method. The protein expression levels of apoptosis protein Bax, anti-apoptotic protein Bcl-2 and PI3K-serine/threonine kinase (Akt) protein PI3K expression and Akt phosphorylation level were detected by Western blotting., Results: According to the inhibition rate curve, the IC 50 of LPS on A549 cells was 11.06 mg/L. Therefore, 10 mg/L was selected as the stimulating concentration of LPS. After stimulation with 10 mg/L LPS, the cell viability first increased and then decreased with the increase in MitoQ pretreatment concentration. According to the cell viability curve, 1 μmol/L was selected as the optimum concentration of MitoQ. Compared with LPS group, after pretreated with 1 μmol/L MitoQ, cell mitochondrial dependent apoptosis was significantly attenuated, which was characterized by the apoptosis rate was significantly decreased [flow cytometry: (8.73±0.25)% vs. (18.10±0.70)%, TUNEL: (12.30±0.82)% vs. (21.43±0.86)%, both P < 0.05], the expression of Bax was significantly down-regulated (Bax/β-actin: 0.58±0.03 vs. 1.06±0.10, P < 0.05) and Bcl-2 level was significantly up-regulated (Bcl-2/β-actin: 1.03±0.06 vs. 0.53±0.07, P < 0.05), meanwhile the expression of PI3K and Akt phosphorylation level were significantly increased [PI3K protein (PI3K/β-actin): 1.20±0.02 vs. 0.96±0.04, phosphorylated Akt (p-Akt) protein (p-Akt/t-Akt): 1.22±0.08 vs. 0.92±0.04, both P < 0.05]. Pretreatment with LY294002 could inhibit the anti-apoptotic effect of MitoQ on cells, it was characterized by the apoptotic rate was significantly increased as compared with MitoQ+LPS group [flow cytometry: (14.50±0.57)% vs. (8.73±0.25)%, TUNEL: (16.50±0.53)% vs. (12.30±0.82)%, both P < 0.05], the expression of Bax was significantly up-regulated (Bax/β-actin: 0.95±0.03 vs. 0.58±0.03, P < 0.05) and Bcl-2 level was significantly down-regulated (Bcl-2/β-actin: 0.62±0.03 vs. 1.03±0.06, P < 0.05), meanwhile the expression of PI3K and Akt phosphorylation level were significantly decreased [PI3K protein (PI3K/β-actin): 0.90±0.05 vs. 1.20±0.02, p-Akt protein (p-Akt/t-Akt): 0.89±0.02 vs. 1.22±0.08, both P < 0.05]., Conclusions: MitoQ improved LPS induced mitochondria-dependent apoptosis of A549 cells by significantly activating PI3K/Akt signal pathway, which provided a new treatment for LPS induced ALI.

Background: Lidocaine is a local anesthetic that wildly used in surgical treatment and postoperative medical care for lung cancers. We hypothesized that lidocaine at clinical plasma concentration can inhibit CXCL12/CXCR4 axis-regulated cytoskeletal remodeling thereby reduce the migration of Non-small-cell lung cancers (NSCLC) cells., Methods: We determined the effect of lidocaine at clinical plasma concentration on CXCL12-induced cell viability, apoptosis, cell death, monolayer cell wound healing rate, individual cell migration indicators, expression of CXCR4, CD44, and ICAM-1, intracellular Ca2+ level, and filamentous actin level alteration of NSCLC cells A549 and CXCR4-knocked down A549 cells using CCK-8, Bcl-2 ELISA, Cell death ELISA, wound healing assay, chemotaxis assay, western blotting, QPCR, Fura-2-based intracellular Ca2+ assay, and Fluorescein Phalloidin staining respectively., Results: Lidocaine did not affect cell viability, apoptosis, and cell death but inhibited CXCL12-induced migration, intracellular Ca2+ releasing, and filamentous actin increase. Lidocaine decreased expression of CXCR4, increased CD44, but had no effect on ICAM-1. CXCL12 induced the increase of CD44 and ICAM-1 but did not affect CD44 in the presence of lidocaine. The knockdown of CXCR4 eliminated all the effects of lidocaine. The overexpression of CXCR4 promoted migration but the migration was inhibited by lidocaine., Conclusion: Lidocaine at clinical plasma concentrations inhibited CXCL12-induced CXCR4 activation, thereby reduced the intracellular Ca2+-dependent cytoskeleton remodeling, resulting in slower migration of A549 cells.

SETD3 was recently identified as the histidine methyltransferase responsible for N 3 -methylation of His73 of β-actin in humans. Overexpression of SETD3 is associated with several diseases, including breast cancer. Here, we report a development of actin-based peptidomimetics as inhibitors of recombinantly expressed human SETD3. Substitution of His73 by simple natural and unnatural amino acids led to selected β-actin peptides with high potency against SETD3 in MALDI-TOF MS assays. The selenomethionine-containing β-actin peptide was found to be the most potent SETD3 inhibitor (IC 50 =161 nM). Supporting our inhibition assays, a combination of computational docking and molecular dynamics simulations revealed that the His73 binding pocket for β-actin in SETD3 is rigid and accommodates the inhibitor peptides with similar binding modes. Collectively, our work demonstrates that actin-based peptidomimetics can act as potent SETD3 inhibitors and provide a basis for further development of highly potent and selective inhibitors of SETD3., (© 2021 Wiley-VCH GmbH.)

Objectives: We investigated the effect of apelin-13 on the cellular model of AD, amyloid-β (Aβ) treated SH-SY5Y cells in rats., Methods: The SH-SY5Y cells were pretreated with different doses of apelin-13 (1, 2.5, 5, and 10 μg/mL), half an hour before adding 50% Aβ treatment. After 24 h, cells were evaluated for survival, oxidative stress, mitochondrial calcium release, caspase-3, and cytochrome c levels, compared to control group (beta-actin). Statistical analysis was performed by SPSS 16., Results: Apelin-13 at the dose of 2.5 μg/mL protected against IC50 Aβ (p<0.001). Apelin-13 at doses of 1, 2.5, and 5 μg/mL showed protective effects against the reactive oxygen species (ROS) produced by Aβ (p<0.001). Apelin-13 at doses of 2.5 and 5 μg/mL reduced calcium release, caspase-3, and cytochrome c (all p<0.001)., Conclusions: Apelin-13 prevented apoptosis, oxidative stress, and mitochondrial toxicity and can be a suitable option for treatment of AD. The appropriate treatment strategy for humans has to be investigated in future studies., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

Damage-associated molecular patterns (DAMPs) are molecules exposed or released by dead cells that trigger or modulate immunity and tissue repair. In vertebrates, the cytoskeletal component F-actin is a DAMP specifically recognised by DNGR-1, an innate immune receptor. Previously we suggested that actin is also a DAMP in Drosophila melanogaster by inducing STAT-dependent genes (Srinivasan et al., 2016). Here, we revise that conclusion and report that α-actinin is far more potent than actin at inducing the same STAT response and can be found in trace amounts in actin preparations. Recombinant expression of actin or α-actinin in bacteria demonstrated that only α-actinin could drive the expression of STAT target genes in Drosophila . The response to injected α-actinin required the same signalling cascade that we had identified in our previous work using actin preparations. Taken together, these data indicate that α-actinin rather than actin drives STAT activation when injected into Drosophila ., Competing Interests: OG, CH, NS, SA, AF, SD, PC, DP, RG, SK, DF, AS, RV, CS, LT, BT, MD, WW, CR No competing interests declared, (© 2018, Gordon et al.)

A series of novel matrinic thiadiazole derivatives were designed, synthesized and evaluated for their inhibitory effect on COL1A1 promotor. The SAR indicated that: (i) the introduction of a thiadiazole on the 11-side chain was beneficial for activity; (ii) a 12- N -benzyl moiety was favorable for activity. Among them, compound 6n displayed a high activity with an inhibitory rate of 39.7% at a concentration of 40 μM. It also effectively inhibited the expression of two representative collagen proteins (COL1A1 and α-SMA) on both the mRNA and protein levels and showed a high safety profile in vivo, indicating its great promise as an anti-liver fibrosis agent. Further study indicated that it might repress hepatic fibrogenesis via the TGFβ/Smad pathway. This study provided powerful information for further strategic optimization and the top compound 6n was selected for further study as an ideal liver fibrosis lead for next investigation.

Sympathetic innervations exist in mammalian CL. The action of catecholaminergic system on luteal cells has been the focus of a variety of studies. Norepinephrine (NE) increased progesterone secretion of cattle luteal cells by activating β-adrenoceptors. In this study, murine luteal cells were treated with NE and isoprenaline (ISO). We found that NE increased the viability of murine luteal cells and ISO decreased the viability of luteal cells. Both NE and ISO promoted the progesterone production. Nonselective β-adrenergic antagonist, propranolol reversed the effect of ISO on cell viability but did not reverse the effect of NE on cell viability. Propranolol blocked the influence of NE and ISO on progesterone production. These results reveal that the increase of luteal cell viability induced by NE is not dependent on β-adrenergic activation. α-Adrenergic activation possibly contributes to it. Both NE and ISO increased progesterone production through activating β-adrenergic receptor. Further study showed that CyclinD2 is involved in the increase of luteal cell induced by NE. 3β-Hydroxysteroid dehydrogenase, LHR, steroidogenic acute regulatory protein (StAR), and PGF2α contribute to the progesterone production induced by NE and ISO., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Thymosin β4 (Tβ4) is a 43-amino acid signature motif peptide that defines the beta-thymosin (βT) family of proteins. βTs are intrinsically unstructured in their free states and undergo disorder-to-order transitions in carrying out their biological functions. This property poses challenges in determining their 3D structures, mainly favoring structural studies on the complexes formed between βTs and their interaction partners. One of the βTs' primary binding partners is monomeric actin, a major component of the cytoskeleton in eukaryotic cells. Tβ4's role in this system is to maintain the highly concentrated pool of monomeric actin that can be accessed through profilin by actin filament nucleating machineries. Here, we give an account of the structures of βTs that have been illuminated by nuclear magnetic resonance (NMR) and X-ray crystallography. NMR has been the method of choice for probing regions that have intrinsic conformational preference within the largely disordered βTs in their native states in solution. X-ray crystallography has demonstrated at atomic detail how βTs interact with actin. Detailed analysis of these structures highlights the disorder-to-order transition of Tβ4 in binding to actin and its isoform specificity., (© 2016 Elsevier Inc. All rights reserved.)

Treatment with thymosin beta 4 (Tβ4) reduces infarct volume and preserves cardiac function in preclinical models of cardiac ischemic injury. These effects stem in part from decreased infarct size, but additional benefits are likely due to specific antifibrotic and proangiogenic activities. Injected or transgenic Tβ4 increase blood vessel growth in large and small animal models, consistent with Tβ4 converting hibernating myocardium to an actively contractile state following ischemia. Tβ4 and its degradation products have antifibrotic effects in in vitro assays and in animal models of fibrosis not related to cardiac injury. This large number of pleiotropic effects results from Tβ4's many interactions with cellular signaling pathways, particularly indirect regulation of cellular motility and movement via the SRF-MRTF-G-actin transcriptional pathway. Variation in effects and effect sizes in animal models may potentially be due to variable distribution of Tβ4. Preclinical studies of PK/PD relationships and a reliable pharmacodynamic biomarker would facilitate clinical development of Tβ4., (© 2016 Elsevier Inc. All rights reserved.)

Background: Biofilm formation is associated with various aspects of bacterial and fungal infection. This study was designed to assess the impact of diverse natural polyelectrolytes, such as DNA, F-actin, neurofilaments (NFs), vimentin and purified Pf1 bacteriophage on biofilm formation and swarming motility of select pathogens including Pseudomonas aeruginosa associated with lung infections in CF patients., Results: The bacteriophage Pf1 (1 mg/ml) significantly increased biofilm mass produced by Pseudomonas aeruginosa P14, Escherichia coli RS218 and Bacillus subtilis ATCC6051. DNA, F-actin, NFs and Pf1 also increased biofilm mass of the fungal C. albicans 1409 strain. Addition of F-actin, DNA or Pf1 bacteriophage to 0.5% agar plates increased swarming motility of Pseudomonas aeruginosa Xen5., Conclusions: The presence of polyelectrolytes at infection sites is likely to promote biofilm growth and bacterial swarming.

Objective: To investigate the effect of cytoskeleton modification on the adipogenic differentiation of rat Achilles-derived tendon stem cells (TSCs) in vitro., Methods: TSCs were isolated from the tendon tissue of male Sprague Dawley rats (aged 3 weeks) by enzymatic digestion method and cultured for 3 passages. After the 3rd passage cells were cultured with DMEM medium containing 15% fetal bovine serum and cytochalasin D (CYD) at the concentrations of 0, 50, 100, 500, and 1 000 ng/mL, the cell survival condition and morphology changes were observed by inverted phase contrast microscope, the cytoskeleton was observed through fibrous actin (F-actin) staining, and the ratio of F-actin/ soluble globular actin (G-actin) was detected and calculated through Western blot. According to the above results, the effective concentration of CYD was selected and used for next experiments. After TSCs were cultured for 3 and 7 days respectively with adipogenic induction media (induction group), adipogenic induction media containing CYD (CYD+induction group), ordinary medium (ordinary group), and ordinary medium containing CYD (CYD+ordinary group), the real-time quantitative PCR (qRT-PCR) and Western blot were carried out to measure the mRNA and protein expressions of adipogenic differentiation-related markers, including peroxisome proliferator-activated receptor y (PPARγ), lipoprotein lipase (LPL), and fatty acid binding protein (aP2)., Results: The final CYD concentration of 100 ng/mL can inhibit effectively G-actin polymerization into F-actin, but could not affect TSCs survival, which was used for next experiments. qRT-PCR and Western blot suggested that the mRNA expressions of PPARγ, LPL, and aP2 and the protein expressions of PPARγ and aP2 were increased significantly in the CYD+induction group at 3 and 7 days when compared with the induction group (P < 0.05). In the CYD+ordinary group, there still was a significant increase in the mRNA expressions of PPARγ, LPL, and aP2 when compared with the ordinary group (P < 0.05)., Conclusion: Inhibition of F-actin polymerization can increase adipogenic differentiation of rat Achilles-derived TSCs in vitro, and cytoskeleton modification is a pre-requisite for TSCs differentiation into adipocytes, which might have important implications for the mechanism research of tendinopathy.

The barrier morphology of skin provides major obstacles for the application of siRNA for gene silencing, which current delivery technologies do not effectively overcome. Emerging technologies utilise microprojection array devices to penetrate into the skin epidermis and dermis for delivery of drug payloads. Delivery of siRNA by such devices has been proven in principle, yet requires optimisation for clinical applications. Herein, we demonstrate the use of Nanopatch™ microprojection arrays to deliver liposome-encapsulated siRNA to overcome skin barrier, and in vivo siRNA delivery hurdles. This application provided effective silencing of CXCL1 expression induced by the co-delivery of Fluvax 2012® by microprojection array. Liposomes encapsulating siRNA were dry-coated onto microprojection arrays, and remained intact after elution from arrays in vitro. Microprojection arrays facilitated the delivery of fluorescently-labelled nucleic acids through murine ear stratum corneum to the epidermis and dermis, with diffusion from microprojections into adjacent skin evident within 30s. CXCL1 mRNA, induced by delivery of Fluvax by microprojection array, was reduced by 75% up to 20 h post-treatment by co-delivery of liposome-encapsulated CXCL1-specific siRNA, but not by arrays co-delivering liposome-encapsulated control siRNA. CXCL1 protein expression in explant cultures from skin treated with arrays bearing CXCL1 specific or control siRNA was similarly reduced. These results as a test case have many implications for gene silencing in skin and inflammation, with the benefit of targeted delivery using microprojection arrays to deliver liposome-encapsulated siRNA., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: In vitro studies have shown that the active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), can regulate differentiation of CD4+ T cells by inhibiting Th1 and Th17 cell differentiation and promoting Th2 and Treg cell differentiation. However, the serum concentration of 1,25(OH)2D3 is far below the effective concentration of 1,25(OH)2D3 found in in vitro studies, and it has been suggested that 1,25(OH)2D3 must be produced locally from the inactive precursor 25-hydroxyvitamin D3 (25(OH)D3) to affect ongoing immune responses in vivo. Although it has been reported that activated T cells express the 25(OH)D-1α-hydroxylase CYP27B1 that converts 25(OH)D3 to 1,25(OH)2D3, it is still controversial whether activated T cells have the capacity to produce sufficient amounts of 1,25(OH)2D3 to affect vitamin D-responsive genes. Furthermore, it is not known how the vitamin D-binding protein (DBP) found in high concentrations in serum affects T cell responses to 25(OH)D3., Results: We found that activated T cells express CYP27B1 and have the capacity to produce sufficient 1,25(OH)2D3 to affect vitamin D-responsive genes when cultured with physiological concentrations of 25(OH)D3 in serum-free medium. However, if the medium was supplemented with serum or purified DBP, DBP strictly inhibited the production of 1,25(OH)2D3 and 25(OH)D3-induced T cell responses. In contrast, DBP did not inhibit the effect of exogenous 1,25(OH)2D3. Actin, arachidonic acid and albumin did not affect the sequestration of 25(OH)D3 by DBP, whereas carbonylation of DBP did., Conclusions: Activated T cells express CYP27B1 and can convert 25(OH)D3 to 1,25(OH)2D3 in sufficiently high concentrations to affect vitamin D-responsive genes when cultured in serum-free medium. However, DBP sequesters 25(OH)D3 and inhibits the production of 1,25(OH)2D3 in T cells. To fully exploit the immune-regulatory potential of vitamin D, future studies of the mechanisms that enable the immune system to exploit 25(OH)D3 and convert it to 1,25(OH)2D3 in vivo are required.

Purpose: Molecular therapeutics often require an effective nanoparticle-based delivery strategy to transport them to cytosolic organelles to be functional. Recently, a cytosolic delivery strategy based on the scavenger receptor class B type I (SR-BI) mediated pathway has shown great potential for the effective delivery of theranostics agents into the cytoplasm of cells without detrimental endosomal entrapment. This study elucidates this unique delivery mechanism for improving cytosolic drug delivery., Methods: Multifluorophore-labeled HDL-mimicking peptide phospholipid scaffold (HPPS) nanoparticles were developed. Fluorescence imaging was utilized to examine HPPS transporting payloads into cells step by step through sequential inhibition studies., Results: HPPS specifically recognizes and binds to SR-BI, then interacts with SR-BI, which results in direct transport of payload molecules into the cell cytoplasm without entire particles internalization. The cytosolic transport of payloads occurred through a temperature- and energy-independent pathway, and was also different from actin- and clathrin-mediated endocytosis. Furthermore, this transport was significantly inhibited by disruption of lipid rafts using filipin or methyl-β-cyclodextrin., Conclusions: The cytosolic delivery of payloads by HPPS via SR-BI targeting is predominately mediated through a lipid rafts/caveolae-like pathway. This cytosolic delivery strategy can be utilized for transporting molecular therapeutics that require their action sites to be within cytosolic organelles to enhance therapeutic effect.

Intramolecular communication within myosin is essential for its function as motor, but the specific amino acid residue interactions required are unexplored within muscle cells. Using Drosophila melanogaster skeletal muscle myosin, we performed a novel in vivo molecular suppression analysis to define the importance of three relay loop amino acid residues (Ile(508), Asn(509), and Asp(511)) in communicating with converter domain residue Arg(759). We found that the N509K relay mutation suppressed defects in myosin ATPase, in vitro motility, myofibril stability, and muscle function associated with the R759E converter mutation. Through molecular modeling, we define a mechanism for this interaction and suggest why the I508K and D511K relay mutations fail to suppress R759E. Interestingly, I508K disabled motor function and myofibril assembly, suggesting that productive relay-converter interaction is essential for both processes. We conclude that the putative relay-converter interaction mediated by myosin residues 509 and 759 is critical for the biochemical and biophysical function of skeletal muscle myosin and the normal ultrastructural and mechanical properties of muscle.

Mechanical stimulation and estrogen have been proven to be two important factors in promoting mesenchymal stem cell activity, which is closely associated with bone formation, mass maintenance and remodeling. However, the superposition effects of mechanical stimulation and estrogen on stem cells remain unknown. It is also unclear if the estrogen receptor (ER) plays only a key role in estrogen signaling or if it is also involved in the mechanotransduction of stem cells. To investigate the role of estrogen and its receptors in the mechanobiological effects in bone mesenchymal stem cells (BMSCs), isolated mesenchymal stem cells from bone marrow were exposed to mechanical pressure under additional estrogen treatment or ER blockade. Cell proliferation was examined using an MTT assay and alkaline phosphatase (ALP) activity was determined by a modified enzyme kinetic method. Alignment of the cytoskeleton was observed by Coomassie brilliant blue staining and F-actin fluorescent staining. Cellular ultrastructure was observed under transmission electron microscope. Expression of ERα was investigated using Western blot analysis. Results indicated that mechanical pressure promoted cell proliferation, ALP activity, ERα expression and F-actin stress fiber formation. Overall, this effect was enhanced by the addition of estrogen and inhibited by ER blockade. We concluded that pressure stimulated proliferation and differentiation capability via F-actin transduction in BMSCs. The effects were enhanced by the addition of estrogen, and the ER plays an important role in regulating mechanobiological effects and the mechanotransduction processes of BMSCs., (Copyright © 2011 S. Karger AG, Basel.)

Methyl CpG-binding protein-2 (MeCP2) is a transcriptional regulator that binds to methylated DNA at CpG sites and functions to silence DNA transcription. MeCP2 is subject to the phosphorylation modification at serine 421 (S421), which releases MeCP2 from DNA and thus facilitates gene expression. As a transcriptional repressor densely expressed in limbic reward circuits of adult mammalian brains, MeCP2 is recently emerging as a critical epigenetic factor in experience-dependent neural plasticity and psychostimulant addiction. In this study, we investigated the regulation of MeCP2 phosphorylation in the rat striatum by the psychostimulant cocaine in vivo. We found that acute systemic injection of cocaine increased MeCP2 phosphorylation at S421 in the rat striatum, including both the caudate putamen and the nucleus accumbens, while cocaine did not affect MeCP2 phosphorylation in the medial prefrontal cortex. The cocaine-stimulated MeCP2 phosphorylation in the nucleus accumbens was a rapid and transient event, as it was evident at 20 min and returned to normal levels 3h after drug injection. The cocaine effect in the caudate putamen was however relatively delayed. Reliable induction of MeCP2 phosphorylation in this region was detected at 60 min. Pretreatment with an N-methyl-d-aspartate (NMDA) glutamate receptor antagonist significantly reduced the cocaine-stimulated MeCP2 phosphorylation in the caudate putamen, although not in the nucleus accumbens. Our data support that MeCP2 is a sensitive target of psychostimulants. Its phosphorylation status is regulated by psychostimulant exposure. NMDA receptors play a region-specific role in linking cocaine to MeCP2 phosphorylation in striatal neurons in vivo., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The goal of this study was to investigate the possible role of reactive oxygen species (ROS) in signaling, in modulation of the cytoskeleton, and in differentiation of fibroblasts. For this purpose, we have applied a novel mitochondria-targeted antioxidant: plastoquinone conjugated with decyltriphenylphosphonium (SkQ1). This antioxidant at nanomolar concentration prevented ROS accumulation and cell death induced by H(2)O(2) in fibroblasts. We found that scavenging of ROS produced by mitochondria activated the Rho/ROCK/LIMK signaling pathway that was followed by phosphorylation of cofilin and stabilization of actin stress fibers. The mitochondria-targeted antioxidant induced differentiation of human subcutaneous fibroblasts to myofibroblasts as revealed by expression of fibronectin isoform (EDA-FN) and smooth muscle actin (α-SMA). This effect was shown to be mediated by transforming growth factor β1 (TGFβ1), which was activated by matrix metalloprotease 9 (MMP9) in the culture medium. Scavenging of ROS stimulated secretion of MMP9 rather than its processing. The same effect was achieved by the nontargeted antioxidant Trolox at higher concentration, but the thiol antioxidant N-acetylcysteine (NAC) inhibited MMP activity and was not able to induce myofibroblast differentiation. The myofibroblast phenotype was supported due to autocrine TGFβ1-dependent stimulation after removal of SkQ1. It is concluded that ROS scavenging in mitochondria induces TGFβ1-dependent myofibroblast differentiation.

To induce tumor cell apoptosis, a modified 15 kDa actin linked with a peptide NGR "homing" into tumor or tumor vessels was expressed in Escherichia coli. After refolding and purification, this fusion protein NGR-15actin was labeled with FITC to testify whether NGR-15actin could integrate into the cytoskeleton. It was found that this targeted peptide could induce HepG2 and HeLa cells apoptosis through its effect on the cytoskeleton function by binding to cytoskeleton protein. Thus, targeted NGR-15actin could be a candidate molecule for the therapy of cancer.

Troponin regulates contraction of vertebrate striated muscle in a Ca(2+)-dependent manner. More specifically, it acts as an inhibitor of actin-myosin interaction in the absence of Ca(2+) during contraction. In vertebrates, this regulatory mechanism is unlike that in some less highly derived taxa. Troponin in the smooth muscle of the protochordate ascidian species Halocynthia roretzi regulates actinmyosin contraction as an activator in the presence of Ca(2+), not as an inhibitor in the absence of Ca(2+) as is the case in vertebrates. In this study, contractile regulation of striated muscle from another protochordate, the amphioxus Branchiostoma belcheri, was analyzed using recombinant troponin components TnT, TnI, and TnC that were produced in an Escherichia coli expression system to further elucidate their roles in Ca(2+)-dependent regulation of the actin-myosin interaction. Combination of these troponin components in an actin-myosin ATPase activity assay showed that troponin in amphioxus striated muscle functions in a similar manner to troponin in vertebrate striated muscle, and differently from ascidian smooth muscle troponin. Thus, troponin function appears to have evolved differently in different protochordate muscles.

Although the role of the actin cytoskeleton has become increasingly elucidated, the role of actin polymerization in inflammatory processes remains poorly understood. Here, we examine the role of the actin cytoskeleton during LPS-mediated inflammatory events in RAW264.7 cells and peritoneal macrophages. We observed that actin cytoskeleton disruption by cytochalasin B and siRNA to cytoplasmic actin strongly down-regulated LPS-mediated inflammatory responses such as NO production, PGE(2) release, and TNF-alpha secretion. Actin cytoskeleton disruption by cytochalasin B down-regulated a series of signaling cascades including PI3K, Akt, and IKK, but not MAPKs, necessary for NF-kappaB activation without down-regulating total forms of the proteins as assessed by measuring their phosphorylation levels. In particular, cytochalasin B significantly inhibited LPS-induced both phosphorylation and kinase activity of Src without altering total level, implying that Src may be a potential pharmacological target of actin cytoskeleton rearrangement. Moreover, the direct association of Src with actin was actin polymerization-dependent according to immunoprecipitation analysis performed with a GFP-actin wild type and HA-tagged Src. Therefore, our data suggest that actin cytoskeleton rearrangement may be a key event during the regulation of inflammatory responses that modulates the activity of Src and its downstream signaling molecules.

The aim of this study was to investigate the role of RhoA/mDia1 pathway in the process of thrombin-induced platelet aggregation and regulatory effect of PI3K inhibitor on this process. The human platelets were isolated from peripheral blood, the activation of RhoA, Rac1 and Cdc42 in the platelet aggregation was detected by GST pull-down assay and immune co-precipitation, the interaction of RhoA, Rac1 and Cdc42 with mDia1 and the formation of complex in the process of platelet aggregation were determined by immune coprecipitation, and the effect of PI3K inhibitor (wortmannin) on above-mentioned process was assayed. The results showed that thrombin elevated the activity of RhoA and the binding capability of RhoA with mDia1 during thrombin-induced platelet aggregation and spreading on Fg coated coverslips. Wortmannin inhibited the rising of RhoA activity and the binding level of RhoA with mDia1 induced by thrombin. Thrombin elevated the activity of Rac1 and Cdc42 during thrombin-induced platelet aggregation, but could not induce binding of Rac1 or Cdc42 with mDia1. Wortmannin could not inhibit the rising of Rac1 and Cdc42 activity induced by thrombin. It is concluded that the PI3-kinase regulates the thrombin-induced actin cytoskeleton reconstitution in platelets by RhoA-mDia1 pathway.

Background: To compare the effectiveness of liposomal tobramycin or polymyxin B against Pseudomonas aeruginosa in the Cystic Fibrosis (CF) sputum and its inhibition by common polyanionic components such as DNA, F-actin, lipopolysaccharides (LPS), and lipoteichoic acid (LTA)., Methodology: Liposomal formulations were prepared from a mixture of 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) or 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC) and Cholesterol (Chol), respectively. Stability of the formulations in different biological milieus and antibacterial activities compared to conventional forms in the presence of the aforementioned inhibitory factors or CF sputum were evaluated., Results: The formulations were stable in all conditions tested with no significant differences compared to the controls. Inhibition of antibiotic formulations by DNA/F-actin and LPS/LTA was concentration dependent. DNA/F-actin (125 to 1000 mg/L) and LPS/LTA (1 to 1000 mg/L) inhibited conventional tobramycin bioactivity, whereas, liposome-entrapped tobramycin was inhibited at higher concentrations--DNA/F-actin (500 to 1000 mg/L) and LPS/LTA (100 to 1000 mg/L). Neither polymyxin B formulation was inactivated by DNA/F-actin, but LPS/LTA (1 to 1000 mg/L) inhibited the drug in conventional form completely and higher concentrations of the inhibitors (100 to 1000 mg/L) was required to inhibit the liposome-entrapped polymyxin B. Co-incubation with inhibitory factors (1000 mg/L) increased conventional (16-fold) and liposomal (4-fold) tobramycin minimum bactericidal concentrations (MBCs), while both polymyxin B formulations were inhibited 64-fold., Conclusions: Liposome-entrapment reduced antibiotic inhibition up to 100-fold and the CFU of endogenous P. aeruginosa in sputum by 4-fold compared to the conventional antibiotic, suggesting their potential applications in CF lung infections.

Full-length Drosophila myosin 7a (myosin 7a-FL) has a complex tail containing a short predicted coiled coil followed by a MyTH4-FERM domain, an SH3 domain, and a C-terminal MyTH4-FERM domain. Myosin 7a-FL expressed in Sf9 cells is monomeric despite the predicted coiled coil. We showed previously that Subfragment-1 (S1) from this myosin has MgATPase of V(max) approximately 1 s(-1) and K(ATPase) approximately 1 microM actin. We find that myosin 7a-FL has V(max) similar to S1 but K(ATPase) approximately 30 microM. Thus, at low actin concentrations (5 microM), the MgATPase of S1 is fully activated, whereas that of myosin 7a-FL is low, suggesting that the tail regulates activity. Electron microscopy of myosin 7a-FL with ATP shows the tail is tightly bent back against the motor domain. Myosin 7a-FL extends at either high ionic strength or without ATP, revealing the motor domain, lever, and tail. A series of C-terminal truncations show that deletion of 99 aa (the MyTH7 subdomain of the C-terminal FERM domain) is sufficient to abolish bending, and the K(ATPase) is then similar to S1. This region is highly conserved in myosin 7a. We found that a double mutation in it, R2140A-K2143A, abolishes bending and reduces K(ATPase) to S1 levels. In addition, the expressed C-terminal FERM domain binds actin with K(d) approximately 30 microM regardless of ATP, similar to the K(ATPase) value for myosin 7a-FL. We propose that at low cellular actin concentrations, myosin 7a-FL is bent and inactive, but at high actin concentrations, it is unfolded and active because the C-terminal FERM domain binds to actin.

The effect of resveratrol on activity and expression of nitric oxide synthase (NOS) in the spinal cord of neuropathic rats was assessed. Spinal nerve ligation produced tactile allodynia along with a reduction of catalytic activity of the constitutive Ca(2+)-dependent NOS (eNOS and nNOS isoforms) in the ipsilateral dorsal horn, but not contralateral dorsal or ipsilateral or contralateral ventral, spinal cord at 1, 5, 10 and 15 days after surgery compared to naïve and sham-operated animals. Nerve ligation also induced a reduction of nNOS expression in the ipsilateral dorsal horn spinal cord at 10 and 15 days after surgery. Intrathecal resveratrol reduced allodynia and reversed the reduction of constitutive Ca(2+)-dependent NOS activity in the ipsilateral dorsal spinal cord. Moreover, resveratrol significantly reversed the reduction of nNOS expression in the ipsilateral dorsal horn spinal cord. Results show that spinal nerve ligation leads to development of tactile allodynia along with a reduction in constitutive Ca(2+)-dependent NOS activity and nNOS isoform expression in the ipsilateral dorsal horn. Data suggest that resveratrol may reduce tactile allodynia in neuropathic rats by restoring altered NOS activity and expression.

It has been reported that F-actin is transported to the presumptive cleavage furrow along the cortex during anaphase-cytokinesis, an event termed cortical actin flow in animal cultured cells. The motor source has remained unknown. We reported that Ca2+ stores with IP3 receptor (IP3R) was re-distributed from the polar cortex during metaphase to the presumptive cleavage furrow just before the onset of furrowing, and that Ca2+ stores with IP3R microinjected into dividing newt eggs moved toward the presumptive cleavage furrow during anaphase-cytokinesis in a microtubule-dependent manner, and that Ca2+ store-enriched microsome fractions induced the cleavage furrow as the putative cleavage stimulus. Because the distribution of F-actin and Ca2+ stores with IP3R during metaphase to cytokinesis is similar, we considered that this cortical actin flow may be powered by transportation of Ca2+ stores with IP3R. Purified F-actin labeled with phalloidin-rhodamine was microinjected into the dividing newt eggs and the eggs observed under a confocal microscope. We found that the microinjected F-actin moved linearly toward the next cleavage furrow and that this movement was blocked by nocodazole, microtubule-depolarizing agent and AMP-PNP, a blocking agent of microtubule motors. Co-microinjected rhodamine-labeled F-actin and sacro/endoplasmic reticulum Ca2+-ATPase (SERCA)-GFP-labeled Ca2+ stores with IP3R co-moved and co-accumulated to the next cleavage furrow. These results strongly suggest that Ca2+ stores with IP3R, which is transferred by microtubule-based motility as cleavage stimulus, act as an F-actin translocator.

Twenty-two fragments of beta-actin and beta-actin-related protein were isolated from the acidic extracts of rat spleen tissue. beta-Actin fragments (75-90), (78-89), and (78-88), 0.01-1 microM, decreased live cell number of L929 murine tumor fibroblasts by 80-90%, with maximal cytotoxic effect of 30-40%. The fragments of (78-90) segment and the fragment of beta-actin-related protein (69-77) were less active (inhibitory effect up to 55%, cytotoxic-up to 25%)., (Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.)

These studies relate to a working hypothesis that glycogen storage is facilitated in resting muscle by inhibiting glycolysis via inhibition of LDH, AK, and PFK-1 by ascorbate; when muscle is active, these isozymes combine with muscle proteins and are released and protected from inhibition by ascorbate and glycolysis proceeds. Focus in these studies is on the ability of G-actin and aldolase to prevent PFK-1 inhibition by ascorbate. We found that inhibition by ascorbate was PFK-1 concentration dependent; ascorbate does not inhibit above 200 nM PFK-1. We conclude that ascorbate inhibits PFK-1 dimers (and perhaps monomers) but not PFK-1 tetramers. Separation of PFK-1 dimers from tetramers was achieved with centrifugal filter devices and differences in their sensitivity to ascorbate inhibition were demonstrated. Some comparisons are made with attributes of AK inhibitions by ascorbate that, like PFK-1, are also enzyme concentration dependent. Discussions relate findings to cellular infrastructure and the role of ascorbate in glycogen synthesis.

A spectroscopic assay using pyrene-labeled fission yeast Arp2/3 complex revealed that the complex binds to and dissociates from actin filaments extremely slowly with or without the nucleation-promoting factor fission yeast Wsp1-VCA. Wsp1-VCA binds both Arp2/3 complex and actin monomers with high affinity. These two ligands have only modest impacts on the interaction of the other ligand with VCA. Simulations of a mathematical model based on the kinetic parameters determined in this study and elsewhere account for the full time course of actin polymerization in the presence of Arp2/3 complex and Wsp1-VCA and show that an activation step, postulated to follow binding of a ternary complex of Arp2/3 complex, a bound nucleation-promoting factor, and an actin monomer to an actin filament, has a rate constant at least 0.15 s(-1). Kinetic parameters determined in this study constrain the process of actin filament branch formation during cellular motility to one main pathway.