[Porphyromonas gingivalis outer membrane vesicles activate Toll-like receptor 2 to promote osteoclast differentiation by carrying lipopolysaccharide].

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.

Boldine protects against carbon tetrachloride-induced chronic liver injury by regulating NF-κB signaling pathway.

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 (CCl4 )-induced chronic liver injury in rats. CCl4 (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 ß (IL-1ß), and α-smooth muscle actin (α-SMA) gene and protein expressions were evaluated. CCl4 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 CCl4 -induced elevation of transaminase levels in serum, restored enzymic and non-enzymic antioxidants, and downregulated NF-κB, TNF-α, Cox-2 and IL-1ß 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.

Promotion of myofibroblast differentiation through repeated treatment of fibroblasts to low concentrations of PM2.5.

Exposure to particulate matter ≤ 2.5 µm (PM2.5) is a risk factor for many lung diseases. Although the toxicologic effects of PM2.5 on airway epithelium are well-described, the effects of PM2.5 on fibroblasts in the lung are less studied. Here, we sought to examine the effects of PM2.5 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 PM2.5 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 PM2.5 to induce myofibroblast differentiation. These data demonstrate that potential impact of PM2.5 to fibroblast activation and fibrosis and support the importance of utilizing low concentrations and varying exposure protocols to toxicologic studies.

Involvement of transcribed lncRNA uc.291 in hyperproliferative skin disorders.

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.

The Effects of ROCK Inhibitor on Prevention of Dexamethasone-Induced Glaucoma Phenotype in Human Trabecular Meshwork Cells.

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.

Vascular endothelial growth factor induces the migration of human airway smooth muscle cells by activating the RhoA/ROCK pathway.

BACKGROUND: Airway remodeling due to increased airway smooth muscle cell (ASMC) mass, likely due to enhanced proliferation, hypertrophy, and migration, has been proven to be highly correlated with decreased lung function in asthma patients. Vascular endothelial growth factor (VEGF) mediates vascular and extravascular remodeling and inflammation and has been proven to be involved in the progression of asthma. Previous studies have focused on the effects of VEGF on ASMC proliferation, but few researchers have focused on the effects of VEGF on human ASMC migration. The purpose of this study was to explore the effect of VEGF on the migration of ASMCs and its related signaling pathway mechanism to provide evidence for the treatment of airway remodeling. METHODS: We examined the effects of VEGF induction on ASMC migration and explored the mechanisms involved in ASMC migration. RESULTS: We found by wound healing and Transwell assays that VEGF promoted ASMC migration. Through the Cell Counting Kit-8 (CCK-8) experiment, we found that VEGF had no significant effect on the proliferation of ASMCs, which excluded the involvement of cell proliferation in the process of wound healing. Moreover, a cellular immunofluorescence assay showed that VEGF promoted F-actin reorganization, and Western blotting showed that VEGF improved RhoA activation and myosin phosphatase targeting subunit-1 (MYPT1) and myosin light chain (MLC) phosphorylation in ASMCs. Treatment with the ROCK inhibitor Y27632 significantly attenuated the effects of VEGF on MYPT1/MLC activation and cell migration. CONCLUSION: In conclusion, the results suggest that the promigratory function of VEGF activates the RhoA/ROCK pathway, induces F-actin reorganization, improves the migration of ASMCs, and provides a better rationale for targeting the RhoA/ROCK pathway for therapeutic approaches in airway remodeling.

Role of alpha smooth muscle actin in odontogenic differentiation of dental pulp stem cells.

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-ß1). 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.

Low-dose albumin-coated gold nanorods induce intercellular gaps on vascular endothelium by causing the contraction of cytoskeletal actin.

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.

On the significance of membrane unfolding in mechanosensitive cell spreading: Its individual and synergistic effects.

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.

EphrinB2 promotes the human aortic smooth muscle cell growth and migration via mediating F-actin remodeling.

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.

Cold Plasma Treatment Promotes Full-thickness Healing of Skin Wounds in Murine Models.

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.

Asiaticoside conveys an antifibrotic effect by inhibiting activation of hepatic stellate cells via the Jagged-1/Notch-1 pathway.

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 (CCl4)-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.

Involvement of integrin αvß3 in thyroid hormone-induced dendritogenesis.

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 αvß3 in neurons and glia. Integrin αvß3-mediated signal transduction mediates various cellular events during development including morphogenesis, migration, synaptogenesis, and intracellular metabolism. However, the involvement of integrin αvß3-mediated TH action during brain development remains poorly understood. Thus, we examined the integrin αvß3-mediated effects of TH (T3, T4, and rT3) 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 αvß3, as well as TRα and TRß. A selective integrin αvß3 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 TRß. 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 αvß3 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.

Histological functions of parathyroid hormone on bone formation and bone blood vessels.

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.

Cell-to-cell transmission of HIV-1 from provirus-activated cells to resting naïve and memory human primary CD4 T cells is highly efficient and requires CD4 and F-actin but not chemokine receptors.

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.

[Mitochondrial coenzyme Q attenuates lipopolysaccharide-induced mitochondria-dependent apoptosis in type II alveolar epithelial cells via phosphatidylinositol 3-kinase/Akt pathway].

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 (IC50). 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 IC50 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/ß-actin: 0.58±0.03 vs. 1.06±0.10, P < 0.05) and Bcl-2 level was significantly up-regulated (Bcl-2/ß-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/ß-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/ß-actin: 0.95±0.03 vs. 0.58±0.03, P < 0.05) and Bcl-2 level was significantly down-regulated (Bcl-2/ß-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/ß-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.

Positional effects of double-stranded RNAs targeting ß-Actin gene affect RNA interference efficiency in Colorado potato beetle.

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) ß-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, ß-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.

[Effect of Hypoxia Inducible Factor-1α on Chemosensitivity of B-ALL Cells to Vincristine and Its Mechanism].

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 (CoCl2 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 ß-actin protein expression. Quantitative real-time fluorescent PCR (qRT-PCR) was used to detect BAX and ß-actin mRNA levels. RESULTS: CoCl2 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.

[Effect of Hypoxia Inducible Factor-1α on Chemosensitivity of B-ALL Cells to Vincristine and Its Mechanism].

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 (CoCl2 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 ß-actin protein expression. Quantitative real-time fluorescent PCR (qRT-PCR) was used to detect BAX and ß-actin mRNA levels. RESULTS: CoCl2 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.

Effect of dimethyl alpha-ketoglutarate supplementation on the in vitro developmental competences of ovine oocytes.

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 H2O2 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 H2O2 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.