Oncogenic lncRNA downregulates cancer cell antigen presentation and intrinsic tumor suppression.

How tumor cells genetically lose antigenicity and evade immune checkpoints remains largely elusive. We report that tissue-specific expression of the human long noncoding RNA LINK-A in mouse mammary glands initiates metastatic mammary gland tumors, which phenotypically resemble human triple-negative breast cancer (TNBC). LINK-A expression facilitated crosstalk between phosphatidylinositol-(3,4,5)-trisphosphate and inhibitory G-protein-coupled receptor (GPCR) pathways, attenuating protein kinase A-mediated phosphorylation of the E3 ubiquitin ligase TRIM71. Consequently, LINK-A expression enhanced K48-polyubiquitination-mediated degradation of the antigen peptide-loading complex (PLC) and intrinsic tumor suppressors Rb and p53. Treatment with LINK-A locked nucleic acids or GPCR antagonists stabilized the PLC components, Rb and p53, and sensitized mammary gland tumors to immune checkpoint blockers. Patients with programmed ccll death protein-1(PD-1) blockade-resistant TNBC exhibited elevated LINK-A levels and downregulated PLC components. Hence we demonstrate lncRNA-dependent downregulation of antigenicity and intrinsic tumor suppression, which provides the basis for developing combinational immunotherapy treatment regimens and early TNBC prevention.

Colloidal Synthesis of Hollow Double Perovskite Nanocrystals and Their Applications in X-ray Imaging.

Rare earth-based halide double perovskites are regarded as an emerging class of X-ray scintillation materials. However, the majority of related scintillator applications are still focused on single crystal and powder systems; the application of nanocrystal (NC) scintillators is rarely reported. Here, we present the synthesis of high-purity Cs2NaTbCl6 NCs by an improved hot-injection method. Interestingly, hollow Cs2NaTbCl6 NCs are observed, the monitoring of the growth process indicates that micrometer-sized NaCl is the initial product, and then the NaCl would convert into Cs2NaTbCl6 NCs through the diffusion of Cs+ and Tb3+ into NaCl lattice, and the faster outward diffusion of Na+ results in the formation of hollow NCs. The double perovskite NCs exhibit green light emission, and the photoluminescence intensity can be significantly enhanced through Ce3+ doping. In particular, the Cs2NaTbCl6:5%Ce3+ scintillator exhibits a linear response and a low detection limit of 79.09 nGy/s when exposed to X-rays. Furthermore, a flexible scintillator film for X-ray imaging is prepared by mixing NCs with polymer, showing a high spatial resolution imaging capability of 10 lp/mm. This work provides a new strategy for hollow perovskite NCs and may shed light on the synthesis of related hollow NCs and their applications in X-ray detection.

Identification of a tumour immune barrier in the HCC microenvironment that determines the efficacy of immunotherapy.

BACKGROUND & AIMS: The tumour microenvironment (TME) is a crucial mediator of cancer progression and therapeutic outcome. The TME subtype correlates with patient response to immunotherapy in multiple cancers. Most previous studies have focused on the role of different cellular components in the TME associated with immunotherapy efficacy. However, the specific structure of the TME and its role in immunotherapy efficacy remain largely unknown. METHODS: We combined spatial transcriptomics with single-cell RNA-sequencing and multiplexed immunofluorescence to identify the specific spatial structures in the TME that determine the efficacy of immunotherapy in patients with hepatocellular carcinoma (HCC) receiving anti-PD-1 treatment. RESULTS: We identified a tumour immune barrier (TIB) structure, a spatial niche composed of SPP1+ macrophages and cancer-associated fibroblasts (CAFs) located near the tumour boundary, which is associated with the efficacy of immune checkpoint blockade. Furthermore, we dissected ligand‒receptor networks among malignant cells, SPP1+ macrophages, and CAFs; that is, the hypoxic microenvironment promotes SPP1 expression, and SPP1+ macrophages interact with CAFs to stimulate extracellular matrix remodelling and promote TIB structure formation, thereby limiting immune infiltration in the tumour core. Preclinically, the blockade of SPP1 or macrophage-specific deletion of Spp1 in mice led to enhanced efficacy of anti-PD-1 treatment in mouse liver cancer, accompanied by reduced CAF infiltration and increased cytotoxic T-cell infiltration. CONCLUSIONS: We identified that the TIB structure formed by the interaction of SPP1+ macrophages and CAFs is related to immunotherapy efficacy. Therefore, disruption of the TIB structure by blocking SPP1 may be considered a relevant therapeutic approach to enhance the therapeutic effect of immune checkpoint blockade in HCC. IMPACT AND IMPLICATIONS: Only a limited number of patients with hepatocellular carcinoma (HCC) benefit from tumour immunotherapy, which significantly hinders its application. Herein, we used multiomics to identify the spatial structure of the tumour immune barrier (TIB), which is formed by the interaction of SPP1+ macrophages and cancer-associated fibroblasts in the HCC microenvironment. This structure constrains immunotherapy efficacy by limiting immune cell infiltration into malignant regions. Preclinically, we revealed that blocking SPP1 or macrophage-specific deletion of Spp1 in mice could destroy the TIB structure and sensitize HCC cells to immunotherapy. These results provide the first key steps towards finding more effective therapies for HCC and have implications for physicians, scientists, and drug developers in the field of HCC.

Spherical Gauss-Laguerre beam propagation in 4D space-time.

In this paper, what we believe to be a novel class of beams, which are referred to as the spherical Gauss-Laguerre beams, are proposed. The beams propagate stably in the anomalous dispersive media, within which the second order derivative with respect to t could be combined with the two-dimensional (2D) Laplacian operator in the transverse direction and forms a three-dimensional (3D) Laplacian operator, which describes the beam propagation in the z direction within the four-dimensional (4D) x-y-z-t space-time. The wave equation is solved by the variable separation method and the analytical expression for the spherical Gauss-Laguerre beams is derived. The beams have a 3D Gaussian field distribution with a variable beam waist with respect to the propagation distance. Unlike any 2D spatial vortex beams, the 3D beams could possess either the spatial vortex or the spatiotemporal optical vortex (STOV) by choosing the vortex plane in the 3D x-y-t space-time. The derived spherical Gauss-Laguerre beam expression in the 4D space-time is verified by the numerical simulations with excellent agreement.

Highly Effective Hybrid Copper(I) Iodide Cluster Emitter with Negative Thermal Quenched Phosphorescence for X-Ray Imaging.

The low efficiency triplet emission of hybrid copper(I) iodide clusters is a critical obstacle to their further practical optoelectronic application. Herein, we present an efficient hybrid copper(I) iodide cluster emitter (DBA)4 Cu4 I4 , where the cooperation of excited state structure reorganization and the metallophilicity interaction enables ultra-bright triplet yellow-orange emission with a photoluminescence quantum yield over 94.9 %, and the phonon-assisted de-trapping process of exciton induces the negative thermal quenching effect at 80-300 K. We also investigate the potential of this emitter for X-ray imaging. The (DBA)4 Cu4 I4 wafer demonstrates a light yield higher than 104  photons MeV-1 and a high spatial resolution of ≈5.0 lp mm-1 , showing great potential in practical X-ray imaging applications. Our new copper(I) iodide cluster emitter can serve as a model for investigating the thermodynamic mechanism of photoluminescence in hybrid copper(I) halide phosphorescence materials.

Nonlinear Fourier transform receiver based on a time domain diffractive deep neural network.

A diffractive deep neural network (D2NN) is proposed to distinguish the inverse nonlinear Fourier transform (INFT) symbols. Different from other recently proposed D2NNs, the D2NN is fiber based, and it is in the time domain rather than the spatial domain. The D2NN is composed of multiple cascaded dispersive elements and phase modulators. An all-optical back-propagation algorithm is proposed to optimize the phase. The fiber-based time domain D2NN acts as a powerful tool for signal conversion and recognition, and it is used in a receiver to recognize the INFT symbols all optically. After the symbol conversion by the D2NN, simple phase and amplitude measurement will determine the correct symbol while avoiding the time-consuming NFT. The proposed device can not only be implemented in the NFT transmission system, but also in other areas which require all optical time domain signal transformation and recognition, like sensing, signal coding and decoding, beam distortion compensation and image recognition.

Fall Detection for Shipboard Seafarers Based on Optimized BlazePose and LSTM.

Aiming to avoid personal injury caused by the failure of timely medical assistance following a fall by seafarer members working on ships, research on the detection of seafarer's falls and timely warnings to safety officers can reduce the loss and severe consequences of falls to seafarers. To improve the detection accuracy and real-time performance of the seafarer fall detection algorithm, a seafarer fall detection algorithm based on BlazePose-LSTM is proposed. This algorithm can automatically extract the human body key point information from the video image obtained by the vision sensor, analyze its internal data correlation characteristics, and realize the process from RGB camera image processing to seafarer fall detection. This fall detection algorithm extracts the human body key point information through the optimized BlazePose human body key point information extraction network. In this section, a new method for human bounding-box acquisition is proposed. In this study, a head detector based on the Vitruvian theory was used to replace the pre-trained SSD body detector in the BlazePose preheating module. Simultaneously, an offset vector is proposed to update the bounding box obtained. This method can reduce the frequency of repeated use of the head detection module. The algorithm then uses the long short-term memory neural network to detect seafarer falls. After extracting fall and related behavior data from the URFall public data set and FDD public data set to enrich the self-made data set, the experimental results show that the algorithm can achieve 100% accuracy and 98.5% specificity for the seafarer's falling behavior, indicating that the algorithm has reasonable practicability and strong generalization ability. The detection frame rate can reach 29 fps on a CPU, which can meet the effect of real-time detection. The proposed method can be deployed on common vision sensors.

Characterization of a Thermostable and Surfactant-Tolerant Chondroitinase B from a Marine Bacterium Microbulbifer sp. ALW1.

Chondroitinase plays an important role in structural and functional studies of chondroitin sulfate (CS). In this study, a new member of chondroitinase B of PL6 family, namely ChSase B6, was cloned from marine bacterium Microbulbifer sp. ALW1 and subjected to enzymatic and structural characterization. The recombinant ChSase B6 showed optimum activity at 40 °C and pH 8.0, with enzyme kinetic parameters of Km and Vmax against chondroitin sulfate B (CSB) to be 7.85 µg/mL and 1.21 U/mg, respectively. ChSase B6 demonstrated thermostability under 60 °C for 2 h with about 50% residual activity and good pH stability under 4.0-10.0 for 1 h with above 60% residual activity. In addition, ChSase B6 displayed excellent stability against the surfactants including Tween-20, Tween-80, Trion X-100, and CTAB. The degradation products of ChSase B6-treated CSB exhibited improved antioxidant ability as a hydroxyl radical scavenger. Structural analysis and site-directed mutagenesis suggested that the conserved residues Lys248 and Arg269 were important for the activity of ChSase B6. Characterization, structure, and molecular dynamics simulation of ChSase B6 provided a guide for further tailoring for its industrial application for chondroitin sulfate bioresource development.

Importance of ß2AR elevation for re-endothelialization capacity mediated by late endothelial progenitor cells in hypertensive patients.

Dysfunction of late endothelial progenitor cells (EPCs) has been suggested to be associated with hypertension. ß2-Adrenergic receptor (ß2AR) is a novel and key target for EPC homing. Here, we proposed that attenuated ß2AR signaling contributes to EPCs dysfunction, whereas enhanced ß2AR signaling restores EPCs' functions in hypertension. EPCs derived from hypertensive patients exhibited reduced cell number, impaired in vitro migratory and adhesion abilities, and impaired re-endothelialization after transplantation in nude mice with carotid artery injury. ß2AR expression of EPCs from hypertensive patients was markedly downregulated, whereas the phosphorylation of the p38 mitogen-activated protein kinase (p38-MAPK) was elevated. The cleaved caspase-3 levels were elevated in EPCs. The overexpression of ß2AR in EPCs from hypertensive patients inhibited p38-MAPK signaling, whereas it enhanced in vitro EPC proliferation, migration, and adhesion and in vivo re-endothelialization. The ß2AR-mediated effects were attenuated by treating the EPCs with a neutralizing monoclonal antibody against ß2AR, which could be partially antagonized by the p38-MAPK inhibitor SB203580. Moreover, shear stress stimulation, a classic nonpharmacological intervention, increased the phosphorylation levels of ß2AR and enhanced the in vitro and in vivo functions of EPCs from hypertensive patients. Collectively, the current investigation demonstrated that impaired ß2AR/p38-MAPK/caspase-3 signaling at least partially reduced the re-endothelialization capacity of EPCs from hypertensive patients. Restoration of ß2AR expression and shear stress treatment could improve their endothelial repair capacity by regulating the p38-MAPK/caspase-3 signaling pathway. The clinical significance of ß2AR in endothelium repair still requires further investigation.NEW & NOTEWORTHY Impaired ß2-adrenergic receptor (ß2AR) expression with an elevation of p38-MAPK/caspase-3 signaling at least partially contributes to the decline of re-endothelialization capacity of late endothelial progenitor cells (EPCs) from hypertensive patients. ß2AR gene transfer and shear stress treatment improve the late EPC-mediated enhancement of the re-endothelialization capacity in hypertensive patients through activating ß2AR/p38-MAPK/caspase-3 signaling. The present study is the first to reveal the potential molecular mechanism of the impaired endothelium-reparative capacity of late EPCs in hypertension after vascular injury and strongly suggests that ß2AR is a novel and crucial therapeutic target for increasing EPC-mediated re-endothelialization capacity in hypertension.

TDP-43 loss of function increases TFEB activity and blocks autophagosome-lysosome fusion.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by selective loss of motor neurons in brain and spinal cord. TAR DNA-binding protein 43 (TDP-43) was identified as a major component of disease pathogenesis in ALS, frontotemporal lobar degeneration (FTLD), and other neurodegenerative disease. Despite the fact that TDP-43 is a multi-functional protein involved in RNA processing and a large number of TDP-43 RNA targets have been discovered, the initial toxic effect and the pathogenic mechanism underlying TDP-43-linked neurodegeneration remain elusive. In this study, we found that loss of TDP-43 strongly induced a nuclear translocation of TFEB, the master regulator of lysosomal biogenesis and autophagy, through targeting the mTORC1 key component raptor. This regulation in turn enhanced global gene expressions in the autophagy-lysosome pathway (ALP) and increased autophagosomal and lysosomal biogenesis. However, loss of TDP-43 also impaired the fusion of autophagosomes with lysosomes through dynactin 1 downregulation, leading to accumulation of immature autophagic vesicles and overwhelmed ALP function. Importantly, inhibition of mTORC1 signaling by rapamycin treatment aggravated the neurodegenerative phenotype in a TDP-43-depleted Drosophila model, whereas activation of mTORC1 signaling by PA treatment ameliorated the neurodegenerative phenotype. Taken together, our data indicate that impaired mTORC1 signaling and influenced ALP may contribute to TDP-43-mediated neurodegeneration.

LncRNAs as tumor cell intrinsic factors that affect cancer immunotherapy.

Long noncoding RNAs (lncRNAs) have been found to associate with all major types of malignancies and play important roles in regulating several hallmarks of cancer by interacting with proteins, DNA, and RNA. The possible functions of lncRNAs and their roles in the regulation of tumour growth will be reported and discussed in the present review. In our recent report, based on genetic mice models and a series of systematic analyses, we suggested that lncRNAs also play critical roles in the regulation of antigen presentation in tumour cells and allow tumour cells to escape immune surveillance, which further broadens the scope of understanding lncRNA functions and how they relate to cancer immunotherapy resistance.

Tunable Color Temperatures and Efficient White Emission from Cs2 Ag1- x Nax In1- y Biy Cl6 Double Perovskite Nanocrystals.

Recently, Bi-doped Cs2 Ag0.6 Na0.4 InCl6 lead-free double perovskites demonstrating efficient warm-white emission have been reported. To enable the solution processing and enrich the application fields of this promising material, here a colloidal synthesis of Cs2 Ag1- x Nax In1- y Biy Cl6 nanocrystals is further developed. Different from its bulk states, the emission color temperatures of the nanocrystal can be tuned from 9759.7 to 4429.2 K by Na+ and Bi3+ incorporation. Furthermore, the newly developed nanocrystals can break the wavefunction symmetry of the self-trapped excitons by partial replacement of Ag+ ions with Na+ ions and consequently allow radiative recombination. Assisted with Bi3+ ions doping and ligand passivation, the photoluminescence quantum yield of the Cs2 Ag0.17 Na0.83 In0.88 Bi0.12 Cl6 nanocrystals is further promoted to 64%, which is the highest value for lead-free perovskite nanocrystals at present. The new colloidal nanocrystals with tunable color temperature and efficient photoluminescence are expected to greatly advance the research progress of lead-free perovskites in single-emitter-based white emitting materials and devices.

Folliculin, a tumor suppressor associated with Birt-Hogg-Dubé (BHD) syndrome, is a novel modifier of TDP-43 cytoplasmic translocation and aggregation.

TDP-43 was identified as the major component of ubiquitin and autophagosome-positive cytoplasmic inclusions in neurons in the large majority of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD) patients. It has been shown that a loss of nuclear TDP-43 in combination with enhanced cytoplasmic mislocalization of TDP-43, which is associated with accumulation of TDP-43 aggregates in the cytosol, is an early and key event in TDP-43-mediated neurodegeneration. However, the mechanism underlying TDP-43 nucleocytoplasmic shuttling is still not clear. Here, we show that the tumor suppressor folliculin (FLCN) is a novel positive regulator of TDP-43 cytoplasmic translocation. FLCN directly interacts with TDP-43. The amino acids 202-299 of FLCN and RNA-recognition motif domains of TDP-43 are necessary for their interaction. In addition, both exogenous and endogenous FLCNs are required for TDP-43 cytoplasmic accumulation, protein aggregation and stress granule formation. Overall, our study suggests that FLCN may play an important role in the regulation of TDP-43 nucleocytoplasmic shuttling and TDP-43-mediated proteinopathy.

The mitochondrial protein BNIP3L is the substrate of PARK2 and mediates mitophagy in PINK1/PARK2 pathway.

Mitochondrial dysfunction plays important roles in Parkinson's disease (PD) and the degradation of the damaged mitochondria by the mitochondria quality control system is important for dopaminergic (DA) neuronal survival. BNIP3L/Nix is a mitochondrial outer membrane protein that is required for the selective clearance of mitochondria. Here, we found that the mitochondrial protein BNIP3L acts downstream of the PINK1/PARK2 pathway to induce mitophagy. BNIP3L is a substrate of PARK2 to drive PARK2-mediated mitophagy. The ubiquitination of BNIP3L by PARK2 recruits NBR1 to mitochondria, thereby targeting mitochondria for degradation. BNIP3L rescues mitochondrial defects in pink1 mutant Drosophila but not in park mutant Drosophila, indicating that the clearance of mitochondria induced by BNIP3L depends on the presence of PARK2. In cells intoxicated with mitochondrial complex I inhibitors rotenone, 6-OHDA or MPP(+), the disrupted mitochondria are not appropriately eliminated by mitophagy due to the improper degradation of BNIP3L. Thus, our study demonstrates that BNIP3L, as a substrate of PARK2, promotes mitophagy in the PINK1/PARK2 pathway associated with PD pathogenesis.

Hydrogen Sulfide-Preconditioning of Human Endothelial Progenitor Cells Transplantation Improves Re-Endothelialization in Nude Mice with Carotid Artery Injury.

BACKGROUND/AIMS: The aim of present study was to test the hypothesis that preconditioning with sodium hydrosulfide (NaHS) could enhance the capacity of migration, adhesion and proliferation of endothelial progenitor cells (EPCs) in vitro, and also could improve the efficacy of EPCs transplantation for re-endothelialization in nude mice with carotid artery injury. The paper further addressed the underlying mechanisms. METHODS: EPCs were isolated from peripheral blood mononuclear cells of healthy male volunteers and the markers of EPCs were analyzed by flow cytometry. Thereafter, different concentrations of NaHS (25, 50, 100, 200 and 500 uM) were used for preconditioning EPCs. In vitro and in vivo migration, adhesion and proliferation as well as nitric oxide (NO) production of EPCs were evaluated. Carotid artery injury model was produced in nude mice and thereafter, NaHS-preconditioned EPCs were transplanted in order to evaluate their capacity of re-endothelialization. RESULTS: Cellular immuno-staining showed that isolated cells expressed the key markers of EPCs. In vitro, EPCs proliferation rates and NO production were gradually increased in a NaHS-concentration dependent manner, while these benefits were blocked at a concentration of 500 uM NaHS. Similarly, the migration and adhesion rates of EPCs were also increased the most prominently at a concentration of 200 µM NaHS. In vivo, compared to the control group, treatment with NaHS-preconditioned EPCs significantly enhanced the capacity of re-endothelialization of EPCs. Fluorescent microscope revealed that there were more EPCs homing to the injury vessels in the NaHS-preconditioned EPCs group than the non-preconditioned group. With the administration of AMPK or eNOS inhibitors respectively, the above benefits of NaHS-preconditioning were abrogated. CONCLUSION: These results suggested that NaHS-preconditioning enhanced the biological function and re-endothelialization of EPCs through the AMPK/eNOS signaling pathway.

Vitamin K2 suppresses rotenone-induced microglial activation in vitro.

AIM: Increasing evidence has shown that environmental factors such as rotenone and paraquat induce neuroinflammation, which contributes to the pathogenesis of Parkinson's disease (PD). In this study, we investigated the molecular mechanisms underlying the repression by menaquinone-4 (MK-4), a subtype of vitamin K2, of rotenone-induced microglial activation in vitro. METHODS: A microglial cell line (BV2) was exposed to rotenone (1 µmol/L) with or without MK-4 treatment. The levels of TNF-α or IL-1ß in 100 µL of cultured media of BV2 cells were measured using ELISA kits. BV2 cells treated with rotenone with or without MK4 were subjected to mitochondrial membrane potential, ROS production, immunofluorescence or immunoblot assays. The neuroblastoma SH-SY5Y cells were treated with conditioned media (CM) of BV2 cells that were exposed to rotenone with or without MK-4 treatment, and the cell viability was assessed using MTT assay. RESULTS: In rotenone-treated BV2 cells, MK-4 (0.5-20 µmol/L) dose-dependently suppressed the upregulation in the expression of iNOS and COX-2 in the cells, as well as the production of TNF-α and IL-1ß in the cultured media. MK-4 (5-20 µmol/L) significantly inhibited rotenone-induced nuclear translocation of NF-κB in BV2 cells. MK-4 (5-20 µmol/L) significantly inhibited rotenone-induced p38 activation, ROS production, and caspase-1 activation in BV2 cells. MK-4 (5-20 µmol/L) also restored the mitochondrial membrane potential that had been damaged by rotenone. Exposure to CM from rotenone-treated BV2 cells markedly decreased the viability of SH-SY5Y cells. However, this rotenone-activated microglia-mediated death of SH-SY5Y cells was significantly attenuated when the BV2 cells were co-treated with MK-4 (5-20 µmol/L). CONCLUSION: Vitamin K2 can directly suppress rotenone-induced activation of microglial BV2 cells in vitro by repressing ROS production and p38 activation.

[Study on Hydrothermal Preparation and Luminescence Properties of Luminescent Material BaSrMg(PO4)2:Eu³âº].

Eu³âº doped BaSrMg (PO4)2 were prepared by a hydrothermal method. The crystal structure and morphology of BaSrMg(PO4)2:Eu³âº phosphor were characterized by X-ray powder diffraction (XRD) and field emission scanning electron microscopy (FESEM). The effects of different pH values (5, 6, 7 and 8) and different reaction temperatures (120, 140, 160, 180 and 200 °C) on the crystal structure and morphology of BaSrMg(PO4)2:Eu³âº phosphor were studied in this paper. The results of XRD indicate that diffraction peaks are sharp and strong only when pH value is 6, meanwhile the FESEM shows the morphology is regular-shaped. The XRD patterns show amorphous halos superimposed with several weak sharp peaks for the samples preparing under the pH values of 5, 7 and 8. It indicates that these three samples are solid solution or mixed phases, which are in accord with the results of FESEM. From the fluorescence spectra, the peaks in the excitation spectra were assigned to the transition from 7F0 to 5D4, 5L8, 5L6 and 5D2, while the peaks of emission spectra corresponding to the transition of 5D1 --> 7F1 and 5D0-->7Fj (J = 0, 1, 2, 3 and 4). The strongest emission peak of the optimized phosphor located at 613 nm (5D0--> 7F2), excited by the main excitation peak with wavelength of 394 nm. The splitting of the emission peaks changes depends on pH values and temperatures, which indicating that luminescence properties is closely related to the crystal structure and morphology of particles.

Dyssynchronous pacing triggers endothelial-mesenchymal transition through heterogeneity of mechanical stretch in a canine model.

BACKGROUND: Endothelial-mesenchymal transition (EndMT) plays a pivotal role in cardiac fibrosis. However, it is unclear whether EndMT is involved in dyssynchronous heart failure (DHF). METHODS AND RESULTS: Twelve dogs received 3-week rapid right ventricular pacing (RVP) to develop DHF and then were randomly divided into a RVP group (n=6; RVP for another 3 weeks) and a biventricular pacing (BiVP) group (n=6; BiVP for 3 weeks), and another 6 dogs were in the control group. Contractile function in BiVP group was a little better than that in RVP group (P<0.05), but significant heart failure remained in 2 groups. RVP induced more significant cardiac fibrosis and higher collagen 1A2 expression in the left ventricular lateral wall (late-contracting and high-stress) than that in the anterior wall, and for those in the BiVP group, it was much lower. CD31, S100A4, α-smooth muscle actin and collagen 1A2 were used to evaluate EndMT. EndMT levels, transforming growth factor-ß (TGF-ß)/snail signaling, collagen 1A2 and integrin ß1 expression were much higher in the endothelial cells from the RVP lateral wall than that from BiVP. In this in vitro study, cyclic stretch could independently induce EndMT and enhance the pro-EndMT effect of TGF-ß in HUVECs, which could be partly blocked by integrin ß1 siRNA. CONCLUSIONS: RVP-induced DHF could aggravate fibrosis due to regional heterogeneity of mechanical stress, and it was better in the BiVP group where mechanical stress-induced EndMT might play a pivotal role through the integrin ß1 pathway.

Protease Omi facilitates neurite outgrowth in mouse neuroblastoma N2a cells by cleaving transcription factor E2F1.

AIM: Omi is an ATP-independent serine protease that is necessary for neuronal function and survival. The aim of this study was to investigate the role of protease Omi in regulating differentiation of mouse neuroblastoma cells and to identify the substrate of Omi involved in this process. METHODS: Mouse neuroblastoma N2a cells and Omi protease-deficient mnd2 mice were used in this study. To modulate Omi and E2F1 expression, N2a cells were transfected with expression plasmids, shRNA plasmids or siRNA. Protein levels were detected using immunoblot assays. The interaction between Omi and E2F1 was studied using immunoprecipitation, GST pulldown and in vitro cleavage assays. N2a cells were treated with 20 µmol/L retinoic acid (RA) and 1% fetal bovine serum to induce neurite outgrowth, which was measured using Image J software. RESULTS: E2F1 was significantly increased in Omi knockdown cells and in brain lysates of mnd2 mice, and was decreased in cells overexpressing wild-type Omi, but not inactive Omi S276C. In brain lysates of mnd2 mice, endogenous E2F1 was co-immunoprecipitated with endogenous Omi. In vitro cleavage assay demonstrated that Omi directly cleaved E2F1. Treatment of N2a cells with RA induced marked differentiation and neurite outgrowth accompanied by significantly increased Omi and decreased E2F1 levels, which were suppressed by pretreatment with the specific Omi inhibitor UCF-101. Knockdown of Omi in N2a cells suppressed RA-induced neurite outgrowth, which was partially restored by knockdown of E2F1. CONCLUSION: Protease Omi facilitates neurite outgrowth by cleaving the transcription factor E2F1 in differentiated neuroblastoma cells; E2F1 is a substrate of Omi.

Parkin represses 6-hydroxydopamine-induced apoptosis via stabilizing scaffold protein p62 in PC12 cells.

AIM: Parkin has been shown to exert protective effects against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in different models of Parkinson disease. In the present study we investigated the molecular mechanisms underlying the neuroprotective action of parkin in vitro. METHODS: HEK293, HeLa and PC12 cells were transfected with parkin, parkin mutants, p62 or si-p62. Protein expression and ubiquitination were assessed using immunoblot analysis. Immunoprecipitation assay was performed to identify the interaction between parkin and scaffold protein p62. PC12 and SH-SY5Y cells were treated with 6-OHDA (200 µmol/L), and cell apoptosis was detected using PI and Hoechst staining. RESULTS: In HEK293 cells co-transfected with parkin and p62, parkin was co-immunoprecipitated with p62, and parkin overexpression increased p62 protein levels. In parkin-deficient HeLa cells, transfection with wild-type pakin, but not with ligase activity-deficient pakin mutants, significantly increased p62 levels, suggesting that parkin stabilized p62 through its E3 ligase activity. Transfection with parkin or p62 significantly repressed ERK1/2 phosphorylation in HeLa cells, but transfection with parkin did not repress ERK1/2 phosphorylation in p62-knockdown HeLa cells, suggesting that p62 was involved in parkin-induced inhibition on ERK1/2 phosphorylation. Overexpression of parkin or p62 significantly repressed 6-OHDA-induced ERK1/2 phosphorylation in PC12 cells, and parkin overexpression inhibited 6-OHDA-induced apoptosis in PC12 and SH-SY5Y cells. CONCLUSION: Parkin protects PC12 cells against 6-OHDA-induced apoptosis via ubiquitinating and stabilizing scaffold protein p62, and repressing ERK1/2 activation.