Podocytes are the major target in proteinuric kidney diseases such as diabetic nephropathy. The underlying molecular mechanisms by which high glucose (HG) results in podocyte damage remain unclear. This study investigated the regulatory role of Smad3, ezrin, and protein kinase A (PKA) in NADPH oxidase (Nox4) expression, reactive oxidative species (ROS) production, and apoptosis in HG-treated podocytes. A human podocyte cell line was cultured and differentiated, then treated with 30 mM HG. Apoptosis and intracellular ROS levels were assessed using TUNEL and DCF assays, respectively. Expressions of Nox4, phospho-Smad3(Ser423/425), phospho-PKA(Thr197), and phospho-ezrin(Thr567) were evaluated using western blotting. ELISA was used to quantify intracellular cAMP concentration and PKA activity. Knockdown assay was used to inhibit the expressions of Smad3, Nox4, and ezrin by lentiviral shRNA. In HG-treated podocytes, the level of phospho-Smad3(Ser423/425) and phosphoezrin(Thr567) was increased significantly, which was accompanied by the reduction of cAMP and phospho-PKA(Thr197). HG-induced apoptosis was significantly prevented by the Smad3-inhibitor SIS3 or shRNA-Smad3. In podocytes expressing shRNA-ezrin or shRNA-Nox4, apoptosis was remarkably mitigated following HG treatment. HG-induced upregulation of phospho-ezrin(Thr567) and downregulation of phospho-PKA(Thr197) was significantly prevented by SIS3, shRNA-ezrin or shRNA-Smad3. Forskolin, a PKA activator, significantly inhibited HG-mediated upregulation of Nox4 expression, ROS generation, and apoptosis. Additionally, an increase in the ROS level was prohibited in HG-treated podocytes with the knockdown of Nox4, Smad3, or ezrin. Taken together, our findings provided evidence that Smad3-mediated ezrin activation upregulates Nox4 expression and ROS production, by suppressing PKA activity, which may at least in part contribute to HG-induced podocyte apoptosis.

Retraction: "Effects of microRNA-494 on proliferation, migration, invasion, and apoptosis of medulloblastoma cells by mediating c-myc through the p38 MAPK signaling pathway," by Xiao-Heng Xu, Si-Jin Zhang, Qi-Bo Hu, Xing-Yu Song, Wei Pan, J Cell Biochem. 2019; 2594-2606: The above article, published online on 10 October 2018 in Wiley Online Library (), has been retracted by agreement between the journal's Editor in Chief, Prof. Dr. Christian Behl, and Wiley Periodicals LLC. The retraction has been agreed following an investigation based on allegations raised by a third party. Several flaws and inconsistencies between results presented and experimental methods described were found. Thus, the editors consider the conclusions of this article to be invalid.

Rett syndrome (RTT) is a rare neurodevelopmental disorder that results in multiple disabilities. Exosomal microRNA (miRs) from urine-derived stem cells (USCs) have been shown to induce neurogenesis and aid in functional recovery from brain ischemia. In the present study, we sought to determine whether that exosomal miR-21-5p from USCs could promote early neural formation in a model of RTT. USCs were isolated and evaluated by flow cytometry. Exosomes were analyzed by transmission electron microscopy, tunable resistive pulse sensing (TRPS), and western blotting. PKH26 fluorescent dyes were used to observe intake of exosomes in vivo and in vitro. An RTT mouse model was treated with exosomes for behavioral studies. Dual‐luciferase report gene assays were conducted to evaluate the relationship between miR-21-5p and Eph receptor A4 (EphA4). In vitro, treatment with exosomes from human urine‐derived stem cells (USC-Exos) increased the percentage of neuron-specific class III beta-tubulin (Tuj1) + nerve cells as well as the transcription levels of β-III tubulin and doublecortin (DCX). A higher level of miR-21-5p was observed in USC-Exos, which promoted differentiation in NSCs by targeting the EPha4/TEK axis. In vivo, exosomal miR-21-5p improved the behavior, motor coordination, and cognitive ability of mice, facilitated the differentiation of NSCs in the subventricular zone of the lateral ventricle and promoted a marked rise in the number of DCX + cells. Our data provide evidence that exosomal miR-21-5p from human USCs facilitate early nerve formation by regulating the EPha4/TEK axis.

We investigated the role of microRNA (miR)-485 and its downstream signaling molecules on mediating epilepsy in cellular and rat models. We established a cellular epilepsy model by exposing hippocampal neurons to magnesium and a rat model by treating ICR mice with lithium chloride (127 mg/kg) and pilocarpine (30 mg/kg). We confirmed that miR-485 could bind and inhibit histone deacetylase 5 (HDAC5) and then measured expression of miR-485 and in mice and cells. Cells were transfected with overexpression or knockdown of miR-485, HDAC5, hypoxia-inducible factor-1alpha (HIF1α), or 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 enzyme (PFKFB3) to verify their roles in apoptosis, oxidative stress, and inflammation in epileptic hippocampal neurons. Binding relationship between miR-485, HDAC5, HIF1α, and PFKFB3 was verified. Oxidative stress and inflammation marker levels in epilepsy model mice were assessed. miR-485 was downregulated and HDAC5 was upregulated in cell and animal model of epilepsy. Seizure, neuronal apoptosis, oxidative stress (increased SOD and GSH-Px expression and decreased MDA and 8-OHdG expression) and inflammation (reduced IL-1β, TNF-α, and IL-6 expression) were reduced by miR-485 in epileptic cells. HIF1α and PFKFB3 expression was reduced by HDAC5 knockdown in cells, which was recapitulated in vivo. Thus, miR-485 alleviates neuronal damage and epilepsy by inhibiting HDAC5, HIF1α, and PFKFB3.

Aims: Type 1 diabetes (T1D) is the most common autoimmune disease that affects a global scale. Accumulating evidence has indicated, nuclear factor kappa B (NF-kappa B) and some microRNAs (miRNAs) as important biomarkers participating in the development of T1D. Thus, we aimed to determine the role of NF-kappa B and miR-150 in the development of T1D and to unravel the molecular mechanism. Main methods: Non-obese diabetic mice were used for the T1D model establishment by injecting with streptozotocin. Besides, pancreatic islet beta cells, separated from T1D mice, were induced by interferon-gamma and tumor necrosis factor-alpha for 3 days to mimic T1D damage. The expression of NF-kappa B p65, miR-150, and p53 up-regulated modulator of apoptosis (PUMA) was evaluated by RT-qPCR, while the expression of PUMA, p65, and apoptotic proteins in pancreatic islet beta cells were determined by western blot analysis. Besides, inflammatory factors IL-17A, IL-2, IFN-gamma, and IL-4 were detected by ELISA. The relationship among NF-kappa B, miR-150, and PUMA was analyzed by the dual-luciferase reporter gene, chromatin- and RNA-immunoprecipitation assays, respectively. Key findings: Restoration of NF-kappa B reduced the incidence of T1D in mice. Over-expressed NF-kappa B inhibited the release of inflammatory factors and apoptosis in pancreatic islet beta cells. PUMA was confirmed to be a potential target gene of miR-150. miR-150 suppressed PUMA to inhibit the T1D-induced inflammation and beta cell apoptosis whereas NF-kappa B activated the miR-150 expression by binding to the miR-150 promoter, thereby preventing the T1D-induced inflammation and beta cell apoptosis. Significance: NF-kappa B/miR-150/PUMA may serve as potential therapeutic targets for T1D.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy)This article has been retracted at the request of the Editor-in-Chief.Concern was raised about the reliability of the Western blot results in Figures 1D,E+H, 2E+H, 3F,H+K, and 4B+E which appear to have a similar phenotype as many other publications, as detailed here: https://pubpeer.com/publications/C6FD5C041268DBBCDA521AEC112FA4 and here: https://docs.google.com/spreadsheets/d/1r0MyIYpagBc58BRF9c3luWNlCX8VUvUuPyYYXzxWvgY/edit#gid=262337249. The journal requested the corresponding author comment on these concerns and provide the raw Western blot data. However, the authors were not able to satisfactorily fulfill this request and therefore the Editor-in-Chief decided to retract the article.

Medulloblastoma (MB) represents a fatal malignancy often occurring in children. Angiogenesis is a hallmark of the progression of MB. Over the past decade, investigators have attempted to develop more effective and less toxic anti-angiogenic strategies to treat MB. Thrombospondin (TSP) family is observed to be a key regulator of angiogenesis. Thus, the current study aimed to elucidate the function of TSP2 in patients with MB and the underlying mechanism. The expression of TSP2, Notch1 and VEGF in MB and adjacent tissues collected from clinical samples as well as a MB cell line (Daoy) was examined. The results demonstrated that in the MB tissues and Daoy cells, TSP2 was downregulated, while Notch1 and VEGF were upregulated. Then, after the Daoy cells were treated with TSP2 silencing, TSP2 overexpression, or Notch signaling pathway inhibition, a series of in vitro cell experiments were performed to verify the interaction between TSP2 and Notch signaling pathway, and to examine the abilities of cell proliferation, migration, invasion, and tube formation. Upregulation of TSP2 was observed to lead to the downregulation of the Notch signaling pathway. Moreover, cells overexpressing TSP2 exhibited diminished proliferation, invasion, migration, and tube formation. In addition, a significant attenuation of tumor growth and angiogenesis was identified in vivo in the Daoy cells overexpressing TSP2 inoculated in nude mice. Taken together, the key findings of this study revealed the inhibitory role of TSP2 in the development of MB via blockade of the Notch signaling pathway, highlighting its potential as a treatment target for MB treatment.

Medulloblastoma (MB) is the most prevalent brain tumor that occurs during childhood and originates from cerebellar granule cell precursors. Based on recent studies, the differential expression of several microRNAs is involved in MB, while the role of microRNA-494 (miR-494) in MB remains unclear. Therefore, we conducted this study to investigate the regulative role of miR-494 in MB cells via the p38 mitogen-activated protein kinase (MAPK) signaling pathway by mediating c-myc. In the current study, MB cells were collected and transfected with miR-494 mimic, miR-494 inhibitor, siRNA- c-myc, and miR-494 inhibitor + siRNA-c-myc. The expressions of miR-494, c-myc, p38 MAPK, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), interleukin-6 (IL-6), metadherin (MTDH), phosphatase and tensin homolog (PTEN) and survivin were determined. Cell proliferation, cell-cycle distribution, apoptosis, migration, and invasion were evaluated. The results revealed that there was a poor expression of miR-494 and high expression of c-myc in MB tissues. C-myc was determined as the target gene of miR-494. In response to miR-494 mimic, MB cells were found to have increased Bax and PTEN expressions, as well as cell number in G1 phase and cell apoptosis and decreased c-myc, p38 MAPK, Bcl-2, MTDH, IL-6, and survivin expression and cell number count in the S phase, cell proliferation, migration, and invasion. In conclusion, the results demonstrated that the upregulation of miR-494 results in the suppression of cell proliferation, migration, and invasion, while it promotes apoptosis of MB cells through the negative mediation of c-myc, which in turn inactivates the p38 MAPK pathway.

The intracellular pathogen Brucella abortus (B. abortus) survives and replicates inside host cells within the Brucella-containing vacuole, in which membrane contains a small GTPase Rab1. Here, we reported that Rab1 mediates B. abortus intracellular growth. Furthermore, B. abortus DnaK was identified to interact with Rab1 using GST pull-down and mass spectrometry analysis. This interaction was confirmed by co-immunoprecipitation and immunofluorescence. Through DnaK–CyaA fusion protein translocation assay and immunofluorescence confocal microscopy, the B. abortus DnaK was proved to be a virB-dependent translocated substrate.

ABSTRACT AMP-activated protein kinase (AMPK) is a serine/threonine kinase that is well conserved during evolution. AMPK activation inhibits production of reactive oxygen species (ROS) in cells via suppression of NADPH oxidase. However, the role of AMPK during the process of Brucella infection remains unknown. Our data demonstrate that B. abortus infection induces AMPK activation in HeLa cells in a time-dependent manner. The known AMPK kinases LKB1, CAMKKβ, and TAK1 are not required for the activation of AMPK by B. abortus infection. Instead, this activation is dependent on the RNase activity of inositol-requiring enzyme 1 (IRE1). Moreover, we also found that B. abortus infection-induced IRE1-dependent activation of AMPK promotes B. abortus intracellular growth with peritoneal macrophages via suppression of NADPH-derived ROS production. IMPORTANCE Previous studies showed that B. abortus infection does not promote any oxidative burst regulated by NADPH oxidase. However, the underlying mechanism remains elusive. We report for the first time that AMPK activation caused by B. abortus infection plays important role in NADPH oxidase-derived ROS production.

Our previous study showed that the downstream of kinase 3 (DOK3) is degraded during macrophage stimulation with CpG. However, the underlying mechanism and role in Toll-like receptor 9 (TLR9) signaling remains elusive. In this study, we demonstrate that CpG treatment leads to ubiquitin-mediated degradation of DOK3 via interaction with an E3 ligase TNFR-associated factor 6 (TRAF6). We also identified the 27th amino acid (lysine) of DOK3 is responsible for Ly48 polyubiquitination of DOK3. Furthermore, reintroduction of DOK3 (K27R) into DOK3-deficient macrophages abolishes DOK3 degradation induced by CpG and suppresses the production of IL-6 and TNFα. More importantly, our study uncovers a novel role of an E3 ligase TRAF6, namely, TRAF6 is also able to catalyse Lys 48 polyubiquitylation of target protein except for Lys 63 polyubiquitylation.

Telomerase is a widely accepted cancer biomarker. The conventional method for telomerase activity assay, the telomeric repeat amplification protocol (TRAP), is time-consuming and susceptible to contaminants. Therefore, development of simple and sensitive strategies for telomerase detection is still a challenging subject. Here we develop a highly sensitive method for telomerase detection based on primer-modified gold nanoparticles (GNPs) manipulated by exonuclease I (Exo I). In the absence of telomerase, Exo I digests the substrate nucleic acid on the surface of GNPs, inducing the GNPs' aggregation. In the presence of telomerase, the telomerase elongation products which fold into G-quadruplex are resistant to the digestion of Exo I, and protect the GNPs from aggregation. By using this method, we can detect telomerase activity in 100 HL-60 cancer cells mL−1 by naked eyes, and the detection limit is 29 HL-60 cells mL−1. This method is very simple and reliable, without any separation and amplification procedure. We also demonstrate the feasibility of this protocol for screening of telomerase inhibitors as anticancer agents. This method is promising to be applied in early clinical diagnosis and drug discovery.

Annexin A2 (ANXA2) is a well-known calcium-dependent phospholipid binding protein widely distributed in the nucleus, cytoplasm and extracellular surface of various eukaryotic cells. It has been recognized as a pleiotropic protein affecting a wide range of molecular and cellular processes. Dysregulation and abnormal expression of ANXA2 are linked to a large number of prevalent diseases, including autoimmune and neurodegenerative disease, antiphospholipid syndrome, inflammation, diabetes mellitus and a series of cancers. Accumulating data suggest that ANXA2 is aberrantly expressed in a wide spectrum of cancers, and exerts profound effects on tumor cell adhesion, proliferation, apoptosis, invasion and metastasis as well as tumor neovascularization via different modes of action. However, despite significant research, our knowledge of the mechanism by which ANXA2 participates in cancer development remains fragmented. The present review systematically summarizes the effects of ANXA2 on tumor progression, in an attempt to gain an improved understanding of the underlying mechanisms and to provide a potential effective target for cancer therapy.

Triggering receptor expressed on myeloid cells-2 (TREM-2) is a cell surface receptor primarily expressed on macrophages and dendritic cells. TREM-2 functions as a phagocytic receptor for bacteria as well as an inhibitor of Toll like receptors (TLR) induced inflammatory cytokines. However, the role of TREM-2 in Brucella intracellular growth remains unknown. To investigate whether TREM-2 is involved in Brucella intracellular survival, we chose bone marrow derived macrophages (BMDMs), in which TREM-2 is stably expressed, as cell model. Colony formation Units (CFUs) assay suggests that TREM-2 is involved in the internalization of Brucella abortus (B. abortus) by macrophages, while silencing of TREM-2 decreases intracellular survival of B. abortus. To further study the underlying mechanisms of TREM-2-mediated bacterial intracellular survival, we examined the activation of B. abortus-infected macrophages through determining the kinetics of activation of the three MAPKs, including ERK, JNK and p38, and measuring TNFα production in response to lipopolysaccharide (LPS) of Brucella (BrLPS) or B. abortus stimulation. Our data show that TREM-2 deficiency promotes activation of Brucella-infected macrophages. Moreover, our data also demonstrate that macrophage activation promotes killing of Brucella by enhancing nitric oxygen (NO), but not reactive oxygen species (ROS) production, macrophage apoptosis or cellular death. Taken together, these findings provide a novel interpretation of Brucella intracellular growth through inhibition of NO production produced by TREM-2-mediated activated macrophages.

The calcium-dependent protease calpain2 is involved in macrophages apoptosis. Brucella infection-induced up-regulation of intracellular calcium level is an essential factor for the intracellular survival of Brucella within macrophages. Here, we hypothesize that calcium-dependent E3 ubiquitin ligase Nedd4 ubiquitinates calpain2 and inhibits Brucella infection-induced macrophage apoptosis via degradation of calpain2.Our results reveal that Brucella infection induces increases in Nedd4 activity in an intracellular calcium dependent manner. Furthermore, Brucella infection-induced degradation of calpain2 is mediated by Nedd4 ubiquitination of calpain2. Brucella infection-induced calpain2 degradation inhibited macrophages apoptosis. Treatment of Brucella infected macrophages with calcium chelator BAPTA or Nedd4 knock-down decreased Nedd4 activity, prevented calpain2 degradation, and resulted in macrophages apoptosis.

The zinc-finger protein A20 has crucial physiological functions as a dual inhibitor of macrophage activation and apoptosis in tumor necrosis factor receptor1 (TNFR1) signaling pathway. Brucella infection can induce A20 expression in macrophages. Here, we hypothesize that A20 promotes Brucella intracellular growth via inhibition of activation and apoptosis of macrophages. To test this hypothesis, we stably incorporated mouse A20-shRNA into the RAW264.7 cells by lentiviral gene transfer to successfully knockdown A20. A20-deficient RAW264.7 cells were subsequently challenged with Brucella abortus and colony formation units (CFUs) of bacteria, TNFα production, NF-kB activation, macrophages apoptosis and cell death were evaluated. The A20 knockdown was shown to effectively promote B. abortus-stimulated TNFα release, NF-kB activation and macrophage cell death, which suppressed B. abortus intracellular replication. Unexpectedly, deficiency of A20 failed to lead to B. abortus-induced macrophage apoptosis. A20 deficiency coupled NF-kB inhibition promoted caspase-8 dependent B. abortus-induced macrophage apoptosis. These findings provide a novel mechanism by which Brucella intracellular growth within macrophages occurs through up-regulation of A20 thereby limiting activation and macrophages cell death.

We investigated an acute outbreak of the cutaneous form of fowlpox among chickens in China in November 2009. Using pathologic and virologic methods, we identified a novel type of fowlpox virus that carried an integrated genomic sequence of reticuloendotheliosis virus. This highly pathogenic virus could lead to severe ecologic effects and economic losses.