Construction of a novel circRNA-miRNA-ferroptosis related mRNA network in ischemic stroke.

Molecule alterations are important to explore the pathological mechanism of ischemic stroke (IS). Ferroptosis, a newly recognized type of regulated cell death, is related to IS. Identification of the interactions between circular RNA (circRNA), microRNA (miRNA) and ferroptosis related mRNA may be useful to understand the molecular mechanism of IS. The circRNA, miRNA and mRNA transcriptome data in IS, downloaded from the Gene Expression Omnibus (GEO) database, was used for differential expression analysis. Ferroptosis related mRNAs were identified from the FerrDb database, followed by construction of circRNA-miRNA-ferroptosis related mRNA network. Enrichment and protein-protein interaction analysis of mRNAs in circRNA-miRNA-mRNA network was performed, followed by expression validation by reverse transcriptase polymerase chain reaction and online dataset. A total of 694, 41 and 104 differentially expressed circRNAs, miRNAs and mRNAs were respectively identified in IS. Among which, dual specificity phosphatase 1 (DUSP1), nuclear receptor coactivator 4 (NCOA4) and solute carrier family 2 member 3 (SLC2A3) were the only three up-regulated ferroptosis related mRNAs. Moreover, DUSP1, NCOA4 and SLC2A3 were significantly up-regulated in IS after 3, 5 and 24 h of the attack. Based on these three ferroptosis related mRNAs, 4 circRNA-miRNA-ferroptosis related mRNA regulatory relationship pairs were identified in IS, including hsa_circ_0071036/hsa_circ_0039365/hsa_circ_0079347/hsa_circ_0008857-hsa-miR-122-5p-DUSP1, hsa_circ_0067717/hsa_circ_0003956/hsa_circ_0013729-hsa-miR-4446-3p-SLC2A3, hsa_circ_0059347/hsa_circ_0001414/hsa_circ_0049637-hsa-miR-885-3p-SLC2A3, and hsa_circ_0005633/hsa_circ_0004479-hsa-miR-4435-NCOA4. In addition, DUSP1 is involved in the signaling pathway of fluid shear stress and atherosclerosis. Relationship of regulatory action between circRNAs, miRNAs and ferroptosis related mRNAs may be associated with the development of IS.

The evaluation and treatment of acute anterior circulation occlusion stroke with high clot burden: Progressive stratified aspiration thrombectomy vs. stent retriever thrombectomy.

OBJECTIVE: To evaluate the safety and effectiveness of the progressive stratified aspiration thrombectomy (PSAT) in treatment of patients with acute ischemic stroke and large vessel occlusion (AIS-LVO). METHODS: 117 AIS-LVO patients with high clot burden who underwent emergency endovascular treatment were included. All patients were divided into two groups according to surgical technique: PSAT group, stent retriever thrombectomy (SRT) group. The primary outcome was the 90-day mRS, the secondary outcomes included recanalization rate, the 24-h and 7-day NIHSS, the 7-day symptomatic intracranial hemorrhage (SICH) rate and 90-days mortality. RESULTS: 65 patients underwent PSAT, and 52 patients underwent SRT. The PSAT group performed better than SRT group regarding the successful recanalization rate (86.3 % vs. 71.2 %, P < 0.05) and time from puncture to recanalization (70 min [IQR, 58-87 min] vs. 87 min [IQR, 68-103 min], P < 0.05). The 7-day NIHSS score of the PSAT group was lower than that of the SRT group (12 [10-18] vs. 12 [8-25], P < 0.05). It was worth noting that at the 90-day follow-up, the favorable functional outcome (mRS 0-2) rate of PSAT group was higher (P < 0.05). There was no significant difference in terms of the 24-h NIHSS score after surgery (15 [10-18] vs. 15 [10-22], P > 0.05), SICH (23.1 % vs. 26.9 %, P > 0.05) and mortality rate between the two groups (13.4 % vs. 19.2 %, P > 0.05). CONCLUSIONS: It is safe and effective to treat high clot burden AIS-LVO patients with PSAT, which has a better reperfusion rate and prognostic outcome than SRT.

Identification of a novel amphioxus leucine-rich repeat receptor involved in phagocytosis reveals a role for Slit2-N-type LRR in bacterial elimination.

The basal chordate amphioxus is a model for tracing the origin and evolution of vertebrate immunity. To explore the evolution of immunoreceptor signaling pathways, we searched the associated receptors of the amphioxus Branchiostoma belcheri (Bb) homolog of immunoreceptor signaling adaptor protein Grb2. Mass-spectrum analysis of BbGrb2 immunoprecipitates from B. belcheri intestine lysates revealed a folate receptor (FR) domain- and leucine-rich repeat (LRR)-containing protein (FrLRR). Sequence and structural analysis showed that FrLRR is a membrane protein with a predicted curved solenoid structure. The N-terminal Fr domain contains very few folate-binding sites; the following LRR region is a Slit2-type LRR, and a GPI-anchored site was predicted at the C-terminus. RT-PCR analysis showed FrLRR is a transcription-mediated fusion gene of BbFR-like and BbSlit2-N-like genes. Genomic DNA structure analysis implied the B. belcheri FrLRR gene locus and the corresponding locus in Branchiostoma floridae might be generated by exon shuffling of a Slit2-N-like gene into an FR gene. RT-qPCR, immunostaining, and immunoblot results showed that FrLRR was primarily distributed in B. belcheri intestinal tissue. We further demonstrated that FrLRR localized to the cell membrane and lysosomes. Functionally, FrLRR mediated and promoted bacteria-binding and phagocytosis, and FrLRR antibody blocking or Grb2 knockdown inhibited FrLRR-mediated phagocytosis. Interestingly, we found that human Slit2-N (hSlit2-N) also mediated direct bacteria-binding and phagocytosis which was inhibited by Slit2-N antibody blocking or Grb2 knockdown. Together, these results indicate FrLRR and hSlit2-N may function as phagocytotic-receptors to promote phagocytosis through Grb2, implying the Slit2-N-type-LRR-containing proteins play a role in bacterial binding and elimination.

Identification and Characterization of the Amphioxus Lck and Its Associated Tyrosine Phosphorylation-Dependent Inhibitory LRR Receptor.

Amphioxus (e.g., Branchiostoma belcheri, Bb) has recently emerged as a new model for studying the origin and evolution of vertebrate immunity. Mammalian lymphocyte-specific tyrosine kinase (Lck) plays crucial roles in T cell activation, differentiation and homeostasis, and is reported to phosphorylate both the ITIM and ITSM of PD-1 to induce the recruitment of phosphatases and thus the inhibitory function of PD-1. Here, we identified and cloned the amphioxus homolog of human Lck. By generating and using an antibody against BbLck, we found that BbLck is expressed in the amphioxus gut and gill. Through overexpression of BbLck in Jurkat T cells, we found that upon TCR stimulation, BbLck was subjected to tyrosine phosphorylation and could partially rescue Lck-dependent tyrosine phosphorylation in Lck-knockdown T cells. Mass spectrometric analysis of BbLck immunoprecipitates from immunostimulants-treated amphioxus, revealed a BbLck-associated membrane-bound receptor LRR (BbLcLRR). By overexpressing BbLcLRR in Jurkat T cells, we demonstrated that BbLcLRR was tyrosine phosphorylated upon TCR stimulation, which was inhibited by Lck knockdown and was rescued by overexpression of BbLck. By mutating single tyrosine to phenylalanine (Y-F), we identified three tyrosine residues (Y539, Y655, and Y690) (3Y) of BbLcLRR as the major Lck phosphorylation sites. Reporter gene assays showed that overexpression of BbLcLRR but not the BbLcLRR-3YF mutant inhibited TCR-induced NF-κB activation. In Lck-knockdown T cells, the decline of TCR-induced IL-2 production was reversed by overexpression of BbLck, and this reversion was inhibited by co-expression of BbLcLRR but not the BbLcLRR-3YF mutant. Sequence analysis showed that the three tyrosine-containing sequences were conserved with the tyrosine-based inhibition motifs (ITIMs) or ITIM-like motifs. And TCR stimulation induced the association of BbLcLRR with tyrosine phosphatases SHIP1 and to a lesser extent with SHP1/2. Moreover, overexpression of wild-type BbLcLRR but not its 3YF mutant inhibited TCR-induced tyrosine phosphorylation of multiple signaling proteins probably via recruiting SHIP1. Thus, we identified a novel immunoreceptor BbLcLRR, which is phosphorylated by Lck and then exerts a phosphorylation-dependent inhibitory role in TCR-mediated T-cell activation, implying a mechanism for the maintenance of self-tolerance and homeostasis of amphioxus immune system and the evolutionary conservatism of Lck-regulated inhibitory receptor pathway.

Sevoflurane Suppresses Colon Cancer Cell Malignancy by Regulating circ-PI4KA.

PURPOSE: To explore the effect of SEV on colon cancer cells through circ-PI4KA. METHODS: The RNA level of circular RNA_0062389, microRNA-331-3p and LIM and SH3 protein 1 was determined by quantitative real-time polymerase chain reaction. Protein expression was detected by Western blot. Cell proliferation was investigated by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide, cell colony formation and 5-ethynyl-29-deoxyuridine assays. Cell apoptosis was demonstrated using Annexin V-fluorescein isothiocyanate/propidium iodide double staining assay. Cell migration and invasion were detected by transwell assay. The target relationship between miR-331-3p and circ-PI4KA or LASP1 was predicted by starBase v2.0 online database, and identified by a dual-luciferase reporter assay. The effects between SEV treatment and circ-PI4KA knockdown on tumor formation were presented by in vivo tumor formation assay. RESULTS: Circ-PI4KA and LASP1 expressions were dramatically upregulated, while miR-331-3p was downregulated in colon cancer tissues and cells, respectively. SEV exposure significantly decreased the expression of circ-PI4KA and LASP1, but increased miR-331-3p expression. SEV inhibited cell proliferation, migration and invasion, and induced cell apoptosis by regulating circ-PI4KA. Furthermore, circ-PI4KA interacted with miR-331-3p, and miR-331-3p interacted with LASP1. SEV inhibited tumor growth by controlling circ-PI4KA in vivo. CONCLUSION: Circ-PI4KA attenuated SEV-treated colon cancer cell malignancy by upregulating LASP1 through binding to miR-331-3p, which provided a new mechanism for studying surgery-mediated therapy of colon cancer.

Effects of Percutaneous Neuromuscular Electrical Stimulation for Neck Pain in Patients With Cervical Spondylosis.

CONTEXT: Cervical spondylosis (CS) is a very common, age-related, chronic, disc-degeneration condition. Alternative medicine has been widely used to treat neck pain in CS. However, no randomized controlled trials have focused on the effects and safety of percutaneous neuromuscular electrical stimulation (PNMES) for neck-pain relief in patients with CS. OBJECTIVE: The study aimed to evaluate the effects and safety of PNMES for treating neck pain in patients with cervical spondylosis (CS). DESIGN: The research team designed a two-arm, double-blinded, randomized, sham-controlled trial. SETTING: The study was conducted at the People's Hospital of Yan'an in Yan'an, China. PARTICIPANTS: Participants were 124 patients with neck pain from CS at the hospital. INTERVENTION: Participants were randomly divided into an intervention group and a control group in a ratio of 1:1. The intervention group received PNMES (PNMES group), and the control group received sham PNMES for 30 minutes daily 3 times weekly, for 12 weeks. OUTCOME MEASURES: The outcome measures included: (1) a visual analog scale (VAS), (2) a test of cervical range of motion (ROM), and (3) the neck disability index (NDI) score. All outcome measurements were measured immediately postintervention and in a follow-up at 4 weeks postintervention. In addition, AEs were also recorded duration the period of treatment. RESULTS: Immediately postintervention and at the follow-up, the PNMES group exhibited decreases in the mean VAS (P < .01) and NDI score (P < .01) that were significantly greater than those of the control group. Additionally, the increase in the mean ROM was significantly higher in the PNMES group than that in the control group, both immediately postintervention and at the follow-up (P < .01). No AEs were found in either group. CONCLUSIONS: The results of this study have demonstrated that PNMES is more effective than sham PNMES for neck-pain relief in patients with CS.

Liraglutide and Insulin Have Contrary Effects on Adipogenesis of Human Adipose-Derived Stem Cells via Wnt Pathway.

BACKGROUND: Glucagon-like peptide-1 (GLP-1) has been reported to have beneficial impacts on improving human's metabolism and ameliorating insulin resistance. While insulin is another important and conventional drug in diabetes treatment, but it has an adverse effect on weight gain. PURPOSE: To make sure whether GLP-1 and insulin play different roles in human adipose-derived stem cells (hADSCs). METHODS: We examined the in vitro roles and molecular mechanisms of liraglutide, a GLP-1 analogue, and human insulin on hADSCs isolated from subcutaneous adipose tissue. Different concentrations (0, 0.1, 1, 10, 100nM) of liraglutide and insulin were added to proliferation and differentiation medium of hADSCs, respectively. RESULTS: Liraglutide inhibits while insulin promotes the proliferation and differentiation at the concentration of 100nM. Moreover, the levels of GSK-3 increase during differentiation and liraglutide could down-regulate it when compared with insulin. We also find that the activation of phosphorylated GSK-3α and GSK-3ß is involved in the differentiation roles. And classical and non-classical Wnt pathways all play roles in the differentiation, which are characterized with the up/down-regulation of the expression of adipogenesis genes such as PPAR-γ and CEBP-α. CONCLUSION: Liraglutide and insulin have contrary effects on the proliferation and adipogenesis via Wnt pathway in primary cultured ADSCs. Those effects could partly explain the different roles of GLP-1 and insulin on weight gain and insulin resistance.

Antifungal itraconazole ameliorates experimental autoimmune encephalomyelitis through a novel mechanism of action.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune disease characterized by a loss of myelin, limb disabilities and dysregulation of gene expression. Unfortunately, there still is no treatment to cure MS. OBJECTIVES: To explore a novel way to treat MS using currently available antifungal drugs. MATERIAL AND METHODS: We built an experimental autoimmune encephalomyelitis (EAE) model to mimic MS and tested the effect of an antifungal drug - itraconazole - on EAE by comparing it with a phosphate-buffered saline (PBS) control group. We assessed the animal limb deficits with Weaver's scoring and used histology staining (including luxol fast blue (LFB) and hematoxylin & eosin (H&E) methods) to determine the demyelination in the spinal tissues. We also performed western blotting to quantify the expression changes of proteins related to endoplasmic reticulum (ER) stress response and apoptosis. RESULTS: The limb disabilities were greatly diminished and the demyelination in the spinal tissues of the EAE mice was mostly reduced following itraconazole treatment. The hyperactivation of the ER stress response and apoptosis pathway in EAE was also significantly diminished by the itraconazole treatment. In addition, the AMPK pathway was downregulated in EAE, its expression level bi-directionally affected the activity of the ER stress response, and its downregulation removed the beneficial effect of itraconazole. CONCLUSIONS: Our study revealed a new method for treating MS using currently approved antifungal drugs.

A Novel Polyclonal Antiserum against Toxoplasma gondii Sodium Hydrogen Exchanger 1.

The sodium hydrogen exchanger 1 (NHE1), which functions in maintaining the ratio of Na(+) and H(+) ions, is widely distributed in cell plasma membranes. It plays a prominent role in pH balancing, cell proliferation, differentiation, adhesion, and migration. However, its exact subcellular location and biological functions in Toxoplasma gondii are largely unclear. In this study, we cloned the C-terminal sequence of T. gondii NHE1 (TgNHE1) incorporating the C-terminal peptide of NHE1 (C-NHE1) into the pGEX4T-1 expression plasmid. The peptide sequence was predicted to have good antigenicity based on the information obtained from an immune epitope database. After induction of heterologous gene expression with isopropyl-b-D-thiogalactoside, the recombinant C-NHE1 protein successfully expressed in a soluble form was purified by glutathione sepharose beads as an immunogen for production of a rabbit polyclonal antiserum. The specificity of this antiserum was confirmed by western blotting and immunofluorescence. The antiserum could reduce T. gondii invasion into host cells, indicated by the decreased TgNHE1 expression in T. gondii parasites that were pre-incubated with antiserum in the process of cell entry. Furthermore, the antiserum reduced the virulence of T. gondii parasites to host cells in vitro, possibly by blocking the release of Ca(2+). In this regard, this antiserum has potential to be a valuable tool for further studies of TgNHE1.