A novel protein FNDC3B-267aa encoded by circ0003692 inhibits gastric cancer metastasis via promoting proteasomal degradation of c-Myc.

BACKGROUND: Gastric cancer (GC) ranks fifth in global cancer incidence and third in mortality rate among all cancer types. Circular RNAs (circRNAs) have been extensively demonstrated to regulate multiple malignant biological behaviors in GC. Emerging evidence suggests that several circRNAs derived from FNDC3B play pivotal roles in cancer. However, the role of circFNDC3B in GC remains elusive. METHODS: We initially screened circFNDC3B with translation potential via bioinformatics algorithm prediction. Subsequently, Sanger sequencing, qRT-PCR, RNase R, RNA-FISH and nuclear-cytoplasmic fractionation assays were explored to assess the identification and localization of circ0003692, a circRNA derived from FNDC3B. qRT-PCR and ISH were performed to quantify expression of circ0003692 in human GC tissues and adjacent normal tissues. The protein-encoding ability of circ0003692 was investigated through dual-luciferase reporter assay and LC/MS. The biological behavior of circ0003692 in GC was confirmed via in vivo and in vitro experiments. Additionally, Co-IP and rescue experiments were performed to elucidate the interaction between the encoded protein and c-Myc. RESULTS: We found that circ0003692 was significantly downregulated in GC tissues. Circ0003692 had the potential to encode a novel protein FNDC3B-267aa, which was downregulated in GC cells. We verified that FNDC3B-267aa, rather than circ0003692, inhibited GC migration in vitro and in vivo. Mechanistically, FNDC3B-267aa directly interacted with c-Myc and promoted proteasomal degradation of c-Myc, resulting in the downregulation of c-Myc-Snail/Slug axis. CONCLUSIONS: Our study revealed that the novel protein FNDC3B-267aa encoded by circ0003692 suppressed GC metastasis through binding to c-Myc and enhancing proteasome-mediated degradation of c-Myc. The study offers the potential applications of circ0003692 or FNDC3B-267aa as therapeutic targets for GC.

RNA-binding protein IGF2BP2 suppresses metastasis of clear cell renal cell carcinoma by enhancing CKB mRNA stability and expression.

Clear cell renal cell carcinoma (ccRCC) is the most prevalent kidney cancer, with a highly aggressive phenotype and poor prognosis. RNA binding proteins (RBPs) play crucial roles in post-transcriptional gene regulation and have been implicated in tumorigenesis. RBPs have the potential to become a new therapeutic target for ccRCC. In this study, we screened and validated that insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) as an RBP, was down-regulated in ccRCC tissues and cell lines. Functionally, we verified that IGF2BP2 significantly suppressed the migration and invasion ability of ccRCC in vitro and in vivo. Mechanistically, RIP-seq and actinomycin D experiments results showed that IGF2BP2 enhanced the expression of Creatine Kinase B (CKB) by binding to CKB mRNA and enhancing its mRNA stability. Thus, IGF2BP2 inhibited ccRCC metastasis through enhancing the expression of CKB. Taken together, these finding suggests that IGF2BP2 is a novel metastasis suppressor of ccRCC and may serve as a potential therapeutic target.

A novel peptide PDHK1-241aa encoded by circPDHK1 promotes ccRCC progression via interacting with PPP1CA to inhibit AKT dephosphorylation and activate the AKT-mTOR signaling pathway.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most prevalent kidney cancer with high aggressive phenotype and poor prognosis. Accumulating evidence suggests that circRNAs have been identified as pivotal mediators in cancers. However, the role of circRNAs in ccRCC progression remains elusive. METHODS: The differentially expressed circRNAs in 4 paired human ccRCC and adjacent noncancerous tissues ccRCC were screened using circRNA microarrays and the candidate target was selected based on circRNA expression level using weighted gene correlation network analysis (WGCNA) and the gene expression omnibus (GEO) database. CircPDHK1 expression in ccRCC and adjacent noncancerous tissues (n = 148) were evaluated along with clinically relevant information. RT-qPCR, RNase R digestion, and actinomycin D (ActD) stability test were conducted to identify the characteristics of circPDHK1. The subcellular distribution of circPDHK1 was analyzed by subcellular fractionation assay and fluorescence in situ hybridization (FISH). Immunoprecipitation-mass spectrometry (IP-MS) and immunofluorescence (IF) were employed to evaluate the protein-coding ability of circPDHK1. ccRCC cells were transfected with siRNAs, plasmids or lentivirus approach, and cell proliferation, migration and invasion, as well as tumorigenesis and metastasis in nude mice were assessed to clarify the functional roles of circPDHK1 and its encoded peptide PDHK1-241aa. RNA-sequencing, western blot analysis, immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP) assays were further employed to identify the underlying mechanisms regulated by PDHK1-241aa. RESULTS: CircPDHK1 was upregulated in ccRCC tissues and closely related to WHO/ISUP stage, T stage, distant metastasis, VHL mutation and Ki-67 levels. CircPDHK1 had a functional internal ribosome entry site (IRES) and encoded a novel peptide PDHK1-241aa. Functionally, we confirmed that PDHK1-241aa and not the circPDHK1 promoted the proliferation, migration and invasion of ccRCC. Mechanistically, circPDHK1 was activated by HIF-2A at the transcriptional level. PDHK1-241aa was upregulated and interacted with PPP1CA, causing the relocation of PPP1CA to the nucleus. This thereby inhibited AKT dephosphorylation and activated the AKT-mTOR signaling pathway. CONCLUSIONS: Our data indicated that circPDHK1-encoded PDHK1-241aa promotes ccRCC progression by interacting with PPP1CA to inhibit AKT dephosphorylation. This study provides novel insights into the multiplicity of circRNAs and highlights the potential use of circPDHK1 or PDHK1-241aa as a therapeutic target for ccRCC.

RNA Binding Protein PTBP1 Promotes the Metastasis of Gastric Cancer by Stabilizing PGK1 mRNA.

Gastric cancer (GC) is the most common type of malignant tumor within the gastrointestinal tract, and GC metastasis is associated with poor prognosis. Polypyrimidine tract binding protein 1 (PTBP1) is an RNA-binding protein implicated in various types of tumor development and metastasis. However, the role of PTBP1 in GC metastasis remains elusive. In this study, we verified that PTBP1 was upregulated in GC tissues and cell lines, and higher PTBP1 level was associated with poorer prognosis. It was shown that PTBP1 knockdown in vitro inhibited GC cell migration, whereas PTBP1 overexpression promoted the migration of GC cells. In vivo, the knockdown of PTBP1 notably reduced both the size and occurrence of metastatic nodules in a nude mice liver metastasis model. We identified phosphoglycerate kinase 1 (PGK1) as a downstream target of PTBP1 and found that PTBP1 increased the stability of PGK1 by directly binding to its mRNA. Furthermore, the PGK1/SNAIL axis could be required for PTBP1's function in the promotion of GC cell migration. These discoveries suggest that PTBP1 could be a promising therapeutic target for GC.

Construction of a prognostic model based on genes associated with mitochondrial energy metabolic pathway in colon adenocarcinoma and its clinical significance.

Mitochondria are the main sites of oxidative metabolism and energy release of sugars, fats and amino acids in the body. According to studies, malignant tumor occurrence and development have been linked to abnormal mitochondrial energy metabolism (MEM). However, the feasible role of abnormal MEM in colon adenocarcinoma (COAD) is poorly understood. In this work, we obtained COAD patient data from The Cancer Genome Atlas (TCGA) as the training set, and GSE103479 from Gene Expression Omnibus (GEO) as the validation set. Combined with the mitochondrial energy metabolic pathway (MEMP)-related genes in Kyoto Encyclopedia of Genes and Genomes (KEGG) database, a risk prognostic model was constructed by utilizing Cox regression analysis to identify 6 feature genes (CYP4A11, PGM2, PKLR, PPARGC1A, CPT2 and ACAT2) that were significantly associated with MEMP in COAD. By stratifying the samples based on riskscore, two distinct groups, namely the high- and low-risk groups, were identified. The model demonstrated accurate assessment of the prognosis risk in COAD patients and exhibited independent prognostic capability, as evidenced by the survival curve and receiver operating characteristic (ROC) curve analysis. A nomogram was plotted based on clinical information and riskscore. We proved it could predict the survival time of COAD patients effectively combined with the calibration curve of risk prediction. Subsequently, based on the immune evaluation and mutation frequency analysis performed on COAD patients, patients in high-risk group had observably higher immune scores, immune activity and PDCD1 expression level than low-risk group. In general, the prognostic model developed using MEMP-related genes served as a valuable biomarker for forecasting the prognosis of COAD patients, which offered a reference for the prognosis evaluation and clinical cure of COAD patients.

CircNFATC3 promotes the proliferation of gastric cancer through binding to IGF2BP3 and restricting its ubiquitination to enhance CCND1 mRNA stability.

BACKGROUND: Insulin like growth factor II mRNA binding protein 3 (IGF2BP3) is an RNA binding protein with multiple roles in regulation of gene expression at the post-transcriptional level and is implicated in tumorigenesis and progression of numerous cancers including gastric cancer (GC). Circular RNAs (circRNAs) are a diverse endogenous noncoding RNA population that have important regulatory roles in cancer. However, circRNAs that regulate the expression of IGF2BP3 in GC is largely unknown. METHODS: CircRNAs that bound to IGF2BP3 were screened in GC cells using RNA immunoprecipitation and sequencing (RIP-seq). The identification and localization of circular nuclear factor of activated T cells 3 (circNFATC3) were identified using Sanger sequencing, RNase R assays, qRT-PCR, nuclear-cytoplasmic fractionation and RNA-FISH assays. CircNFATC3 expression in human GC tissues and adjacent normal tissues were measured by qRT-PCR and ISH. The biological role of circNFATC3 in GC was confirmed by in vivo and in vitro experiments. Furthermore, RIP, RNA-FISH/IF, IP and rescue experiments were performed to uncover interactions between circNFATC3, IGF2BP3 and cyclin D1 (CCND1). RESULTS: We identified a GC-associated circRNA, circNFATC3, that interacted with IGF2BP3. CircNFATC3 was significantly overexpressed in GC tissues and was positively associated with tumor volume. Functionally, the proliferation of GC cells decreased significantly after circNFATC3 knockdown in vivo and in vitro. Mechanistically, circNFATC3 bound to IGF2BP3 in the cytoplasm, which enhanced the stability of IGF2BP3 by preventing ubiquitin E3 ligase TRIM25-mediated ubiquitination, thereby enhancing the regulatory axis of IGF2BP3-CCND1 and promoting CCND1 mRNA stability. CONCLUSIONS: Our findings demonstrate that circNFATC3 promotes GC proliferation by stabilizing IGF2BP3 protein to enhance CCND1 mRNA stability. Therefore, circNFATC3 is a potential novel target for the treatment of GC.

Desolvation Synergy of Multiple H/Li-Bonds on an Iron-Dextran-Based Catalyst Stimulates Lithium-Sulfur Cascade Catalysis.

Traditional lithium-sulfur battery catalysts are still facing substantial challenges in solving sulfur redox reactions, which involve multistep electron transfer and multiphase transformations. Here, inspired by the combination of iron dextran (INFeD) and ascorbic acid (VC) as a blood tonic for the treatment of anemia, a highly efficient VC@INFeD catalyst is developed in the sulfur cathode, accomplishing the desolvation and enrichment of high-concentration solvated lithium polysulfides at the cathode/electrolyte interface with the assistance of multiple H/Li-bonds and resolving subsequent sulfur transformations through gradient catalysis sites where the INFeD promotes long-chain lithium polysulfide conversions and VC accelerates short-chain lithium polysulfide conversions. Comprehensive characterizations reveal that the VC@INFeD can substantially reduce the energy barrier of each sulfur redox step, inhibit shuttle effects, and endow the lithium-sulfur battery with high sulfur utilization and superior cycling stability even under a high sulfur loading (5.2 mg cm-2 ) and lean electrolyte (electrolyte/sulfur ratio, ≈7 µL mg-1 ) condition.

CircARID1A binds to IGF2BP3 in gastric cancer and promotes cancer proliferation by forming a circARID1A-IGF2BP3-SLC7A5 RNA-protein ternary complex.

BACKGROUND: Gastric cancer (GC) is one of the most common malignant tumors in China. Circular RNAs (circRNAs) are novel non-coding RNAs with important regulatory roles in cancer progression. IGF2BP3 has been found to play oncogenic roles in various cancers including GC, while the exact mechanism of IGF2BP3 is largely unknown. METHODS: The expression of IGF2BP3 in GC was evaluated by Western Blot and bioinformatics analysis. CircRNA expression profiles were screened via IGF2BP3 RIP-seq in GC. Sanger sequencing, RNase R digestion, nucleo-plasmic separation and RNA-FISH assays were used to detect the existence and expression of circARID1A. RNA ISH assay was employed to test the expression of circARID1A in paraffin-embedded GC tissues. Moreover, the function of circARID1A on cellular proliferation was assessed by CCK-8, plate colony formation, EdU assays and GC xenograft mouse model in vivo. Furthermore, the location or binding of circARID1A, IGF2BP3 protein and SLC7A5 in GC was evaluated by RNA-FISH/IF or RNA pull-down assays. RESULTS: We identified a novel circRNA, circARID1A, that can bind to IGF2BP3 protein. CircARID1A was significantly upregulated in GC tissues compared with noncancerous tissues and positively correlated with tumor length, tumor volume, and TNM stage. CircARID1A knockdown inhibited the proliferation of GC cells in vitro and in vivo and circARID1A played an important role in the oncogenic function of IGF2BP3. Mechanistically, circARID1A served as a scaffold to facilitate the interaction between IGF2BP3 and SLC7A5 mRNA, finally increasing SLC7A5 mRNA stability. Additionally, circARID1A was able to directly bind SLC7A5 mRNA through complementary base-pairing and then formed the circARID1A-IGF2BP3-SLC7A5 RNA-protein ternary complex and promoted the proliferation of GC via regulating AKT/mTOR pathway. CONCLUSIONS: Altogether, our data suggest that circARID1A is involved in the function of IGF2BP3 and GC proliferation, and the circARID1A-IGF2BP3-SLC7A5 axis has the potential to serve as a novel therapeutic target for GC.

Circular RNA circ-TNPO3 inhibits clear cell renal cell carcinoma metastasis by binding to IGF2BP2 and destabilizing SERPINH1 mRNA.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is a common malignant tumour of the urinary tract. The major causes of poor prognosis are the lack of early diagnosis and metastasis. Accumulating research reveals that circular RNAs (circRNAs) can play key roles in the development and the progression of cancer. However, the role of circRNAs in ccRCC is still uncertain. METHODS: The circRNAs microarray (n = 4) was performed to investigate the circRNAs with differential expression in ccRCC tissues. The candidate circRNA was selected based on the cut-off criteria, such as circRNA expression abundance, circRNA size and the design of divergent primers. The circ-transportin-3 (TNPO3) levels in ccRCC tissues were tested by quantitative real-time (qRT)-PCR (n = 110). The characteristics and subcellular localization of circ-TNPO3 were identified via RNase R assay, qRT-PCR and fluorescence in situ hybridization (FISH). Then, we explored the biological roles of circ-TNPO3 in ccRCC via the function experiments in vitro and in vivo. RNA pull-down, RNA immunoprecipitation, bioinformatic analysis, RNA-FISH assays and rescue assays were applied to validate the interactions between circ-TNPO3, insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) and serpin family H member 1 (SERPINH1) to uncover the underlying molecular mechanisms of circ-TNPO3. RESULTS: We detected the obvious downregulation of circ-TNPO3 in ccRCC compared to matched adjacent normal tissues (n = 110). The lower circ-TNPO3 expression was found in ccRCC patients with distant metastasis, higher World Health Organization/International Society of Urologic Pathologists (WHO/ISUP) grade and more advanced tumour T stage. In vitro and in vivo, circ-TNPO3 significantly suppressed the proliferation and migration of ccRCC cells. Mechanistically, we elucidated that circ-TNPO3 directly bound to IGF2BP2 protein and then destabilized SERPINH1 mRNA. Moreover, IGF2BP2/SERPINH1 axis was responsible for circ-TNPO3's function of inhibiting ccRCC metastasis. Epithelial splicing regulatory protein 1 (ESRP1) was probably involved in the biogenesis of circ-TNPO3. CONCLUSIONS: Circ-TNPO3 can suppress ccRCC progression and metastasis via directly binding to IGF2BP2 protein and destabilizing SERPINH1 mRNA. Circ-TNPO3 may act as a potential target for ccRCC treatment.

Multistage pH-responsive codelivery liposomal platform for synergistic cancer therapy.

BACKGROUND: Small interfering RNA (siRNA) is utilized as a potent agent for cancer therapy through regulating the expression of genes associated with tumors. While the widely application of siRNAs in cancer treatment is severely limited by their insufficient biological stability and its poor ability to penetrate cell membranes. Targeted delivery systems hold great promise to selectively deliver loaded drug to tumor site and reduce toxic side effect. However, the elevated tumor interstitial fluid pressure and efficient cytoplasmic release are still two significant obstacles to siRNA delivery. Co-delivery of chemotherapeutic drugs and siRNA represents a potential strategy which may achieve synergistic anticancer effect. Herein, we designed and synthesized a dual pH-responsive peptide (DPRP), which includes three units, a cell-penetrating domain (polyarginine), a polyanionic shielding domain (ehG)n, and an imine linkage between them. Based on the DPRP surface modification, we developed a pH-responsive liposomal system for co-delivering polo-like kinase-1 (PLK-1) specific siRNA and anticancer agent docetaxel (DTX), D-Lsi/DTX, to synergistically exhibit anti-tumor effect. RESULTS: In contrast to the results at the physiological pH (7.4), D-Lsi/DTX lead to the enhanced penetration into tumor spheroid, the facilitated cellular uptake, the promoted escape from endosomes/lysosomes, the improved distribution into cytoplasm, and the increased cellular apoptosis under mildly acidic condition (pH 6.5). Moreover, both in vitro and in vivo study indicated that D-Lsi/DTX had a therapeutic advantage over other control liposomes. We provided clear evidence that liposomal system co-delivering siPLK-1 and DTX could significantly downregulate expression of PLK-1 and inhibit tumor growth without detectable toxic side effect, compared with siPLK-1-loaded liposomes, DTX-loaded liposomes, and the combinatorial administration. CONCLUSION: These results demonstrate great potential of the combined chemo/gene therapy based on the multistage pH-responsive codelivery liposomal platform for synergistic tumor treatment.

Modified Renshen Yangrong decoction enhances angiogenesis in ischemic stroke through promotion of MicroRNA-210 expression by regulating the HIF/VEGF/Notch signaling pathway.

OBJECTIVE: This study aims to investigate the efficacy of modified Ginseng Yangrong decoction (GSYRD) promoting angiogenesis after ischemic stroke. METHODS: In an in vivo study, rats that survived surgery were allocated into four groups: the control group and model group were treated with normal saline, the GSYRD group was treated with 18.9 mg/kg of GSYRD daily, and the positive control group was treated with Tongxinluo (TXL) (1 g/kg/d). At the end of the seven-day treatment, the area of cerebral infarction, the expression changes of miRNA-210 and ephrin A3 were determined. In an in vitro study, HUVECs were divided into a normal control serum group (NC group), normal control serum OGD group (Oxygen Glucose Deprivation group) (OGD group), OGD + drug-containing serum group (OGD+GSYRD group), and OGD + drug-containing serum + ES group (Endostatin group) (OGD+GSYRD+ES group). The cells in all groups except the NC group were cultured in a sugar-free DMEM medium under hypoxia for 48 h. Cell proliferation, angiogenic structure formation ability, the expression changes of miRNA-210, ephrin A3, and the HIF/VEGF/Notch signaling pathway-related molecules were determined. RESULTS: In vivo, GSYRD significantly reduced infarct size (p < .01), the expression of miRNA-210 and ephrin A3 were decreased in the GSYRD group (p < .05). In vitro, the cell proliferation and tube formation ability were significantly increased in the GSYRD group (p < .05), and the expression of miRNA-210 and ephrin A3 was decreased (p < .05). In addition, in the GSYRD group, the expression of the HIF/VEGF/Notch signaling pathway-related molecules was significantly increased (p < .01 or p < .05). CONCLUSION: GSYRD promotes cerebral protection following angiogenesis and ischemic brain injury. The specific mechanism was activating the HIF/VEGF/Notch signaling pathway via miRNA-210.

Extraordinary Superhydrophobic Polycaprolactone-Based Composite Membrane with an Alternated Micro-Nano Hierarchical Structure as an Eco-friendly Oil/Water Separator.

Extraordinary superhydrophobic polycaprolactone (PCL) composite membranes with an alternated hierarchical micro-nano structure were designed by addition of SiO2 aerogel. The highest water contact angle (WCA) of 166.8 ± 1.5° was obtained when SiO2 aerogel content was 0.5% (PCL/SiO2-a0.5) in the PCL composite membrane, which was higher than other reported polymer-based membranes. SiO2 aerogel lowered PCL composite membrane's surface energy. The triple curvature structure composed of microspheres, nanospheres, and nanofibers produced on PCL/SiO2-a0.5 membranes endowed the excellent roughness of the surface. Also, the inner structure of the PCL/SiO2-a0.5 composite membrane composed of micro-nano spheres, nanofibers, and microfibers increased the porosity of the separation membrane, which would provide more adsorption space. The PCL/SiO2-a0.5 composite membrane as a separator for surfactant-stabilized emulsions of water-in-oil showed ultrahigh separation flux and efficiency. Meanwhile, the PCL/SiO2-a0.5 composite membrane had an outstanding chemical resistance, self-cleaning ability, and good reusability. The composite membranes reported in this work as eco-friendly separation materials possessed all these characters in oil/water separation. This research proposed a very simple method to design eco-friendly high-efficiency separators through the construction of the alternated micro-nano hierarchical structure.

Astragaloside IV improves neurobehavior and promotes hippocampal neurogenesis in MCAO rats though BDNF-TrkB signaling pathway.

Astragaloside IV (AST) as the main active ingredient of Astragalus membranaceus. Clinical and laboratory-based studies have demonstrated the effects of AST on cerebral protection and angiogenesis after ischemia stroke. In addition, several reports investigated the effect of AST on proliferation of neural stem cells. The current study was aimed to evaluate the influence of AST on neurogenesis in hippocampal dentate gyrus (DG) of MCAO rats and to explore the possible mechanisms. In this study, the neurobehavioral tests (Ludmila Belayev 12-point scoring, Screen test, fore limb placing test) had been employed to investigate the effect of AST treatment against functional deficit of MCAO rats. The immunofluorescence staining, western-blot and qRT-PCR was performed to evaluate the effects of AST on proliferation, differentiation and maturity of neural stemr cells in hippocampus. Moreover, we investigated the possible mechanism of the AST treatment in promoting neurogenesis after ischemic stroke. The findings indicated that AST treatment ameliorated the neurobehavior of MCAO rats. The results indicated that AST treatment possessed the potential to improve proprioceptive sense and motor function of MCAO rats. AST treatment sustained neuronal viability and stimulates sensorimotor integration functional recovery in MCAO rats. The results suggested that AST improved neurobehavior deficit after ischemic stroke. Furthermore, AST promoted neurogenesis through upregulating the expressing of BNDF/TrkB signaling pathway. Therefore AST might be a promising therapeutic agent for ischemic stroke.

Astragaloside IV regulates the HIF/VEGF/Notch signaling pathway through miRNA-210 to promote angiogenesis after ischemic stroke.

BACKGROUND: Astragaloside IV (AS-IV) is one of the main active ingredients of Astragalusmembranaceus. Studies have shown that AS-IV stimulates angiogenesis, including cell proliferation, migration, and neovascularization. However, the relevant mechanism remains unclear. OBJECTIVE: This study aims to investigate whether AS-IV activates the HIF/VEGF/Notch signaling pathway through miRNA-210 to promote angiogenesisafter ischemic stroke. METHODS: The present study established a rat model of middle cerebral artery occlusion (MCAO) and cultured human umbilical vein endothelial cells (HUVECs) under hypoxic conditions in vitro to investigate the role of AS-IV in promoting angiogenesis and reveal its underlying mechanism. Through in vivo studies, the area of cerebral infarction was determined by 2,3,5-triPhenyltetrazolium chloride (TTC) staining. Immunofluorescence staining and RT-qPCR were used to detect the expression changes of miRNA-210 and ephrinA3 in the ischemic cortex after ischemia. Through in vitro studies, cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Subsequently, angiogenesis experiments were performed to observe the angiogenic ability. RESULTS: Results revealed that AS-IV significantly reduced infarct size, promoted cell proliferation and ductal formation, and inhibited the expression of the target gene ephrinA3 by increasing the expression of miRNA-210 and inducing activation of the HIF-VEGF-Notch signaling pathway. CONCLUSIONS: AS-IV promotes cerebral protection following angiogenesis and ischemic brain injury. The specific mechanism was activating the HIF/VEGF/Notch signaling pathway via miRNA-210.

Delineating an extracellular redox-sensitive module in T-type Ca2+ channels.

T-type (Cav3) Ca2+ channels are important regulators of excitability and rhythmic activity of excitable cells. Among other voltage-gated Ca2+ channels, Cav3 channels are uniquely sensitive to oxidation and zinc. Using recombinant protein expression in HEK293 cells, patch clamp electrophysiology, site-directed mutagenesis, and homology modeling, we report here that modulation of Cav3.2 by redox agents and zinc is mediated by a unique extracellular module containing a high-affinity metal-binding site formed by the extracellular IS1-IS2 and IS3-IS4 loops of domain I and a cluster of extracellular cysteines in the IS1-IS2 loop. Patch clamp recording of recombinant Cav3.2 currents revealed that two cysteine-modifying agents, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) and N-ethylmaleimide, as well as a reactive oxygen species-producing neuropeptide, substance P (SP), inhibit Cav3.2 current to similar degrees and that this inhibition is reversed by a reducing agent and a zinc chelator. Pre-application of MTSES prevented further SP-mediated current inhibition. Substitution of the zinc-binding residue His191 in Cav3.2 reduced the channel's sensitivity to MTSES, and introduction of the corresponding histidine into Cav3.1 sensitized it to MTSES. Removal of extracellular cysteines from the IS1-IS2 loop of Cav3.2 reduced its sensitivity to MTSES and SP. We hypothesize that oxidative modification of IS1-IS2 loop cysteines induces allosteric changes in the zinc-binding site of Cav3.2 so that it becomes sensitive to ambient zinc.

Structural and functional analysis of the Hsp70/Hsp40 chaperone system.

As one of the most abundant and highly conserved molecular chaperones, the 70-kDa heat shock proteins (Hsp70s) play a key role in maintaining cellular protein homeostasis (proteostasis), one of the most fundamental tasks for every living organism. In this role, Hsp70s are inextricably linked to many human diseases, most notably cancers and neurodegenerative diseases, and are increasingly recognized as important drug targets for developing novel therapeutics for these diseases. Hsp40s are a class of essential and universal partners for Hsp70s in almost all aspects of proteostasis. Thus, Hsp70s and Hsp40s together constitute one of the most important chaperone systems across all kingdoms of life. In recent years, we have witnessed significant progress in understanding the molecular mechanism of this chaperone system through structural and functional analysis. This review will focus on this recent progress, mainly from a structural perspective.

Intracellular zinc activates KCNQ channels by reducing their dependence on phosphatidylinositol 4,5-bisphosphate.

M-type (Kv7, KCNQ) potassium channels are proteins that control the excitability of neurons and muscle cells. Many physiological and pathological mechanisms of excitation operate via the suppression of M channel activity or expression. Conversely, pharmacological augmentation of M channel activity is a recognized strategy for the treatment of hyperexcitability disorders such as pain and epilepsy. However, physiological mechanisms resulting in M channel potentiation are rare. Here we report that intracellular free zinc directly and reversibly augments the activity of recombinant and native M channels. This effect is mechanistically distinct from the known redox-dependent KCNQ channel potentiation. Interestingly, the effect of zinc cannot be attributed to a single histidine- or cysteine-containing zinc-binding site within KCNQ channels. Instead, zinc dramatically reduces KCNQ channel dependence on its obligatory physiological activator, phosphatidylinositol 4,5-bisphosphate (PIP2). We hypothesize that zinc facilitates interactions of the lipid-facing interface of a KCNQ protein with the inner leaflet of the plasma membrane in a way similar to that promoted by PIP2 Because zinc is increasingly recognized as a ubiquitous intracellular second messenger, this discovery might represent a hitherto unknown native pathway of M channel modulation and provide a fresh strategy for the design of M channel activators for therapeutic purposes.

Inflammatory mediator bradykinin increases population of sensory neurons expressing functional T-type Ca(2+) channels.

T-type Ca(2+) channels are important regulators of peripheral sensory neuron excitability. Accordingly, T-type Ca(2+) currents are often increased in various pathological pain conditions, such as inflammation or nerve injury. Here we investigated effects of inflammation on functional expression of T-type Ca(2+) channels in small-diameter cultured dorsal root ganglion (DRG) neurons. We found that overnight treatment of DRG cultures with a cocktail of inflammatory mediators bradykinin (BK), adenosine triphosphate (ATP), norepinephrine (NE) and prostaglandin E2 (PGE2) strongly increased the population size of the small-diameter neurons displaying low-voltage activated (LVA, T-type) Ca(2+) currents while having no effect on the peak LVA current amplitude. When applied individually, BK and ATP also increased the population size of LVA-positive neurons while NE and PGE2 had no effect. The PLC inhibitor U-73122 and B2 receptor antagonist, Hoe-140, both abolished the increase of the population of LVA-positive DRG neurons. Inflammatory treatment did not affect CaV3.2 mRNA or protein levels in DRG cultures. Furthermore, an ubiquitination inhibitor, MG132, did not increase the population of LVA-positive neurons. Our data suggest that inflammatory mediators BK and ATP increase the abundance of LVA-positive DRG neurons in total neuronal population by stimulating the recruitment of a 'reserve pool' of CaV3.2 channels, particularly in neurons that do not display measurable LVA currents under control conditions.

[Exposure of human hepatoma cells to nitrite and ammonia promotes invasive activity through activation of ROS/ODC pathway].

Recent studies have demonstrated that nitrite and ammonia levels are higher in the tumor environment, but their effects on cancer cells remains unclear. The present study was designed to determine the effects of nitrite and ammonia on tumor invasion and the role of reactive oxygen (ROS)/ornithine decarboxylase (ODC) pathway. SMMC-7721 cells were treated with sodium nitrite, ammonium chloride, sodium nitrite and ammonium chloride mixture for 24 h, the cell viability was analyzed using the MTT assay, cell invasion was analyzed with the transwell assay, the intracellular ROS levels were detected with a reactive oxygen species (ROS) test kits, the expression of intracellular ODC was examined with immunofluorescence and Western blot, the expression of matrix metallopeptidase-2 (MMP-2) and MMP-9 were analyzed by Western blot. Compared with the control group, SMMC-7721 cells exhibited an increase in cell viability, invasion ability, ROS levels and ODC protein after exposure to 150 µmol·L(-1) sodium nitrite and ammonium chloride mixture for 24 h. The invasive activity was reduced by ROS scavenger N-acetycysteine (NAC) in SMMC-7721 cells. The specific ODC inhibitor difluoromethylornithine (DFMO) increased ROS levels and weakened the ability of sodium nitrite and ammonium chloride mixture in the regulation of invasion of SMMC-7721 cells. These data demonstrated that sodium nitrite and ammonium chloride mixture promote invasion of SMMC-7721 cells by enhancing ROS/ODC pathway.

Characterization of a bio-oil from pyrolysis of rice husk by detailed compositional analysis and structural investigation of lignin.

Detailed compositional analysis of a bio-oil (BO) from pyrolysis of rice husk was carried out. The BO was extracted sequentially with n-hexane, CCl(4), CS(2), benzene and CH(2)Cl(2). In total, 167 organic species were identified with GC/MS in the extracts and classified into alkanes, alcohols, hydroxybenzenes, alkoxybenzenes, dioxolanes, aldehydes, ketones, carboxylic acids, esters, nitrogen-containing organic compounds and other species. The benzene ring-containing species (BRCCs) were attributed to the degradation of lignin while most of the rests were derived from the degradation of cellulose and hemicellulose. Along with guaiacyl and p-hydroxyphenyl units as the main components, a new type of linkage was suggested, i.e., C(ar)-CH(2)-C(ar) in 4,4'-methylenebis(2,6-dimethoxyphenol). Based on the species identified, a possible macromolecular structure of the lignin and the mechanism for its pyrolysis are proposed. The BO was also extracted with petroleum ether in ca. 17.8% of the extract yield and about 82.1% of the extracted components are BRCCs.