Chemoproteomic capture of RNA binding activity in living cells.

Proteomic methods for RNA interactome capture (RIC) rely principally on crosslinking native or labeled cellular RNA to enrich and investigate RNA-binding protein (RBP) composition and function in cells. The ability to measure RBP activity at individual binding sites by RIC, however, has been more challenging due to the heterogenous nature of peptide adducts derived from the RNA-protein crosslinked site. Here, we present an orthogonal strategy that utilizes clickable electrophilic purines to directly quantify protein-RNA interactions on proteins through photoaffinity competition with 4-thiouridine (4SU)-labeled RNA in cells. Our photo-activatable-competition and chemoproteomic enrichment (PACCE) method facilitated detection of >5500 cysteine sites across ~3000 proteins displaying RNA-sensitive alterations in probe binding. Importantly, PACCE enabled functional profiling of canonical RNA-binding domains as well as discovery of moonlighting RNA binding activity in the human proteome. Collectively, we present a chemoproteomic platform for global quantification of protein-RNA binding activity in living cells.

Keratin 19 binds and regulates cytoplasmic HNRNPK mRNA targets in triple-negative breast cancer.

BACKGROUND: Heterogeneous nuclear ribonucleoprotein K (HNRNPK) regulates pre-mRNA processing and long non-coding RNA localization in the nucleus. It was previously shown that shuttling of HNRNPK to the cytoplasm promotes cell proliferation and cancer metastasis. However, the mechanism of HNRNPK cytoplasmic localization, its cytoplasmic RNA ligands, and impact on post-transcriptional gene regulation remain uncharacterized. RESULTS: Here we show that the intermediate filament protein Keratin 19 (K19) directly interacts with HNRNPK and sequesters it in the cytoplasm. Correspondingly, in K19 knockout breast cancer cells, HNRNPK does not localize in the cytoplasm, resulting in reduced cell proliferation. We comprehensively mapped HNRNPK binding sites on mRNAs and showed that, in the cytoplasm, K19-mediated HNRNPK-retention increases the abundance of target mRNAs bound to the 3' untranslated region (3'UTR) at the expected cytidine-rich (C-rich) sequence elements. Furthermore, these mRNAs protected by HNRNPK in the cytoplasm are typically involved in cancer progression and include the p53 signaling pathway that is dysregulated upon HNRNPK knockdown (HNRNPK KD) or K19 knockout (KRT19 KO). CONCLUSIONS: This study identifies how a cytoskeletal protein can directly regulate gene expression by controlling the subcellular localization of RNA-binding proteins to support pathways involved in cancer progression.

Generation of a TBX20-knockout human embryonic stem line by CRISPR/Cas9 system.

The TBX20 gene plays a crucial role in embryonic development and has been involved in various diseases, such as heart defects, intellectual disability, and cancer. Herein, we have established a TBX20-knockout human embryonic stem cell line (WAe009-A-84) that maintains stem cell-like features, pluripotency, a normal karyotype, and the ability to differentiate into all three germ layers in vivo. This cell line will be a valuable resource for exploring TBX20's role in human development and could have significant implications for regenerative medicine and disease modeling.

p38 MAPK and MKP-1 control the glycolytic program via the bifunctional glycolysis regulator PFKFB3 during sepsis.

Hyperlactatemia often occurs in critically ill patients during severe sepsis/septic shock and is a powerful predictor of mortality. Lactate is the end product of glycolysis. While hypoxia due to inadequate oxygen delivery may result in anaerobic glycolysis, sepsis also enhances glycolysis under hyperdynamic circulation with adequate oxygen delivery. However, the molecular mechanisms involved are not fully understood. Mitogen-activated protein kinase (MAPK) families regulate many aspects of the immune response during microbial infections. MAPK phosphatase (MKP)-1 serves as a feedback control mechanism for p38 and JNK MAPK activities via dephosphorylation. Here, we found that mice deficient in Mkp-1 exhibited substantially enhanced expression and phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB) 3, a key enzyme that regulates glycolysis following systemic Escherichia coli infection. Enhanced PFKFB3 expression was observed in a variety of tissues and cell types, including hepatocytes, macrophages, and epithelial cells. In bone marrow-derived macrophages, Pfkfb3 was robustly induced by both E. coli and lipopolysaccharide, and Mkp-1 deficiency enhanced PFKFB3 expression with no effect on Pfkfb3 mRNA stability. PFKFB3 induction was correlated with lactate production in both WT and Mkp-1-/- bone marrow-derived macrophage following lipopolysaccharide stimulation. Furthermore, we determined that a PFKFB3 inhibitor markedly attenuated lactate production, highlighting the critical role of PFKFB3 in the glycolysis program. Finally, pharmacological inhibition of p38 MAPK, but not JNK, substantially attenuated PFKFB3 expression and lactate production. Taken together, our studies suggest a critical role of p38 MAPK and MKP-1 in the regulation of glycolysis during sepsis.

Upregulation of miR-29b-3p alleviates coronary microembolization-induced myocardial injury via regulating BMF and GSK-3ß.

Coronary microembolization (CME) is an intractable complication results from acute coronary syndrome. CME-induced myocardial apoptosis was associated with progressive cardiac contractile dysfunction. miR-29b-3p has been reported implicated in variety cardiovascular diseases, but its function in CME-induced myocardial injury is yet unknown. Herein, a rat model of CME was established by injecting microspheres into the left ventricle and found that the expression level of miR-29b-3p was markedly decreased in the CME rat heart tissues. By using echocardiography, CD31 immunohistochemistry staining, hematoxylin basic fuchsin picric acid (HBFP) staining, TUNEL staining, and western blotting analysis after CME, it was found that upregulating miR-29b-3p improved cardiac dysfunction, promoted angiogenesis, decreased myocardial microinfarct area, and inhibited myocardial apoptosis. Additionally, miR-29b-3p inhibition can reverse the protective benefits of miR-29b-3p overexpression. Mechanistically, the target genes of miR-29b-3p were identified as glycogen synthase kinase 3 (GSK-3ß) and Bcl-2 modifying factor (BMF) by bioinformatics analysis and luciferase reporter experiment. Overall, our findings imply that induction of miR-29b-3p, which negatively regulates GSK-3ß and BMF expression, attenuates CME-induced myocardial injury, suggesting a novel potential therapeutic target for cardioprotective after CME.

Inflammation and Oxidative Stress Role of S100A12 as a Potential Diagnostic and Therapeutic Biomarker in Acute Myocardial Infarction.

Acute myocardial infarction (AMI) is one of the most serious cardiovascular diseases with high morbidity and mortality. Numerous studies have indicated that S100A12 may has an essential role in the occurrence and development of AMI, and in-depth studies are currently lacking. The purpose of this study is to investigate the effect of S100A12 on inflammation and oxidative stress and to determine its clinical applicability in AMI. Here, AMI datasets used to explore the expression pattern of S100A12 in AMI were derived from the Gene Expression Omnibus (GEO) database. The pooled standard average deviation (SMD) was calculated to further determine S100A12 expression. The overlapping differentially expressed genes (DEGs) contained in all included datasets were recognized by the GEO2R tool. Then, functional enrichment analyses, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, were carried out to determine the molecular function of overlapping DEGs. Gene set enrichment analysis (GSEA) was conducted to determine unrevealed mechanisms of S100A12. Summary receiver operating characteristic (SROC) curve analysis and receiver operating characteristic (ROC) curve analysis were carried out to identify the diagnostic capabilities of S100A12. Moreover, we screened miRNAs targeting S100A12 using three online databases (miRWalk, TargetScan, and miRDB). In addition, by comprehensively using enzyme-linked immunosorbent assay (ELISA), real-time quantitative PCR (RT-qPCR), Western blotting (WB) methods, etc., we used the AC16 cells to validate the expression and underlying mechanism of S100A12. In our study, five datasets related to AMI, GSE24519, GSE60993, GSE66360, GSE97320, and GSE48060 were included; 412 overlapping DEGs were identified. Protein-protein interaction (PPI) network and functional analyses showed that S100A12 was a pivotal gene related to inflammation and oxidative stress. Then, S100A12 overexpression was identified based on the included datasets. The pooled standard average deviation (SMD) also showed that S100A12 was upregulated in AMI (SMD = 1.36, 95% CI: 0.70-2.03, p = 0.024). The SROC curve analysis result suggested that S100A12 had remarkable diagnostic ability in AMI (AUC = 0.90, 95% CI: 0.87-0.92). And nine miRNAs targeting S100A12 were also identified. Additionally, the overexpression of S100A12 was further confirmed that it maybe promote inflammation and oxidative stress in AMI through comprehensive in vitro experiments. In summary, our study suggests that overexpressed S100A12 may be a latent diagnostic biomarker and therapeutic target of AMI that induces excessive inflammation and oxidative stress. Nine miRNAs targeting S100A12 may play a crucial role in AMI, but further studies are still needed. Our work provides a positive inspiration for the in-depth study of S100A12 in AMI.

Mitogen-activated protein kinase phosphatase-1 controls PD-L1 expression by regulating type I interferon during systemic Escherichia coli infection.

Mitogen-activated protein kinase phosphatase 1 (Mkp-1) KO mice produce elevated cytokines and exhibit increased mortality and bacterial burden following systemic Escherichia coli infection. To understand how Mkp-1 affects immune defense, we analyzed the RNA-Seq datasets previously generated from control and E. coli-infected Mkp-1+/+ and Mkp-1-/- mice. We found that E. coli infection markedly induced programmed death-ligand 1 (PD-L1) expression and that Mkp-1 deficiency further amplified PD-L1 expression. Administration of a PD-L1-neutralizing monoclonal antibody (mAb) to Mkp-1-/- mice increased the mortality of the animals following E. coli infection, although bacterial burden was decreased. In addition, the PD-L1-neutralizing mAb increased serum interferon (IFN)-γ and tumor necrosis factor alpha, as well as lung- and liver-inducible nitric oxide synthase levels, suggesting an enhanced inflammatory response. Interestingly, neutralization of IFN-α/ß receptor 1 blocked PD-L1 induction in Mkp-1-/- mice following E. coli infection. PD-L1 was potently induced in macrophages by E. coli and lipopolysaccharide in vitro, and Mkp-1 deficiency exacerbated PD-L1 induction with little effect on the half-life of PD-L1 mRNA. In contrast, inhibitors of Janus kinase 1/2 and tyrosine kinase 2, as well as the IFN-α/ß receptor 1-neutralizing mAb, markedly attenuated PD-L1 induction. These results suggest that the beneficial effect of type I IFNs in E. coli-infected Mkp-1-/- mice is, at least in part, mediated by Janus kinase/signal transducer and activator of transcription-driven PD-L1 induction. Our studies also support the notion that enhanced PD-L1 expression contributes to the bactericidal defect of Mkp-1-/- mice.

The nuclear transcription factor, TAF7, is a cytoplasmic regulator of protein synthesis.

The TFIID component, TAF7, has been extensively characterized as essential for transcription and is critical for cell proliferation and differentiation. Here, we report that TAF7 is a previously unknown RNA chaperone that contributes to the regulation of protein synthesis. Mechanistically, TAF7 binds RNAs in the nucleus and delivers them to cytoplasmic polysomes. A broad spectrum of target RNA species, including the HIV-1 transactivation response element, binds TAF7 through consensus CUG motifs within the 3' untranslated region. Export to the cytoplasm depends on a TAF7 nuclear export signal and occurs by an exportin 1­dependent pathway. Notably, disrupting either TAF7's RNA binding or its export from the nucleus results in retention of target messenger RNAs in the nucleus and reduced levels of the protein products of TAF7-target RNAs. Thus, TAF7, an essential transcription factor, plays a key role in the regulation of RNA translation, thereby potentially connecting these processes.

MKP-1 modulates ubiquitination/phosphorylation of TLR signaling.

Ubiquitination and phosphorylation are reversible posttranslational protein modifications regulating physiological and pathological processes. MAPK phosphatase (MKP)-1 regulates innate and adaptive immunity. The multifaceted roles of MKP-1 were attributed to dephosphorylation of p38 and JNK MAPKs. We show that the lack of MKP-1 modulates the landscape of ubiquitin ligases and deubiquitinase enzymes (DUBs). MKP-1-/- showed an aberrant regulation of several DUBs and increased expression of proteins and genes involved in IL-1/TLR signaling upstream of MAPK, including IL-1R1, IRAK1, TRAF6, phosphorylated TAK1, and an increased K63 polyubiquitination on TRAF6. Increased K63 polyubiquitination on TRAF6 was associated with an enhanced phosphorylated form of A20. Among abundant DUBs, ubiquitin-specific protease-13 (USP13), which cleaves polyubiquitin-chains on client proteins, was substantially enhanced in murine MKP-1-deficient BMDMs. An inhibitor of USP13 decreased the K63 polyubiquitination on TRAF6, TAK1 phosphorylation, IL-1ß, and TNF-α induction in response to LPS in BMDMs. Our data show for the first time that MKP-1 modulates the ligase activity of TRAF6 through modulation of specific DUBs.

The miR-26 family regulates neural differentiation-associated microRNAs and mRNAs by directly targeting REST.

Repressor element 1-silencing transcription factor (REST) plays a crucial role in the differentiation of neural progenitor cells (NPCs). C-terminal domain small phosphatases (CTDSPs) are REST effector proteins that reduce RNA polymerase II activity on genes required for neurogenesis. miR-26b regulates neurogenesis in zebrafish by targeting ctdsp2 mRNA, but the molecular events triggered by this microRNA (miR) remain unknown. Here, we show in a murine embryonic stem cell differentiation paradigm that inactivation of miR-26 family members disrupts the formation of neurons and astroglia and arrests neurogenesis at the neural progenitor level. Furthermore, we show that miR-26 directly targets Rest, thereby inducing the expression of a large set of REST complex-repressed neuronal genes, including miRs required for induction of the neuronal gene expression program. Our data identify the miR-26 family as the trigger of a self-amplifying system required for neural differentiation that acts upstream of REST-controlled miRs.

Knockout of MAPK Phosphatase-1 Exaggerates Type I IFN Response during Systemic Escherichia coli Infection.

We have previously shown that Mkp-1-deficient mice produce elevated TNF-α, IL-6, and IL-10 following systemic Escherichia coli infection, and they exhibited increased mortality, elevated bacterial burden, and profound metabolic alterations. To understand the function of Mkp-1 during bacterial infection, we performed RNA-sequencing analysis to compare the global gene expression between E. coli-infected wild-type and Mkp-1 -/- mice. A large number of IFN-stimulated genes were more robustly expressed in E. coli-infected Mkp-1 -/- mice than in wild-type mice. Multiplex analysis of the serum cytokine levels revealed profound increases in IFN-ß, IFN-γ, TNF-α, IL-1α and ß, IL-6, IL-10, IL-17A, IL-27, and GMSF levels in E. coli-infected Mkp-1 -/- mice relative to wild-type mice. Administration of a neutralizing Ab against the receptor for type I IFN to Mkp-1 -/- mice prior to E. coli infection augmented mortality and disease severity. Mkp-1 -/- bone marrow-derived macrophages (BMDM) produced higher levels of IFN-ß mRNA and protein than did wild-type BMDM upon treatment with LPS, E. coli, polyinosinic:polycytidylic acid, and herring sperm DNA. Augmented IFN-ß induction in Mkp-1 -/- BMDM was blocked by a p38 inhibitor but not by an JNK inhibitor. Enhanced Mkp-1 expression abolished IFN-ß induction by both LPS and E. coli but had little effect on the IFN-ß promoter activity in LPS-stimulated RAW264.7 cells. Mkp-1 deficiency did not have an overt effect on IRF3/7 phosphorylation or IKK activation but modestly enhanced IFN-ß mRNA stability in LPS-stimulated BMDM. Our results suggest that Mkp-1 regulates IFN-ß production primarily through a p38-mediated mechanism and that IFN-ß plays a beneficial role in E. coli-induced sepsis.

A miR-375/YAP axis regulates neuroendocrine differentiation and tumorigenesis in lung carcinoid cells.

Lung carcinoids are variably aggressive and mechanistically understudied neuroendocrine neoplasms (NENs). Here, we identified and elucidated the function of a miR-375/yes-associated protein (YAP) axis in lung carcinoid (H727) cells. miR-375 and YAP are respectively high and low expressed in wild-type H727 cells. Following lentiviral CRISPR/Cas9-mediated miR-375 depletion, we identified distinct transcriptomic changes including dramatic YAP upregulation. We also observed a significant decrease in neuroendocrine differentiation and substantial reductions in cell proliferation, transformation, and tumor growth in cell culture and xenograft mouse disease models. Similarly, YAP overexpression resulted in distinct and partially overlapping transcriptomic changes, phenocopying the effects of miR-375 depletion in the same models as above. Transient YAP knockdown in miR-375-depleted cells reversed the effects of miR-375 on neuroendocrine differentiation and cell proliferation. Pathways analysis and confirmatory real-time PCR studies of shared dysregulated target genes indicate that this axis controls neuroendocrine related functions such as neural differentiation, exocytosis, and secretion. Taken together, we provide compelling evidence that a miR-375/YAP axis is a critical mediator of neuroendocrine differentiation and tumorigenesis in lung carcinoid cells.

The p53-induced RNA-binding protein ZMAT3 is a splicing regulator that inhibits the splicing of oncogenic CD44 variants in colorectal carcinoma.

p53 is an intensely studied tumor-suppressive transcription factor. Recent studies suggest that the RNA-binding protein (RBP) ZMAT3 is important in mediating the tumor-suppressive effects of p53. Here, we globally identify ZMAT3-regulated RNAs and their binding sites at nucleotide resolution in intact colorectal cancer (CRC) cells. ZMAT3 binds to thousands of mRNA precursors, mainly at intronic uridine-rich sequences and affects their splicing. The strongest alternatively spliced ZMAT3 target was CD44, a cell adhesion gene and stem cell marker that controls tumorigenesis. Silencing ZMAT3 increased inclusion of CD44 variant exons, resulting in significant up-regulation of oncogenic CD44 isoforms (CD44v) and increased CRC cell growth that was rescued by concurrent knockdown of CD44v Silencing p53 phenocopied the loss of ZMAT3 with respect to CD44 alternative splicing, suggesting that ZMAT3-mediated regulation of CD44 splicing is vital for p53 function. Collectively, our findings uncover a p53-ZMAT3-CD44 axis in growth suppression in CRC cells.

Alprostadil Injection Attenuates Coronary Microembolization-Induced Myocardial Injury Through GSK-3ß/Nrf2/HO-1 Signaling-Mediated Apoptosis Inhibition.

OBJECTIVE: Coronary microembolization (CME) results in progressive contractile dysfunction associated with cardiomyocyte apoptosis. Alprostadil injection improves microcirculation, which is effective in treating various cardiovascular disorders. However, the therapeutic effects of alprostadil in CME-induced myocardia injury remain unknown. Therefore, we evaluated the effects of alprostadil injection on cardiac protection in a rat model of CME and explored the underlying mechanisms. METHODS: A rat model of CME was established by injecting polyethylene microspheres into the left ventricle. After injection of microspheres, rats in the alprostadil group received alprostadil via tail vein within 2 minutes. Cardiac function, histological alterations in myocardium, serum c-troponin I (cTnI) levels, myocardium adenosine triphosphate (ATP) concentrations, the activity of superoxide dismutase (SOD) and malondialdehyde (MDA) content in myocardium, and myocardial apoptosis-related proteins were detected 12 hours after CME modeling. RESULTS: Compared with the Sham group, ATP concentrations, SOD activity in the myocardium, and cardiac function were significantly decreased in a rat model of CME. In addition, serum cTnI levels, MDA content, expression levels of pro-apoptotic proteins, and the number of TUNEL-positive nuclei were remarkably higher in CME group than those in the Sham group. However, alprostadil treatment notably reduced serum cTnI levels and expression levels of pro-apoptotic proteins, while noticeably improved cardiac function, and accelerated SOD activity in the myocardium following CME. Additionally, it was unveiled that the protective effects of alprostadil injection inhibit CME-induced myocardial apoptosis in the myocardium potentially through regulation of the GSK-3ß/Nrf2/HO-1 signaling pathway. CONCLUSION: Alprostadil injection seems to significantly suppress oxidative stress, alleviate myocardial apoptosis in the myocardium, and improve cardiac systolic and diastolic functions following CME by regulating the GSK-3ß/Nrf2/HO-1 signaling pathway.

Correction: Intellectual capital and the efficiency of SMEs in the transition economy China; Do financial resources strengthen the routes?

[This corrects the article DOI: 10.1371/journal.pone.0235462.].

Generation of a human embryonic stem cell line (WAe009-A-43) carrying a Long QT syndrome related mutation in KCNH2 gene.

The hereditary Long QT syndrome (LQTS) is a life-threaten channelopathy of the heart characterized by prolonged QT intervals and predisposition to occur polymorphic ventricular tachyarrhythmias. LQTS type 2 is the second most prevalent type of LQTS and more than 200 putative disease-causing mutations have been identified for KCNH2. Herein, we have generated a human embryonic stem cell line (WAe009-A-43) carrying a LQTS related mutation in KCNH2 (WAe009-A-43). The WAe009-A-43 line maintained stem cell like morphology, pluripotency, normal karyotype and could differentiate into all three germ layers in vivo.

miR-30e-3p Promotes Cardiomyocyte Autophagy and Inhibits Apoptosis via Regulating Egr-1 during Ischemia/Hypoxia.

BACKGROUND: Microvascular obstruction (MVO) can result in coronary microcirculation embolism and myocardial microinfarction. Myocardial injury induced by MVO is characterized by continuous ischemia and hypoxia of cardiomyocytes. Autophagy and apoptosis are closely associated with various cardiovascular diseases. Based on our previous study, we observed a decrease in miR-30e-3p expression and an increase in Egr-1 expression in a rat coronary microembolization model. However, the specific function of miR-30e-3p in regulating autophagy and apoptosis in an ischemia/hypoxia (IH) environment remains to be deciphered. We exposed cardiomyocytes to an IH environment and then determined whether miR-30e-3p was involved in promoting cardiomyocyte autophagy and inhibiting apoptosis by regulating Egr-1. METHODS: Cardiomyocytes were isolated from rats for our in vitro study. miR-30e-3p was either overexpressed or inhibited by transfection with lentiviral vectors into cardiomyocytes. 3-Methyladenine (3-MA) was used to inhibit autophagy. RT-qPCR and western blotting were used to determine the expression levels of miR-30e-3p, Egr-1, and proteins related to the autophagy and apoptosis process. Autophagic vacuoles and autophagic flux were evaluated using transmission electron microscopy (TEM) and confocal microscopy, respectively. Cardiomyocyte viability was evaluated using the MTS assay. Cell injury was assessed by lactate dehydrogenase (LDH) leakage, and apoptosis was determined by flow cytometry. RESULTS: Both miR-30e-3p expression and autophagy were significantly inhibited, and apoptosis was increased in cardiomyocytes after 9 hours of IH exposure. Overexpression of miR-30e-3p increased autophagy and inhibited apoptosis, as well as suppressed Egr-1 expression and decreased cell injury. In addition, inhibition of miR-30e-3p reduced autophagy and increased apoptosis and cell injury. CONCLUSIONS: miR-30e-3p may be involved in promoting cardiomyocyte autophagy and inhibiting apoptosis by indirectly regulating Egr-1 expression in an IH environment.

The variation on nitrogen removal mechanisms and the succession of ammonia oxidizing archaea and ammonia oxidizing bacteria with temperature in biofilm reactors treating saline wastewater.

To reveal nitrogen removal mechanisms under environmental stresses, biofilm reactors were operated at different temperatures (10 °C-35 °C) treating saline wastewater (salinity 3%). The results showed nitrogen removal efficiency was 98.46% at 30 °C and 60.85% at 10 °C, respectively. Both ammonia oxidizing archaea (AOA) and ammonia oxidizing bacteria (AOB) participated in nitrification. 94.9% of the overall ammonia oxidation was attributed to AOA at 10 °C, but only 48.2% of that was undertaken by AOA at 35 °C. AOA had a greater contribution at low temperature, which demonstrated that nitrogen removal pathway varied with temperature. Aerobic denitrification was more stable than anoxic denitrification. High-throughput sequencing showed Crenarchaeota was the dominant AOA (97.02-34.47%), cooperating with various heterotrophic AOB. Real-time PCR indicated that AOA was three orders of magnitude more abundant than AOB. AOA was more resistant to low temperature and high-saline stresses. Ammonia oxidizers had distinct responses to temperature change and showed diverse relationships at different temperatures.

Intellectual capital and the efficiency of SMEs in the transition economy China; Do financial resources strengthen the routes?

Intellectual capital has been grabbed the attention of researchers due to its momentous role in sustainable competitive advantage and organizational success. There is a growing catalog of related assessments, publications and reviews that display the direct and indirect role of intellectual capital in business success and profitability. Despite the bourgeoning literature, studies have not yet unleashed the influence of each dimension of intellectual capital; human capital, structural capital and customer capital on SMEs' efficiency with financial resources as a moderator. The present study fills the gap and assesses if financial resources strengthen the paths between the dimensions of intellectual capital and SMEs' efficiency. A survey method was used and collected evidence from 264 Chinese SMEs. The findings exhibit that human capital directly enhances SMEs' efficiency but the presence of financial resources as a moderator weakens the influence. However, social capital and customer capital do not directly improve SMEs' efficiency but financial resources reinforce the paths social and customer capital and SMEs efficiency. This research recommends that owners and managers of SMEs need to use their financial resources complementary with structural and customer capital while human capital should be used exclusively.

Ischemia/hypoxia inhibits cardiomyocyte autophagy and promotes apoptosis via the Egr-1/Bim/Beclin-1 pathway.

BACKGROUND: Myocardial injury caused by microvascular obstruction (MVO) is characterized by persistent ischemia/hypoxia (IH) of cardiomyocytes after microembolization. Autophagy and Egr-1 were closely associated with various cardiovascular diseases, including MVO. Bim and Beclin-1 are the important genes for autophagy and apoptosis. We aimed to explore whether the Egr-1/Bim/Beclin-1 pathway is involved in regulating autophagy and apoptosis in IH-exposed cardiomyocytes. METHODS: Neonatal rat cardiomyocytes exposed to the IH environment in vitro were transfected with lentivirus expressing Egr-1 or Egr-1 shRNA, or further treated with 3-methyladenine (3-MA). The expressions of autophagy and apoptosis-associated genes were evaluated using RT-qPCR and Western blots assays. Autophagic vacuoles and autophagic flux were detected by transmission electron microscopy (TEM) and confocal microscope, respectively. Cell injury was assessed by lactate dehydrogenase (LDH) leakage, and apoptosis was determined by flow cytometry. RESULTS: IH exposure elevated Egr-1 and Bim expressions, and decreased Beclin-1 expression in rat cardiomyocytes. Egr-1 overexpression in IH-exposed cardiomyocytes significantly up-regulated the levels of Egr-1 and Bim, and down-regulated the level of Beclin-1. Egr-1 knockdown resulted in down-regulated expressions of Egr-1 and Bim, as well as up-regulated expression of Beclin-1. In addition, Egr-1 knockdown induced autophagy was suppressed by 3-MA treatments. TEM and autophagic flux experiments also confirmed that Egr-1 inhibited autophagy progression in IH-exposed cardiomyocytes. Egr-1 suppression protected cardiomyocytes from IH-induced injury, as evidenced by the positive correlations between Egr-1 expression and LDH leakage or apoptosis index in IH-exposed cardiomyocytes. CONCLUSIONS: IH-induced cardiomyocyte autophagy and apoptosis are regulated by the Egr-1/Bim/Beclin-1 pathway, which is a potential target for treating cardiomyocyte injury caused by MVO in the IH environment.