Search

Polysialylation is a process of polysialic acid (polySia) addition to neural cell adhesion molecule (NCAM), which is associated with tumor cell migration and progression in many metastatic cancers and neurocognition. Polysialylation can be catalyzed by two highly homologous mammalian polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST). It has been proposed that two polybasic domains, polybasic region (PBR) and polysialyltransferase domain (PSTD) in polySTs, are possible binding sites for the intermolecular interactions of polyST–NCAM and polyST–polySia, respectively, as well as the intramolecular interaction of PSTD–PBR. In this study, Chou’s wenxiang diagrams of the PSTD and PBR are used to determine the key amino acids of these intermolecular and intramolecular interactions, and thus it may be helpful for the identification of the crucial amino acids in the polyST and for the understanding of the molecular mechanism of NCAM polysialylation by incorporating the wenxiang diagram and molecular modeling into NMR spectroscopy.

The polysialic acid (polySia) is a unique carbohydrate polymer produced on the surface of Neuronal Cell Adhesion Molecule (NCAM) in a number of cancer cells, and strongly correlates with the migration and invasion of tumor cells and with aggressive, metastatic disease and poor clinical prognosis in the clinic. Its synthesis is catalyzed by two polysialyltransferases (polySTs), ST8SiaIV (PST) and ST8SiaII (STX). Selective inhibition of polySTs, therefore, presents a therapeutic opportunity to inhibit tumor invasion and metastasis due to NCAM polysialylation. It has been proposed that NCAM polysialylation could be inhibited by two types of heparin inhibitors, low molecular heparin (LMWH) and heparin tetrasaccharide (DP4). This review summarizes how the interactions between Polysialyltransferase Domain (PSTD) in ST8SiaIV and CMP-Sia, and between the PSTD and polySia take place, and how these interactions are inhibited by LMWH and DP4. Our NMR studies indicate that LMWH is a more effective inhibitor than DP4 for inhibition of NCAM polysialylation. The NMR identification of heparin-binding sites in the PSTD may provide insight into the design of specific inhibitors of polysialylation.

Objective. The aim of this research was to create a new genetic signature of immune checkpoint-associated genes as a prognostic method for pediatric acute myeloid leukemia (AML). Methods. Transcriptome profiles and clinical follow-up details were obtained in Therapeutically Applicable Research to Generate Effective Treatments (TARGET), a database of pediatric tumors. Secondary data was collected from the Gene Expression Omnibus (GEO) to test the observations. In univariate Cox regression and multivariate Cox regression studies, the expression of immune checkpoint-related genes was studied. A three-mRNA signature was developed for predicting pediatric AML patient survival. Furthermore, the GEO cohort was used to confirm the reliability. A bioinformatics method was utilized to identify the diagnostic and prognostic value. Results. A three-gene (STAT1, BATF, EML4) signature was developed to identify patients into two danger categories depending on their OS. A multivariate regression study showed that the immune checkpoint-related signature (STAT1, BATF, EML4) was an independent indicator of pediatric AML. By immune cell subtypes analyses, the signature was correlated with multiple subtypes of immune cells. Conclusion. In summary, our three-gene signature can be a useful tool to predict the OS in AML patients.

In 2019 a Scientific Research & Demonstration Platform was deployed near islands and reefs in South China Sea by a joint research group of 7 institutes and universities in China. It is a simplified small model of a two-module semi-submersible-type VLFS. The test on site has continued for more than one and half years since then for long-term observations to validate the developed key technologies for design and behavior predictions of floating structures deployed near islands and reefs. An integrated information system was set up to continuously collect and inspect the data of the encountered waves, structure responses, connector forces, mooring line forces, anti-corrosion status of the platform, the performance efficiencies of a floating breakwater nearby and a wave energy converter attached on the breakwater. In this paper, the status of the on-site measurements and validations of the key technologies are briefly described.

The propeller cavitation not only affects the propulsive efficiency of a ship but also can cause vibration and noise. Accurate predictions of propeller cavitation are crucial at the design stage. This paper investigates the feasibility of the Reynolds-averaged Navier–Stokes (RANS) method in predicting propeller cavitation in behind-hull conditions, focusing on four aspects: (i) grid sensitivity; (ii) the time step effect; (iii) the turbulence model effect; and (iv) ability to rank two slightly different propellers. The Schnerr-Sauer model is adopted as the cavitation model. A model test is conducted to validate the numerical results. Good agreement on the cavitation pattern is obtained between the model test and computational fluid dynamics. Two propellers are computed, which have similar geometry but slightly different pitch ratios. The results show that RANS is capable of correctly differentiating the cavitation patterns between the two propellers in terms of the occurrence of face cavitation and the extent of sheet cavitation; moreover, time step size is found to slightly affect sheet cavitation and has a significant impact on the survival of the tip vortex cavitation. It is also observed that grid refinement is crucial for capturing tip vortex cavitation and the two-equation turbulence models used – realizable k-ε and shear stress transport (SST) k-ω – yield similar cavitation results.

AarF domain containing kinase 4 (ADCK4) is identified as a candidate gene associated with hereditary nephrotic syndrome (NS). Kruppel-like factor 5 (KLF5) is reported to promote podocyte survival by blocking the ERK/p38 MAPK pathways. Both ADCK4 and KLF5 are involved in the occurrence and development of podocyte disease, but their interaction remains unclear. Firstly, we found that the mRNA levels of ADCK4 and KLF5 decreased in NS patients, and both levels showed an obvious linear relationship. Secondly, we cloned the ADCK4 promoter region and examined its promoter activity in Hela, A549, and HEK 293 cell lines. Deletion analysis showed that the region - 116/- 4 relative to the transcriptional start site (TSS) was the core region of ADCK4 promoter. Thirdly, mutation analysis showed that putative binding sites for KLF5 contributed to the ADCK4 promoter activity. In HEK293 cells, we found that KLF5 upregulated the mRNA and protein levels of ADCK4. Finally, our chromatin immunoprecipitation assay found that KLF5 could bind to the specific region of ADCK4 promoter. These results showed that KLF5 can positively regulate the transcriptional activity of ADCK4.

Respiratory syncytial virus (RSV) infection enhances the cell‑mediated immune responses of type 2 helper T cells and promotes the progression of allergic inflammation and asthma by producing thymic stromal lymphopoietin (TSLP), especially long isoform TSLP (lfTSLP). However, the role of short isoform TSLP (sfTSLP) in RSV infection remains to be elucidated. The present study was designed to demonstrate the role of both lfTSLP and sfTSLP, as transcription regulators, in RSV infection. The expression of lfTSLP and sfTSLP in RSV‑infected Beas‑2B cells was analyzed. Activating protein 2 (AP‑2)α was overexpressed or knocked down to detect the changes in sfTSLP and lfTSLP expression. Luciferase reporter plasmid and chromatin immunoprecipitation experiments demonstrated that AP‑2α bound to the sfTSLP promoter region. LfTSLP and sfTSLP increased while AP‑2α decreased in RSV‑infected Beas‑2B cells. In the Beas‑2B cells, AP‑2α was found to negatively regulate the activity of the sfTSLP promoter and the mRNA level of sfTSLP. AP‑2α also negatively regulated the expression of lfTSLP at both the mRNA and protein levels. The results of the chromatin immunoprecipitation assay indicated that AP‑2α bound to the core promoter region of sfTSLP. These results confirmed that the transcription factor AP‑2α can repress the expression of lfTSLP and sfTSLP in bronchial epithelial cells in RSV infection.

Purpose Lung cancer is the most common malignant tumor in the world, and its incidence and mortality are very high. This study focuses on the mechanism of non-small cell lung cancer to find new therapeutic targets. Methods We used RT-PCR and Western blot to verify the linear relationship between E2F1 and IRF5 in normal lung tissue and lung cancer tissues. Secondly, we used overexpression and knock down E2F1 in cell lines to detect the expression of IRF5. The prime enzyme reporter plasmid verified that E2F1 binds to the core promoter region of IRF5; finally, CHIP experiments demonstrated that E2F1 binds directly to IRF5. Results We verified that E2F1 and IRF5 are decreased in patient tissues, and there is a strong linear relationship between E2F1 and IRF5. Secondly, we used overexpression of E2F1 or E2F1 siRNA transfected into HCC827 cells and found that E2F1 positively regulates the activity of the IRF5 promoter and the mRNA level of IRF5. Finally, the results of a chromatin immunoprecipitation assay demonstrated that E2F1 bound to the promoter region of IRF5 in vitro. These results suggested that the E2F1 transcription factor is the primary determinant for activating the basal transcription of the IRF5. Conclusion The transcription factor E2F1 positively regulates IRF5 in non-small cell lung cancer.

Background:Inhibition of α-amylase activity is an important strategy in the treatment of diabetes mellitus. An important treatment for diabetes mellitus is to reduce the digestion of carbohydrates and blood glucose concentrations. Inhibiting the activity of carbohydrate-degrading enzymes such as α-amylase and glucosidase significantly decreases the blood glucose level. Most inhibitors of α-amylase have serious adverse effects, and the α-amylase inactivation mechanisms for the design of safer inhibitors are yet to be revealed.Objective:In this study, we focused on the inhibitory effect of Zn2+ on the structure and dynamic characteristics of α-amylase from Anoxybacillus sp. GXS-BL (AGXA), which shares the same catalytic residues and similar structures as human pancreatic and salivary α-amylase (HPA and HSA, respectively).Methods:Circular dichroism (CD) spectra of the protein (AGXA) in the absence and presence of Zn2+ were recorded on a Chirascan instrument. The content of different secondary structures of AGXA in the absence and presence of Zn2+ was analyzed using the online SELCON3 program. An AGXA amino acid sequence similarity search was performed on the BLAST online server to find the most similar protein sequence to use as a template for homology modeling. The pocket volume measurer (POVME) program 3.0 was applied to calculate the active site pocket shape and volume, and molecular dynamics simulations were performed with the Amber14 software package.Results:According to circular dichroism experiments, upon Zn2+ binding, the protein secondary structure changed obviously, with the α-helix content decreasing and β-sheet, β-turn and randomcoil content increasing. The structural model of AGXA showed that His217 was near the active site pocket and that Phe178 was at the outer rim of the pocket. Based on the molecular dynamics trajectories, in the free AGXA model, the dihedral angle of C-CA-CB-CG displayed both acute and planar orientations, which corresponded to the open and closed states of the active site pocket, respectively. In the AGXA-Zn model, the dihedral angle of C-CA-CB-CG only showed the planar orientation. As Zn2+ was introduced, the metal center formed a coordination interaction with H217, a cation-π interaction with W244, a coordination interaction with E242 and a cation-π interaction with F178, which prevented F178 from easily rotating to the open state and inhibited the activity of the enzyme.Conclusion:This research may have uncovered a subtle mechanism for inhibiting the activity of α-amylase with transition metal ions, and this finding will help to design more potent and specific inhibitors of α-amylases.

Background:The polysialic acid (polySia) is a unique carbohydrate polymer produced on the surface Of Neuronal Cell Adhesion Molecule (NCAM) in a number of cancer cells, and strongly correlates with the migration and invasion of tumor cells and with aggressive, metastatic disease and poor clinical prognosis in the clinic. Its synthesis is catalyzed by two polysialyltransferases (polySTs), ST8SiaIV (PST) and ST8SiaII (STX). Selective inhibition of polySTs, therefore, presents a therapeutic opportunity to inhibit tumor invasion and metastasis due to NCAM polysialylation. Heparin has been found to be effective in inhibiting the ST8Sia IV activity, but no clear molecular rationale. It has been found that polysialyltransferase domain (PSTD) in polyST plays a significant role in influencing polyST activity, and thus it is critical for NCAM polysialylation based on the previous studies.Objective:To determine whether the three different types of heparin (unfractionated hepain (UFH), low molecular heparin (LMWH) and heparin tetrasaccharide (DP4)) is bound to the PSTD; and if so, what are the critical residues of the PSTD for these binding complexes?Methods:Fluorescence quenching analysis, the Circular Dichroism (CD) spectroscopy, and NMR spectroscopy were used to determine and analyze interactions of PSTD-UFH, PSTD-LMWH, and PSTD-DP4.Results:The fluorescence quenching analysis indicates that the PSTD-UFH binding is the strongest and the PSTD-DP4 binding is the weakest among these three types of the binding; the CD spectra showed that mainly the PSTD-heparin interactions caused a reduction in signal intensity but not marked decrease in α-helix content; the NMR data of the PSTD-DP4 and the PSTDLMWH interactions showed that the different types of heparin shared 12 common binding sites at N247, V251, R252, T253, S257, R265, Y267, W268, L269, V273, I275, and K276, which were mainly distributed in the long α-helix of the PSTD and the short 3-residue loop of the C-terminal PSTD. In addition, three residues K246, K250 and A254 were bound to the LMWH, but not to DP4. This suggests that the PSTD-LMWH binding is stronger than the PSTD-DP4 binding, and the LMWH is a more effective inhibitor than DP4.Conclusion:The findings in the present study demonstrate that PSTD domain is a potential target of heparin and may provide new insights into the molecular rationale of heparin-inhibiting NCAM polysialylation.

Juvenile idiopathic arthritis (JIA) is the most common rheumatic disease in childhood driven by aberrant pathways of T-cell activation. T helper 17 (Th17)/regulatory T cell (Treg) imbalance plays critical roles in the pathogenesis of arthritis. MicroRNA-125b (miR-125b) was upregulated after the activation of the initial CD4+ T cells, and could regulate the differentiation of CD4+ T cells. However, the effects of miR-125b on Th17/Treg imbalance and differentiation of Th17/Treg cells remain unknown. In this study, we evaluated the expression of miR-125b in the peripheral blood mononuclear cells (PBMCs) of children with JIA, and the relationship of miR-125b with Th17/Treg imbalance. Then, we used lentivirus vector-mediated overexpression technology to investigate the regulatory function of miR-125b in CD4+ T cells or dendritic cell/CD4+ T co-culture system. Decreased miR-125b expression in PBMCs and CD4+ T cells of JIA patients was negatively correlated with the ratio of Th17/Treg cells. It also correlated negatively with retinoic acid receptor-related orphan receptor γt but positively with Forkhead box protein 3 at transcriptional levels. Furthermore, we found that miR-125b overexpression inhibited Th17 cell differentiation, whereas facilitated the differentiation of Treg cells. MiR-125b upregulation led to the decrease of Th17-secreting cytokines but the increase of the Treg-secreting cytokines. Our results demonstrate that miR-125b participated in regulating Th17/Treg cell differentiation and imbalance in JIA patients. These findings provide novel insight into the critical role of miR-125b in the Th17/Treg imbalance of JIA, and raise the distinct possibility that miR-125b may prove to be a potential therapeutic target for JIA.

Di-n-butyl phthalate (DBP) is a kind of ubiquitous chemical linked to hormonal disruptions that affects male reproductive system. However, the mechanism of DBP-induced germ cells toxicity remains unclear. Here, we demonstrate that DBP induces reduction of proliferation, increase of apoptosis and DNA damage dependent on the PTEN/AKT pathway. Mechanistically, DBP decreases PTEN promoter methylation and increases its transcriptional activity, leading to increased PTEN expression. Notably, DNMT3b is confirmed as a target of miR-29b and miR-29b-mediated status of PTEN methylation is involved in the effects of DBP treatment. Meanwhile, DBP decreases AKT pathway expression via increasing PTEN expression. In addition, the fact that DBP decreases the sperm number and the percentage of motile and progressive sperm is associated with downregulated AKT pathway and sperm flagellum-related genes. Collectively, these findings indicate that DBP induces aberrant PTEN demethylation, leading to inhibition of the AKT pathway, which contributes to the reproductive toxicity.

Evidence has been emerging of the importance of long non‐coding RNAs (lncRNAs) in genome instability. However, no study has established how to classify such lncRNAs linked to genomic instability, and whether that connection poses a therapeutic significance. Here, we established a computational frame derived from mutator hypothesis by combining profiles of lncRNA expression and those of somatic mutations in a tumor genome, and identified 185 candidate lncRNAs associated with genomic instability in lung adenocarcinoma (LUAD). Through further studies, we established a six lncRNA‐based signature, which assigned patients to the high‐ and low‐risk groups with different prognosis. Further validation of this signature was performed in a number of separate cohorts of LUAD patients. In addition, the signature was found closely linked to genomic mutation rates in patients, indicating it could be a useful way to quantify genomic instability. In summary, this research offered a novel method by through which more studies may explore the function of lncRNAs and presented a possible new way for detecting biomarkers associated with genomic instability in cancers.

Acute myeloid leukemia (AML) is the most prevalent form of acute leukemia. Patients with AML often have poor clinical prognoses. Hypoxia can activate a series of immunosuppressive processes in tumors, resulting in diseases and poor clinical prognoses. However, how to evaluate the severity of hypoxia in tumor immune microenvironment remains unknown. In this study, we downloaded the profiles of RNA sequence and clinicopathological data of pediatric AML patients from Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database, as well as those of AML patients from Gene Expression Omnibus (GEO). In order to explore the immune microenvironment in AML, we established a risk signature to predict clinical prognosis. Our data showed that patients with high hypoxia risk score had shorter overall survival, indicating that higher hypoxia risk scores was significantly linked to immunosuppressive microenvironment in AML. Further analysis showed that the hypoxia could be used to serve as an independent prognostic indicator for AML patients. Moreover, we found gene sets enriched in high-risk AML group participated in the carcinogenesis. In summary, the established hypoxia-related risk model could act as an independent predictor for the clinical prognosis of AML, and also reflect the response intensity of the immune microenvironment in AML.

Background: Dysregulation of RNA binding proteins (RBPs) has been identified in multiple malignant tumors correlated with tumor progression and occurrence. However, the function of RBPs is not well understood in hepatocellular carcinoma (HCC). Methods: The RNA sequence data of HCC was extracted out of the Cancer Genome Atlas (TCGA) database and different RBPs were calculated between regular and cancerous tissue. The study explored the expression and predictive value of the RBPs systemically with a series of bioinformatic analyzes. Results: A total of 330 RBPs, including 208 up-regulated and 122 down-regulated RBPs, were classified differently. Four RBPs (MRPL54, EZH2, PPARGC1A, EIF2AK4) were defined as the forecast related hub gene and used to construct a model for prediction. Further study showed that the high-risk subgroup is poor survived (OS) compared to the model-based low-risk subgroup. The area of the prognostic model under the time-dependent receiver operator characteristic (ROC) curve is 0.814 in TCGA training group and 0.729 in validation group, indicating a strong prognostic model. We also created a predictive nomogram and a web-based calculator ( https://dxyjiang.shinyapps.io/RBPpredict/ ) based on the 4 RBPs and internal validation in the TCGA cohort, which displayed a beneficial predictive ability for HCC. Conclusions: Our results provide new insights into HCC pathogenesis. The 4-RBP gene signature showed a reliable HCC prediction ability with possible applications in therapeutic decision making and personalized therapy.

Sepsis is a systemic inflammatory response syndrome caused by infection, resulting in organ dysfunction. Sepsis‐induced acute kidney injury (AKI) is one of the most common potential complications. Increasing reports have shown that M1 and M2 macrophages both take part in the progress of AKI by influencing the level of inflammatory factors and the cell death, including pyroptosis. However, whether M1 and M2 macrophages regulate AKI by secreting exosome remains unknown. In the present study, we isolated the exosomes from M1 and M2 macrophages and used Western blot and enzyme‐linked immunosorbent assay (ELISA) to investigate the effect of M1 and M2 exosomes on cell pyroptosis. miRNA sequencing was used to identify the different miRNA in M1 and M2 exosomes. Luciferase reporter assay was used to verify the target gene of miRNA. We confirmed that exosomes excreted by macrophages regulated cell pyroptosis in vitro by using Western blot and ELISA. miRNA sequencing revealed the differentially expressed level of miRNAs in M1 and M2 exosomes, among which miR‐93‐5p was involved in the regulation of pyroptosis. By using bioinformatics predictions and luciferase reporter assay, we found that thioredoxin–interacting protein (TXNIP) was a direct target of miR‐93‐5p. Further in vitro and in vivo experiments indicated that exosomal miR‐93‐5p regulated the TXNIP directly to influence the pyroptosis in renal epithelial cells, which explained the functional difference between different phenotypes of macrophages. This study might provide new targets for the treatment of sepsis‐induced AKI.

Asthma is an inflammatory disease of the airways, characterized by lung eosinophilia, mucus hypersecretion by goblet cells and airway hyper‑responsiveness to inhaled allergens. The present study aimed to identify the function of microRNA (miR/miRNA)‑106b‑5p in TGF‑β1‑induced pulmonary fibrosis and epithelial‑mesenchymal transition (EMT) via targeting sine oculis homeobox homolog 1 (SIX1) through regulation of E2F transcription factor 1 (E2F1) in asthma. Asthmatic mouse models were induced with ovalbumin. miRNA expression was evaluated using reverse transcription‑quantitative PCR. Transfection experiments using bronchial epithelial cells were performed to determine the target genes. A luciferase reporter assay system was applied to identify the target gene of miR‑106b‑5p. The present study revealed downregulated miR‑106b‑5p expression and upregulated SIX1 expression in asthmatic mice and TGF‑β1‑induced BEAS‑2B cells. Moreover, miR‑106b‑5p overexpression inhibited TGF‑β1‑induced fibrosis and EMT in BEAS‑2B cells, while miR‑106b‑5p‑knockdown produced the opposite effects. Subsequently, miR‑106b‑5p was found to regulate SIX1 through indirect regulation of E2F1. Additionally, E2F1‑ and SIX1‑knockdown blocked TGF‑β1‑induced fibrosis and EMT in BEAS‑2B cells. In addition, miR‑106b‑5p negatively regulated SIX1 via E2F1 in BEAS‑2B cells. The present study demonstrated that the miR‑106b‑5p/E2F1/SIX1 signaling pathway may provide potential therapeutic targets for asthma.

Background. The research analyzed a group of patients to develop a statistical nomogram and a web-based survival rate predictor for the comprehensive estimate of the overall survival (OS) of children with acute myeloid leukemia. Methods. Between 1999 to 2015, we used the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database to evaluate and randomly divide 440 children diagnosed with AML into the population of training ( n = 309 ) and validation ( n = 131 ). The analysis of Lasso Cox was used to identify separate predictive variables. We have used essential forecasting considerations to construct a nomogram and a web-based calculator focused on Cox regression analysis. Nomogram validation was tested through discrimination and calibration. Results. Compared to the multivariate training cohort models, a nomogram integrating gender, age of diagnose, WBC at diagnosis, bone marrow leukemic blast percentage, and chromosomal abnormalities [ t (8; 21), inv(16)] were designed for the prediction of OS. We also developed a predictive survival nomogram and a web-based calculator. C-indexes validated internally and checked externally were 0.747 and 0.716. The calibration curves have shown that the nomogram might accurately forecast 3-year and 5-year OS. Conclusions. A nomogram effectively predicts survival in children with AML. This prognostic model can be used in clinical practice.

Oxygen therapy is a common treatment in neonatal intensive care units, but long-term continuous hyperoxia ventilation may induce acute lung injury (ALI). Gasdermin D (GSDMD)-mediated pyroptosis participates in various diseases including ALI, but the role of GSDMD in hyperoxia-induced ALI is yet understood. Here, we showed a significant increase in GSDMD after exposure to high oxygen. To elucidate the molecular mechanisms involved in GSDMD regulation, we identified the core promoter of GSDMD, −98 ~ −12 bp relative to the transcriptional start site (TSS). The results of mutational analysis, overexpression or siRNA interference, EMSA and ChIP demonstrated that E2F4 and TFAP2A positively regulate the transcriptional activity of the GSDMD by binding to its promoter. However, only TFAP2A showed a regulatory effect on the expression of GSDMD. Moreover, TFAP2A was increased in the lung tissues of rats exposed to hyperoxia and showed a strong linear correlation with GSDMD. Our results indicated that TFAP2A positively regulates the GSDMD expression via binding to the promoter region of GSDMD.

The expression changes of CD2-associated protein (CD2AP) can lead to kidney diseases with proteinuria, including nephrotic syndrome (NS). A recent study reported that miRNAs may be important transcriptional regulators. In this study, we found increased expression of miR-939-5p and decreased expression of CD2AP in the peripheral blood of patients with NS. However, miR-939-5p did not show a regulatory effect on the 3'-untranslated region of CD2AP. The expression levels of specific protein 1 and adenovirus E2 promoter-binding factor 1, important transcription regulators in the promoter region of CD2AP, were also not affected by microRNA (miR)-939-5p. We confirmed that miR-939-5p is in the nucleus by fluorescent in situ hybridization and cytoplasmic separation polymerase chain reaction. The promoter plasmid and miR-939-5p were cotransfected into HEK-293 cells, and the luciferase reporter gene assay was used to analyze the promoter activity. We found that miR-939-5p binds to a specific sequence in the CD2AP promoter. miR-939-5p was confirmed to reduce the recruitment of RNA polymerase II to the CD2AP promoter region by chromatin immunoprecipitation. These findings improve our understanding of the mechanism of miR-939-5p in NS and provide potential molecular therapeutic targets for NS.

Background: α-Amylases are starch-degrading enzymes and used widely, the study on thermostability of α-amylase is a central requirement for its application in life science and biotechnology. Objective: In this article, our motivation is to study how the effect of Ca2+ ions on the structure and thermal characterization of α-amylase (AGXA) from thermophilic Anoxybacillus sp.GXS-BL. Methods: α-Amylase activity was assayed with soluble starch as the substrate, and the amount of sugar released was determined by DNS method. For AGXA with calcium ions and without calcium ions, optimum temperature (Topt), half-inactivation temperature (T50) and thermal inactivation (halflife, t1/2) was evaluated. The thermal denaturation of the enzymes was determined by DSC and CD methods. 3D structure of AGXA was homology modeled with α-amylase (5A2A) as the template. Results: With calcium ions, the values of Topt, T50, t1/2, Tm and ΔH in AGXA were significantly higher than those of AGXA without calcium ions, showing calcium ions had stabilizing effects on α-amylase structure with the increased temperature. Based on DSC measurements AGXA underwent thermal denaturation by adopting two-state irreversible unfolding processes. Based on the CD spectra, AGXA without calcium ions exhibited two transition states upon unfolding, including α- helical contents increasing, and the transition from α-helices to β-sheet structures, which was obviously different in AGXA with Ca2+ ions, and up to 4 Ca2+ ions were located on the inter-domain or intra-domain regions according to the modeling structure. Conclusion: These results reveal that Ca2+ ions have pronounced influences on the thermostability of AGXA structure.

To investigate the effect of dihydroartemisinin (DHA) on multidrug resistance of human oral squamous cell carcinoma cell line KBV200 and to explore its relationship with ROS-MAPK pathway.MTT assay was used to detect the effects of different concentrations of DHA on proliferation of KB and KBV200 cells. MTT assay was used to detect the inhibition rates of cisplatin (DDP), vincristine (VCR), adriamycin (ADM), etoposide (VP-16) on proliferations of KB and KBV200 cells and the changes after DHA addition, combined with ROS inducer 3-AT, ERK inhibitor UO126, JNK inhibitor SP600125, and p38MAPK inhibitor SB203580, respectively, the difference of reversal before and after intervention was observed; the fluorescence intensity of ROS was measured by flow cytometry. SPSS 17.0 software package was used to analyze the data.Compared with KB cells, drug resistances of KBV200 cells to VCR, VP-16 and ADM were significantly increased(P0.05), after 10, 20, and 30 μg/mL DHA treatment; the IC50 of KBV200 cells to VCR, VP-16 and ADM was decreased significantly in a concentration-dependent manner(P0.05). Compared with KB cells, ROS fluorescence intensity of KBV200 cells decreased(P0.05); after DHA treatment, ROS fluorescence intensity increased significantly, IC50 of VCR, VP-16, ADM decreased significantly(P0.05), which was consistent with ROS promoter effect. Compared with KB cells, the levels of p-ERK/ERK, p-JNK/JNK, p-p38MAPK/p38MAPK in KBV200 cells increased significantly (P0.05); after DHA treatment, the levels of p-ERK/ERK, p-JNK/JNK, p-p38MAPK/p38MAPK decreased significantly, ICDihydroartemisinin may reverse multidrug resistance of KBV2001 cells by promoting ROS production and inhibiting MAPK pathway.

Bronchopulmonary dysplasia (BPD) is a severe complication of preterm infants characterized by increased alveolarization and inflammation. Premature exposure to hyperoxia is believed to be a key contributor to the pathogenesis of BPD. No effective preventive or therapeutic agents have been created. Stimulator of interferon gene (STING) is associated with inflammation and apoptosis in various lung diseases. Long non-coding RNA MALAT1 has been reported to be involved in BPD. However, how MALAT1 regulates STING expression remains unknown. In this study, we assessed that STING and MALAT1 were up-regulated in the lung tissue from BPD neonates, hyperoxia-based rat models and lung epithelial cell lines. Then, using the flow cytometry and cell proliferation assay, we found that down-regulating of STING or MALAT1 inhibited the apoptosis and promoted the proliferation of hyperoxia-treated cells. Subsequently, qRT-PCR, Western blotting and dual-luciferase reporter assays showed that suppressing MALAT1 decreased the expression and promoter activity of STING. Moreover, transcription factor CREB showed its regulatory role in the transcription of STING via a chromatin immunoprecipitation. In conclusion, MALAT1 interacts with CREB to regulate STING transcription in BPD neonates. STING, CREB and MALAT1 may be promising therapeutic targets in the prevention and treatment of BPD.

Orosomucoid like-3 (ORMDL3) has been identified to be associated with the development of asthma according to previous studies. However, the definite role of ORMDL3 in the pathogenesis of asthma remains unclear. In this study, we found ORMDL3 was highly expressed in PBMC specimens from childhood asthma patients. Cytokines production and p-ERK/MMP-9 pathway expression was also increased in childhood asthma patients compared with controls. In addition, ORMDL3 overexpression induced IL-6 and IL-8 release and activated p-ERK/MMP-9 pathway in vitro. Increased ORMDL3 expression was observed after treated with 5-Aza-CdR. 5-Aza-CdR decreased the percentage of the CpG island in the ORMDL3 promoter region and increased its promoter activity. In addition, 5-Aza-CdR significantly increased IL-6 and IL-8 levels in NHBE cells while there was no obvious alteration after knocking down ORMDL3. Knockdown of ORMDL3 also significantly decreased the expression of p-ERK/MMP-9 pathway in the presence or absence of 5-Aza-CdR. In conclusion, our study provided novel evidence for the association between ORMDL3 and asthma-associated cytokines. Moreover, DNA methylation plays an important role in ORMDL3-mediated increased IL-6 and IL-8 levels and p-ERK/MMP-9 pathway expression.

CD80 has been known to be a co-stimulatory factor expressed in antigen presenting cells (APCs) involved in T-cell activation. It can be expressed in non-bone marrow-derived cells induced by lipopolysaccharide (LPS) or other stimulators, and directly involved in the pathogenesis following T cell activation. However, the intricate transcription mechanisms that underlying the upregulation of CD80 expression levels in LPS-activated non-bone marrow-derived cells is still unknown. LncRNAs are confirmed to be involved in transcriptional regulation by influencing transcription factors. Therefore, we wanted to determine whether the lncRNA MALAT1, which has been found to be related to LPS-induced inflammation, could regulate the transcription of CD80. Our results showed that CD80 was upregulated in low-dose LPS-activated A549 cells in a NF-κB-dependent manner. The lncRNA MALAT1 can interfere with NF-κB activation and disrupt its binding efficiency with the CD80 promoter. Thus, the lncRNA MALAT1 can regulate CD80 transcription via the NF-κB signaling pathway in the LPS-activated A549 cell line.

// Lu-Lu Wang 1 , Lan-Bo Zhou 2 , Jin Shu 3 , Nan-Nan Li 1 , Hui-Wen Zhang 1 , Rui Jin 1 , Li-Li Zhuang 1 and Guo-Ping Zhou 1 1 Department of Pediatrics, The First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu Province, China 2 Grade 2013 Clinical Class 7, The First School of Clinical Medicine, Nanjing Medical University, Nanjing, Jiangsu Province, China 3 Department of Pediatric Respiration, Affiliated Wuxi People’s Hospital, Nanjing Medical University, Wuxi, Jiangsu, China Correspondence to: Guo-Ping Zhou, email: guopingzhou@aliyun.com Keywords: interferon regulatroy factor 3, GATA-1, acetylation, histone deacetylase inhibitor, lung adenocarcinoma Received: December 20, 2016 Accepted: April 21, 2017 Published: June 06, 2017 ABSTRACT Interferon regulatory factor 3 (IRF-3) is an important transcription factor for interferon genes. Although its functional activation by viral infection has been widely explicated, the regulatory mechanism of IRF-3 gene expression in cancer cells is poorly understood. In this study, we demonstrated treatment of lung adenocarcinoma A549 cells with trichostatin A (TSA) and valproic acid (VPA), two different classes of histone deacetylase inhibitors, strongly stimulated IRF-3 gene expression. Truncated and mutated IRF-3 promoter indicated that a specific GATA-1 element was responsible for TSA-induced activation of IRF-3 promoter. Chromatin immunoprecipitation and electrophoretic mobility shift assay showed that TSA treatment increased the binding affinity of GATA-1 to IRF-3 promoter. Using immunoprecipitation assay and immunoblotting, we demonstrated that TSA increased the level of acetylated GATA-1 in A549 cells. In summary, our study implied that TSA enhanced IRF-3 gene expression through increased GATA-1 recruitment to IRF-3 promoter and the acetylation level of GATA-1 in lung adenocarcinoma A549 cells.

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis in infancy, which is a major risk factor for recurrent wheezing and asthma. Orosomucoid 1-like protein 3 (ORMDL3) has been reported to associate with virus-triggered recurrent wheezing and asthma in children. However, little is known about how ORMDL3 is involved into RSV infection. In this study, we showed that the mRNA expression of ORMDL3 is significantly increased in the peripheral blood lymphocytes of infants with RSV-induced bronchiolitis compared with uninfected controls, also increased in bronchial epithelial cells and lung fibroblasts following RSV infection in vitro. To investigate the underlying mechanisms of RSV-induced ORMDL3 expression, we performed in silico analysis of the binding sites of several transcription factors in the ORMDL3 promoter. The proximal interferon-regulatory factor-3 (IRF-3) binding site positively regulated ORMDL3 transcription following exposure to RSV, as determined through mutational analysis. Overexpression and RNA interference experiments targeting IRF-3 showed that it regulates the expression of ORMDL3 following RSV exposure. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay showed that IRF-3 binds directly to the promoter of the ORMDL3 gene. Furthermore, we confirmed that expression of IRF-3 is significantly increased and shows a strong linear correlation with increased ORMDL3 in the peripheral blood lymphocytes from infants with RSV-induced bronchiolitis. Our results indicate that IRF-3 is an important regulator of ORMDL3 induction following RSV infection by binding directly to the promoter of ORMDL3, which may be implicated in the inflammatory and immune reactions involved in bronchiolitis and wheezing diseases.

Interferon regulatory factor 5 (IRF5) plays a critical role in the induction of type I interferon, proinflammatory cytokines and chemokines, and participates in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). However, the relationship between IRF5 and childhood-onset SLE remains elusive. In the present study, we demonstrated that levels of mRNA expression of IRF5, IFN-α, and Sp1 were significantly increased in childhood-onset SLE, as seen on quantitative real-time PCR, and the expression of Sp1 and IFN-α was positively correlated with IRF5. In addition to being used as antitumor drugs, a number of histone deacetylase inhibitors (HDACi) display potent anti-inflammatory properties; however, their effects on IRF5 expression remain unclear. In this study, we identified that HDACi trichostatin A (TSA) and histone acetyltransferase (HAT)-p300 downregulated IRF5 promoter activity, mRNA expression, and protein level, whereas the HAT-p300/CBP-associated factor had no effect. Moreover, TSA inhibited the production of TNF-α and IL-6 in differentiated THP-1cells. Furthermore, chromatin immunoprecipitation assays revealed that TSA inhibited DNA binding of Sp1, RNA polymerase II, HDAC3, and p300 to the core promoter region of IRF5. Our results suggest that HDACi may have therapeutic potential in patients with autoimmune diseases such as SLE through repression of IRF5 expression.

Stimulator of Interferon Gene (STING) is a key mediator of innate immune signaling. STING plays a pivotal role in the pathogenesis of many diseases including infectious diseases, auto-immune diseases and cancer. Many studies have been carried out recently in the field of STING-regulated pathway, however, rarely of transcriptional mechanisms. To characterize the murine STING (mSTING) promoter, we cloned a series of different nucleotide sequences of the 5′-flanking region of the mSTING gene. Transient transfection of promoter-reporter recombinant plasmids and luciferase assay illustrated the region (−77/+177) relative to the transcription start site (TSS) of the mSTING gene was sufficient for full promoter activity. This region contains GATA1, IK2, Sp1/Sp3 and STAT putative transcription factor binding sites. Mutation of GATA1 or Sp1/Sp3 sites led to obvious decrease of the mSTING promoter activity. Overexpression of GATA1 and Sp3 enhanced the mSTING promoter activity, whereas knockdown of GATA1 and Sp3 by a siRNA strategy significantly reduced the transcription activity. Chromatin immunoprecipitation assays demonstrated that GATA1 and Sp3 interact with the mSTING promoter in vivo. These results provided the first analysis of mSTING promoter and demonstrated that transcription factor GATA1 and Sp3 positively regulate the basal transcription of the mSTING gene.

Polysialic acid (polySia) is an unusual glycan that posttranslational modifies neural cell adhesion molecule (NCAM) proteins in mammalian cells. The up-regulated expression of polySia-NCAM is associated with tumor progression in many metastatic human cancers and in neurocognitive processes. Two members of the ST8Sia family of &alpha, 2,8-polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST) both catalyze synthesis of polySia when activated cytidine monophosphate(CMP)-Sialic acid (CMP-Sia) is translocate into the lumen of the Golgi apparatus. Two key polybasic domains in the polySTs, the polybasic region (PBR) and the polysialyltransferase domain (PSTD) areessential forpolysialylation of the NCAM proteins. However, the precise molecular details to describe the interactions required for polysialylation remain unknown. In this study, we hypothesize that PSTD interacts with both CMP-Sia and polySia to catalyze polysialylation of the NCAM proteins. To test this hypothesis, we synthesized a 35-amino acid-PSTD peptide derived from the ST8Sia IV gene sequence and used it to study its interaction with CMP-Sia, and polySia. Our results showed for the PSTD-CMP-Sia interaction,the largest chemical-shift perturbations (CSP) were in amino acid residues V251 to A254 in the short H1 helix, located near the N-terminus of PSTD. However, larger CSP values for the PSTD-polySia interaction were observed in amino acid residues R259 to T270 in the long H2 helix. These differences suggest that CMP-Sia preferentially binds to the domain between the short H1 helix and the longer H2 helix. In contrast, polySia was principally bound to the long H2 helix of PSTD. For the PSTD-polySia interaction, a significant decrease in peak intensity was observed in the 20 amino acid residues located between the N-and C-termini of the long H2 helix in PSTD, suggesting a slower motion in these residues when polySia bound to PSTD. Specific features of the interactions between PSTD-CMP-Sia, and PSTD-polySia were further confirmed by comparing their 800 MHz-derived HSQC spectra with that of PSTD-Sia, PSTD-TriSia (DP 3) and PSTD-polySia. Based on the interactions between PSTD-CMP-Sia, PSTD-polySia, PBR-NCAM and PSTD-PBR, these findingsprovide a greater understanding of the molecular mechanisms underlying polySia-NCAM polysialylation, and thus provides a new perspective for translational pharmacological applications and development by targeting the two polysialyltransferases.