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Biofilm formation is easily found in patients suffered from ventilator-associated pneumonia (VAP) in neonatal intensive care unit (NICU) and makes the VAP infections not only harder to be treated but easier to relapse. In order to find some novel ways to inhibit biofilm formation, this study describe a previously unrecognized role for the human umbilical cord mesenchymal stem cells (hUCMSCs). In addition to multiple differentiation, hUCMSCs have the ability to prevent the biofilms formation in vitro by secreting antibacterial peptides (LL-37 and hBD-2). This occurred while P. aeruginosa PA27853 and hUCMSCs were cocultured, and the filtrated medium, which was the supernatant containing antibacterial peptides (5.9 ng/ml of LL-37, 1.77 ng/ml of hBD-2), and inhibited the growth of the bacterial biofilm on the surface of tracheal tube (2.5#, for preterm infant). Using microarrays, we were able to demonstrate that the antibacterial peptides from hUCMSC affected biofilm formation by downregulating the gene-encoded polysaccharide biosynthesis protein. In addition, in order to find out the most suitable concentration of hUCMSCs, P. aeruginosa was cocultured with eight-level concentrations of hUCMSCs, and we found that the concentration of LL-37 was positively correlated with the concentration of hUCMSCs. Meanwhile, the concentration of LL-37 became stable while the hUCMSC concentration reaches higher than 5 × 10 6 cells / ml . But the concentration of hBD-2 had no significant correlation with hUCMSCs. The collection of these stem cells is not only limited by ethics but also reduces host rejection. This makes it possible to use autologous hUCMSCs to treat neonatal VAP.

Odorant-binding proteins (OBPs) play a key role in the olfactory system and are essential for mating and oviposition host selection. Tirathaba rufivena, a serious lepidopterous insect pest of the palm area in recent years, has threatened cultivations of Areca catechu in Hainan. Female-biased odorant-binding protein 4 of T. rufivena (TrufOBP4) expression was hypothesized to participate in the process of oviposition host recognition and localization. In this study, we cloned and analyzed the cDNA sequence of TrufOBP4. The predicted mature protein TrufOBP4 is a small, soluble, secretory protein and belongs to a classic OBP subfamily. Fluorescence binding assay results showed that TrufOBP4 had high binding abilities with the host plant volatiles, octyl methoxycinnamate, dibutyl phthalate, myristic acid and palmitic acid. These four components tend to dock in the same binding pocket based on the molecular docking result. The interactions and contributions of key amino acid residues were also characterized. This research provides evidence that TrufOBP4 might participate in the chemoreception of volatile compounds from inflorescences of A. catechu and can contribute to the integrated management of T. rufivena.

k nearest neighbor ( k NN) queries are frequently used in location-based services (LBSs), by which we wish to get k closest points of interest (POIs) given a certain point. Since the cloud computing is developing fast, LBS providers are tended to outsource spatial databases to the cloud. However, cloud servers are often untrusty, so that ensuring the spatial query integrity as well as the spatial query privacy is critical. We present a verifiable privacy-preserving k NN query scheme, which can be used on road networks. Our work makes use of the network Voronoi diagram and several cryptographic primitives including pseudo-random functions, Paillier cryptosystem, condensed RSA digital signature, and so on. It can simultaneously preserve the privacy of spatial data and k NN queries, and verify the reliability of query results. The effectiveness and practicability of our scheme are validated by our experimental results. We further analyzed the security of our scheme under the adaptive chosen-query attack via rigorous proof.

ObjectiveEarly life bronchiolitis has been hypothesised to be associated with the subsequent risk of persistent wheezing or asthma. However, the link remains controversial. The objective of our study was to evaluate the association between bronchiolitis before 2 years of age and the late-onset wheezing/asthma.DesignSystematic review and meta-analysis.MethodsPubMed, Embase and Web of Science databases were systematically searched for studies published between 1955 and January 2020. Meanwhile, we also checked through the reference lists of relevant articles to see whether these references included reports of other studies that might be eligible for the review. Cohort and case–control studies assessing the association between early-life bronchiolitis and late-onset wheezing/asthma were included in this meta-analysis. Data were extracted by two independent reviewers. Results were pooled using a random-effects model or fixed-effects model according to the heterogeneity among studies.Results32 original articles with 292 844 participants, which met the criteria, were included in this meta-analysis. Bronchiolitis before 2 years of age was associated with an increased risk of subsequent wheezing/asthma (relative risk=2.46, 95% CI 2.14 to 2.82, pConclusionsThe meta-analysis indicates an association between bronchiolitis before 2 years of age and the wheezing/asthma in later life. Well-designed and highly standardised prospective studies that better address bias due to potential confounding factors are needed to validate the risk identified in our meta-analysis.PROSPERO registration numberCRD42018089453.

Background: Minimal manifestation status (MMS) is an important landmark in the treatment of myasthenia gravis (MG), and predictors of MMS induction have rarely been identified in previous studies. Objective: The objective of this study is to evaluate the clinical factors associated with MMS induction among patients with MG. Design: This two-step retrospective cohort study with a single center investigated the factors that may be associated with MMS induction and retested these predictors in a test cohort. Methods: A total of 388 diagnosed MG patients who visited Xiangya Hospital between 1 July 2015 and 1 July 2019 were involved. We performed detailed chart reviews and recorded all cases achieving MMS. Demographics and clinical characteristics were also collected and their relationships to achieving MMS were investigated. Results: MMS was achieved in 124 patients (50.2%), and the median time to achieve MMS was 26 months. Several factors were found to be associated with MMS induction in exploring cohort, including muscle-specific tyrosine-protein kinase receptor (MuSK) antibody positivity (adjusted hazard ratio, HR = 4.333, 95% confidence interval, CI: 1.862–10.082), isolated ocular involvement (adjusted HR = 1.95, 95% CI: 1.284–2.961), and low baseline quantitative myasthenia gravis score (QMG score; adjusted HR = 2.022, 95% CI: 1.086–3.764). These factors were then retested in the test cohort. Isolated ocular involvement or low baseline QMG scores were factors found to be beneficial for MMS induction were confirmed. Conclusion: Isolated ocular involvement and low baseline QMG score are predictors of MMS induction in MG patients.

Aim: The purpose of the present study was to explore the function and mechanism of tensin 1 (TNS1) in non-small cell lung cancer (NSCLC) progression. Methods: The expression of TNS1 in NSCLC cells and tissues was assessed by RT-PCR and Western blot. Besides, Kaplan–Meier survival analysis was recruited to explore the association between TNS1 and NSCLC. Cell growth was analyzed by MTT and flow cytometry assay, while cell metastasis was determined by wound healing and transwell assays. The targeting relationship between TNS1 and miR-152 was assessed by luciferase activity assays. And Western blot was employed to determine the expression of related proteins of Akt/mTOR/RhoA pathway. Results: TNS1 level was boosted in NSCLC cells and tissues, related to the prognosis of NSCLC patients. Furthermore, it was proved that TNS1 promoted the growth and metastasis of NSCLC cells via Akt/mTOR/RhoA pathway. And miR-152 targeted TNS1 to affect the progression of NSCLC. Conclusion: miR-152/TNS1 axis inhibits the progression of NSCLC by Akt/mTOR/RhoA pathway.

In order to better understand effects of metal cationization on protonation and reduction in MALDI process, mass spectra of riboflavin complexes with different alkali metal ions generated by a series of laser shots at a fixed target spot were consecutively recorded with the matrix of 2,5-dihydroxybenzoic acid (DHB). Progressive changes in the spectra, including the decrease of reduction extent for the metalized molecule ions and the increase of relative intensities of metalized species compared to protonated species, were observed with the increase of laser shots. Covariance mapping analysis was also performed for the data. Results show that different metalized ions are positively correlated each other; while metalized ions and protonated species are negatively correlated. These results not only reflect competition between metalization and protonation/reduction in laser plume, but also show that the winner of it might be changed during the process of sample consumption by laser irradiation.

Key points Gestational hypoxia represses ten-eleven translocation methylcytosine dioxygenase 1 (TET1) expression in uterine arteries, which is recovered by inhibiting endogenous miR-210. Inhibition of miR-210 rescues BKCa channel expression and current in uterine arteries of pregnant animals acclimatized to high altitude hypoxia in a TET-dependent manner. miR-210 blockade restores BKCa channel-mediated relaxations and attenuates pressure-dependent myogenic tone in uterine arteries of pregnant animals acclimatized to high altitude. Abstract Gestational hypoxia at high altitude has profound adverse effects on the uteroplacental circulation, and is associated with increased incidence of preeclampsia and fetal intrauterine growth restriction. Previous studies demonstrated that suppression of large-conductance Ca2+ -activated K+ (BKCa ) channel function played a critical role in the maladaptation of uteroplacental circulation caused by gestational hypoxia. Yet, the mechanisms underlying gestational hypoxia-induced BKCa channel repression remain undetermined. The present study investigated a causal role of microRNA-210 (miR-210) in hypoxia-mediated repression of BKCa channel expression and function in uterine arteries using a sheep model. The results revealed that gestational hypoxia significantly decreased ten-eleven translocation methylcytosine dioxygenase 1 (TET1) expression in uterine arteries, which was recovered by inhibiting endogenous miR-210 with miR-210 locked nucleic acid (miR-210-LNA). Of importance, miR-210-LNA restored BKCa channel β1 subunit expression in uterine arteries, which was blocked by a competitive TET inhibitor, fumarate, thus functionally linking miR-210 to the TET1-BKCa channel cascade. In addition, miR-210-LNA reversed hypoxia-mediated suppression of BKCa channel function and rescued the effect of steroid hormones in upregulating BKCa channel expression and function in uterine arteries, which were also ablated by fumarate. Collectively, the present study demonstrates a causative effect of miR-210 in the downregulation of TET1 and subsequent repression of BKCa channel expression and function, providing a novel mechanistic insight into the regulation of BKCa channel function and the molecular basis underlying the maladaptation of uterine vascular function in gestational hypoxia.

SIRT1 regulates cell senescence. We investigated a novel role of SIRT1 in the regulation of cardiomyocyte terminal differentiation in the developing heart. Retinoic acid (RA)-induced binucleation of H9c2 cells was associated with increased SIRT1 expression. Inhibition of SIRT1 activity or expression significantly decreased RA-induced binucleation. SIRT1 expression was minimal in the fetal heart and significantly upregulated in the hearts of postnatal day 7 (P7) rat pups. In contrast, heart-specific miR-133a expression was high in the fetal heart but significantly reduced in P7 pup hearts. The miR-133a promoter contains a canonical HRE element and hypoxia upregulated miR-133a gene expression in the heart. SIRT1 mRNA 3'UTR has miR-133a binding sequences and miR-133a and hypoxia suppressed SIRT1 expression in cardiomyocytes. Of importance, inhibition of SIRT1 significantly reduced binucleated cardiomyocytes in the hearts of P7 pups. Taken together, the present study reveals a novel role of SIRT1 and its regulation by miR-133a in cardiomyocyte terminal differentiation of the developing heart, and suggests a potential therapeutic strategy that may impact cardiac function later in life.

Gestational hypoxia inhibits large conductance Ca 2+ -activated K + (BK Ca ) channel expression and function in uterine arterial adaptation to pregnancy. Given the findings that microRNA-210 (miR-210) is increased in hypoxia during gestation and preeclampsia, the present study sought to investigate the role of miR-210 in the regulation of BK Ca channel adaptation in the uterine artery. Gestational hypoxia significantly increased uterine vascular resistance and blood pressure in pregnant sheep and upregulated miR-210 in uterine arteries. MiR-210 bound to ovine ten-eleven translocation methylcytosine dioxygenase 1 mRNA 3′ untranslated region and decreased ten-eleven translocation methylcytosine dioxygenase 1 mRNA and protein abundance in uterine arteries of pregnant sheep, as well as abrogated steroid hormone–induced upregulation of ten-eleven translocation methylcytosine dioxygenase 1 expression in uterine arteries of nonpregnant animals. In accordance, miR-210 blocked pregnancy- and steroid hormone–induced upregulation of BK Ca channel β1 subunit expression in uterine arteries. Functionally, miR-210 suppressed BK Ca channel current density in uterine arterial myocytes of pregnant sheep and inhibited steroid hormone–induced increases in BK Ca channel currents in uterine arteries of nonpregnant animals. Blockade of endogenous miR-210 inhibited hypoxia-induced suppression of BK Ca channel activity. In addition, miR-210 decreased BK Ca channel–mediated relaxations and increased pressure-dependent myogenic tone of uterine arteries. Together, the results demonstrate that miR-210 plays an important role in the downregulation of ten-eleven translocation methylcytosine dioxygenase 1 and repression of BK Ca channel function in uterine arteries, revealing a novel mechanism of epigenetic regulation in the maladaptation of uterine hemodynamics in gestational hypoxia and preeclampsia.

A new strategy to obtain IRMPD spectra of metal cationized ions by combining MALDI and IRMPD methods is presented here, in which graphene is selected to be the matrix to generate the metal cationized ions. [Arg + Rb]+ were studied here as the sample ions. Based on the experiential IRMPD spectrum and theoretical calculations, it is suggested that the ions of [Arg + Rb]+ generated here had more internal energies than those generated by ESI method reported previously by Williams et al. (J. Phys. Chem. A 2007, 111, 11759–11770). The method is also combined with the H/D exchange to identify the chromophores for the observed IR peak. The method provides a new way to obtain structural information of metal cationized ions, and also makes the identification of isomeric compounds feasible.

The large-conductance Ca 2+ -activated K + (BK Ca ) channel is of critical importance in pregnancy-mediated increase in uterine artery vasodilation and blood flow. The present study tested the hypothesis that active DNA demethylation plays a key role in pregnancy-induced reprogramming and upregulation of BK Ca channel β1 subunit (BKβ1) in uterine arteries. Uterine arteries were isolated from nonpregnant and near-term pregnant sheep. Pregnancy significantly increased the expression of ten-eleven translocation methylcytosine dioxygenase 1 (TET1) in uterine arteries. A half-palindromic estrogen response element was identified at the TET1 promoter, and estrogen treatment increased TET1 promoter activity and TET1 expression in uterine arteries. In accordance, pregnancy and steroid hormone treatment resulted in demethylation of BKβ1 promoter by increasing 5-hydroxymethylcytosine and decreasing 5-methylcytosine at the CpG in the Sp1 -380 binding site that is of critical importance in the regulation of the promoter activity and BKβ1 expression. Inhibition of TET1 with fumarate significantly decreased BKβ1 expression in uterine arteries of pregnant animals and blocked steroid hormone–induced upregulation of BKβ1. Functionally, fumarate treatment inhibited pregnancy and steroid hormone–induced increases in BK Ca channel current density and BK Ca channel–mediated relaxations. In addition, fumarate blocked pregnancy and steroid hormone–induced decrease in pressure-dependent myogenic tone of the uterine artery. The results demonstrate a novel mechanism of estrogen-mediated active DNA demethylation in reprogramming of BK Ca channel expression and function in the adaption of uterine circulation during pregnancy.

Introduction: Cytosine methylation at CpG dinucleotides is a chief mechanism in epigenetic modification of gene expression patterns. Previous studies demonstrated that increased CpG methylation of Sp1 sites at -268 and -346 of protein kinase C e promoter repressed the gene expression. Materials & Methods: The present study investigated the impact of CpG methylation on the Sp1 binding via molecular modeling and electrophoretic mobility shift assay. Each of the Sp1 sites contain two CpGs. Methylation of either CpG lowered the binding affinity of Sp1, whereas methylation of both CpGs produced a greater decrease in the binding affinity. Computation of van der Waals (VDW) energy of Sp1 in complex with the Sp1 sites demonstrated increased VDW values from one to two sites of CpG methylation. Molecular modeling indicated that single CpG methylation caused underwinding of the DNA fragment, with the phosphate groups at C1, C4 and C5 reoriented from their original positions. Methylation of both CpGs pinched the minor groove and increased the helical twist concomitant with a shallow, hydrophobic major groove. Additionally, double methylation eliminated hydrogen bonds on recognition helix residues located at positions -1 and 1, which were essential for interaction with O6/N7 of G-bases. Bonding from linker residues Arg565, Lys595 and Lys596 were also reduced. Methylation of single or both CpGs significantly affected hydrogen bonding from all three Sp1 DNA binding domains, demonstrating that the consequences of cytosine modification extend beyond the neighboring nucleotides. Results: The results indicate that cytosine methylation causes subtle structural alterations in Sp1 binding sites consequently resulting in inhibition of side chain interactions critical for specific base recognition and reduction of the binding affinity of Sp1.

MicroRNA (miR)-217 serves a pivotal role in the progression of colorectal cancer, renal cell carcinoma and glioma, however, the role of miR-217 in cervical cancer (CC) remains unclear. In the present study, the mechanism of miR-217 in cervical cancer was explored. The mRNA expression of miR-217 and mitogen-activated protein kinase 1 (MAPK1) were assessed using reverse transcription-quantitative polymerase chain reaction analysis. Cell Counting-Kit 8, wound-healing and Transwell assays were performed to detect cell viability, migration and invasion, respectively. Apoptosis and cell cycle were determined by flow cytometry. TargetScan 7.2 and dual-luciferase reporter assays were respectively used to determine miR-217 target genes and their binding capacities. The protein expression levels of MAPK1, phosphorylated (p)-extracellular signal-regulated kinase 1/2 (ERK1/2)/ERK1/2, Bcl-2, Bax and cleaved caspase-3 were quantified by western blotting. It was found that miR-217 was downregulated in patients with CC and in CC cells. The viability, migration and invasion of cells were suppressed by a miR-217 mimic. It was also found that apoptosis was increased and cell cycle was inhibited by the miR-217mimic, which was supported by changes in Bcl-2, Bax and cleaved caspase-3. MAPK1 was upregulated in patients with CC and was a target gene of miR-217. MAPK1 reversed the inhibition of miR-217 on cell viability, migration, invasion and apoptosis. The protein levels of MAPK1 and p-ERK1/2, which were higher in the mimic MAPK1 group than those in the control or mimic groups, were ameliorated by PD98059. The results of the present study demonstrated that miR-217 had an anti-CC effect and may be effectively used in the treatment of CC.

Background Helios is important for functional and phenotype stability of regulatory T cells (Tregs). However, the role of Helios in autoimmune diseases and its regulation remains unclear. This study aimed to investigate the role of Helios+ Tregs in myasthenia gravis (MG) and glucocorticoid-induced tumor necrosis factor receptor (GITR) and its ligand (GITRL) in the modulation of Helios. Method Multicolor flow cytometry was performed to analyze Helios+ Tregs in peripheral blood from MG patients and healthy donors (HDs). Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of soluble GITRL/GITR in plasma. Tregs were isolated via magnetic separation and treated with recombinant GITRL and GITR-Fc. Membrane GITRL on Tregs and expression of Helios and other markers (FOXP3, CD25, CD39, CTLA-4, PD-L1 and IL-10) involved in immunosuppressive activity were determined by flow cytometry. Result Both Helios+ Tregs and soluble GITR were decreased in generalized MG (GMG) patients (n = 14), compared with HDs (n = 14) and ocular MG (OMG) patients (n = 16). Helios+ Tregs possessed greater immunosuppressive capacity compared to Helios− Tregs. Further analysis indicates soluble GITR was negatively correlated with quantitative MG score and promoted Helios expression and enhanced function of Tregs independently of membrane GITRL. Conclusion This work demonstrates abnormal changes in Helios+ Tregs and soluble GITR in MG, as well as direct regulation of Helios by GITR in the context of Tregs. This work provides new insight into the role of GITR in the regulatory pathway of Helios and pathogenesis of MG.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has shown its great success in the qualitative analysis of a wide range of organic and biological molecules. However, its application in quantitative analysis is still limited by the difficulty in the availability of isotope-labeled internal standards. The present work investigates the relationship between the correlation coefficient of the peak intensities of analyte and candidate internal standard ions and the linearity of possible quantitative analysis. Based on the two analyte examples, ciprofloxacin and substance P, the results show that the performance of the selected nonisotope-labeled internal standard is greatly related to the correlation coefficient. A high positive correlation coefficient (>0.7) between the ions of analyte and candidate standard can result in a good linearity (R2 > 0.98) and vice versa. The results provide a new way to select nonisotope-labeled internal standards for MALDI analysis and thus can be potentially applied in the rapid quantitative mass spectrometry.

Malignant peripheral nerve sheath tumor (MPNST) is a rare tumor that can develop on the lining of nerves and within the network of nerve fibers in different organs, and it is commonly found in the head and neck, limbs, and trunk. These tumors can occur in patients of any age. They most commonly occur in adults aged 20 to 50 years; however, fewer cases of this tumor in children have been reported. To date, no neonatal case of MPNST in the nasal cavity has been reported. Here, we report the case of a 4-day-old female newborn who presented with a nasal mass that re-enlarged after surgery and was diagnosed as MPNST of the nasal cavity on the basis of pathological results. This is the first report of MPNST in the nasal cavity of a neonate. Differential diagnosis and treatment of nasal masses have been proposed in the related literature.

Chronic hypoxia during gestation suppresses large-conductance Ca2+-activated K+ (BKCa) channel function and impedes uterine arterial adaptation to pregnancy. This study tested the hypothesis that chronic hypoxia has a direct effect in upregulating DNA methyltransferase (DNMT) and epigenetically repressing BKCa channel beta-1 subunit (KCNMB1) expression in uterine arteries. Resistance-sized uterine arteries were isolated from near-term pregnant sheep maintained at ∼300 m above sea level or animals acclimatized to high-altitude (3,801 m) hypoxia for 110 days during gestation. For ex vivo hypoxia treatment, uterine arteries from normoxic animals were treated with 21.0% O2 or 10.5% O2 for 48 h. High-altitude hypoxia significantly upregulated DNMT3b expression and enzyme activity in uterine arteries. Similarly, ex vivo hypoxia treatment upregulated DNMT3b expression and enzyme activity that was blocked by a DNMT inhibitor 5-aza-2'-deoxycytidine (5-Aza). Of importance, 5-Aza inhibited hypoxia-induced hypermethylation of specificity protein (SP) 1 binding site at the KCNMB1 promoter and restored transcription factor binding to the KCNMB1 promoter, resulting in the recovery of KCNMB1 gene expression in uterine arteries. Furthermore, 5-Aza blocked the effect of hypoxia and rescued BKCa channel activity and reversed hypoxia-induced decrease in BKCa channel-mediated relaxations and increase in myogenic tone of uterine arteries. Collectively, these results suggest that chronic hypoxia during gestation upregulates DNMT expression and activity, resulting in hypermethylation and repression of KCNMB1 gene and BKCa channel function, impeding uterine arterial adaptation to pregnancy.

The geometric and thermodynamic stability of the M2C100 (M = La, Y, and Sc) series was systematically investigated using density functional theory calculations on the level of B3LYP/6-31G(d) ∼ Lanl2dz. In all the cases, M2@D5(285913)-C100 isomers are the lowest-energy species. However, carbide endohedral fullerenes M2C2@C1(230933)-C98 present excellent thermodynamic stabilities, except for those with La metal. The main product in electric arc experiments at temperatures lower than 3500 K for La2C100 should be La2@D5(285913)-C100, which was successfully synthesized previously; for Y and Sc, the predicted main products in these experiments should be M2C2@C1(230933)-C98. Further analysis of the geometric structures of the M2C100 series showed that the dimetallofullerenes M2@C100 have greater effects on the shapes of cages than M2C2@C98. These results provide some valuable guidance for the synthesis and characterization of large endohedral fullerenes including La, Y or Sc.

Phosphorus cluster anions were generated and investigated using collision-induced dissociation mass spectrometry. Results show that the primary dissociation channels of P 2 m + 1 − (3 ≤ m ≤ 11) are generally characterized by the loss of a P 2 unit, which distinguishes them from their counterpart cations. However, P 11 − and P 13 − are two exceptions. The former is characterized by the loss of a P 4 unit, and the latter is characterized by two parallel dissociation channels, which lead to the loss of P 2 and P 6 units. The dissociation behaviors of larger cluster anions of P 8 k + 1 − ( k = 3–5) are more complex. The primary dissociation pathway of P 25 − is the loss of a P 8 unit, and those of P 33 − and P 41 − are the loss of P 16 and P 2 units respectively. Theoretical calculations were performed for anions of P 2 m + 1 − (3 ≤ m ≤ 9). Results show that their dissociation channels characterized by the loss of the P 2 unit are more thermodynamically favored, which is generally consistent with the experimental results.

Previous in vivo study demonstrated that chronic hypoxia during gestation was associated with estrogen receptor-α (ER-α) gene repression in ovine uterine arteries. Yet, it remains undetermined whether hypoxia had a direct effect and if DNA methylation played a causal role in hypoxia-mediated ER-α gene repression. Thus, this study tested the hypothesis that prolonged hypoxia has a direct effect and increases promoter methylation resulting in ER-α gene repression and inhibition of estrogen-mediated adaptation of uterine vascular tone. Uterine arteries isolated from nonpregnant and pregnant sheep were treated ex vivo with 21.0% O 2 and 10.5% O 2 for 48 hours. Hypoxia significantly increased ER-α promoter methylation at both specificity protein-1 and upstream stimulatory factor binding sites, decreased specificity protein-1 and upstream stimulatory factor binding to the promoter, and suppressed ER-α expression in uterine arteries of pregnant animals. Of importance, the effects of hypoxia were blocked by a methylation inhibitor 5-aza-2′-deoxycytidine. In addition, hypoxia abrogated steroid hormone–mediated increase in ER-α expression and inhibited the hormone-induced increase in large-conductance Ca 2+ -activated K + channel activity and decrease in myogenic tone in uterine arteries of nonpregnant animals, which were reversed by 5-aza-2′-deoxycytidine. The results provide novel evidence of a direct effect of hypoxia on heightened promoter methylation that plays a causal role in ER-α gene repression and ablation of steroid hormone–mediated adaptation of uterine arterial large conductance Ca 2+ -activated K + channel activity and myogenic tone in pregnancy.

A class of adhesion protein that occurs in the membrane with both extracellular and intracellular domain and play vital role in maintaining multicellularity is TRASK, also called CUB-domain containing protein1, CD318 (CDCP1). Specifically, in the current study, documented aggressive grades of lung cancers and distant metastatic tissues were examined for protein interactions of Trask and compared with lung cancer variants in situ. The intracellular domain of Trask has the ability to undergo tyrosine phosphorylation and thereafter undergo increased genomic expression, as well as interact with cytoskeletal proteins in the cell periphery and other local signal transduction machinery to induce invadopodia formation and distant metastasis. We incorporated proximity ligation assay to examine protein interactions of Trask in metastatic lung cancer tissues and compare with advanced and low-grade lung cancers restricted to the primary site of origins. Here, we provide direct evidence that activated Trask, which is a phosphorylated form, binds with cytoskeletal proteins actin and spectrin. These interactions were not seen in locally growing lung cancer and cancer in situ. These interactions may be responsible for invadopodia formation and breaking free from a multicellular environment. Functional studies demonstrated interaction between Trask and the STOCs Orai1 and Stim1. Calcium release from internal stores was highest in metastatic lung cancers, suggesting this mechanism as an initial stimulus for the cells to respond chaotically to external growth factor stimulation, especially in aggressive metastatic variants of lung cancers. Recently, inhibitors of STOCs have been identified, and preclinical evidence may be obtained whether these drugs may be of benefit in preventing the deadly consequences of lung cancer.

Gestational hypoxia inhibits large conductance Ca

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. The results of the present study demonstrate that high expression of microRNA (miR)-137 and low expression of steroid receptor coactivator-3 (SRC3) had a significant negative correlation in 40 NSCLC tissue samples. In addition, cell colony formation and proliferation was significantly reduced in miR-137-transfected A549 and NCI-H838 cells compared with scramble-transfected NSCLC cell lines. miR-137 was identified to induce G1/S cell cycle arrest and dysregulate the mRNA expression of cell cycle-associated proteins (proliferating cell nuclear antigen, cyclin E, cyclin A1, cyclin A2 and p21) in NSCLC cells. Notably, miR-137 could significantly suppress SRC3 3' untranslated region (UTR) luciferase-reporter activity, an effect that was not detectable when the putative 3'-UTR target-site was mutated, further clarifying the molecular mechanisms underlying the role of miR-137 in NSCLC. In conclusion, the results of the present study suggest that miR-137 suppresses NSCLC cell proliferation by partially targeting SRC3.

To determine the long-term effect of maternal nicotine intake on the lung development of the offspring in adult life, we analyzed the alveolar structure, protein expression in the adult rat offspring lungs.We determined animal body weight (BW), lung weight (LW), lung/body weight ratio (L/BWR), lung volume (LV), radial alveolar count (RAC), alveolar septal thickness (AST) and expression of collagen, AT1R, AT2R, TGF-β1, pSmad3, Smad3 and CTGF proteins.Male offspring lung showed decreased RAC, thickened alveolar septa, increased collagen, AT1R, TGF-β1, pSmad3 and CTGF proteins. In contrast, female offspring lungs had reduced L/BWR, increased LV, and expression of AT2R, resulting in decreased AT1R to AT2R ratio.Maternal nicotine use during development programs abnormal lung development in male rats. This finding links maternal nicotine use to increased susceptibility to interstitial pulmonary fibrosis in adult male but not female offspring, indicating sex-dependent effects of developmental nicotine exposure.

Our previous study demonstrated that increased Ca 2+ -activated K + (BK Ca ) channel activity played a key role in the normal adaptation of reduced myogenic tone of uterine arteries in pregnancy. The present study tested the hypothesis that chronic hypoxia during gestation inhibits pregnancy-induced upregulation of BK Ca channel function in uterine arteries. Resistance-sized uterine arteries were isolated from nonpregnant and near-term pregnant sheep maintained at sea level (≈300 m) or exposed to high-altitude (3801 m) hypoxia for 110 days. Hypoxia during gestation significantly inhibited pregnancy-induced upregulation of BK Ca channel activity and suppressed BK Ca channel current density in pregnant uterine arteries. This was mediated by a selective downregulation of BK Ca channel β1 subunit in the uterine arteries. In accordance, hypoxia abrogated the role of the BK Ca channel in regulating pressure-induced myogenic tone of uterine arteries that was significantly elevated in pregnant animals acclimatized to chronic hypoxia. In addition, hypoxia abolished the steroid hormone-mediated increase in the β1 subunit and BK Ca channel current density observed in nonpregnant uterine arteries. Although the activation of protein kinase C inhibited BK Ca channel current density in pregnant uterine arteries of normoxic sheep, this effect was ablated in the hypoxic animals. The results demonstrate that selectively targeting BK Ca channel β1 subunit plays a critical role in the maladaption of uteroplacental circulation caused by chronic hypoxia, which contributes to the increased incidence of preeclampsia and fetal intrauterine growth restriction associated with gestational hypoxia.

Uterine vascular tone significantly decreases whereas uterine blood flow dramatically increases during pregnancy. However, the complete molecular mechanisms remain elusive. We hypothesized that increased Ca 2+ -activated K + (BK Ca ) channel activity contributes to the decreased myogenic tone of uterine arteries in pregnancy. Resistance-sized uterine arteries were isolated from nonpregnant and near-term pregnant sheep. Electrophysiological studies revealed a greater whole-cell K + current density in pregnant compared with nonpregnant uterine arteries. Tetraethylammonium and iberiotoxin inhibited K + currents to the same extent in uterine arterial myocytes. The BK Ca channel current density was significantly increased in pregnant uterine arteries. In accordance, tetraethylammonium significantly increased pressure-induced myogenic tone in pregnant uterine arteries and abolished the difference in myogenic responses between pregnant and nonpregnant uterine arteries. Activation of protein kinase C produced a similar effect to tetraethylammonium by inhibiting BK Ca channel activity and increasing myogenic tone in pregnant uterine arteries. Chronic treatment of nonpregnant uterine arteries with physiologically relevant concentrations of 17β-estradiol and progesterone caused a significant increase in the BK Ca channel current density. Western blot analyses demonstrated a significant increase of the β1, but not α, subunit of BK Ca channels in pregnant uterine arteries. In accordance, steroid treatment of nonpregnant uterine arteries resulted in an upregulation of the β1, but not α, subunit expression. The results indicate that the steroid hormone-mediated upregulation of the β1 subunit and BK Ca channel activity may play a key role in attenuating myogenic tone of the uterine artery in pregnancy.

BACKGROUND AND PURPOSE Antenatal nicotine exposure causes aberrant vascular reactivity and increased blood pressure in adult male rat offspring in a sex-dependent manner. The present study tested the hypothesis that maternal nicotine administration increases the production of reactive oxygen species resulting in the vascular hypertensive reactivity in male offspring. EXPERIMENTAL APPROACH Nicotine was administered to pregnant rats via subcutaneous osmotic minipumps throughout the gestation. The vascular oxidative damage and dysfunction were determined in 5-month-old male offspring. Contraction studies were performed on isolated aortas and their expression of NADPH oxidase (Nox2)/gp91 and nox4 determined by Western blot analysis. In addition, oxidative damage in the vessel wall was determined by measuring malondialdehyde concentrations, vascular superoxide production and SOD activity. KEY RESULTS Antenatal nicotine significantly increased angiotensin II-induced arterial contractions in the offspring. The exaggerated vascular contractions were inhibited by both apocynin (a Nox inhibitor) and tempol (a SOD mimetic) in a concentration-dependent manner. In addition, ACh-induced relaxations were impaired in aortas isolated from the nicotine-treated offspring, which were restored by both apocynin and tempol in a concentration-dependent manner. The nicotine treatment significantly decreased the superoxide dismutase activity and increased malondialdehyde, superoxide and nitrotyrosine protein levels in the vascular wall. Consistently, antenatal nicotine exposure significantly enhanced the protein expression of NADPH oxidase Nox2/gp91, but not Nox4 in the aorta. CONCLUSIONS AND IMPLICATIONS The present findings suggest that antenatal nicotine exposure results in the programming of heightened oxidative stress and vascular hypertensive reactivity via a Nox2-dependent mechanism, leading to an increased risk of hypertension in adult offspring. LINKED ARTICLE This article is commented on by Lim and Sobey, pp. 1397–1399 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01488.x

Pregnancy is associated with significantly decreased uterine vascular tone and increased uterine blood flow. The present study tested the hypothesis that the downregulation of actin polymerization plays a key role in reduced vascular tone of uterine arteries in the pregnant state. Uterine arteries were isolated from nonpregnant and near-term pregnant sheep. Activation of protein kinase C significantly increased the filamentous:globular actin ratio and contractions in the uterine arteries, which were inhibited by an actin polymerization inhibitor cytochalasin B. The basal levels of filamentous:globular actin were significantly higher in nonpregnant uterine arteries than those in near-term pregnant sheep. Prolonged treatment (48 hours) of nonpregnant sheep with 17β-estradiol (0.3 nmol/L) and progesterone (100.0 nmol/L) caused a significant decrease in the filamentous:globular actin. In accordance, the treatment of near-term pregnant sheep for 48 hours with an estrogen antagonist ICI 182 780 (10.0 μmol/L) and progesterone antagonist RU 486 (1.0 μmol/L) significantly increased the levels of filamentous:globular actin. Increased intraluminal pressure from 20 to 100 mm Hg resulted in an initial increase in uterine arterial diameter and vascular wall Ca 2+ concentrations, followed by a decrease in the diameter at a constant steady-state level of Ca 2+ . Cytochalasin B blocked pressure-induced myogenic constrictions without effect on vascular wall Ca 2+ levels and eliminated the differences in pressure-dependent myogenic tone between nonpregnant sheep and near-term pregnant sheep. The results indicate a key role of actin polymerization in protein kinase C–induced myogenic contractions and suggest a novel mechanism of sex steroid hormone–mediated downregulation of actin polymerization underlying the decreased myogenic tone of uterine arteries in pregnancy.

Previous studies in ovine uterine arteries have demonstrated that sex steroid hormones upregulate extracellular signal–regulated kinase 1/2 expression and downregulate the protein kinase C signaling pathway, resulting in the attenuated myogenic tone in pregnancy. The present study tested the hypothesis that chronic hypoxia during gestation inhibits the sex steroid-mediated adaptation of extracellular signal–regulated kinase 1/2 and protein kinase C signaling pathways and increases the myogenic tone of uterine arteries. Uterine arteries were isolated from nonpregnant and near-term pregnant sheep that had been maintained at sea level (≈300 m) or exposed to high-altitude (3801 m) hypoxia for 110 days. In contrast to the previous findings in normoxic animals, 17β-estradiol and progesterone failed to suppress protein kinase C–induced contractions and the pressure-induced myogenic tone in uterine arteries from hypoxic animals. Western analyses showed that the sex steroids lost their effects on extracellular signal–regulated kinase 1/2 expression and phospho- extracellular signal–regulated kinase 1/2 levels, as well as the activation of protein kinase C isozymes in uterine arteries of hypoxic ewes. In normoxic animals, pregnancy and the sex steroid treatments significantly increased uterine artery estrogen receptor-α and progesterone receptor B expression. Chronic hypoxia selectively downregulated estrogen receptor-α expression in uterine arteries of pregnant animals and eliminated the upregulation of estrogen receptor-α in pregnancy or by the steroid treatments observed in normoxic animals. The results demonstrate that, in the ovine uterine artery, chronic hypoxia in pregnancy inhibits the sex steroid hormone–mediated adaptation of decreased myogenic tone by downregulating estrogen receptor-α expression, providing a mechanism linking hypoxia and maladaptation of uteroplacental circulation and an increased risk of preeclampsia in pregnancy.

Background: Promoter polymorphisms in the apolipoprotein C3 (APOC3) gene are associated with hypertriglyceridemia (HTG), which is a complication of Type 2 Diabetes Mellitus (T2DM). Because there is a strong insulin resistance in T2DM, it is essential to investigate whether the APOC3 promoter variation affects plasma lipid profiles in the population with or without T2DM. Methods: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to detect the genotypes of APOC3. The levels of serum lipid profiles were also studied by biochemical methods. Results: In the control group, the prevalence of the −455CC or −482TT variants was higher in persons with HTG compared to those with normal triglyceride (NTG). Compared with the most common genotype, −455TT or −482CC, the variants had a significantly increased HTG risk in this population. However, a similar association was not found in the T2DM group. Conclusions: There was no relationship between promoter rare allele −455C or −482T of APOC3 gene and HTG in T2DM, but both the rare alleles contributed to an increased risk of HTG in the control group without T2DM.

Cocaine abuse is a significant problem among pregnant women. The present study tested the hypothesis that prenatal cocaine exposure impairs myogenic reactivity of coronary arteries in adult offspring. Pregnant rats received cocaine (30 mg kg −1 day −1 ) or saline from days 15 to 21 of gestational age, and experiments were conducted in 3-month-old offspring. In pressurized coronary septal arteries, the diameter and vessel wall intracellular Ca 2+ concentrations were measured simultaneously in the same tissue as a function of intraluminal pressure. Cocaine did not affect KCl-induced contractions of coronary arteries in either males or females but decreased the distensibility in male vessels. In male offspring, cocaine treatment resulted in a significant decease in pressure-dependent myogenic contractions. Inhibition of eNOS with N G -nitro- l -arginine did not alter the myogenic response in either saline control or cocaine-treated animals. In females, cocaine caused a significant increase in pressure-dependent myogenic contractions. N G -nitro- l -arginine did not affect the myogenic response in the control animals but blocked the cocaine-mediated effect. In both males and females, the pressure-induced increases in vessel wall Ca 2+ concentrations were not significantly different between cocaine and saline groups. The ratio of changes in the diameter to Ca 2+ concentrations in the pressurized arteries was significantly less in male but greater in female offspring after cocaine treatment. The results suggest that prenatal cocaine exposure causes reprogramming of coronary myogenic tone via changes in the Ca 2+ sensitivity in a sex-dependent manner, leading to an increased risk of dysfunction of coronary autoregulation in adult offspring.

Epidemiological studies have shown a clear association of adverse intrauterine environment and an increased risk of cardiovascular diseases and hypertension in adult life. The present study tested the hypothesis that prenatal cocaine exposure causes reprogramming of vascular reactivity, leading to an increased risk of hypertension in adult offspring. Pregnant rats received cocaine (30 mg kg −1 day −1 ) or saline from days 15 to 21 of gestational age, and experiments were conducted in 3-month-old offspring. Cocaine had no effect on the baseline blood pressure but significantly increased norepinephrine-stimulated blood pressure and decreased the baroreflex sensitivity in male but not female offspring. The cocaine treatment significantly increased norepinephrine-induced contractions in pressurized resistance–sized mesenteric arteries but not in aortas, which was primarily because of a loss of endothelial NO synthase–mediated inhibition and an enhanced Ca 2+ sensitivity in mesenteric arteries. In addition, the cocaine treatment significantly attenuated the endothelium-dependent relaxation in mesenteric arteries in male but not female offspring. Endothelial NO synthase protein levels in aortas but not mesenteric arteries were significantly increased in the cocaine-treated animals. However, cocaine significantly decreased phosphorylation levels of endothelial NO synthase in both aortas and mesenteric arteries. The results suggest that prenatal cocaine exposure programs vascular contractility via changes in endothelial NO synthase–regulated Ca 2+ sensitivity of myofilaments in the sex- and tissue-dependent manners in resistance arteries leading to an increased risk of hypertension in male offspring.

In the present study we tested the hypothesis that prenatal nicotine exposure increases heart susceptibility to ischemia/reperfusion (I/R) injury in adult offspring. Nicotine was administered to pregnant rats via subcutaneous osmotic minipumps throughout gestation. Nicotine treatment resulted in a rapid and transient decrease in food-intake and a moderate decrease in maternal body weight gain. Hearts were isolated from adult male and female offspring and subjected to I/R in a Langendorff preparation. Nicotine significantly attenuated left ventricle (LV) developed pressure, heart rate, and coronary flow rate in female but not male hearts at baseline. Additionally, nicotine significantly increased LV infarct size and attenuated postischemic recovery of LV function in both male and female offspring with more pronounced effects in females. In female but not male hearts, nicotine significantly decreased the postischemic coronary flow rate. However, coronary nitric oxide release was decreased in male but not female hearts. Caspase-3, -8, and -9 levels were not significantly changed in either female or male hearts. However, nicotine caused a significant decrease in protein levels of protein kinase (PK) Cepsilon in both male and female hearts and a decrease in PKCdelta levels in female hearts only. Control studies of maternal food restriction showed that a moderate decrease in maternal body weight gain had no effect on female hearts but significantly improved postischemic recovery of LV function in male hearts. The results suggest that prenatal nicotine exposure causes in utero programming of the PKC isozyme gene expression pattern in the developing heart and increases heart susceptibility to I/R injury in adult offspring.

The aim of the present study was to investigate the effects of human papillomavirus (HPV) infection on the gynecological disease of vaginitis and to demonstrate how the small interfering RNA (siRNA) method may be used for HPV prevention in the clinic. Human vaginal epithelial cells were transfected with HPV-11 L1 expression vector and siRNA-HPV-11 L1 vectors and a control group was transfected with scrambled siRNA. Cell proliferation in each group was analyzed using the MTT assay and the expression of apoptosis-associated proteins was measured by western blot analysis. Compared with the control group, HPV-11 L1 mRNA and protein levels were significantly increased following transfection with the HPV-11 L1 expression vector in cells (P