Despite the success of interventional processes such as drug-eluting stents, complete prevention of restenosis is still hindered by impaired or delayed endothelialization or both. Here, we report that 1H-pyrrole-2,5-dione-based small molecule-generated mesenchymal stem cell-derived functional endothelial cells (MDFECs) facilitated rapid transmural coverage of injured blood vessels.
Small molecules that induced CD31 expression were screened by principal component analysis (PCA). Rat mesenchymal stem cells (MSCs) were treated with selected small molecules for up to 16 days, and the expression levels of CD90 and CD31 were examined by immunocytochemistry. In vitro functional assays of MDFECs, including tube formation assays and nitric oxide production assays, were performed. After MDFECs (intravenous, 3×106 cells per animal) were injected into balloon-injured rats, neointima formation was monitored for up to 21 days. The endothelial coverage of denuded blood vessels was evaluated by Evans Blue staining. The functionality of repaired blood vessels was evaluated by measuring vasorelaxation and hemodynamic changes. Additionally, derivatives of the selected small molecules were examined for their ability to induce endothelial markers.
PCA indicated that 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione effectively induced MDFECs. MDFECs inhibited the neointima formation of denuded blood vessels by facilitating more rapid endothelialization. Further examination indicated that derivatives with a 1H-pyrrole-2,5-dione moiety are important for initiating the endothelial cell differentiation of MSCs.
Small molecules with 1H-pyrrole-2,5-dione as a core structure have great potential to improve the efficacy of MSC-based cell therapy for vascular diseases, such as atherosclerosis and restenosis.
Electronic supplementary material
The online version of this article (doi:10.1186/s13287-015-0170-6) contains supplementary material, which is available to authorized users.
AIM: To identify alkyl hydroperoxide reductase subunit C (AhpC) homologs in Bacillus subtilis (B. subtilis) and to characterize their structural and biochemical properties. AhpC is responsible for the detoxification of reactive oxygen species in bacteria.
METHODS: Two AhpC homologs (AhpC_H1 and AhpC_H2) were identified by searching the B. subtilis database; these were then cloned and expressed in Escherichia coli. AhpC mutants carrying substitutions of catalytically important Cys residues (C37S, C47S, C166S, C37/47S, C37/166S, C47/166S, and C37/47/166S for AhpC_H1; C52S, C169S, and C52/169S for AhpC_H2) were obtained by site-directed mutagenesis and purified, and their structure-function relationship was analyzed. The B. subtilis ahpC genes were disrupted by the short flanking homology method, and the phenotypes of the resulting AhpC-deficient bacteria were examined.
RESULTS: Comparative characterization of AhpC homologs indicates that AhpC_H1 contains an extra C37, which forms a disulfide bond with the peroxidatic C47, and behaves like an atypical 2-Cys AhpC, while AhpC_H2 functions like a typical 2-Cys AhpC. Tryptic digestion analysis demonstrated the presence of intramolecular Cys37-Cys47 linkage, which could be reduced by thioredoxin, resulting in the association of the dimer into higher-molecular-mass complexes. Peroxidase activity analysis of Cys→Ser mutants indicated that three Cys residues were involved in the catalysis. AhpC_H1 was resistant to inactivation by peroxide substrates, but had lower activity at physiological H2O2 concentrations compared to AhpC_H2, suggesting that in B. subtilis, the enzymes may be physiologically functional at different substrate concentrations. The exposure to organic peroxides induced AhpC_H1 expression, while AhpC_H1-deficient mutants exhibited growth retardation in the stationary phase, suggesting the role of AhpC_H1 as an antioxidant scavenger of lipid hydroperoxides and a stress-response factor in B. subtilis.
CONCLUSION: AhpC_H1, a novel atypical 2-Cys AhpC, is functionally distinct from AhpC_H2, a typical 2-Cys AhpC.
Cysteine-dependent peroxidase; Thioredoxin; Thiol peroxidase; Peroxiredoxin; Alkyl hydroperoxide; Ortholog; Bacillus subtilis; Oxidative stress
Alzheimer’s disease (AD) is an inexorable neurodegenerative disease that commonly occurs in the elderly. The cognitive impairment caused by AD is associated with abnormal accumulation of amyloid-β (Aβ) and hyperphosphorylated tau, which are accompanied by inflammation. Neural stem cells (NSCs) are self-renewing, multipotential cells that differentiate into distinct neural cells. When transplanted into a diseased brain, NSCs repair and replace injured tissues after migration toward and engraftment within lesions. We investigated the therapeutic effects in an AD mouse model of human NSCs (hNSCs) that derived from an aborted human fetal telencephalon at 13 weeks of gestation. Cells were transplanted into the cerebral lateral ventricles of neuron-specific enolase promoter-controlled APPsw-expressing (NSE/APPsw) transgenic mice at 13 months of age.
Implanted cells extensively migrated and engrafted, and some differentiated into neuronal and glial cells, although most hNSCs remained immature. The hNSC transplantation improved spatial memory in these mice, which also showed decreased tau phosphorylation and Aβ42 levels and attenuated microgliosis and astrogliosis. The hNSC transplantation reduced tau phosphorylation via Trk-dependent Akt/GSK3β signaling, down-regulated Aβ production through an Akt/GSK3β signaling-mediated decrease in BACE1, and decreased expression of inflammatory mediators through deactivation of microglia that was mediated by cell-to-cell contact, secretion of anti-inflammatory factors generated from hNSCs, or both. The hNSC transplantation also facilitated synaptic plasticity and anti-apoptotic function via trophic supplies. Furthermore, the safety and feasibility of hNSC transplantation are supported.
These findings demonstrate the hNSC transplantation modulates diverse AD pathologies and rescue impaired memory via multiple mechanisms in an AD model. Thus, our data provide tangible preclinical evidence that human NSC transplantation could be a safe and versatile approach for treating AD patients.
Electronic supplementary material
The online version of this article (doi:10.1186/s13024-015-0035-6) contains supplementary material, which is available to authorized users.
Alzheimer’s disease; Human neural stem cells; Transplantation; Trophic factors; Glycogen synthase kinase 3β (GSK3β); Anti-inflammation
Microglia are brain resident macrophages rapidly responding to various stimuli to exert appropriate inflammatory responses. Although they have recently been exploited as an attractive candidate for imaging neuroinflammation, it is still difficult to visualize them at the cellular and molecular levels. Here we imaged activated microglia by establishing intracranial window chamber (ICW) in a mouse model of focal cerebral ischemia by using two-photon microscopy (TPM), in vivo. Intravenous injection of fluorescent antibodies allowed us to detect significantly elevated levels of Iba-1 and CD68 positive activated microglia in the ipsilateral compared to the contralateral side of the infarct. We further observed that indomethacin, a non-steroidal anti-inflammatory drug significantly attenuated CD68-positive microglial activation in ICW, which was further confirmed by qRT-PCR biochemical analyses. In conclusion, we believe that in vivo TPM imaging of ICW would be a useful tool to screen for therapeutic interventions lowering microglial activation hence neuroinflammation.
(000.1430) Biology and medicine; (180.2520) Fluorescence microscopy; (180.4315) Nonlinear microscopy
Fluid overload is a well-known predictor of mortality in patients with acute kidney injury (AKI). Multifrequency bioimpedance analysis (MF-BIA) is a promising tool for quantifying volume status. However, few studies have analyzed the effect of MF-BIA-defined volume status on the mortality of critically ill patients with AKI. This retrospective medical research study aimed to investigate this issue.
We retrospectively reviewed the medical records of patients with AKI who underwent continuous veno-venous hemodiafiltration (CVVHDF) from Jan. 2013 to Feb. 2014. Female patients were excluded to control for sex-based differences. Volume status was measured using MF-BIA (Inbody S20, Seoul, Korea) at the time of CVVHDF initiation, and volume parameters were adjusted with height squared (H2). Binary logistic regression analyses were performed to test independent factors for prediction of in-hospital mortality.
A total of 208 male patients were included in this study. The mean age was 65.19±12.90 years. During the mean ICU stay of 18.29±27.48 days, 40.4% of the patients died. The in-hospital mortality rate increased with increasing total body water (TBW)/H2 quartile. In the multivariable analyses, increased TBW/H2 (OR 1.312(1.009-1.705), p=0.043) and having lower serum albumin (OR 0.564(0.346-0.919, p=0.022) were independently associated with higher in-hospital mortality. When the intracellular water (ICW)/H2 or extracellular water (ECW)/H2 was adjusted instead of the TBW/H2, only excess ICW/H2 was independently associated with increased mortality (OR 1.561(1.012-2.408, p=0.044).
MF-BIA-defined excess TBW/H2 and ICW/H2 are independently associated with higher in-hospital mortality in male patients with AKI undergoing CVVHDF.
This study aimed for a collaborative evaluation of variability in the target volumes for glioblastoma, determined and contoured by different radiotherapy (RT) facilities in Korea.
Fifteen panels of radiation oncologists from independent institutions contoured the gross target volumes (GTVs) and clinical target volumes (CTVs) for 3-dimensional conformal RT or intensity-modulated RT on each simulation CT images, after scrutinizing the enhanced T1-weighted and T2-weighted-fluid-attenuated inversion recovery MR images of 9 different cases of glioblastoma. Degrees of contouring agreement were analyzed by the kappa statistics. Using the algorithm of simultaneous truth and performance level estimation (STAPLE), GTVSTAPLE and CTVSTAPLE contours were derived.
Contour agreement was moderate (mean kappa 0.58) among the GTVs and was substantial (mean kappa 0.65) among the CTVs. However, each panels’ GTVs and modification of CTVs regarding anatomical structures varied. Three-fourth of contoured panels’ CTVs encompassed the peritumoral areas of T2-high signal intensity (T2-HSI). Nine of nine GTVSTAPLE encompased the surgical cavity and the T1-enhanced lesions. Eight of nine CTVSTAPLE encompassed the peritumoral T2-HSI area. The median MARGIN90 and the median MARGIN95 were 1.4 cm and 1.5 cm, respectively.
Moderate to substantial agreement existed in target volumes for 3-dimensional or intensity-modulated RT determined by radiation oncologists in Korea. According to the estimated consensus contours, the initial CTV encompassed the GTV with margin less than 2.0 cm and the whole peritumoral areas of T2-HSI. The findings of our study propose the need for further studies and modified guidelines.
Electronic supplementary material
The online version of this article (doi:10.1186/s13014-015-0439-z) contains supplementary material, which is available to authorized users.
Glioblastoma; Target volume; Peritumoral edema; STAPLE; Margin
This study was conducted in order to validate the radiosensitization effect of valproic acid, a biologically available histone deacetylase inhibitor, for fractionated radiation.
Materials and Methods
Radiosensitization effect of valproic acid was tested for the A549 cell line and U87MG cell line in vitro. Fractionated irradiation of 12 Gy in four fractions was administered on D2-5 with valproic acid, 150 mg/Kg, ip, bid for six consecutive days (D1-6) to A549 and U87MG tumors implanted in BALB/c-nude mice. A growth delay curve was formulated.
Radiosensitization effect of valproic acid was found for both cell lines; A549 at 1.5 mM and 3.0 mM concentration and U87MG at 3.0 mM concentration. In growth delay analysis, a statistically significant radiosensitization effect was observed for both tumors (p < 0.001 for both tumors). Difference for change in slope for control and valproic acid versus radiotherapy and radiotherapy plus valproic acid showed borderline significance for the U87MG cell line (p=0.065), indicating beyond additive effect, whereas this difference was statistically insignificant for A549 tumor (p=0.951), indicating additive effect.
Results of this study indicate that a radiosensitizing effect for fractionated radiotherapy of valproic acid for A549 and U87MG tumors in vivo is evident and that it may be more than additive for U87MG tumors. Further exploitation of histone deacetylase inhibitors in clinical trials is warranted.
Radiation tolerance; Valproic acid; Radiation; Glioblastoma; Non-small-cell lung carcinoma
Apocarotenoids, like the C13-norisoprenoids, are natural compounds that contribute to the flavor and/or aroma of flowers and foods. They are produced in aromatic plants—like raspberries and roses—by the enzymatic cleavage of carotenes. Due to their pleasant aroma and flavour, apocarotenoids have high commercial value for the cosmetic and food industry, but currently their production is mainly assured by chemical synthesis. In the present study, a Saccharomyces cerevisiae strain that synthesizes the apocarotenoid β-ionone was constructed by combining integrative vectors and high copy number episomal vectors, in an engineered strain that accumulates FPP.
Integration of an extra copy of the geranylgeranyl diphosphate synthase gene (BTS1), together with the carotenogenic genes crtYB and crtI from the ascomycete Xanthophyllomyces dendrorhous, resulted in carotenoid producing cells. The additional integration of the carotenoid cleavage dioxygenase gene from the plant Petunia hybrida (PhCCD1) let to the production of low amounts of β-ionone (0.073 ± 0.01 mg/g DCW) and changed the color of the strain from orange to yellow. The expression of the crtYB gene from a high copy number plasmid in this former strain increased β-ionone concentration fivefold (0.34 ± 0.06 mg/g DCW). Additionally, the episomal expression of crtYB together with the PhCCD1 gene in the same vector resulted in a final 8.5-fold increase of β-ionone concentration (0.63 ± 0.02 mg/g DCW). Batch fermentations with this strain resulted in a final specific concentration of 1 mg/g DCW at 50 h, which represents a 15-fold increase.
An efficient β-ionone producing yeast platform was constructed by combining integrative and episomal constructs. By combined expression of the genes BTS1, the carotenogenic crtYB, crtI genes and the plant PhCCD1 gene—the highest β-ionone concentration reported to date by a cell factory was achieved. This microbial cell factory represents a starting point for flavor production by a sustainable and efficient process that could replace current methods.
Electronic supplementary material
The online version of this article (doi:10.1186/s12934-015-0273-x) contains supplementary material, which is available to authorized users.
Metabolic engineering; Carotenoids; Apocarotenoids; Saccharomyces cerevisiae
Odontogenic carcinosarcoma is an extremely rare malignant odontogenic tumor with only a few reported cases. It is characterized by a true mixed tumor showing malignant cytology of both epithelial and mesenchymal components. It has been assumed to arise from pre-existing lesions such as ameloblastoma, ameloblastic fibroma, and ameloblastic fibrosarcoma. To date, the reported cases have exhibited considerably aggressive clinical behavior. The case of an odontogenic carcinosarcoma in the mandible of a 61-year-old male is described herein. The tumor destroyed the cortex of the mandible and invaded the adjacent tissues. Treatment was performed by surgical resection and reconstruction. The purposes of this article are to introduce odontogenic carcinosarcoma through this case study, to distinguish it from related diseases and to discuss features of the tumor in the existing literature.
Carcinosarcoma; Mixed tumor; Ameloblastoma; Fibrosarcoma; Odontogenic tumor
Analysis of gene expression patterns in normal tissues and their perturbations in tumors can help to identify the functional roles of oncogenes or tumor suppressors and identify potential new therapeutic targets. Here, gene expression correlation networks were derived from 92 normal human lung samples and patient-matched adenocarcinomas. The networks from normal lung show that NKX2-1 is linked to the alveolar type 2 lineage, and identify PEBP4 as a novel marker expressed in alveolar type 2 cells. Differential correlation analysis shows that the NKX2-1 network in tumors includes pathways associated with glutamate metabolism, and identifies Vaccinia-related kinase (VRK1) as a potential drug target in a tumor-specific mitotic network. We show that VRK1 inhibition cooperates with inhibition of PARP signaling to inhibit growth of lung tumor cells. Targeting of genes that are recruited into tumor mitotic networks may provide a wider therapeutic window than that seen by inhibition of known mitotic genes.
O-GlcNAcylation is a reversible post-translational modification. O-GlcNAc addition and removal is catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. More recent evidence indicates that regulation of O-GlcNAcylation is important for inflammatory diseases and tumorigenesis. In this study, we revealed that O-GlcNAcylation was increased in the colonic tissues of dextran sodium sulfate (DSS)-induced colitis and azoxymethane (AOM)/DSS-induced colitis-associated cancer (CAC) animal models. Moreover, the O-GlcNAcylation level was elevated in human CAC tissues compared with matched normal counterparts. To investigate the functional role of O-GlcNAcylation in colitis, we used OGA heterozygote mice, which have an increased level of O-GlcNAcylation. OGA+/− mice have higher susceptibility to DSS-induced colitis than OGA+/+ mice. OGA+/− mice exhibited a higher incidence of colon tumors than OGA+/+ mice. In molecular studies, elevated O-GlcNAc levels were shown to enhance the activation of NF-κB signaling through increasing the binding of RelA/p65 to its target promoters. We also found that Thr-322 and Thr352 in the p65-O-GlcNAcylation sites are critical for p65 promoter binding. These results suggest that the elevated O-GlcNAcylation level in colonic tissues contributes to the development of colitis and CAC by disrupting regulation of NF-κB-dependent transcriptional activity.
O-GlcNAcylation; O-GlcNAcase; colitis; colitis-associated cancer
Tubulointerstitial injury plays an important role in the progression of immunoglobulin A nephropathy (IgAN), and neutrophil gelatinase-associated lipocalin (NGAL) is among the most sensitive tubular biomarkers. We investigated whether serum or urine NGAL predicts prognosis in patients with IgAN.
The present study enrolled patients with biopsy-proven IgAN from January 2005 to December 2010, whose serum and urine samples at the time of kidney biopsy were preserved by freezing. We retrospectively reviewed patient clinical data and followed patients until October 2012. Serum and urine NGAL levels were measured using an enzyme-linked immunosorbent assay kit. Renal progression was defined as an estimated glomerular filtration rate decline by > 50% or progression to end-stage renal disease.
There were 121 patients enrolled in this study. During the median follow-up period of 41.49 months, renal progression was found in nine patients (7.4%). Serum or urine NGAL alone could not predict renal progression; however, when serum and urine NGAL levels were combined, belonging to the high NGAL group independently predicted renal progression (hazard ratio [HR], 5.56; 95% confidence interval [CI], 1.42 to 21.73; p = 0.014), along with tubular damage graded according to the Oxford classification as T2 (HR, 8.79; 95% CI, 2.01 to 38.51; p = 0.004). In addition, a Kaplan-Meier curve of renal survival showed significantly higher renal progression in patients in the high NGAL group (log rank, p = 0.004).
In patients with IgAN, high serum and urine NGAL levels at the time of kidney biopsy predict renal progression.
Glomerulonephritis, IGA; Prognosis; Serum neutrophil gelatinase-associated lipocalin; Tubular biomarker; Urine neutrophil gelatinase-associated lipocalin
Nitric oxide (NO)-releasing nanoparticles (NPs) have emerged as a wound healing enhancer and a novel antibacterial agent that can circumvent antibiotic resistance. However, the NO release from NPs over extended periods of time is still inadequate for clinical application. In this study, we developed NO-releasing poly(lactic-co-glycolic acid)-polyethylenimine (PEI) NPs (NO/PPNPs) composed of poly(lactic-co-glycolic acid) and PEI/diazeniumdiolate (PEI/NONOate) for prolonged NO release, antibacterial efficacy, and wound healing activity. Successful preparation of PEI/NONOate was confirmed by proton nuclear magnetic resonance, Fourier transform infrared spectroscopy, and ultraviolet/visible spectrophotometry. NO/PPNPs were characterized by particle size, surface charge, and NO loading. The NO/PPNPs showed a prolonged NO release profile over 6 days without any burst release. The NO/PPNPs exhibited potent bactericidal efficacy against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa concentration-dependently and showed the ability to bind on the surface of the bacteria. We also found that the NO released from the NO/PPNPs mediates bactericidal efficacy and is not toxic to healthy fibroblast cells. Furthermore, NO/PPNPs accelerated wound healing and epithelialization in a mouse model of a MRSA-infected wound. Therefore, our results suggest that the NO/PPNPs presented in this study could be a suitable approach for treating wounds and various skin infections.
nitric oxide-releasing nanoparticles; PLGA; PEI; antimicrobial; wound healing
Textural features of edge-enhanced fluence were analysed to quantify modulation degree of volumetric modulated arc therapy (VMAT) plans.
Twenty prostate and twenty head and neck VMAT plans were retrospectively selected. Fluences of VMAT plans were generated by integration of monitor units shaped by multi-leaf collimators (MLCs) at each control point. When generating fluences, the values of pixels representing MLC tips were doubled to prevent smearing out of small or irregular fields (edge-enhancement). Six kinds of textural features, including angular second moment, inverse difference moment, contrast, variance, correlation and entropy, were calculated with particular displacement distances (d) of 1, 5 and 10. Plan delivery accuracy was evaluated by gamma-index method, mechanical parameter differences between plan and delivery and differences in dose-volumetric parameters between plan and delivery. Spearman’s correlation coefficients (rs) were calculated between the values of textural features and VMAT delivery accuracy.
The rs values of contrast (d = 1) with edge-enhancement to global gamma passing rates with 2%/2 mm, 1%/2 mm and 2%/1 mm were 0.546 (p < 0.001), 0.744 (p < 0.001) and 0.487 (p = 0.001), respectively. Those with local 2%/2 mm, 1%/2 mm and 2%/1 mm were 0.588, 0.640 and 0.644, respectively (all with p < 0.001). The rs values of contrast (d = 1) to MLC and gantry angle errors were -0.853 and 0.655, respectively (all with p < 0.001). The contrast (d = 1) showed statistically significant rs values in 11 dose-volumetric parameter differences from a total of 35 cases, and generally showed better correlations to plan delivery accuracy than did previously suggested textural features with non-edge-enhanced fluences, as well as conventional modulation indices.
Contrast (d = 1) with edge-enhanced fluences could be used as modulation index for VMAT.
Texture analysis; Volumetric modulated arc therapy; Modulation index; Fluence
Lymphomatoid granulomatosis (LYG) is an angiocentric and angiodestructive neoplastic proliferation of B and T lymphocytes commonly involving the lungs. Epstein-Barr virus is commonly detected in lesional cells. We report a case of a 54-year-old female with underlying monoclonal gammopathy of unknown significance who presented with a 4 week history of dyspnea and cough. Computed tomography scan of the chest showed multiple lung nodules as well as endobronchial narrowing causing atelectasis at the left upper lobe. Bronchoscopic findings revealed obstruction at the lingula segment due to endobronchial mass as a rare presentation. Bronchoscopic biopsy was diagnosed with LYG grade 1. After treatment, the endobronchial mass and lung lesions were completely resolved. However, the patient eventually evolved to malignant lymphoma after 1 year.
Lymphomatoid Granulomatosis; Lymphoma
The melanin-inducing properties of cirsimaritin were investigated in murine B16F10 cells. Cirsimaritin is an active flavone with methoxy groups, which is isolated from the branches of Lithocarpus dealbatus. Tyrosinase activity and melanin content in murine B16F10 melanoma cells were increased by cirsimaritin in a dose-dependent manner. Western blot analysis revealed that tyrosinase, tyrosinase-related protein (TRP) 1, TRP2 protein levels were enhanced after treatment with cirsimaritin for 48 h. Cirsimaritin also upregulated the expression of microphthalmia-associated transcription factor (MITF) after 24 h of treatment. Furthermore, cirsimaritin induced phosphorylation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) in a dose-dependent manner after treatment for 15 min. The cirsimaritin-mediated increase of tyrosinase activity was significantly attenuated by H89, a cAMP-dependent protein kinase A inhibitor. These findings indicate that cirsimaritin stimulates melanogenesis in B16F10 cells by activation of CREB as well as upregulation of MITF and tyrosinase expression, which was activated by cAMP signaling. Finally, the melanogenic effect of cirsimaritin was confirmed in human epidermal melanocytes. These results support the putative application of cirsimaritin in ultraviolet photoprotection and hair coloration treatments.
cirsimaritin; melanogenesis; melanoma; tyrosinase; MITF
Ethyl pyruvate (EP), a simple aliphatic ester of pyruvic acid, has been shown to have antiinflammatory effects and to confer protective effects in various pathological conditions. Recently, a number of studies have reported EP inhibits high mobility group box 1 (HMGB1) secretion and suggest this might contribute to its antiinflammatory effect. Since EP is used in a calcium-containing balanced salt solution (Ringer solution), we wondered if EP directly chelates Ca2+ and if it is related to the EP-mediated suppression of HMGB1 release. Calcium imaging assays revealed that EP significantly and dose-dependently suppressed high K+-induced transient [Ca2+]i surges in primary cortical neurons and, similarly, fluorometric assays showed that EP directly scavenges Ca2+ as the peak of fluorescence emission intensities of Mag-Fura-2 (a low-affinity Ca2+ indicator) was shifted in the presence of EP at concentrations of ≥7 mmol/L. Furthermore, EP markedly suppressed the A23187-induced intracellular Ca2+ surge in BV2 cells and, under this condition, A23187-induced activations of Ca2+-mediated kinases (protein kinase Cα and calcium/calmodulin-dependent protein kinase IV), HMGB1 phosphorylation and subsequent secretion of HMGB1 also were suppressed. (A23187 is a calcium ionophore and BV2 cells are a microglia cell line.) Moreover, the above-mentioned EP-mediated effects were obtained independent of cell death or survival, which suggests that they are direct effects of EP. Together, these results indicate that EP directly chelates Ca2+, and that it is, at least in part, responsible for the suppression of HMGB1 release by EP.
HHT shows clinical variability within and between families. Organ site and prevalence of arteriovenous malformations (AVMs) depend on the HHT causative gene and on environmental and genetic modifiers. We tested whether variation in the functional ENG allele, inherited from the unaffected parent, alters risk for pulmonary AVM in HHT1 mutation carriers who are ENG haploinsufficient. Genetic association was found between rs10987746 of the wild type ENG allele and presence of pulmonary AVM [relative risk = 1.3 (1.0018–1.7424)]. The rs10987746-C at-risk allele associated with lower expression of ENG RNA in a panel of human lymphoblastoid cell lines (P = 0.004). Moreover, in angiogenically active human lung adenocarcinoma tissue, but not in uninvolved quiescent lung, rs10987746-C was correlated with expression of PTPN14 (P = 0.004), another modifier of HHT. Quantitative TAQMAN expression analysis in a panel of normal lung tissues from 69 genetically heterogeneous inter-specific backcross mice, demonstrated strong correlation between expression levels of Eng, Acvrl1, and Ptpn14 (r2 = 0.75–0.9, P < 1 × 10−12), further suggesting a direct or indirect interaction between these three genes in lung in vivo. Our data indicate that genetic variation within the single functional ENG gene influences quantitative and/or qualitative differences in ENG expression that contribute to risk of pulmonary AVM in HHT1, and provide correlative support for PTPN14 involvement in endoglin/ALK1 lung biology in vivo. PTPN14 has been shown to be a negative regulator of Yap/Taz signaling, which is implicated in mechanotransduction, providing a possible molecular link between endoglin/ALK1 signaling and mechanical stress. EMILIN2, which showed suggestive genetic association with pulmonary AVM, is also reported to interact with Taz in angiogenesis. Elucidation of the molecular mechanisms regulating these interactions in endothelial cells may ultimately provide more rational choices for HHT therapy.
endoglin; ACVRL1; PTPN14; EMILIN2; lung; arteriovenous malformations
MicroRNAs (miRs) are small, non-coding RNAs that function to post-transcriptionally regulate gene expression. First transcribed as long primary miR transcripts (pri-miRs), they are enzymatically processed in the nucleus by Drosha into hairpin intermediate miRs (pre-miRs) and further processed in the cytoplasm by Dicer into mature miRs where they regulate cellular processes following activation by a variety of signals such as those stimulated by β-adrenergic receptors (βARs). Initially discovered to desensitize βAR signaling, β-arrestins are now appreciated to transduce multiple effector pathways independent of G protein-mediated second messenger accumulation, a concept known as biased signaling. We previously showed that the β-arrestin-biased βAR agonist carvedilol activates cellular pathways in the heart.
Here, we tested whether carvedilol could activate β-arrestin-mediated miR maturation, thereby providing a novel potential mechanism for its cardioprotective effects.
Methods and Results:
In human cells and mouse hearts, carvedilol upregulates a subset of mature and pre-miRs but not their pri-miRs in β1AR-, G protein-coupled receptor kinase 5/6- and β-arrestin1-dependent manner. Mechanistically, β-arrestin1 regulates miR processing by forming a nuclear complex with hnRNPA1 and Drosha on pri-miRs.
Our findings indicate a novel function for β1AR-mediated β-arrestin1 signaling activated by carvedilol in miR biogenesis, which may be linked, in part, to its mechanism for cell survival.
β-arrestin-biased β-adrenergic receptor signaling; carvedilol; heart disease; microRNA biogenesis
To protect patient autonomy when confronting death, the importance of advance directives (ADs) has recently became an issue and gradually accepted in Korea. However, in real practice, ADs were not completed by patients but their families in most cases. To analyze the current situation of performing ADs, we reviewed medical charts of 214 terminal cancer patients admitted to the hospice center from October 2012 to September 2013. Seventy-six (35.5%) patients completed ADs. All ADs were completed by patients themselves. The most common reason for not completing ADs was poor physical and/or mental condition. As a proxy, the majority of patients preferred their spouses (55.3%). Few patients wanted life sustaining treatment (1.3%), however palliative sedation was accepted in 89.5%. The median timing of ADs after admission was three (0-90) days, and duration of survival since ADs was 22 (1-340) days. In conclusion, approximately one third of terminal cancer patients completed ADs by themselves. Considering that patient's poor condition is the main reason for not completing ADs, earlier discussion regarding ADs is necessary to enhance patients' participation.
Advance Directives; Hospice Care; Neoplasms
Tailoring of semiconducting metal oxide nanostructures, which possess controlled pore size and concentration, is of great value to accurately detect various volatile organic compounds in exhaled breath, which act as potential biomarkers for many health conditions. In this work, we have developed a very simple and robust route for controlling both the size and distribution of spherical pores in electrospun WO3 nanofibers (NFs) via a sacrificial templating route using polystyrene colloids with different diameters (200 nm and 500 nm). A tentacle-like structure with randomly distributed pores on the surface of electrospun WO3 NFs were achieved, which exhibited improved surface area as well as porosity. Porous WO3 NFs with enhanced surface area exhibited high gas response (Rair/Rgas = 43.1 at 5 ppm) towards small and light H2S molecules. In contrast, porous WO3 NFs with maximized pore diameter showed a high response (Rair/Rgas = 2.8 at 5 ppm) towards large and heavy acetone molecules. Further enhanced sensing performance (Rair/Rgas = 65.6 at 5 ppm H2S) was achieved by functionalizing porous WO3 NFs with 0.1 wt% non-oxidized graphene (NOGR) flakes by forming a Schottky barrier (ΔΦ = 0.11) at the junction between the WO3 NFs (Φ = 4.56 eV) and NOGR flakes (Φ = 4.67 eV), which showed high potential for the diagnosis of halitosis.
The anesthetic management of patients undergoing endovascular treatment of cerebral aneurysms in the interventional neuroradiology suite can be challenged by hypothermia because of low ambient temperature for operating and maintaining its equipments. We evaluated the efficacy of skin surface warming prior to induction of anesthesia to prevent the decrease in core temperature and reduce the incidence of hypothermia.
Seventy-two patients were randomized to pre-warmed and control group. The patients in pre-warmed group were warmed 30 minutes before induction with a forced-air warming blanket set at 38°C. Pre-induction tympanic temperature (Tpre) was measured using an infrared tympanic thermometer and core temperature was measured at the esophagus immediately after intubation (T0) and recorded at 20 minutes intervals (T20, T40, T60, T80, T100, and T120). The number of patients who became hypothermic at each time was recorded.
Tpre in the control and pre-warmed group were 36.4 ± 0.4°C and 36.6 ± 0.3°C, whereas T0 were 36.5 ± 0.4°C and 36.6 ± 0.2°C. Core temperatures in the pre-warmed group were significantly higher than the control group at T20, T40, T60, T80, T100, and T120 (P < 0.001). Compared to T0, core temperatures at each time were significantly lower in both two groups (P = 0.007 at T20 in pre-warmed group, P < 0.001 at the other times in both groups). The incidence of hypothermia was significantly lower in the pre-warmed group than the control group from T20 to T120 (P = 0.002 at T20, P < 0.001 at the other times).
Pre-warming for 30 minutes at 38°C did not modify the trends of the temperature decrease seen in the INR suite. It just slightly elevated the beginning post intubation base temperature. The rate of decrease was similar from T20 to T120. However, pre-warming considerably reduced the risk of intraprocedural hypothermia.
Clinical Research Information Service (CRiS) Identifier: KCT0001320. Registered December 19th, 2014.
Cerebral aneurysm; Hypothermia; Interventional neuroradiology; Pre-warming