Synthesis of Quercetin-Acid Esters and Its Reduction of H2 O2 -Triggered PC12 Cells Damage by Down-Regulating ROS.

Quercetin is a kind of polyphenolic flavonoid compounds which has perfect antioxidant properties. However, quercetin is not available in many situations due to its poor bioavailability. In this work, the QAEs with better solubility and even stronger antioxidant properties were synthesized, through the esterification between quercetin and the chlorinated cinnamic acid or its derivatives, whose chlorination were achieved by using SOCl2 . The protective effects of the QAEs were evaluated by the H2 O2 -induced apoptosis experiment in rat adrenal pheochromocytoma cells (PC12 cells) and its ability to remove ROS generated by oxidative stress. Compared with the original quercetin group, the QAEs groups showed much improved cell viability and capability of removing ROS, which means their higher bioavailability than the parent.

Weak UVB Irradiation Promotes Macrophage M2 Polarization and Stabilizes Atherosclerosis.

Atherosclerosis (AS) is a chronic cardiovascular disease endangering human health and is one of the most common causes of myocardial infarction and stroke. Macrophage polarization plays a vital role in regulating plaque stability. As an important component of sunlight, ultraviolet B (UVB) has been proven to promote vitamin D and nitric oxide synthesis. This research used an AS model in ApoE-/- mice to study the effects of UVB on macrophage polarization and atherosclerotic plaque stability. In vitro, UVB irradiation increased arginase-I (Arg-I, M2 macrophage) and macrophage mannose receptor (CD206) expression, while the expression of inducible nitric oxide synthase (iNOS) (M1 macrophage) and CD86 was decreased. UVB promoted Akt phosphorylation in vitro. In vivo, UVB irradiation promoted the stabilization of atherosclerotic lesion plaques, while the phenotype of M2 macrophages increased. Our research provides new evidence for UVB in preventing and treating atherosclerosis.

Prognostic factors and survival outcome of primary pulmonary acinar cell carcinoma.

PURPOSE: The objective of our study was to investigate the epidemiologic characteristics and prognostic factors in patients with pulmonary acinar cell carcinoma (PACC). METHODS: PACC patients diagnosed between 1975 and 2016 were identified from the Surveillance, Epidemiology, and End Results (SEER) database. The trend in PACC incidence was assessed using joinpoint regression software. Overall survival (OS) and disease-specific survival (DSS) were evaluated using the Kaplan-Meier method and log-rank test. Univariate and multivariate Cox regression analysis was performed to identify the independent prognostic factors for OS and DSS. Nomograms to predict survival possibilities were constructed based on the identified independent prognostic factors. RESULTS: A total of 2918 patients were identified with PACC. The mean age was 65.2 ± 8.95 years with a female to male of 1.6:1. The incidence of PACC steadily increased by an annual percentage change (APC) of 3.2% (95% CI 2.1-4.4, p < 0.05). Multivariate Cox regression analysis revealed that age, gender, race, stage, grade, tumor size, number of positive lymph nodes, surgery, and chemotherapy were independent prognostic factors for survival. Nomograms specifically for PACC were constructed to predict 1- and 5-year OS and DSS possibility, respectively. The concordance index (C-index) and calibration plots showed the established nomograms had robust and accurate performance. CONCLUSION: PACC was rare but the incidence has been steadily increasing over the past four decades. Survival has improved in recent years. Surgery or chemotherapy could provide better OS and DSS. The established nomograms specifically for PACC were robust and accurate in predicting 1- and 5-year OS and DSS.

Downregulation of DNMT3a expression by RNAi and its effect on NF-κBs expression of thymic epithelial cells.

OBJECTIVE: To understand the characteristics of DNA methyltransferase 3a (DNMT3a) in thymoma associated Myasthenia Gravis reveal its transcriptional regulator network as while as analyze the effect of DNMT3a on Rel/ nuclear factor-kappaB family (RelA/RelB) and its downstream autoimmune regulatory factor (Aire). METHODS: Tissues of 30 patients with thymoma, with or without myasthenia gravis (MG), were collected and the DNMT3a protein expression were evaluated through immunohistochemistry. We performed mRNA expression profiling microarray detection and analysis, and integrated the analysis by constructing protein-protein interaction networks and the integration with other database. We identified molecular difference between low and high DNMT3a in the thymoma by heatmap. We also performed PCR validation in thymoma tissues. The DNMT3a-shRNA plasmid was transfected into TEC cells, and these cells were treated with 5-aza-2-deoxycytidine, a blocker of DNMT3a. After the down-regulation of DNMT3a in TEC cells, the transcript and protein levels of RelA, RelB, Aire, and CHRNA3 were evaluated by western blotting. In addition, changes in gene expression profiles were screened through microarray technology. We performed differential gene analysis in the thymoma cohort by heatmap with R (v.4.3.0) software. RESULTS: In 30 matched tissue specimens, the expression of DNMT3a protein in thymoma with MG was lower than that in thymoma. Through mRNA expression profiling analysis, we constructed a co-expression network of DNMT3a and found direct interaction between IKZF1 and DNMT3a, and this co-expression relationship was overlappted with Cistrome DB database. We found up-regulation of 149 mRNAs and repression of 177 mRNAs in thymoma with MG compared with thymoma. Gene ontology and pathway analysis show the involvement of a multitude of genes in the mis-regulation of MG-related pathways. RNA interference significantly reduced the level of mRNA of DNMT3a, which proved that plasmid DNMT3a was effective. In comparison to the control group, the levels of DNMT3a, Aire, and CHRNA3 mRNA and protein in TEC cells transfected with DNMT3a-shRNA interference plasmid were significantly decreased, while the expression level of RelA and RelA/RelB was significantly increased. CONCLUSIONS: Our study reveals the DNMT3a-NF-κB pathway has a major effect on MG, and can be used as a marker for diagnosis as well as a target for MG treatment.

Dickkopf-1 promotes Vascular Smooth Muscle Cell proliferation and migration through upregulating UHRF1 during Cyclic Stretch application.

Dickkopf-1 (DKK1) was recently shown to play an important role in cardiovascular disease. The aim of this work was to assess the role of DKK1 in the regulation of smooth muscle cell function by mechanical stretch and the mechanisms underlying this process. Methods: Wild-type C57BL/6J mice were subjected to sham or abdominal aortic constriction (AAC) surgery. The expression level of DKK1 was examined by immunohistochemical staining and Western blotting. Analyses of DKK1 function in vascular smooth muscle cell (VSMC) proliferation and migration were performed. Transcriptome sequencing analysis was performed to identify the differentially expressed genes and pathways regulated by DKK1. Smooth muscle-specific Dkk1 knockout mice were used to confirm the function of DKK1 in vivo. Chromatin immunoprecipitation (ChIP) was used to confirm DNA-protein interactions. Promoter luciferase analysis was used to detect transcription factor activity. Results: We found that AAC significantly increased DKK1 protein levels in the thoracic aorta and coronary artery in vivo. In vitro, high-level stretch (18%) induced the expression of DKK1 in VSMCs. Knocking down DKK1 inhibited VSMC proliferation and migration under high-level stretch (18%). We identified ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) as a target gene of DKK1. Knockdown of UHRF1 with small interfering RNAs partially reversed the regulatory effect of recombinant DKK1 on VSMCs. Specific deletion of DKK1 in VSMCs was sufficient to attenuate the AAC-induced upregulation of UHRF1, thickening of arterial media and increase in VSMC proliferation. Furthermore, we found that DKK1 regulated UHRF1 expression through the YAP-TEAD pathway. TEAD1 and TEAD4 bound directly to the promoter of UHRF1, and blocking the YAP-TEAD interaction inhibited UHRF1 upregulation due to DKK1. Conclusions: This study reveals that DKK1 mediates the mechanical stretch regulation of smooth muscle cell function by modulating UHRF1 expression through the YAP-TEAD pathway.

[The role of ZFP580 in the regulation of rat VSMCs migration by ATRA and its mechanism].

OBJECTIVE: To investigate the probable roles of the novel C2H2 zinc finger transcription factor ZFP580 on all-transretinoic acid (ATRA)-regulated VSMCs migration and underlying mechanisms. METHODS: Rat aortic VSMCs were isolated, cultured and identified. VSMCs were treated with ATRA at the concentrations of 0, 5, 10 or 20 µmol/L for 24 hours. The migration ability of VSMCs was observed in each group and compared with control group which was treated by 0 µmol/L ATRA. The mRNA and protein expression levels of ZFP580 were detected by QPCR and Western blot. ZFP580 protein expression in VSMCs was detected under ATRA stimulation when ERK inhibitor PD98059 was used to inhibit the protein expression of ERK. Adenovirus transfection technology was used to obtain VSMCs with overexpression or low expression of ZFP580, and QPCR and Western blot were used to detect the mRNA and protein levels of MMP-2, MMP-9 and ZFP580. RESULTS: On the 10th day of VSMCs culture, immunofluorescence showed that SM22 alpha antibody, as a specific marker of smooth muscle cells, was positive. Compared to the control group, VSMCs migration was reduced by 32%, 43%, and 59% in the group of 5, 10, and 20 µmol/L ATRA pretreatment. Compared with the control group, VSMCs treated by 20 µmol/L ATRA reduced the cell migration by 49%, 36% and 22% at 24, 48 and 72 h. The mRNA and protein expression levels of ZFP580 were increased with the increase of ATRA stimulation solubility and the extension of stimulation time. ERK was increased significantly after 15 min of ATRA stimulation. Pretreatment with ERK inhibitor PD98059 (20 µmol/L) inhibited the expression of ERK protein and reduced the expression of ATRA-induced ZFP580 protein. Overexpression of ZFP580 inhibited the expressions of MMP-2 and MMP-9, whereas down-expression of ZFP580 promoted the expressions of MMP-2 and MMP-9. CONCLUSION: ATRA increased the expression of ZFP580 through the ERK signaling pathway, while ZFP580 was involved in ATRA's inhibition of VSMCs migration by affecting the expression of downstream MMP-2 and MMP-9.

[Differential expression of immediately early gene c-fos in THP-1 macrophage subtype polarization].

Objective: To investigate the expression of immediate early gene c-fos in THP-1 macrophage subtype polarization. Methods: PMA was used to induce the polarization of THP-1 monocytes to macrophages, and the expression of c-fos in the polarization process was observed. After PMA treatment, LPS or IL-4 were used alone to induce the polarization of THP-1 macrophages to the M1 or M2 subtypes. Subsequently, real-time quantitative PCR and western-blot were used to analyze the changes in the expressions of the cell subtype markers CD274, CD86 and CD163. Meanwhile, the expression of c-fos in the polarization process was observed dynamically. Results: The levels of c-fos protein and mRNA expressions were up-regulated during PMA-induced polarization of THP-1 monocytes. The protein and mRNA expressions of c-fos were significantly decreased during the polarization of THP-1 cells into M1 macrophages induced by LPS. The specific markers showed the characteristics of M1 macrophages polarization at 24 h (CD86 protein increased, CD274 and CD163 protein decreased). The protein and mRNA expressions of c-fos were significantly increased during the polarization of THP-1 cells into M2 macrophages induced by IL-4. The specific markers showed the characteristics of M2 macrophages polarization at 24 h (CD86 protein decreased, CD274 and CD163 protein increased). Conclusion: C-fos plays an important role in the polarization of THP-1 monocytes to macrophages. Moreover, it may be involved in the regulation of macrophage subtype polarization, by inhibiting the formation of M1 macrophage and promoting the polarization of macrophages to the M2 subtype.

The role of ZFP580, a novel zinc finger protein, in TGF-mediated cytoprotection against chemical hypoxia­induced apoptosis in H9c2 cardiac myocytes.

Zing finger protein 580 (ZFP580) is a novel Cys2-His2 zinc-finger transcription factor that has an anti-apoptotic role in myocardial cells. It is involved in the endothelial transforming growth factor­ß1 (TGF­ß1) signal transduction pathway as a mothers against decapentaplegic homolog (Smad)2 binding partner. The aim of the present study was to determine the involvement of ZFP580 in TGF­ß1­mediated cytoprotection against chemical hypoxia­induced apoptosis, using H9c2 cardiac myocytes. Hypoxia was chemically induced in H9c2 myocardial cells by exposure to cobalt chloride (CoCl2). In response to hypoxia, cell viability was decreased, whereas the expression levels of hypoxia inducible factor-1α and ZFP580 were increased. Pretreatment with TGF­ß1 attenuated CoCl2­induced cell apoptosis and upregulated ZFP580 protein expression; however, these effects could be suppressed by SB431542, an inhibitor of TGF­ß type I receptor and Smad2/3 phosphorylation. Furthermore, suppression of ZFP580 expression by RNA interference reduced the anti­apoptotic effects of TGF­ß1 and thus increased CoCl2­induced apoptosis. B­cell lymphoma (Bcl)­2­associated X protein/Bcl­2 ratio, reactive oxygen species generation and caspase­3 activation were also increased following ZFP580 inactivation. In conclusion, these results indicate that ZFP580 is a component of the TGF-ß1/Smad signaling pathway, and is involved in the protective effects of TGF­ß1 against chemical hypoxia­induced cell apoptosis, through inhibition of the mitochondrial apoptotic pathway.

SILAC-based proteomic analysis reveals that salidroside antagonizes cobalt chloride-induced hypoxic effects by restoring the tricarboxylic acid cycle in cardiomyocytes.

Hypoxic status alters the energy metabolism and induces cell injury in cardiomyocytes, and it further triggers the occurrence and development of cardiovascular diseases. Our previous studies have shown that salidroside (SAL) exhibits anti-hypoxic activity. However, the mechanisms remain obscure. In the present study, we successfully screened 92 different expression proteins in CoCl2-induced hypoxic conditions, 106 different expression proteins in the SAL-mediated anti-hypoxic group were compared with the hypoxic group using quantitative proteomics strategy, respectively. We confirmed that SAL showed a positive protective function involving the acetyl-CoA metabolic, tricarboxylic acid (TCA) cycle using bioinformatics analysis. We also demonstrated that SAL plays a critical role in restoring the TCA cycle and in protecting cardiomyocytes from oxidative injury via up-regulation expressions of PDHE1-B, ACO2, SUCLG1, SUCLG2 and down-regulation of MDH2. SAL also inhibited H9c2 cell apoptosis by inhibiting the activation of pro-apoptotic molecules caspase 3 and caspase 9 as well as activation of the anti-apoptotic molecular Bcl-2. Additionally, SAL also improved mitochondrial membrane potential (ΔΨm), reduced reactive oxygen species (ROS) and intercellular Ca(2+) concentration ([Ca(2+)]i) accumulation and inhibited the excessive consumption of ATP in H9c2 cells.

Feasibility and efficacy of concurrent chemoradiotherapy in elderly patients with esophageal squamous cell carcinoma: a respective study of 116 cases from a single institution.

BACKGROUND: To evaluate the safety and efficacy of combined chemoradiotherapy or radiotherapy alone in elderly patients with esophageal carcinoma to identify the best method of treatment. MATERIALS AND METHODS: One hundred and sixteen patients with esophageal carcinoma aged 70 and older who received definitive radiotherapy or chemoradiotherapy entered the study. Overall survival (OS), disease-free survival (DFS) and treatment- related toxicities were assessed. RESULTS: The median OS of the overall population was 17.9 months. For patients treated with cCRT, sCRT and radiotherapy alone, the median OS was 22.3 months, 18.0 months and 12.4 months respectively(P=0.044). Median OS for patients treated with radiotherapy dose ≥60Gy and <60Gy was 20.2 months and 10.9 months respectively (p=0.017). By univariate analysis, Chemoradiotherapy (include cCRT and sCRT) and radiotherapy dose ≥60Gy were found to achieve higher survival rates compared with radiotherapy alone and radiotherapy dose <60Gy (P=0.015, P=0.017). By multivariate analysis, chemoradiotherapy (HR=1.645, P=0.022) and radiotherapy dose ≥60Gy (HR=1.642, P=0.025) were identified as independent prognostic factors of OS. CONCLUSIONS: Definitive concurrent chemoradiotherapy could be considered as a feasible and effective treatment in esophageal carcinoma patients aged 70 and older. Radiotherapy dose 60Gy is an effective treatment option compared with standard dose radiotherapy, while higher doses are not beneficial to improve survival.

[Expression of Foxa2 and its early alarm value of cancerous in gastric polyps].

OBJECTIVE: To explore the expression of Foxa2 in different pathological types of gastric polyps and examine the correlation with cancerous risk. METHODS: According to computerize random number, a total of 2000 patients were selected to receive endoscopic biopsy during November 2011 to October 2012. Tissues were harvested from 170 with gastric polyps and suspicious cancerous lesions and their histological types detected. There were hyperplastic polyps(n = 35), adenomatous polyps(n = 31), fundic gland polyps(n = 42), advanced gastric cancer tissues (n = 32)and normal gastric mucosa tissues (n = 30). ABC immunohistochemical staining and reverse transcription(RT)-PCR were employed to detect the expression of Foxa2 in these different types of tissues. Imagepro plus was used for quantitative and statistical analyses. RESULTS: A low-level expression of Foxa2 was 3.6% ± 1.3% in normal gastric mucosa group. And its expreesion gradually higher in proliferative inflammatory polyp group(33.1% ± 8.0%), adenomatous polyp group (71.4% ± 1.7%) and gastric cancer group(96.3% ± 0.9%, all P < 0.05). Its expression was 35.6% ± 5.6% in fundic gland polyps, similar to that of proliferative inflammatory polyp group (P > 0.05), it was markedly lower than the gastric cancer group (P < 0.05) and higher than the normal gastric mucosa group (P < 0.05). Correlation analyses of clinicopathological parameters showed that no significant correlation existed between its expression and patient gender, age, predilection, Helicobacter. pylori infection or proton pump inhibitor used (all P > 0.05). However, the size of polyps was correlated with Foxa2 (rs = 0.69, P < 0.05). CONCLUSION: The expression level of Foxa2 in different types of gastric polyps may be used as a clinical predicator of polyps risk.

The role of microRNAs in lung cancer progression.

Lung cancer is a heterogeneous disease with currently still unknown mechanisms of development. Besides genetic and epigenetic mechanisms, microRNAs (miRNAs) have recently been discovered as one of the crucial players in lung carcinogenesis through posttranscriptional regulation of tumor suppressor and oncogenes. A substantial number of deregulated miRNAs have been revealed in lung cancer, and the biological significance of those miRNAs has been confirmed in multiple functional experiments. A growing number of studies suggest involvement of miRNAs in various steps of lung carcinogenesis. Great biological stability of miRNAs opens novel fields in biomarker research with potential clinical implementation in screening, diagnosis and prediction of prognosis. In this review, we provide the basic knowledge of miRNA biogenesis and discuss extensively the role of miRNAs in lung carcinogenesis, including potential translational clinical implementations.

Decreased platelet miR-223 expression is associated with high on-clopidogrel platelet reactivity.

OBJECTIVES: We aimed to investigate the relationship between platelet microRNA (miR-223 and miR-96) expression and clopidogrel responsiveness in patients with coronary heart disease (CHD). MATERIALS AND METHODS: A total of 33 consecutive non-diabetic CHD patients scheduled for percutaneous coronary intervention were enrolled. Platelet reactivity after clopidogrel loading dose (300 mg) was determined by two methods [platelet reactivity index (PRI), measured by vasodilator-stimulated phosphoprotein (VASP) phosphorylation flow cytometry and ADP-induced platelet aggregation (PAG), measured by light transmission aggregometry]. Total platelet RNA was isolated from purified platelets (CD45 magnetic bead negative selection) to quantify miR-223 and miR-96 expression by real-time PCR. RESULTS: All subjects were dichotomized according to PRI medians (normal-responders: PRI < 56.5%, n = 17 and low-responders: PRI > 56.5%, n = 16) and PAG medians (normal-responders: PAG < 43%, n = 17 and low-responders: PAG > 43%, n = 16). Compared with PRI-determined normal-responders, miR-223 expression, but not miR-96, was significantly decreased in low-responders (P = 0.037). No differential expression of miR-223 and miR-96 was observed via PAG determination between normal- and low-responders. In addition, miR-223 expression, but not miR96, was statistically correlated with PRI (Spearman r = -0.403, P = 0.020). Stepwise binary logistic regression analysis revealed that among factors that potentially influence platelet reactivity (CYP2C19*2 loss-of-function genotypes, use of calcium channel blockers/proton-pump inhibitors, age, obesity, smoking and platelet microRNAs), decreased miR-223 expression was the only independent predictor associated with the presence of PRI-determined low responders to clopidogrel (OR 0.189, 95% CI 0.043 to 0.836, P = 0.028). CONCLUSIONS: The present work identifies decreased platelet miR-223 expression as a novel mechanism involved in blunted platelet response to clopidogrel in a Chinese population.

Global gene expression differentiating pure bronchioloalveolar carcinoma from adenocarcinoma with bronchioloalveolar carcinoma features.

OBJECTIVES: Pure bronchioloalveolar carcinoma (BAC) is considered the early stage of lung adenocarcinoma, and is even regarded as lung adenocarcinoma in situ. This study was designed to investigate the differences in the gene expression of pure BAC and that of adenocarcinoma with bronchioloalveolar features and explore the mechanism of BAC progression to adenocarcinoma with bronchioloalveolar features METHODS: Total RNA was extracted from 16 tissue specimens. Expression analysis was carried out using Agilent 4 × 44 k arrays. Gene ontology analysis was used to define pathways altered in bronchioloalveolar progression. Differentially expressed candidate genes were validated using quantitative real-time PCR. The statistical analysis was carried out according to the methods of the paired t-test. RESULTS: Adenocarcinoma with bronchioloalveolar features demonstrated an increased expression of 23 genes and reduced expression of 20 genes compared with BAC. These genes were considered candidate marker genes for tumour progression and metastasis. Genes overexpressed in adenocarcinoma with bronchioloalveolar features included fibroblast growth factor receptor 1, and CLDN18 (claudin 18), whereas those overexpressed in BAC included ataxia telangiectasia and Rad3 related (ataxia telangiectasia mutated and Rad3-related), and activating transcription factor 2. Mitogen-activated protein kinase (MAPK) pathway seemed dysregulated in adenocarcinoma with bronchioloalveolar features compared with pure BAC. CONCLUSIONS: Microarray-based expression profiling revealed interesting novel candidate genes in BAC and adenocarcinoma with bronchioloalveolar features. The MAPK pathway seemed dysregulated in adenocarcinoma with bronchioloalveolar features compared with the pure BAC pathway, which is worthy of being explored because it could partially explain the mechanism of the progression of BAC to adenocarcinoma with bronchioloalveolar features.

[Establishment of heat acclimatization model in rabbits and its pathophysiological characteristics].

OBJECTIVE: To establish an effective and stable rabbit heat acclimatization model for the experiment of heat acclimatization mechanisms. METHODS: Sixteen healthy male rabbits were divided into heat acclimatization group and control group randomly (n = 8). Heat acclimatization (HA) group was kept in simulation chamber with dry bulb temperature of (36 +/- 1) degrees C, wet bulb temperature of (29 +/- 0.5) degrees C, black-bulb temperature of (40 +/- 1.0) degrees C, 100 min/day for 21 days. Control group was kept in the room with temperature of 20 degrees C and relative humidity < 60% during 20 days, then removed into simulation chamber on day 21 to estimate and monitor the rectal temperature together with the heat acclimatization group. Venous blood of control and heat acclimatization group before and after heat exposure on the 1st day, 11th day and 21st day were collected to detect levels of tumor necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6) and heat shock protein 70 (HSP70) by ELISA analysis. RESULTS: (1) Rectal temperature: There was no significant change in control group during 21 days. In heat acclimatization group, it increased (2.07 +/- 0.43) degrees C after the 1st exposure, and increased (1.78 +/- 0.37) degrees C after the 11th exposure, the range of increasing decreased (0.29 +/- 0.09) degrees C. After the 21st exposure, it increased (1.52 +/- 0.29) degrees C, which was (0.55 +/- 0.14) degrees C lower than that of the 1st (P < 0.05),and (0.53 +/- 0.14) degrees C lower to that of the control group under 1st heat stress (P < 0.05); (2) The level of TNF-alpha after the 1st exposure increased significantly (P < 0.05), but didn't raise along with the exposure times. And fell back to the original level after the 11th and 21st exposure. Compared with control group, the level of IL-6 increased after the 1st, 11th and 21st exposure (P < 0.05), and maintained highly after the 11th and 21st exposure. Compared with the control group, the level of HSP70 increased dramatically with the heat exposure times. Significant increasing of (HSP70) could be detected after the 11th and 21st exposure (P < 0.05), but there was no difference to that of the 1st exposure. CONCLUSION: Prolonged or repeated exposure to heat stressful environmental conditions can reduce the physiological strain, improve heat tolerance, elicits heat acclimatization.

Myosin light-chain kinase is necessary for membrane homeostasis in cochlear inner hair cells.

The structural homeostasis of the cochlear hair cell membrane is critical for all aspects of sensory transduction, but the regulation of its maintenance is not well understood. In this report, we analyzed the cochlear hair cells of mice with specific deletion of myosin light chain kinase (MLCK) in inner hair cells. MLCK-deficient mice showed impaired hearing, with a 5- to 14-dB rise in the auditory brainstem response (ABR) thresholds to clicks and tones of different frequencies and a significant decrease in the amplitude of the ABR waves. The mutant inner hair cells produced several ball-like structures around the hair bundles in vivo, indicating impaired membrane stability. Inner hair cells isolated from the knockout mice consistently displayed less resistance to hypoosmotic solution and less membrane F-actin. Myosin light-chain phosphorylation was also reduced in the mutated inner hair cells. Our results suggest that MLCK is necessary for maintaining the membrane stability of inner hair cells.

[The association between polymorphism of transforming growth factor-ß1 and radiochemotherapy response and survival in esophageal squamous cell carcinoma patients].

OBJECTIVE: To evaluate the association between polymorphism of transforming growth factor-ß1 (TGF-ß1)-509C/T and radiochemotherapy response and survival in esophageal squamous cell carcinoma (ESCC) patients. METHODS: The genotype of TGF-ß1-509C/T was detected by polymerase chain reaction-based restriction fragment length polymorphism assay (PCR-RFLP) in 230 ESCC patients receiving radiotherapy alone or in combination with chemotherapy. Unconditional multivariate logistic regression analysis was done to estimate adjusted odds ratios (ORs) along with the corresponding 95% confidence intervals (CIs) for the polymorphism and radiochemotherapy response. The associations between overall survival time or hazard ratio (HR) of ESCC patients and genetic variation or the clinical data were estimated by applying univariate and multivariate Cox-regression analyses. RESULTS: Among 208 patients with upper gastrointestinal contrast assessment, 87 cases were susceptible to radiochemotherapy treatment and the TGF-ß1-509CC, CT and TT genotype patients were 17 (19.5%), 48 (55.2%) and 22 (25.3%), respectively. Among the patients who were insensitive to radiochemotherapy treatment (n = 121), the TGF-ß1-509CC, CT and TT genotype patients were 39 (32.2%), 54 (44.6%) and 28 (23.2%), respectively. Compared with TGF-ß1-509CC genotype, the CT and TT genotype carriers had a significantly better treatment response (adjusted OR = 2.07, 95%CI, 1.05 - 4.09, P = 0.036). The median survival time of CC genotype patients was 17.0 (95%CI, 12.0 - 23.0) months, CT genotype patients was 22.0 (95%CI, 16.0 - 33.0) months and TT genotype patients was 25.0 (95%CI, 15.0 - 41.0) months. Compared to CC genotype patients, the survival time difference of CT and TT group was close to the statistical break point (P = 0.063). Our data showed that the subjects with CT or TT genotype had an decreased HR respectively as compared with those with CC genotype (CT, adjusted HR = 0.81, 95%CI, 0.52 - 1.24; TT, adjusted HR = 0.86, 95%CI, 0.65 - 1.12), but the difference was not statistically significant (P > 0.05). However, tumor location, clinical stage and radiochemotherapy response affected the overall survival time of the patient significantly (adjusted HR = 1.28, 95%CI: 1.01 - 1.61, P = 0.040; 1.49, 95%CI, 1.17 - 1.88, P = 0.001; 1.55, 95%CI, 1.06 - 2.26, P = 0.023, respectively). CONCLUSION: These results suggest that TGF-ß1-509C/T polymorphisms were associated with radiochemotherapy for esophageal squamous cell carcinoma which might be genetic markers for prediction of the radiochemotherapy response in ESCC patients.

Sphingosine-1-phosphate induces human endothelial VEGF and MMP-2 production via transcription factor ZNF580: novel insights into angiogenesis.

Sphingosine-1-phosphate (S1P)-induced migration and proliferation of endothelial cells are critical for angiogenesis. C2H2-zinc finger (ZNF) proteins usually play an essential role in altering gene expression and regulating the angiogenesis. The aim of this study is to investigate whether a novel human C2H2-zinc finger gene ZNF580 (Gene ID: 51157) is involved in the migration and proliferation of endothelial cells stimulated by S1P. Our study shows that EAhy926 endothelial cells express S1P1, S1P3 and S1P5 receptors. Furthermore, S1P upregulates both ZNF580 mRNA and protein levels in a concentration- and time-dependent manner. SB203580, the specific inhibitor of the p38 mitogen-activated protein kinase (p38 MAPK) pathway, blocks the S1P-induced upregulation of ZNF580. Moreover, overexpression/downexpression of ZNF580 in EAhy926 cells leads to the enhancement/decrease of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) expression as well as the migration and proliferation of EAhy926 endothelial cells. These results elucidate the important role that ZNF580 plays in the process of migration and proliferation of endothelial cells, which provides a foundation for a novel approach to regulate angiogenesis.

Myosin light chain kinase is necessary for tonic airway smooth muscle contraction.

Different interacting signaling modules involving Ca(2+)/calmodulin-dependent myosin light chain kinase, Ca(2+)-independent regulatory light chain phosphorylation, myosin phosphatase inhibition, and actin filament-based proteins are proposed as specific cellular mechanisms involved in the regulation of smooth muscle contraction. However, the relative importance of specific modules is not well defined. By using tamoxifen-activated and smooth muscle-specific knock-out of myosin light chain kinase in mice, we analyzed its role in tonic airway smooth muscle contraction. Knock-out of the kinase in both tracheal and bronchial smooth muscle significantly reduced contraction and myosin phosphorylation responses to K(+)-depolarization and acetylcholine. Kinase-deficient mice lacked bronchial constrictions in normal and asthmatic airways, whereas the asthmatic inflammation response was not affected. These results indicate that myosin light chain kinase acts as a central participant in the contractile signaling module of tonic smooth muscle. Importantly, contractile airway smooth muscles are necessary for physiological and asthmatic airway resistance.

Brain metastasis treated with Cyberknife.

BACKGROUND: Cyberknife can greatly raise the fractional dose of stereotactic radiosurgery, thus improving its clinical efficacy. We retrospectively analyzed clinical outcomes of brain metastasis treated with Cyberknife. METHODS: We analyzed 40 cases of brain metastases treated with Cyberknife in the Tianjin Cancer Hospital from August 1, 2006 to August 1, 2007, for a total of 68 lesions with maximal diameter of 0.4 - 7.5 cm (average 1.88 cm). Total hypofractional radiated dosage was 18 - 36 Gy (5 - 25 Gy/F, 1 - 5 F) by Cyberknife. We evaluated the remission rate of clinical symptoms, correlation factors to new foci, 3-month local control rates, and 3-month and 1-year survival rates. All patients were followed up for more than 14 months. RESULTS: After 1 week, clinical remission was 90.0% (36/40). After 3 months, the local control rate and therapeutic effective rate were 77.9% (53/68) and 94.1% (64/68), respectively, as observed by cranium augmentation CT or MRI. The three-month, six-month and 1-year survival rates were 97.5% (39/40), 82.5% (33/40) and 67.5% (27/40), respectively. Fourteen patients had neopathy outside the original lesion after 3 months. Neopathy was not correlated with age, whole-brain radiotherapy, number of original lesions, maximum diameter of the original lesion, therapeutic dose per fraction, therapeutic frequency or total therapeutic dose. CONCLUSIONS: Cyberknife got perfect clinical outcomes by higher dosage per fraction. It is an appropriate and valid treatment shortcut for brain metastasis.