DNMT3B plays a crucial role in the generation of aberrant methylation during carcinogenesis. Polymorphisms in the DNMT3B gene may influence the DNA methylation enzymatic activity of DNMT3B, thereby modulating the susceptibility to AML. Thus, we investigated the association between SNPs in the DNMT3Bgene and their haplotypes with the risk of AML in the Chinese Han population. The DNMT3B genotype was determined by HRM in 317 de novo AML patients and 406 healthy control subjects matched for age and gender. Among the 5 SNPs investigated in this study, rs2424913 demonstrated no polymorphisms in the Chinese Han populations, rs1569686 and rs2424908 were significantly associated with AML risk. The GG genotype of rs1569686 was associated with increased AML risk (OR: 5.76; 95%CI: 2.60-12.73; P<0.01) compared with the TT genotype, and individuals with a G allele had a significantly increased risk (OR: 1.89; 95%CI: 1.41-2.52; P<0.01) for AML compared with those harboring a C allele, this polymorphism can predict the risk of AML in a minority of patients. While the CC genotype of rs2424908 appeared to reduce the AML risk (OR: 0.57; 95%CI: 0.36-0.91; P=0.01) compared with the TT genotype, individuals with a C allele were associated with a lower risk (OR: 0.79, 95%CI: 0.64-0.97, P=0.03) for developing AML compared with those harboring a T allele. The other 2 SNPs, rs6087990 and rs6119954, had no significant association with AML risk in the study population. The CGGT, CTAT, TGAT, and CGAT haplotypes of rs6087990, rs1569686, rs6119954, and rs2424908 appeared to significantly increase the AML risk, and the TTGC haplotype appeared to significantly reduce the risk. These results suggest that DNMT3B polymorphisms may contribute to the genetic susceptibility to AML; in particular, the G allele of rs1569686 serves as a risk factor for AML, whereas the C allele of rs2424908 represents a potential protective factor.
To evaluate the utility of multi-slice computed tomography (MSCT) in assessing acute non-reperfused myocardial infarct size.
Seven domestic pigs (mean weight 17.3 ± 1.9 kg) underwent ligation of the distal left anterior descending artery to establish a model of acute myocardial infarction (MI). MSCT and triphenyltetrazolium chloride (TTC) staining were performed two hours later. The following data were acquired and analyzed: MI volume (%), CT values of the infarcted region, left ventricular cavity and normal cardiac tissue at various scanning time-points (1, 5, 10, 15, 20 min after contrast injection).
Using MSCT, the overall MI volume showed a time-dependent decrease, with a reduction of 28.87% after 20 min. The greatest reduction occurred at the 5 min time-point. In TTC staining, MI volume was 9.87% ± 2.44%. When MI size, as determined by MSCT, was compared with that by TTC staining in Bland-Altman plots, there was a better agreement at 5, 10, and 15 min time-points at 1 and 20 min.
The study indicates that double-phase scanning examination using MSCT is a useful tool to assess MI size, and the optimal late-phase scanning time-point set within 5–15 min of contrast injection.
Multi-slice computed tomography; Acute non-reperfused myocardial infarction; Time-factors; Animal model
Neovascularization (NV) is a sight-threatening complication of retinal ischemia in diabetes, retinal vein occlusion, and retinopathy of prematurity. Current treatment modalities, including laser photocoagulation and repeated intraocular injection of VEGF antagonists, are invasive and not always effective, and may carry side effects. We studied the use of hyperoxia as an alternative therapeutic strategy for regressing established vitreous NV in a mouse model of oxygen-induced ischemic retinopathy.
Hyperoxia treatment (HT, 75% oxygen) was initiated on postnatal day (P)17 after the onset of vitreous NV. Immunohistochemistry and quantitative PCR were used to assess retinal vascular changes in relation to apoptosis, and expression of VEGFR2 and inflammatory molecules. Effects of intravitreal injections of VEGF-A, VEGF-E, PlGF-1, and VEGF trap were also studied.
HT selectively reduced NV by 70% within 24 hours. It robustly increased the level of cleaved caspase-3 in the vitreous NV between 6 and 18 hours and promoted infiltration of macrophage/microglial cells. The HT-induced apoptosis was preceded by a significant reduction in VEGFR2 expression within the NV and an increase in VEGFR2 within the surrounding neural tissue. Intravitreal VEGF-A and VEGF-E (VEGFR2 agonist) but not PlGF-1 (VEGFR1 agonist) prevented HT-induced apoptosis and regression of NV. In contrast, VEGF trap and VEGFR2 blockers mimicked the effect of HT. However, intravitreal VEGF trap induced increases in inflammatory molecules while HT did not have such unwanted effect.
HT may be clinically useful to specifically treat proliferative NV in ischemic retinopathy.
We found that hyperoxia therapy caused rapid resolution of established vitreous neovascularization in ischemic retinopathy by downregulating the VEGF/VEGFR2 pathway.
To establish a cell model of β2-adrenergic receptor (β2AR) downregulation of murine airway smooth muscle induced by salbutamol to elucidate the molecular and biological mechanisms of β2AR downregulation.
Airway smooth muscle cells (ASMCs) derived from Balb/c mice were primary cultured. Passage 2-5 cells were characterized by cell morphology and indirect immunofluorescence. More than 95% pure cells at passage 3 or 4 were randomly divided into two groups: control and salbutamol-treated groups. β2AR mRNA and protein expression levels were then detected by RT-PCR and western blot analyses.
Primary cultured cells demonstrated a typical “peak and valley”-like growth characteristic. Smooth muscle α-actin filaments paralleled the cell longitudinal axis in the cytoplasm. The origin of the ASMCs was validated and consistent with their morphology and biological characteristics. β2AR mRNA expression in the salbutamol-treated group was lower than that in the control group (P<0.05), and β2AR protein expression was also markedly lower than that in the control group (P<0.05).
We successfully established a cell model of β2AR downregulation in ASMCs, which may provide the foundation for further study of the mechanism of β2AR downregulation in asthmatic patients.
Airway smooth muscle cells (ASMCs); β2-adrenergic receptor (β2AR); salbutamol; cell culture
To assess the efficacy and safety of local anaesthetics for premature ejaculation (PE), a systematic review of the literature was performed using the Cochrane Library, PUBMED and EMBASE. We screened and retrieved the randomized controlled trials on the treatment of PE with local anaesthetics. End points included intravaginal ejaculation latency time (IELT), patient-reported outcome assessments and adverse events. Meta-analyses were conducted with Stata 11.0. In total, seven publications involving 566 patients with local anaesthetics and 388 with placebos strictly met our eligibility criteria. Meta-analyses showed that after the patients were treated with the local anaesthetics, the value of the standardized mean difference of the changes in IELT was 5.02 (95% CI: 3.03–7.00). A higher rate of adverse events occurred compared with placebos (odds ratio: 3.30, 95% CI: 1.71–6.36), but these events were restricted to local side effects. In addition, significantly greater improvement was observed in patient-reported outcomes. In summary, local anaesthetics can prolong IELT and improve ejaculatory control and sexual satisfaction.
local anaesthetics; premature ejaculation (PE); randomized controlled trial; systematic review
To investigate the distribution of nestin-positive cells in pterygium, as well as the relationship between nestin-positive cells and proliferative cells in the pathogenesis of pterygium.
Nine pterygium specimens and 5 normal conjunctiva specimens were investigated. All explanted specimens were immediately immersed in 5-Ethynyl-2′-deoxyuridine, and were subjected to hematoxylin and eosin staining, as well as immunostaining to detect nestin.
Small sub-populations of nestin-expressing cells in both normal and pterygial conjunctiva epithelium were found. These were located at the superficial layer of the epithelium, and were significantly increased (P=0.007) and spread out in the pterygial conjunctiva epithelium, even though these cells were mitotically quiescent.
In pterygium, more nestin-positive cells were present at the superficial layer of the epithelium. With growing scientific evidence that nestin plays an important role in defining various specialized cell types, such as stem cells, cancer cells and angiogenic cells, further investigations on the roles of nestin-expressing cells in pterygium may help to uncover the mechanisms of initiation, development and the prognosis of this disease.
pterygium; nestin; proliferation; distribution; human
Diabetic retinopathy (DR) is one of the most common complications of diabetes. This devastating disease is a leading cause of blindness in people of working age in industrialized countries and affects the daily lives of millions of people. Despite tight glycemic control, blood pressure control, and lipid-lowering therapy, the number of DR patients keeps growing and therapeutic approaches are limited. Moreover, there are significant limitations and side-effects for the current therapies. Thus, there is a great need for development of new strategies for prevention and treatment of DR. Studies have shown that DR has prominent features of chronic, subclinical inflammation. This review will focus on the role of inflammation in DR and summarize the progress of studies of anti-inflammatory strategies for DR.
cytokine; diabetic retinopathy; inflammation; leukostasis; neovascularization; NF-κB; nitric oxide; oxidative stress; vessel degeneration; vessel leakage
Objectives. Acupoint specificity is the foundation of acupuncture treatment. The aim of this study is to investigate whether the acupoint specificity exists in two adjacent acupoints. Design and Setting. Two adjacent real acupoints, LR3 (Taichong) and ST44 (Neiting), and a nearby nonacupoint were selected. Thirty-three health volunteers were divided into three groups in random order, and each group only received acupuncture at one of the three points. While they received acupuncture, fMRI scan was performed. Results. The common cerebral activated areas responding to LR3 and ST44 included the contralateral primary somatosensory area (SI) and ipsilateral cerebellum. Acupuncture at LR3 specifically activated contralateral middle occipital gyrus, ipsilateral medial frontal gyrus, superior parietal lobe, middle temporal gyrus, rostral anterior cingulate cortex (rACC), lentiform nucleus, insula, and contralateral thalamus. Stimulation at ST44 selectively activated ipsilateral secondary somatosensory area (SII), contralateral middle frontal gyrus, inferior frontal gyrus, lingual gyrus, lentiform nucleus, and bilateral posterior cingulate cortex (PCC). Conclusions. Acupuncture at adjacent acupoints elicits distinct cerebral activation patterns, and those specific patterns might be involved in the mechanism of the specific therapeutic effects of different acupoints.
Hyaluronan (HA), a simple disaccharide unit, can polymerize and is considered a primary component of the extracellular matrix, which has a wide range of biological functions. In recent years, HA was found on the surface of tumor cells. According to previous reports, differing HA content on the cell surface of tumor cells is closely related to lymph node metastases, but the mechanisms mediating this process remained unclear. This research intended to study the surface content of HA on tumor cells and analyze cell adhesive changes caused by the interaction between HA and its lymphatic endothelial receptor (LYVE-1). We screened and observed high HA content on HS-578T breast cells and low HA content on MCF-7 breast cells through particle exclusion, immunofluorescence and flow cytometry experiments. The expression of LYVE-1, the lymph-vessel specific HA receptor, was consistent with our previous report and enhanced the adhesion of HAhigh-HS-578T cells to COS-7LYVE-1(+) through HA in cell static adhesion and dynamic parallel plate flow chamber experiments. MCF-7 breast cells contain little HA on the surface; however, our results showed little adhesion difference between MCF-7 cells and COS-7LYVE-1(+) and COS-7LYVE-1(−) cells. Similar results were observed concerning the adhesion of HS-578T cells or MCF-7 cells to SVEC4-10 cells. Furthermore, we observed for the first time that the cell surface HA content of high transfer tumor cells was rich, and we visualized the cross-linking of HA cable structures, which may activate LYVE-1 on lymphatic endothelial cells, promoting tumor adhesion. In summary, high-low cell surface HA content of tumor cells through the interaction with LYVE-1 leads to adhesion differences.
AIM: To assess the prognostic value of serum human relaxin 2 (H2 RLN) level in patients with esophageal squamous cell carcinoma (ESCC).
METHODS: From October 1998 to September 2009, 146 patients with histopathologically confirmed ESCC were enrolled in this study. One hundred patients underwent en bloc esophagectomy, and 46 patients with unresectable tumors underwent palliative surgery. Five of the 146 patients died of surgical complications. Serum levels of H2 RLN were measured by enzyme linked immunosorbent assay. The relationship between serum H2 RLN level and each of the clinicopathological parameters was analyzed using the χ2 test. Patients were classified into two groups according to their H2 RLN level (< 0.462 ng/mL vs ≥ 0.462 ng/mL). When any analysis cell had fewer than five cases, the Fisher’s exact test was used. The statistical difference between groups A and B in each clinicopathological category was determined by the Student’s t test (two-tailed) or analysis of variance. Survival curves were plotted using the Kaplan-Meier method. The statistical difference in survival between the different groups was compared using the log-rank test. Survival correlation with the prognostic factors was further investigated by multivariate analysis using the Cox proportional hazards model with backward stepwise likelihood ratio.
RESULTS: ESCC patients tended to have significantly higher serum H2 RLN concentrations (0.48 ± 0.17 ng/mL, n = 141) compared with the healthy control group (0.342 ± 0.12 ng/mL, n = 112). There was a significant difference between patients with lymph node involvement (0.74 ± 0.15 ng/mL, n = 90), distant metastasis (0.90 ± 0.19 ng/mL, n = 32) and those without lymph node involvement (0.45 ± 0.12 ng/mL, n = 51), and distant metastasis (0.43 ± 0.14 ng/mL, n = 109), respectively (P < 0.01). Patients with high H2 RLN levels (≥ 0.462 ng/mL) had a poorer prognosis than patients with low serum H2 RLN levels (< 0.462 ng/mL; P = 0.0056). The H2 RLN level was also correlated with survival and tumor-node-metastasis staging, but not with age, tumor size, gender, lymphovascular invasion or the histological grade of tumors. Cox regression analysis showed that H2 RLN was an independent variable.
CONCLUSION: Serum concentrations of H2 RLN are frequently elevated in ESCC patients and are correlated with disease metastasis and survival. Serum concentrations of H2 RLN may be an important prognostic marker in ESCC patients.
Esophageal squamous cell carcinoma; Relaxin; Tumor markers; Metastasis
AIM: To investigate human epidermal growth factor receptor 2 (HER2) gene amplification and protein expression in Chinese patients with resectable gastric cancer and the association with clinicopathological characteristics and survival.
METHODS: One hundred and ninety-seven gastric cancer patients who underwent curative surgery procedures were enrolled into this study. HER2 gene amplification and protein expression were examined using fluorescence in-situ hybridization (FISH) and immunohistochemistry (IHC) analysis on formalin-fixed paraffin-embedded gastric cancer samples from all patients. For scoring, Hofmann’s HER2 gastric cancer scoring system was adopted. All cases showing IHC3+ or FISH positivity were defined as HER2 positive. Patient clinicopathological data and survival information were collected. Finally, χ2 statistical analysis was performed to analyze the HER2 positivity rate amongst the subgroups with different clinicopathological characteristics including; gender, age, tumor location, Lauren classification, differentiation, TNM staging, depth of invasion, lymph node metastases and distant metastasis. The probability of survival for different subgroups with different clinicopathological characteristics was calculated using the Kaplan-Meier method and survival curves plotted using log rank inspection.
RESULTS: According to Hofmann’s HER2 gastric cancer scoring criteria, 31 cases (15.74%) were identified as HER2 gene amplified and 19 cases (9.64%) were scored as strongly positive for HER2 membrane staining (3+), 25 cases (12.69%) were moderately positive (2+) and 153 cases (77.66%) were HER2 negative (0/1+). The concordance rate between IHC and FISH analyses was 88.83% (175/197). Thirty-six cases were defined as positive for HER2 gene amplification and/or protein expression, with 24 of these cases being eligible for Herceptin treatment according to United States recommendations, and 29 of these cases eligible according to EU recommendations. Highly consistent results were detected between IHC3+, IHC0/1 and FISH (73.68% and 95.42%), but low consistency was observed between IHC2+ and FISH (40.00%). The positivity rates in intestinal type and well-differentiated gastric cancer were higher than those in diffuse/mixed type and poorly-differentiated gastric cancer respectively (28.57% vs 13.43%, P = 0.0103; 37.25% vs 11.64%, P < 0.0001), but were not correlated with gender, age, tumor location or TNM stage, depth of invasion, lymph node metastases and distant metastasis. In poorly-differentiated gastric cancer patients, those without lymph node metastasis showed a higher HER2 positivity rate than those with lymph node metastasis (26.47% vs 7.14%, P = 0.0021). This association was not present in those patients with well-differentiated gastric cancer (28.57% vs 43.33%, P = 0.2832). Within our patient cohort, 26 cases were lost to follow-up. The median survival time for the remaining 171 patients was 18 mo. The median survival times of the HER2 positive and negative groups were 17 and 18.5 mo respectively. Overall survival was not significantly different between HER2-positive and negative groups (χ2 = 0.9157, P = 0.3386), but in patients presenting well-differentiated tumors, the overall survival of the HER2-positive group was significantly worse than that of the HER2-negative group (P = 0.0123). In contrast, patients with poorly differentiated and diffuse/mixed subtype gastric cancers showed no significant differences in overall survival associated with HER2. Furthermore, the median survival time of the HER2 positive group did not show any statistically significant differences when compared to the subgroups of gender, age, tumor location, TNM classification, lymph node metastases and distant metastasis.
CONCLUSION: Patients with intestinal type gastric cancer (GC), well-differentiated GC and poorly-differentiated GC without lymph node metastasis, may all represent suitable candidates for targeted therapy using Herceptin.
Gastric cancer; Human epidermal growth factor receptor 2; Gene amplification; Protein expression; Clinicopathological characteristics
Endomorphins (EMs) have a very important bridge-function in
cardiovascular, endocrinological, and neurological systems. This
study is to investigate the effects of EMs on the synthesis and
secretion of vasoactive substances induced by advanced glycation
end products in primary cultured human umbilical vein endothelial
cells (HUVECs). Firstly, HUVECs were stimulated with AGEs-bovine
serum albumin (AGEs-BSA), bovine serum albumin (BSA), or both
AGEs-BSA and EMs together, respectively. Then, HUVEC survival rate
was calculated by MTT assay, the levels of NO, endothelial nitric
oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS)
were detected by colorimetric analysis, and the contents of
endothelin-1 (ET-1) were detected by ELISA. The mRNA levels of
eNOS and ET-1 were measured by RT-PCR. The expression of p38
mitogen-activated protein kinase (p38 MAPK) was detected by
immunofluorescence assay. The results showed that the mRNA
expression and secretion of eNOS were significantly enhanced after
incubation with EMs compared to those with AGEs-BSA, while the
secretion of NO and iNOS, mRNA expression, and secretion of ET-1
had opposite changes. The fluorescence intensity of p38MAPK in
nuclear was decreased after pretreatment with EMs compared to
incubation with AGEs-BSA. Conclusion. The present
study suggests that EMs have certain protection effect on
AGEs-BSA-induced injury in HUVEC.
NEDD4L is a candidate gene for hypertension, both functionally and genetically. Recently, studies showed evidence for the association of NEDD4L with obesity, a key intermediate phenotype in hypertension. To further investigate the relationship between NEDD4L and body mass-related phenotypes, we genotyped three common variants (rs2288774, rs3865418 and rs4149601) in a population-based study of 892 unrelated Han Cantonese using the Sequenom MALDI-TOF-MS platform. Allele frequencies and genotype distribution were calculated in lean controls and overweight/obese cases and analyzed for association by the Chi-squared test and Logistic regression. Linear regression analysis was used to analyze the effect of individual genotypes on quantitative traits. Multivariate analyses demonstrated that the minor allele of rs4149601(A = 20.9%) was associated with a 2.60 kg, 2.78 cm and 0.97 kg/m2 decrease per allele copy in weight, waist and BMI, respectively. Carriers of this allele also had a significant lower risk of overweight/obesity (p < 0.0001, OR = 0.52, 95% CI: 0.37–0.74) as compared to non-carriers. However, no significant association between genotypes at rs2288774 and rs3865418 and covariate-adjusted overweight/obesity or any related phenotypes was observed. These results suggested that the functional variant of NEDD4L, rs4149601, may be associated with obesity and related phenotypes, and further genetic and functional studies are required to understand its role in the manifestation of obesity.
NEDD4L; genetic diversity; obesity
Aim: The aim of the present study was to explore the role of receptor tyrosine kinases (RTKs) in the regulation of expression of PTX-3, a protector in atherosclerosis. Methods: Human monocytic U937 cells were infected with a shRNA lentiviral vector library targeting human RTKs upon LPS stimuli and PTX-3 expression was determined by ELISA analysis. The involvement of downstream signaling in the regulation of PTX-3 expression was analyzed by both Western blotting and ELISA assay. Results: We found that knocking down of ERBB2/3, EPHA7, FGFR3 and RET impaired PTX-3 expression without effects on cell growth or viability. Moreover, inhibition of AKT, the downstream effector of ERBB2/3, also reduced PTX-3 expression. Furthermore, we showed that FGFR3 inhibition by anti-cancer drugs attenuated p38 activity, in turn induced a reduction of PTX-3 expression. Conclusion: Altogether, our study demonstrates the role of RTKs in the regulation of PTX-3 expression and uncovers a potential cardiotoxicity effect of RTK inhibitor treatments in cancer patients who have symptoms of atherosclerosis or are at the risk of atherosclerosis.
PTX-3; RNAi screening; RTKs; target therapy; cardiotoxicity; atherosclerosis
We isolated a bovine viral diarrhea virus (BVDV) from commercial fetal bovine serum and designated it HLJ-10. The complete genome is 12,284 nucleotides (nt); the open reading frame is 11,694 nt, coding 3,898 amino acids. Phylogenetic analysis indicated that this strain belongs to BVDV group 2.
Utilizing a classic stroke model in rodents, middle cerebral artery occlusion (MCAo), we describe a novel neuroregenerative approach using the repeated intranasal administration of cocaine- and amphetamine-regulated transcript (CART) peptide starting from day 3 poststroke for enhancing the functional recovery of injured brain. Adult rats were separated into two groups with similar infarction sizes, measured by magnetic resonance imaging on day 2 after MCAo, and were treated with CART or vehicle. The CART treatment increased CART level in the brain, improved behavioral recovery, and reduced neurological scores. In the subventricular zone (SVZ), CART enhanced immunolabeling of bromodeoxyuridine, a neural progenitor cell marker Musashi-1, and the proliferating cell nuclear antigen, as well as upregulated brain-derived neurotrophic factor (BDNF) mRNA. AAV–GFP was locally applied to the SVZ to examine migration of SVZ cells. The CART enhanced migration of GFP(+) cells from SVZ toward the ischemic cortex. In SVZ culture, CART increased the size of neurospheres. The CART-mediated cell migration from SVZ explants was reduced by anti-BDNF blocking antibody. Using 1H-MRS (proton magnetic resonance spectroscopy), increases in N-acetylaspartate levels were found in the lesioned cortex after CART treatment in stroke brain. Cocaine- and amphetamine-regulated transcript increased the expression of GAP43 and fluoro-ruby fluorescence in the lesioned cortex. In conclusion, our data suggest that intranasal CART treatment facilitates neuroregeneration in stroke brain.
BDNF; CART; MRI; neuroregeneration; stroke
The purpose of the research was to find out the factors which influence plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) levels, then to assess whether preoperative plasma NT-proBNP levels could predict postoperative outcomes of cardiac surgery.
Between November 2008 and February 2010,225 patients who underwent cardiac surgery in our department were included in the study. The mean age was 61.25 ± 12.54 years, and 156 (69.3%) patients were male. NT-proBNP, CK-MB, cTnT and creatinine levels were measured preoperatively and 24 hours after operation. Postoperatively outcomes including ventilation time, length of stay in ICU and hospital, and mortality were closely monitored. The endpoints includes: 1) use of inotropic agents or intra-aortic balloon pump ≥24 h; 2) creatinine level elevated to hemodialysis; 3) cardiac events; 4) ICU stay ≥5d; 5) ventilation dependence ≥ 72 h; 6) deaths within 30 days of surgery.
NT-proBNP concentrations (median [interquartile range]) increased from 728.4 pg/ml (IQR 213.5 to 2551 pg/ml) preoperatively to 1940.5 pg/ml (IQR 995.9 to 3892 pg/ml) postoperatively (P = 0.015). Preoperative atrial fibrillation, NYHA class III/IV, ejection fraction, pulmonary arterial pressure, left ventricle end-diastolic diameter (LVEDD), preoperative plasma creatinine and cTnT levels were significantly associated with preoperative NT-proBNP levels in univariate analysis. The preoperative NT-proBNP was closely related to ventilation time (P = 0.009), length of stay in ICU (P = 0.004) and length of stay in hospital (P = 0.019). Receiver operating characteristic curves demonstrated a cut-off value above 2773.5 pg/ml was the best cutoff (sensitivity of 63.6% and specificity of 80.8%) to predict the mortality within 30d of surgery.
Preoperative plasma NT-proBNP level presents a high individual variability in patients undergoing cardiac surgery. NYHA classification, ejection fraction, pulmonary arterial pressure, LVEDD, atrial fibrillation, preoperative plasma creatinine, and cTnT levels are significantly associated with preoperative NT-proBNP levels. Preoperative NT-proBNP is a valuable marker in predicting postoperative outcomes.
N-terminal pro-brain natriuretic peptide; Cardiac surgery; Prognosis
AIM: To investigate whether the expression of kallikrein 12 (KLK12) is related to the development of gastric cancer (GC) and to determine the role of KLK12 in gastric cancer cells growth, invasion and migration.
METHODS: Between September 2007 and March 2008, 133 patients with histologically confirmed GC were recruited for the study. Expression of KLK12 was detected in samples from GC patients by quantitative real-time reverse transcription polymerase chain reaction and immunohistochemistry. The relationship between KLK12 protein expression and clinicopathological features of GC was analyzed. The difference in 5-year survival rates between the high KLK12 protein expression group and the low KLK12 expression group was compared. Additionally, the expression of KLK12 was examined in various human GC cell lines, including MKN-28, SGC-7901 and MKN-45. Small interfering RNA (siRNA) was used to inhibit KLK12 expression in MKN-45 cells. Cell clones stably transfected with KLK12 siRNA were tested for KLK12 expression by quantitative real-time reverse transcription-polymerase chain reaction and Western blotting. Furthermore, a series of functional assays were performed in this study to assess the biological features of transfected cells. Cell proliferation was assessed using the methylthiazolyltetrazoliumassay. Finally, cell migration and invasion were assessed using transwell chamber assays.
RESULTS: Of the 133 GC patients included in the study, 126 (94.7%) showed a higher expression level of KLK12 mRNA when compared to noncancerous tissue specimens. Expression of KLK12 mRNA was significantly higher in GC tissues than in normal tissue (P < 0.001). KLK12 protein expression was detected in 96 of 133 (72.2%) GC samples with moderate or strong staining primarily in the cytoplasm. In contrast, negative immunostaining for KLK12 protein was observed in the corresponding normal gastric mucosal tissue. Overexpression of KLK12 protein was significantly associated with lymph node metastasis (P = 0.001), histological type (P < 0.001) and tumor-node-metastasis stage (P = 0.005), while no significant correlation was observed between expression of KLK12 protein and sex, age, depth of invasion, tumor size or lymphatic invasion. Furthermore, patients with high KLK12 expression had a significantly poorer 5-year survival rate than those with low KLK12 expression (P = 0.002). Expression of KLK12 mRNA was significantly higher in MKN-45 GC cells compared to normal mucosal cells or two other GC cell lines (P < 0.01). Expression of KLK12 in MKN-45 cells was downregulated after transfection with siRNA. Knockdown of KLK12 markedly decreased the proliferation of MKN-45 cells when compared with parent or mock-transfected cells (P = 0.001), especially from the 3rd to the 5th day of the assay. In migration assays, fewer KLK12 siRNA cells migrated through the chambers (22.00 ± 1.81) when compared to the parent (46.47 ± 2.42) or mock-transfected cells (45.40 ± 1.99); these differences were statistically significant (P < 0.001). However, in the invasion assay, the number of KLK12 siRNA cells that invaded the chambers was 18.40 ± 1.12, closely similar to both the parent (18.67 ± 0.98) and mock-transfected cells (18.53 ± 0.92). There was no significantly difference between the three groups in the invasion assay (P = 0.054).
CONCLUSION: The KLK12 gene is markedly overexpressed in GC tissue, and its expression status may be a powerful prognostic indicator for patients with GC. KLK12 might serve as a novel diagnosis and prognosis biomarker in GC.
Gastric cancer; Human kallikrein 12; Immunohistochemistry; Prognosis; Small interfering RNA; Migration; Invasion
Liraglutide is a glucagon-like peptide-1 analogue that stimulates insulin secretion and improves β-cell function. However, it is not clear whether liraglutide achieves its glucose lowering effect only by its known effects or whether other as yet unknown mechanisms are involved. The aim of this study was to examine the effects of liraglutide on Fibroblast growth factor-21 (FGF-21) activity in High-fat diet (HFD) fed ApoE−/− mice with adiponectin (Acrp30) knockdown.
HFD-fed ApoE−/− mice were treated with adenovirus vectors expressing shAcrp30 to produce insulin resistance. Hyperinsulinemic-euglycemic clamp studies were performed to evaluate insulin sensitivity of the mouse model. QRT-PCR and Western blot were used to measure the mRNA and protein expression of the target genes.
The combination of HFD, ApoE deficiency, and hypoadiponectinemia resulted in an additive effect on insulin resistance. FGF-21 mRNA expressions in both liver and adipose tissues were significantly increased while FGF-21 receptor 1 (FGFR-1) and β-Klotho mRNA levels in adipose tissue, as well as FGFR-1-3 and β-Klotho mRNA levels in liver were significantly decreased in this model. Liraglutide treatment markedly improved insulin resistance and increased FGF-21 expression in liver and FGFR-3 in adipose tissue, restored β-Klotho mRNA expression in adipose tissue as well as FGFR-1-3, β-Klotho levels and phosphorylation of FGFR1 up to the levels observed in control mice in liver. Liraglutide treatment also further increased FGF-21 proteins in liver and plasma. In addition, as shown by hyperinsulinemic-euglycemic clamp, liraglutide treatment also markedly improved glucose metabolism and insulin sensitivity in these animals.
These findings demonstrate an additive effect of HFD, ApoE deficiency, and adiponectin knockdown on insulin resistance and unveil that the regulation of glucose metabolism and insulin sensitivity by liraglutide may be partly mediated via increased FGF-21 and its receptors action.
In acupuncture brain imaging trials, there are many non-acupuncture factors confounding the neuronal mapping. The modality of the placebo, subjects’ psychological attitude to acupuncture and their physical state are the three most confounding factors.
To obtain more precise and accurate cerebral fMRI mapping of acupuncture.
Design and Setting
A 2×2 randomized, controlled, participant-blinded cross-over factorial acupuncture trial was conducted at Xuanwu Hospital in Beijing, China.
Forty-one college students with myopia were recruited to participate in our study and were allocated randomly to four groups, Group A, Group B, Group C and Group D.
Group A received real acupuncture (RA) and treatment instruction (TI); Group B received RA and non-treatment instruction (NI); Group C received sham acupuncture (SA) and TI; Group D received SA and NI.
Stimulation at LR3 activated some areas of the visual cortex, and the cerebral response to non-acupuncture factors was complex and occurred in multiple areas.
The results provide more evidence regarding the credibility of acupuncture therapy and suggest that more precise experimental designs are needed to eliminate sources of bias in acupuncture controlled trials and to obtain sound results.
Thimerosal, a mercury-containing preservative, is one of the most widely used preservatives and found in a variety of biological products. Concerns over its possible toxicity have reemerged recently due to its use in vaccines. Thimerosal has also been reported to be markedly cytotoxic to neural tissue. However, little is known regarding thimerosal-induced toxicity in muscle tissue. Therefore, we investigated the cytotoxic effect of thimerosal and its possible mechanisms on mouse C2C12 myoblast cells.
The study showed that C2C12 myoblast cells underwent inhibition of proliferation and apoptosis after exposure to thimerosal (125–500 nM) for 24, 48 and 72 h. Thimerosal caused S phase arrest and induced apoptosis as assessed by flow cytometric analysis, Hoechst staining and immunoblotting. The data revealed that thimerosal could trigger the leakage of cytochrome c from mitochondria, followed by cleavage of caspase-9 and caspase-3, and that an inhibitor of caspase could suppress thimerosal-induced apoptosis. Thimerosal inhibited the phosphorylation of Aktser473 and survivin expression. Wortmannin, a PI3K inhibitor, inhibited Akt activity and decreased survivin expression, resulting in increased thimerosal-induced apoptosis in C2C12 cells, while the activation of PI3K/Akt pathway by mIGF-I (50 ng/ml) increased the expression of survivin and attenuated apoptosis. Furthermore, the inhibition of survivin expression by siRNA enhanced thimerosal-induced cell apoptosis, while overexpression of survivin prevented thimerosal-induced apoptosis. Taken together, the data show that the PI3K/Akt/survivin pathway plays an important role in the thimerosal-induced apoptosis in C2C12 cells.
Our results suggest that in C2C12 myoblast cells, thimerosal induces S phase arrest and finally causes apoptosis via inhibition of PI3K/Akt/survivin signaling followed by activation of the mitochondrial apoptotic pathway.
Background: The feature of CD44 binding with native high molecular weight hyaluronan (nHA) and hyaluronan oligosaccharides (oHA) is different.
Results: nHA induces but oHA reduces CD44 clustering.
Conclusion: nHA and oHA have distinct effects on CD44 clustering.
Significance: The study provides direct evidence for the different characteristics of CD44 binding with nHA and oHA in vivo.
CD44 is a major cell surface receptor for the glycosaminoglycan hyaluronan (HA). Native high molecular weight hyaluronan (nHA) and oligosaccharides of hyaluronan (oHA) provoke distinct biological effects upon binding to CD44. Despite the importance of such interactions, however, the feature of binding with CD44 at the cell surface and the molecular basis for functional distinction between different sizes of HA is still unclear. In this study we investigated the effects of high and low molecular weight hyaluronan on CD44 clustering. For the first time, we provided direct evidence for a strong relationship between HA size and CD44 clustering in vivo. In CD44-transfected COS-7 cells, we showed that exogenous nHA stimulated CD44 clustering, which was disrupted by oHA. Moreover, naturally expressed CD44 was distributed into clusters due to abundantly expressed nHA in HK-2 cells (human renal proximal tubule cells) and BT549 cells (human breast cancer cell line) without exogenous stimulation. Our results suggest that native HA binding to CD44 selectively induces CD44 clustering, which could be inhibited by oHA. Finally, we demonstrated that HA regulates cell adhesion in a manner specifically dependent on its size. oHA promoted cell adhesion while nHA showed no effects. Our results might elucidate a molecular- and/or cellular-based mechanism for the diverse biological activities of nHA and oHA.
Cd44; Fluorescence Resonance Energy Transfer (FRET); Glycobiology; Hyaluronate; Oligosaccharide
Splenectomy is the most effective treatment for patients with primary immune thrombocytopenia (ITP) who fail to respond to steroid therapy. Thus far, there is no effective means to predict the long-term haematological response of the procedure. The purpose of this study was to identify serum biomarkers as predictors of long-term response based on a proteomics approach.
The serum samples of ITP patients were collected before splenectomy and seven days after surgery. After depletion of the abundant serum proteins, pooled preoperative serum samples from four responders to splenectomy, four nonresponders and four healthy controls were subjected to two-dimensional gel electrophoresis (2-DE). Nine protein spots with at least a five-fold alteration in expression between responders and nonresponders were all identified as haptoglobin (Hp) by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometer (MS) analysis. The validation of serum Hp expression was performed using enzyme-linked immunosorbent assays (ELISA) in thirty-seven responders, thirteen nonresponders and twenty-one healthy controls.
The preoperative serum levels of Hp in the nonresponders (925.9 ± 293.5 μg/ml) were significantly lower than those in the responders (1417.4 ± 315.0 μg/ml, p <0.001) and the healthy controls (1409.1 ± 354.2 μg/ml, p <0.001), while there was no significant difference between the latter two groups. The postoperative serum levels of Hp in responders and nonresponders were (1414.1 ± 225.0 μg/ml) and (952.9 ± 202.4 μg/ml), respectively. There were no significant differences between the serum Hp levels before and after surgery in both responders and nonresponders (p>0.05). The preoperative serum levels of Hp did not significantly correlate with preoperative platelet count of the same blood samples (r = 0.244, p = 0.087), while it positively correlated with postoperative peak platelet count (r = 0.622, p < 0.001). The optimal cutoff value of preoperative serum Hp levels (1173.80 μg/ml) derived from the receiver operating characteristic (ROC) curve led to 78.4% sensitivity and 84.6% specificity.
These results suggest that serum Hp levels may serve as a favourable predictor for the long-term response to splenectomy in ITP and may help to understand the pathophysiological differences between responders and nonresponders.
Primary immune thrombocytopenia; Splenectomy; Proteomics; Biomarkers; Haptoglobin
Human induced pluripotent stem cells (iPSCs) are potential renewable sources of hepatocytes for drug development and cell therapy. Differentiation of human iPSCs into different developmental stages of hepatic cells has been achieved and improved during the last several years. We have recently demonstrated the liver engraftment and regenerative capabilities of human iPSC-derived multistage hepatic cells in vivo. Here we describe the in vitro and in vivo activities of hepatic cells derived from patientspecific iPSCs, including multiple lines established from either inherited or acquired liver diseases, and discuss basic and clinical applications of these cells for disease modeling, drug screening and discovery, gene therapy and cell replacement therapy.
induced pluripotent stem cells (iPSCs); hepatic differentiation; liver ngraftment; disease modeling; drug testing; alpha-1 antitrypsin; liver cirrhosis; hepatocellular carcinoma; cell therapy
Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine with keto-enol tautomerase activity, rises rapidly in response to inflammation, and is elevated in many chronic diseases. Isothiocyanates, such as sulforaphane from broccoli, are very potent inactivators of MIF tautomerase activity. A simple rapid method for determining this activity in tissues and body fluids may therefore be valuable for assessing severity of inflammation and efficacy of intervention.
Existing spectrophotometric assays of MIF, based on conversion of methyl L-dopachrome to methyl 5,6-dihydroxyindole-2-carboxylate and associated loss of absorption at 475 nm, lack sensitivity. Assay sensitivity and efficiency were markedly improved by reducing the nonenzymatic rate, by lowering pH to 6.2, replacing phosphate (which catalyzes the reaction) with Bis-Tris buffer, and converting to a microtiter plate format.
A structure-potency study of MIF tautomerase inactivation by isothiocyanates showed that sulforaphane, benzyl, n-hexyl, and phenethyl isothiocyanates were especially potent. MIF tautomerase could be readily quantified in human urine concentrated by ultrafiltration. This activity comprised: (i) a heat-labile, sulforaphane-inactivated macromolecular fraction (presumably MIF) that was concentrated during ultrafiltration; (ii) a flow-through fraction, with constant activity during filtration, that was heat-stable, and insensitive to sulforaphane. Administration of the sulforaphane precursor glucoraphanin to human volunteers almost completely abolished urinary tautomerase activity, which was recovered over many hours.
A simple, rapid, quantitative MIF tautomerase assay has been developed as a potential biomarker for assessing inflammatory severity and effectiveness of intervention.
An improved assay for measuring MIF tautomerase activity and its applications are described.
dopachrome; glucoraphanin; isotiocyanates; RAW 264.7 cells