Search

Background and aim Astragalus membranaceus (AM) is a major Chinese herb used in the treatment of stroke. Astragaloside IV (AS)is a component of AM. This study investigated the effects of AM on the protein expression through proteomics analysis in ischemia-reperfusion injured Sprague Dawley rats. Experimental procedure An animal model of ischemia-reperfusion injury by occlusion of the right middle cerebral artery for 90 min followed by reperfusion for 24 h. The rats were intraperitoneally injected with AM or AS three times at 30 min, 1 day, and 2 days prior to the occlusion of the cerebral blood flow. Results Aldolase C was overexpressed in the cortex, and Dihydrolipoamide dehydrogenase and Triose-phosphate isomerase were overexpressed in the hippocampus. Conclusion Pretreatment with AM or AS can induce the overexpression of Aldolase C in the cerebral cortex and that of Dihydrolipoamide dehydrogenase and Triose-phosphate isomerase in the hippocampus, suggesting that both AM and AS may act as neuroprotectors through regulating the expression of Aldolase C, Dihydrolipoamide dehydrogenase and Triose-phosphate isomerase. However, the underlying neuroprotective mechanisms need more studies.

Elevated urinary albumin excretion is a clinical manifestation of early-stage diabetic nephropathy (DN).To investigate effect of acupressure at Sanyinjiao on albuminuria in patients with early DN.Total included 53 patients with DN and albuminuria; 21 were assigned to the sham group without acupressure, and 32 were assigned to the experimental group with acupressure at Sanyinjiao (SP6) for 8weeks. The experimental group was divided into experiment A (acupressure45 days) and experiment B (acupressure ≥45 days). The primary outcome measure was the urine albuminuria/creatinine ratio (UACR) or logarithmic transformed urine microalbumin creatinine ratio (log-UACR) changes, and the secondary outcome measures were the estimated glomerular filtration rate and hemoglobin A1c.The difference in UACR and log-UACR before and after the study was higher in the experiment B group than in the experiment A and sham groups.Acupressure at Sanyinjiao for 8 weeks may reduce albuminuria in patients with DN. However, this study was a preliminary design.

Background and aim Astragalus membranaceus (AM) is a major Chinese herb used in the treatment of stroke. Astragaloside IV (AS)is a component of AM. This study investigated the effects of AM on the protein expression through proteomics analysis in ischemia-reperfusion injured Sprague Dawley rats. Experimental procedure An animal model of ischemia-reperfusion injury by occlusion of the right middle cerebral artery for 90 min followed by reperfusion for 24 h. The rats were intraperitoneally injected with AM or AS three times at 30 min, 1 day, and 2 days prior to the occlusion of the cerebral blood flow. Results Aldolase C was overexpressed in the cortex, and Dihydrolipoamide dehydrogenase and Triose-phosphate isomerase were overexpressed in the hippocampus. Conclusion Pretreatment with AM or AS can induce the overexpression of Aldolase C in the cerebral cortex and that of Dihydrolipoamide dehydrogenase and Triose-phosphate isomerase in the hippocampus, suggesting that both AM and AS may act as neuroprotectors through regulating the expression of Aldolase C, Dihydrolipoamide dehydrogenase and Triose-phosphate isomerase. However, the underlying neuroprotective mechanisms need more studies., Graphical abstract Image 1, Highlights • Astragalus membranaceus(AM) is a famous Chinese herb for treating stroke. • This study used Proteomics analysis to investigate the protein biomarkers of brain tissue in AM and its component of Astragaloside (AS) treating ischemic stroke. • The present study found that Aldolase C in the cortex, and Dihydrolipoamide dehydrogenase and Triose- in the hippocampus were overexpression.

Nucleus pulposus (NP) cells locate in the center of avascular intervertebral discs, and thus have presumably adapted to a hypoxic environment. The purpose of this study was to investigate the influences of hypoxic condition, during isolation-expansion of human NP cells, on the cellular proliferation and extracellular matrix (ECM) synthesis in later three-dimensional cultures. Human NP tissues were obtained from patients who underwent lumbar disc surgeries. Immediately after retrieval, NP tissues from each patient were divided into two aliquots for in vitro cultivation either under classical normoxic (21% O2 ) or hypoxic (3.5% O2 ) condition. After isolation-expansion processes, microtissues of NP cells were formed and the analysis was performed after one-week culture. Experiments of pretreatment with TGF-β1 or lovastatin were designed to investigate if the isolation-expansion conditions affect the responsiveness to later exogenous treatments. Hypoxic isolation-expansion stimulated NP cell proliferation during monolayer culture. Hypoxia also upregulated mRNA levels of SOX9 and HIF-1α but downregulated type X collagen as well as improved aggrecan and type II collagen synthesis. Although TGF-β1 had no substantial effect, lovastatin pretreatment showed a greater enhancement on type II collagen expression in hypoxic group. Normoxia negatively affected the biochemical composition of regenerated ECM attributable to downregulation of SOX9 and HIF-1α, while hypoxia enhanced cellular proliferation, improved matrix production, and maintained a functional phenotype of NP cells. Hypoxic isolation-expansion of human NP cells is important to achieve better regenerative cells for later cultivation or cell transplantation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1575-1582, 2017.

Acupuncture, one of the most popular complementary therapies, is best known for its ability to provide pain relief. Accumulating evidence suggests that acupuncture may also be beneficial in depression, although its effectiveness remains uncertain in this condition. We conducted a meta-analysis of randomized trials in which the effects of acupuncture combined with antidepressant medications were compared with those of antidepressant medications alone in adults with a diagnosed depressive disorder. Thirteen randomized controlled trials involving 1046 subjects were included in the meta-analysis. Our results confirmed that the pooled standardized mean difference of the 'endpoint scores of the 17-item Hamilton rating scale for depression' was -3.74 (95% CI, -4.77 to -2.70, p

Introduction The phenotypic changes in thrombin-stimulated endothelial cells include alterations in permeability, cell shape, vasomotor tone, leukocyte trafficking, migration, proliferation, and angiogenesis. Previous studies regarding the pleotropic effects of thrombin on the endothelium used human umbilical vein endothelial cells (HUVECs)—cells derived from fetal tissue that does not exist in adults. Only a few groups have used screening approaches such as microarrays to profile the global effects of thrombin on endothelial cells. Moreover, the proteomic changes of thrombin-stimulated human aortic endothelial cells (HAECs) have not been elucidated. Materials and methods HAECs were stimulated with 2 units/mL thrombin for 5h and their proteome was investigated using isobaric tags for the relative and absolute quantification (iTRAQ) and the MetaCore TM software. Results A total of 627 (experiment A) and 622 proteins (experiment B) were quantified in the duplicated iTRAQ analyses. MetaCore TM pathway analysis identified cell adhesion as a dominant phenotype in thrombin-stimulated HAECs. Replicated iTRAQ data revealed that "Cell adhesion_Chemokines and adhesion," "Cell adhesion_Histamine H1 receptor signaling in the interruption of cell barrier integrity," and "Cell adhesion_Integrin-mediated cell adhesion and migration" were among the top 10 statistically significant pathways. The cell adhesion phenotype was verified by increased THP-1 adhesion to thrombin-stimulated HAECs. In addition, the expression of ICAM-1 , VCAM-1 , and SELE was significantly upregulated in thrombin-stimulated HAECs. Conclusions Several regulatory pathways are altered in thrombin-stimulated HAECs, with cell adhesion being the dominant altered phenotype. Our findings show the feasibility of the iTRAQ technique for evaluating cellular responses to acute stimulation.

Opioid addiction influences many physiological functions including reactions of the immune system. The objective of this study was to investigate the immune system function in heroin addicted patients undergoing methadone maintenance treatment (MMT) compared to healthy controls. We tested the cytokine production of IL-1β, IL-6, IL-8, IL-10 and tumor necrosis factor (TNF)-α from a group of heroin addicts (n=34) and healthy controls (n=20). The results show that production of IL-1β, IL-6 and IL-8 was significantly higher in the group of methadone-maintained patients than in the healthy control group. Plasma TNF-α and IL-6 levels were significantly correlated with the dairy methadone dosage administered, and the IL-1β level was significantly correlated with the duration of methadone maintenance treatment. These findings suggest that methadone maintenance treatment influences the immune system functions of opioid-dependent patients and may also induce long-term systemic inflammation.

Background Gastric adenocarcinoma originates from an abnormal epithelium. The aim of this study was to investigate the expression of sodium-potassium ATPase (NKA), a transmembrane protein located in the epithelium for Na+ and K+ transportation, and E-cadherin, which are both crucial for the epithelium and adherens junction, as potential gastric cancer biomarkers. Methods 45 patients diagnosed with gastric adenocarcinoma were recruited. Immunohistochemistry and immunofluorescence were conducted to for localization of NKA α1-, β1-isoform, and E-cadherin. NKA enzyme activity was determined by NADH-linked methods and immunoblotting of NKA α1-, β1-isoform, and E-cadherin were performed to evaluate protein expression. Results Immunostaining revealed that NKA was co-localized with E-cadherin in the glands of the gastric epithelium. Both NKA activity and α1-isoform protein expression were reduced in the study group (P < 0.05), indicating impaired NKA functions. In the adherens junctions, the NKA β1-isoform and E-cadherin were significantly reduced in the study groups (P < 0.05), indicating the adhesion force between cells may have been weakened. Conclusions A significant decrease in NKA function (protein and activity) and E-cadherin in tumor lesions appear promising biomarker for gastric adenocarcinoma. Therefore, developing screening methods for detecting NKA function may be beneficial for the early diagnosis of gastric cancer. In our knowledge, this study was the first to investigate the NKA and E-cadherin expression in the relation of gastric adenocarcinoma in human patients.

The milkfish (Chanos chanos), an important aquaculture species, is intolerant to cold environments. Temperature fluctuations in the environment affect the physiological response, behavior, and survival rate of the fish. The warm-temperature-acclimation associated 65-kDa protein (Wap65) of teleosts was identified after heat shock treatment and has two isoforms. Both the isoforms were involved in the induction of immune responses in fish. They showed high degree of sequence conservation with the mammalian hemopexin and had high affinity for heme, which helped in the neutralization of free-heme and its transport to the liver. In this study, we isolated and characterized the two isoforms of wap65 genes (Ccwap65-1 and Ccwap65-2) from the liver of milkfish. The Ccwap65-1 and Ccwap65-2 are mainly expressed in livers of milkfish. In hypothermal treatment, the expression levels of Ccwap65-2 in the livers of SW and FW milkfish were up-regulated after exposure to low temperature (18 °C) for 12 h and 96 h compared to those in the normal temperature (28 °C) group, respectively. After intraperitoneal injection of lipopolysaccharide (LPS), the expression of Ccwap65-2 was elevated in both SW and FW milkfish, whereas that of Ccwap65-1 was not affected in both the groups. Thus, Ccwap65-2 expressed in the milkfish liver under hypothermal stress was identified as a novel immune biomarker. In addition, according to the transcriptome database, up-regulation of the other immune-response genes indicated increased pathogen infection status under hypothermal stress. Acute increase in the expression of hepatic Ccwap65-2 in response to pathogen infection might lead to better cold tolerance of SW milkfish compared to that of the FW individuals upon cold challenge.

Background: Colorectal cancer (CRC) is the highest leading cause of cancer-related mortality in Taiwan. Macrophage migration inhibitory factor (MIF) has recently been defined as a novel protumorigenic factor that promotes cell proliferation, migration, and invasion. The aim of the present study is to identify the association between MIF gene polymorphism and CRC. Methods: A case‐control study was designed to test the hypothesis. A total of 192 biopsydiagnosed CRC patients (CRC) and 256 healthy subjects (control) were recruited. Genotyping of four single nucleotide polymorphism (SNPs; rs755662, rs11548059, rs1049829, rs1803976) at chromosome positions 755662 (5 � UTR), 11548059 (exon2), 1049829 (exon2), 1803976 (exon3) was performed using a Taqman SNP genotyping assay. Results: There is a significant difference in genotype frequency distribution of rs755662 polymorphism between CRC patients and controls (P = 0.011). No significant difference was found in the frequency distribution of rs11548059, rs1049829, rs1803976 polymorphism in CRC patients and controls (P = 0.660, P = 0.700, and P =0.959,respectively).Moreover,theMIF173 SNP was also significantly associated with young patients (age < 50 years, P = 0.026) late stage (Stage IV, P = 0.038) and poor differentiation group (P = 0.040). Compared to the control group, the MIF173 SNP also significantly associated with patients with stages III and IV (P = 0.034 and 0.003, respectively). Conclusion: The presence of MIF-173 (G/C) gene polymorphism (rs755662) was associated with susceptibility, patient age, and stages of CRC in Taiwanese. J. Clin. Lab. Anal. 00:1‐7, 2014. C � 2014 Wiley Periodicals, Inc.

Background Childhood acute lymphoblastic leukemia (ALL), a heterogeneous disease that includes multiple subtypes is defined by cell lineage and chromosome anomalies. Previous genome-wide association studies have reported several ARID5B and IKZF1 single nucleotide polymorphisms (SNPs) associated with the incidence of ALL. High-resolution melting (HRM) analysis is a rapid and convenient technique to detect SNPs; we thereby detected SNPs in ARID5B and IKZF1 genes. Methods We enrolled 79 pediatric ALL patients and 80 healthy controls. Polymorphic variants of IKZF1 (rs6964823, rs4132601, and rs6944602) and ARID5B (rs7073837, rs10740055, and rs7089424) were detected by HRM, and SNPs were analyzed for association with childhood ALL. Results The distribution of genotype rs7073837 in ARID5B significantly differed between ALL and controls (P = 0.046), while those of IKZF1 (rs6964823, rs4132601, and rs6944602) and ARID5B (rs10740055 and rs7089424) did not. We analyzed the association for SNPs with B lineage ALL to find rs7073837 in ARID5B, conferring a higher risk for B lineage ALL (odds ratio, OR = 1.70, 95% confidence interval, CI = 1.01–2.87, P = 0.049). Conclusion HRM is a practical method to detect SNPs in ARID5B and IKZF1 genes. We found that rs7073837 in ARID5B correlated with a risk for childhood B lineage ALL.

Background Gastric cancer is a major public health concern as the fourth most common cancer, and it is of particular relevance as the second most common cause of cancer death worldwide. We caparisoned the urinary nucleoside concentrations between the gastric patients and healthy volunteers that try to evaluate the diagnostic value in the gastric cancer. Method Urinary nucleosides from 49 gastric patients and 40 healthy volunteers were evaluated by high-performance liquid chromatography/electrospray ionization–tandem mass spectrometry (HPLC/ESI–MS/MS) under optimized conditions as determined in our previous study. Results The mean concentrations of 5 urinary nucleosides, cytidine, 3-methylcytidine (m3C), 1-methyladenosine (m1A), adenosine, and inosine, were found to be elevated in cancer patients, but only cytidine showed a significant elevation. Moreover, cytidine concentrations were significantly elevated by an average of 1.42-fold in patients with late stage (S3 + 4) disease. Combining the determined concentrations of preoperative serum alpha-fetoprotein (AFP, cutoff of 20 μg/l) or carbohydrate antigen 19-9 (CA19-9, cutoff of 37 U/ml) with the mean urinary cytidine concentration was shown to improve the diagnostic ratio (sensitivity) for gastric cancer from 16.3% (8/49 patients) to 38.8% (8 + 11/49 patients) or from 28.6% (14/49 patients) to 51.0% (14 + 11/49 patients), respectively. Conclusions Urinary cytidine may be an important adjunct biomarker for gastric cancer.

The angiotensin-converting enzyme 2/angiotensin-(1-7)/Mas axis is a pathway that acts against the detrimental effects of the renin-angiotensin system. However, the effects of angiotensin-(1-7) on endothelial protein expression and the related phenotypes are unclear. We performed a duplicate of iTRAQ quantitative proteomic analysis on human aortic endothelial cells (HAECs) treated with angiotensin-(1-7) for 6 hours. Cofilin-1 was identified as a highly abundant candidate with consistent >30% coverage and >1.2-fold overexpression in the angiotensin-(1-7)-treated group. Gene ontology analysis showed that the “regulation_of_mitosis” was significantly altered, and cell cycle analysis indicated that the 6-hour angiotensin-(1-7) treatment significantly induced G0/G1 arrest. Knockdown of the cofilin-1 (CFL1) gene suggested the G0/G1 phase arrest was mediated by the modulation of p27 and the p21/Cyclin/CDK complex by Cofilin-1. Interestingly, quiescent HAECs escaped G0/G1 arrest upon angiotensin-(1-7) treatment for 24 hours, and angiotensin-(1-7) induced autophagy by upregulating Beclin-1 and microtubule-associated protein 1 light chain 3b-II expression, which was also attenuated by A779 pre-treatment and CFL1 knockdown. After pre-treatment with 3-methyladenine (3MA), treatment with angiotensin-(1-7) for 24 h induced significant G0/G1 phase arrest and apoptosis, suggesting a pro-survival role of autophagy in this context. In conclusion, Cofilin-1 plays a dominant role in angiotensin-(1-7)-induced G0/G1 arrest and autophagy to maintain cellular homeostasis in HAECs.

Colorectal cancer (CRC) is the third most common type of cancer worldwide. Carcinoembryonic antigen (CEA) assays usually give false negative results. To improve the diagnosis of primary sporadic CRC, there is an urgent need to identify new biomarkers.We used laser pressure catapulting and proteomics to analyze overexpressed cancer associated proteins from 48 sporadic CRC patients with low preoperative serum CEA (LPSC) (5 ng/ml). Real-time Q-PCR was used to identify the target gene transcripts. Immunoblots were carried out to validate the biomarkers.Alpha-enolase, HSP27 and macrophage migration inhibitory factor (MIF) were overexpressed in all tumor tissues from 48 LPSC CRC patients, as assessed by 2DE image analysis. The genes were also overexpressed at the transcript level in all tumor tissues from the same patients. In the immunoblot assay, only serum levels of Alpha-enolase and MIF were significantly overexpressed in the LPSC group compared to the mean levels in the control group. Combined with the determinations of preoperative CEA levels, screening for serum Alpha-enolase and MIF were shown to improve the diagnosis of primary CRC.Serum Alpha-enolase and MIF may be potential biomarkers that can be used to improve clinical predication of primary CRC with LPSC.

Since the introduction of laparoscopic colectomy, improved short-term surgical results have been noted in the literature. Therefore, efforts have shifted to reducing the invasiveness of laparoscopic surgery, resulting in the invention of single-incision laparoscopic surgery (SILS). Due to its comparable capabilities and feasibility, the implementation of SILS has rapidly grown in different fields. However, few studies discuss its true benefit compared with conventional laparoscopy. This study is the first to use SILS colectomy as an approach for malignant colon cancer. The goal of this cohort series is to compare the short-term surgical outcomes between SILS and conventional right hemicolectomy.This was a case-control study comparing SILS right hemicolectomy patients to traditional laparoscopic right hemicolectomy. The inclusion criteria were only ascending colon cecal lesions. Cases of obstruction or perforation that required emergent operation or previous abdominal surgery were excluded. These patients were specifically matched in regard to patient's age, gender, perioperative condition, surgical indication, and tumor size. No consideration or analysis of operative parameters and outcomes was made until this group was definitively selected as the best comparison cohort based on preoperative variables only.A total of 18 patients were included for SILS and the other 21 patients were completed by conventional laparoscopic right hemicolectomy. The SILS and traditional laparoscopic groups were similar in regard to age, gender, body mass index, and perioperation outcomes. Initial oncologic results were no different, including equal length of distal cut margin, numbers of harvested lymph nodes, and TMN stage. Three patients in the SILS colectomy group were converted (16.6%), and there were no conversions in the traditional laparoscopic colectomy group.Our preliminary experience with SILS right hemicolectomy demonstrated the safety of the procedure and its feasibility in malignant colon cancer. Although SILS right hemicolectomy may provide a subjective cosmetic advantage, there was no benefit in the short-term surgical outcomes. SILS is very situational, requires more effort from the surgeon, and may not offer more patient comfort. More experience with SILS and prospective trials are needed to validate it as a more favorable alternative to conventional laparoscopic colectomy.

In this paper, we propose a new test method to detect write disturbance fault (WDF) for magnetic RAM (MRAM). Furthermore, an adaptive diagnosis algorithm (ADA) is also introduced to identify and diagnose the WDF for MRAM. The proposed test method can evaluate process stability and uniformity. We also develop a built-in self-test (BIST) circuit that supports the proposed WDF diagnosis test method. A 1-Mb toggle MRAM prototype chip with the proposed BIST circuit has been designed and fabricated using a special 0.15-μm CMOS technology. The BIST circuit overhead is only about 0.05% with respect to the 1-Mb MRAM. The test time is reduced by about 30% as compared with the test method without using the decision write mechanism. The chip measurement results show the efficiency of our proposed method.

Ferulic acid (4-hydroxy-3-methoxycinnamic acid, FA) provides neuroprotection against apoptosis in a transient middle cerebral artery occlusion (MCAo) model. This study was to further investigate the anti-apoptotic effect of FA during reperfusion after cerebral ischemia.Rats were subjected to 90 min of cerebral ischemia followed by 3 or 24 h of reperfusion after which they were sacrificed.Intravenous FA (100 mg/kg) administered immediately after middle cerebral artery occlusion (MCAo) or 2 h after reperfusion effectively abrogated the elevation of postsynaptic density-95 (PSD-95), neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS), nitrotyrosine, and cleaved caspase-3 levels as well as apoptosis in the ischemic cortex at 24 h of reperfusion. FA further inhibited Bax translocation, cytochrome c release, and p38 mitogen-activated protein (MAP) kinase phosphorylation. Moreover, FA enhanced the expression of gamma-aminobutyric acid type B receptor subunit 1 (GABA(B1)) in the ischemic cortex at 3 and 24 h of reperfusion. In addition, nitrotyrosine-positive cells colocalized with cleaved caspase-3-positive cells, and phospho-p38 MAP kinase-positive cells colocalized with nitrotyrosine- and Bax-positive cells, indicating a positive relationship among the expression of nitrotyrosine, phospho-p38 MAP kinase, Bax, and cleaved caspase-3. The mutually exclusive expression of GABA(B1) and nitrotyrosine revealed that there is a negative correlation between GABA(B1) and nitrotyrosine expression profiles. Additionally, pretreatment with saclofen, a GABA(B) receptor antagonist, abolished the neuroprotection of FA against nitric oxide (NO)-induced apoptosis.FA significantly enhances GABA(B1) receptor expression at early reperfusion and thereby provides neuroprotection against p38 MAP kinase-mediated NO-induced apoptosis at 24 h of reperfusion.

Background Membranous glomerulonephritis (MGN) is one of the most common causes of nephrotic syndrome in adults. NPHS1 encoding nephrin is a transmembrane protein of the immunoglobulin family. We clarified the relationship between NPHS1 gene polymorphisms and the susceptibility or progression of MGN. Methods We recruited a cohort of 132 biopsy-diagnosed MGN patients and 257 healthy subjects. Genotyping of three SNPs (rs401824, rs437168 and rs3814995) at chromosome positions 41034749 (5′UTR), 41026259(exon17) and 41034052 (exon 3) was performed using a Taqman SNP genotyping assay. Results There was a significant difference in genotype frequency distribution of rs437168 polymorphism between MGN patients and controls. The results also showed that the frequency of the G allele was significantly higher in the patient group. Among the polymorphisms rs437168, rs401824 and rs3814995, no significant haplotype was shown in MGN patients. A stratified analysis revealed that a high disease progression in the AA genotype of rs401824 and GG genotype of rs437168 patients were associated with a low rate of remission. Conclusions The presence of the different genotypes of NPHS1 was associated with susceptibility of MGN and the remission of proteinuria during disease progression after the therapy.

Enhanced O-linked N-acetylglucosamine (O-GlcNAc) modification by O-GlcNAc transferase (OGT) is associated with diabetic complications. Increase oxidative stress causes endothelial inflammation in diabetes. Members of microRNA-200 (miR-200) family are sensitive to oxidative stress. Here, we examined

Patients with paclitaxel-eluting stents are concerned with stent thrombosis caused by premature discontinuation of dual antiplatelet therapy or clopidogrel resistance. This study investigates the effect of (−)-epigallocatechin-3-gallate (EGCG) on the expression of thrombin/paclitaxel-induced endothelial tissue factor (TF) expressions in human aortic endothelial cells (HAECs). EGCG was nontoxic to HAECs at 6 h up to a concentration of 25 μmol/L. At a concentration of 25 μmol/L, EGCG pretreatment potently inhibited both thrombin-stimulated and thrombin/paclitaxel-stimulated endothelial TF protein expression. Thrombin and thrombin/paclitaxel-induced 2.6-fold and 2.9-fold increases in TF activity compared with the control. EGCG pretreatment caused a 29% and 38% decrease in TF activity on thrombin and thrombin/paclitaxel treatment, respectively. Real-time polymerase chain reaction (PCR) showed that thrombin and thrombin/paclitaxel-induced 3.0-fold and 4.6-fold TF mRNA expressions compared with the control. EGCG pretreatment caused an 82% and 72% decrease in TF mRNA expression on thrombin and thrombin/paclitaxel treatment, respectively. The c-Jun terminal NH2 kinase (JNK) inhibitor SP600125 reduced thrombin/paclitaxel-induced TF expression. Furthermore, EGCG significantly inhibited the phosphorylation of JNK to 49% of thrombin/paclitaxel-stimulated HAECs at 60 min. Immunofluorescence assay did not show an inhibitory effect of EGCG on P65 NF-κB nuclear translocation in the thrombin/paclitaxel-stimulated endothelial cells. In conclusion, EGCG can inhibit TF expression in thrombin/paclitaxel-stimulated endothelial cells via the inhibition of JNK phosphorylation. The unique property of EGCG may be used to develop a new drug-eluting stent by co-coating EGCG and paclitaxel.

Uncaria rhynchophylla (Miq) Jack (UR) is a traditional Chinese herb and is used for the treatment of convulsive disorders, including epilepsy. Our previous study has shown that UR, as well as its major component rhynchophylline (RH), has an anticonvulsive effect and this effect is closely related to its scavenging activities of oxygen free radicals. The purpose of the present study was to investigate the effect of (UR) on the expression of proteins using a proteomics analysis in Sprague-Dawley (SD) rats with kainic acid (KA)-induced epileptic seizures. We profiled the differentially expressed proteins on two-dimensional electrophoresis (2-DE) maps derived from the frontal cortex and hippocampus of rat brain tissue 24 hours after KA-induced epileptic seizures. The results indicated that macrophage migration inhibitory factor (MIF) and cyclophilin A were under expressed in frontal cortex by an average of 0.19- and 0.23-fold, respectively. In the frontal cortex, MIF and cyclophilin A were significantly decreased in the KA group and these decreases were confirmed by the Western blots. However, in the hippocampus, only cyclophilin A was significantly decreased in the KA group. In addition, in real-time quantitative PCR (Q-PCR), MIF and cyclophilin A gene expressions were also significantly under expressed in the frontal cortex, and only the cyclophilin A gene was also significantly under expressed in the hippocampus in the KA group. These under expressions of MIF and cyclophilin A could be overcome by the treatment of UR and RH. In conclusion, the under expressions of MIF and cyclophilin A in the frontal cortex and hippocampus in KA-treated rats, which were overcome by both UR and UH treatment, suggesting that both MIF and cyclophilin A at least partly participate in the anticonvulsive effect of UR.

Hepatocellular carcinoma (HCC) is a common neoplasm in Taiwan, for which early diagnosis is difficult and the prognosis is usually poor. HCC is usually diagnosed by abdominal sonography and serum alpha-fetoprotein (AFP) detection. Modified nucleosides, regarded as indicators for the whole-body turnover of RNAs, are excreted in abnormal amounts in the urine of patients with malignancies and can serve as tumor markers. We analyzed the excretion patterns of urinary nucleosides from 25 HCC patients and 20 healthy volunteers by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) under optimized conditions. The HPLC/ESI-MS/MS approach with selective reaction monitoring (SRM) allowed for the sensitive determination of nucleosides in human urine samples. The mean levels of the urinary nucleosides adenosine, cytidine, and inosine were significantly higher in HCC patients than healthy volunteers (average of 1.78-, 2.26-, and 1.47-fold, respectively). However, the mean levels of urinary 1-methyladenosine, 3-methylcytidine, uridine, and 2'-deoxyguanosine were not significantly different. Combined with the determination of serum AFP levels, the higher levels of urinary adenosine, cytidine, and inosine may be additional diagnosis markers for HCC in Taiwanese patients.

Increased levels of modified nucleosides have been observed in urine from patients suffering from several cancers. In this study, we evaluated whether urinary nucleosides can serve as potential tumor markers for colorectal cancer by high performance liquid chromatography-electrospray/tandem mass spectrometry (HPLC/ESI-MS/MS).A simple and specific method based on HPLC/ESI-MS/MS was developed to determine the urinary nucleosides from patients with colorectal cancer. We studied the excretion patterns of nucleosides in urine from 26 patients with colorectal cancer and 18 healthy controls.The LC/MS/MS approach with selective reaction monitoring (SRM) allowed for the sensitive determination of nucleosides in human urine samples with colorectal cancer. The mean levels of 5 urinary nucleosides (adenosine, cytidine, N(2),N(2)-dimethylguanine, 8-hydroxy-2'-deoxyguanosine and uridine) were significantly higher in the patients with colorectal cancer than in the healthy adults.The results indicate that urinary nucleosides determined by LC/MS/MS may be useful as biological markers for colorectal cancer. Our findings suggest that LC/MS/MS is a highly specific and sensitive method for rapidly screening a large number of nucleoside that may be useful as markers for cancer in humans.

Background and objectives: The current pharmacological management of diabetic gastroparesis remains difficult. Acupuncture has been widely used for gastrointestinal symptoms. The aim of this study was to investigate the effects of electroacupuncture (EA) on solid gastric emptying time, serum gastrin, motilin, pancreatic polypeptide (PP), fasting and postprandial blood glucose, and symptoms in patients with diabetic gastroparesis. Interventions: EA at the Zusanli (ST 36) and Hegu (LI 4) points and sham EA as control were administered by an experienced and licensed acupuncturist. Design: This was a pilot study with a randomized, single-blinded design. Subjects and setting: Nineteen (19) patients with type 2 diabetes who had had symptoms of gastroparesis for more than 3 months were included in the trial and randomized into two groups. Each group received EA (n � 9) or sham EA (n � 10) consisting of 4 sessions over 2 weeks. Outcome measures: Symptom severity was evaluated using the Gastroparesis Cardinal Symptom Index (GCSI) at baseline, at the end of treatment, and 2 weeks after the end of the trial; solid-phase gastric half-emptying time was measured by scintigraphy; in addition, serum gastrin, motilin, PP, fasting, and postprandial blood glucose levels were also measured. Results: Gastric half-emptying time in 9 patients with diabetic gastroparesis was significantly shortened by EA treatment (143.8 � 55.9 minutes versus 98.8 � 28.6 minutes, p � 0.03). Half-emptying time did not change (98.9 � 26.4 minutes versus 90.9 � 24.8 minutes, p � 0.05) in the sham EA group. Symptom severity, as measured by GCSI total score, improved significantly both at the end of treatment (2.38 � 0.56 versus 1.48 � 0.19, p � 0.001) and 2 weeks after the end of the trial (2.38 � 0.56 versus 1.65 � 0.44, p � 0.01) when compared with the baseline in the EA group, but did not change from baseline with sham EA treatment. There were no significant changes in fasting and postprandial blood glucose, serum gastrin, motilin, and PP in both groups. No significant adverse events occurred. Conclusions: This study demonstrates that short-term EA at the Zusanli and Hegu points effectively reduces the dyspeptic symptoms of diabetic gastroparesis and accelerates solid gastric emptying. Sustained improvement in dyspeptic symptoms was observed at 2 weeks after the end of the trial. Its potential for treating gastroparesis may be explored, and a larger trial is required to draw definitive conclusions.

Milkfish is an important aquaculture species in Taiwan, and its high mortality during cold snaps in winter usually causes huge economic losses. To understand the effect of hypothermal stress and the corresponding compensatory stress response in milkfish, this study aimed to compare liver and gill protein levels between milkfish exposed to nonlethal (18°C), lethal (16°C), and control (28°C) temperatures. Using a proteomics approach based on two-dimensional electrophoresis and nano-LC-MS/MS analysis, this study identified thirty unique protein spots from milkfish livers and gills for which protein abundance was significantly different between nonlethal, lethal, and control temperature groups. Proteins identified in the liver were classified into three different categories according to their cellular function: (1) anti-oxidative stress, (2) apoptotic pathway, and (3) cytoskeleton. Similarly, proteins identified in the gill were sorted in five different functional categories: (1) cytoskeleton, (2) immune response, (3) protein quality control, (4) energy production, and (5) intracellular homeostasis. Based on functional information derived from the identified proteins, we assumed that different levels of hypothermal stress had a different effect and induced a different cellular response. Upon nonlethal hypothermal stress, the identified proteins were involved in anti-oxidative stress and anti-inflammation pathways, suggesting that milkfish had high levels of oxidative stress in the liver and exhibited inflammation response in the gill. Upon lethal hypothermal stress, however, identified proteins were associated with apoptosis in the liver and regulation of intracellular homeostasis in the gill. The present study provided evidence to illustrate different multi-physiological responses to nonlethal and lethal hypothermal stress in milkfish livers and gills.

The purpose of this work is to differentiate between the Human papillomaviruses 18 positive (HPV18+) and negative (HPV18-) oral squamous cell carcinomas (OSCC) in oral cancer patients with cancer-associated oral habits (betel quid chewing, cigarette smoking, and alcohol drinking). Both gene and protein expression profiles of HPV18+ and HPV18- OSCC were compared: we then further explored the biological effect of HPV in oral cancer. Suppression subtraction hybridization (SSH), clinical proteomics analysis, and immunohistochemistry (IHC) staining were carried out in the HPV18+ and HPV18- OSCC groups. HPV typing detection revealed that 11 OSCC tissues from 82 patients were positive for HPV18. The SSH experiment showed that 4 cancer-associated genes were highly transcribed within 11 cDNA libraries of HPV18+ OSCC, including poly(ADP-ribose)polymerase I (PARP1), replication protein A2 (RPA2), S100A8, and S100A2. Clinical proteomics analysis indicated that there was over 10-fold overexpression of Stratifin, F-actin capping protein alpha-1 subunit (CapZ alpha-1), Apolipoprotein A-1 (ApoA-1), Heat-shock protein 27 (HSP27), Arginase-1, p16INK4A, and S100 calcium-binding protein A8 (S100A8) in HPV18+ OSCC. Interestingly, the results from SSH and protemics analysis showed that S100A8 was overexpressed in HPV18+ OSCC. Moreover, IHC staining demonstrated that S100A8 was up-regulated in HPV18+ OSCC tissues. Our results suggest that S100A8 plays an important role in oral carcinogenesis following HPV18 infection; therefore, S100A8 may be a powerful biomarker of HPV18 as well as a potential therapeutic target for HPV18+ OSCC patients. The study is the first to identify S100A8 as a biomarker in HPV-associated cancer. Furthermore, this is also the first study to discover a biomarker by combining SSH, clinical proteomics, and IHC stain analysis in oral cancer-associated research.

Background Oral cancer is a worldwide problem. It is a universal aggressive disease in the population of smoking and drinking. The oral cancer mortality has been ranked 5th place in Taiwan in male cancer patients. A number of protein markers for oral cancer are still not applicable in large populations. Proteomic technologies provide excellent tools for rapid screening of a large number of potential biomarkers in malignant cells. Method Proteomics and real-time quantitative RT-PCR were used to analyze over-expressed proteins in 10 OSCC patients. Result Forty-one proteins were identified as commonly over-expressed in OSCC tissues. In OSCC tissues, αB-crystallin, tropomyosin 2, myosin light chain 1, heat shock protein 27 (HSP27), stratifin, thioredoxin-dependent peroxide reductase, flavin reductase, vimentin, rho GDP-dissociation inhibitor 2 (rho GDI-2), glutathione S-transferase Pi (GST-pi) and superoxide dismutase [Mn] (MnSOD) were significantly over-expressed (an average of 7.2, 6.0, 5.7, 4.3, 3.6, 3.4, 3.0, 3.0, 2.6, 2.5, 2.1-fold, respectively). In real-time quantitative RT-PCR analysis, the gene expressions of αB-crystallin, HSP27 and MnSOD were also increased in the cancer tissues, consistent with proteomic results. Conclusion The identified proteins in this experiment may be used in future studies of carcinogenesis or as diagnostic markers and therapeutic targets for OSCC.

Nucleic acid sequence-based amplification (NASBA) reactions have been demonstrated to successfully synthesize new sequences based on deletion and insertion reactions. Two RNA internal standards were synthesized for use in competitive amplification reactions in which quantitative analysis can be achieved by coamplifying the internal standard with the wild type sample. The sequences were created in two consecutive NASBA reactions using the E. coli clpB mRNA sequence as model analyte. The primer sequences of the wild type sequence were maintained, and a 20-nt-long segment inside the amplicon region was exchanged for a new segment of similar GC content and melting temperature. The new RNA sequence was thus amplifiable using the wild type primers and detectable via a new inserted sequence. In the first reaction, the forwarding primer and an additional 20-nt-long sequence was deleted and replaced by a new 20-nt-long sequence. In the second reaction, a forwarding primer containing as 5' overhang sequence the wild type primer sequence was used. The presence of pure internal standard was verified using electrochemiluminescence and RNA lateral-flow biosensor analysis. Additional sequence deletion in order to shorten the internal standard amplicons and thus generate higher detection signals was found not to be required. Finally, a competitive NASBA reaction between one internal standard and the wild type sequence was carried out proving its functionality. This new rapid construction method via NASBA provides advantages over the traditional techniques since it requires no traditional cloning procedures, no thermocyclers, and can be completed in less than 4 h.

An end-point quantitative nucleic acid sequence-based amplification (NASBA) reaction with two exogenous internal standards for the detection of the model analyte E. coli clpB mRNA was developed and statistically analyzed. Electrochemiluminescence was chosen as a highly sensitive detection means allowing careful evaluation of the internal standards used. The two internal standards examined had been designed previously using a novel and rapid NASBA-based method. Initially, each standard was used separately in a NASBA reaction; subsequently, two internal standards were added into one reaction at different concentrations. The accuracy and precision of the data obtained were analyzed using linear and multiple regression analysis. In the case of single-standard reactions, the accuracy was >95% and the precision >98.5%. In the case of double-standard reactions, the accuracy increased to >97%. With a single internal standard, 3 orders of magnitude of target sequence could be quantified; using three different concentrations of one internal standard, the dynamic range increased to 5 orders of magnitude. In both cases, a detection limit as low as 0.14 pg of target sequence was obtained. In the case of double-internal standard reactions, a dynamic range with 5 orders of magnitude and a detection limit of 1.76 pg was determined. The high-performance quality of the internal standards was assumed to be in part due to the unique synthesis process using two NASBA reactions rather than traditional cloning techniques.

The label-free detection of oligonucleotides of 12–14 bases at a concentration of 10−7M (double helix) was achieved by using surface enhanced Raman scattering (SERS) and spermine as the aggregant of Ag sol. The wavenumbers of the produced SERS spectra of DNA are similar to those in the corresponding normal Raman spectra of free DNA, allowing the detailed assignment of the vibrational modes. The conformation of the adsorbed DNA, the adsorption geometry, and a molecular model of the interactions among DNA, spermine, and Ag nanoparticle are derived from the SERS spectra. The results show that the protonated amine groups of spermine interact with phosphodioxygens of DNA and N7s of dA and dG from the major groove. The protonated amines are also attracted to the negatively charged Ag surface and thus induce the adsorption of DNA on the metal surface. The DNA remains mostly in the B-conformation with a variation in the C5′O dihedral angle. DNA lies flat with the bases perpendicular to the metal surface. Copyright © 2006 John Wiley & Sons, Ltd.

Methadone maintenance therapy is an effective treatment for opiate dependence, but more than three-quarters of persons receiving the treatment report sleep quality disturbances. In this double-blind, randomized, controlled trial, we recruited 90 individuals receiving methadone for at least one month who reported sleep disturbances and had Pittsburgh Sleep Quality Index (PSQI) scores > 5. The purpose of this study was to determine whether Suan Zao Ren Tang, one of the most commonly prescribed traditional Chinese medications for treatment of insomnia, improves subjective sleep among methadone-maintained persons with disturbed sleep quality. Ninety patients were randomly assigned to intervention group (n=45) and placebo group (n=45), and all participants were analyzed. Compared with placebo treatment, Suan Zao Ren Tang treatment for four weeks produced a statistically significant improvement in the mean total PSQI scores (P=0.007) and average sleep efficiency (P=0.017). All adverse events (e.g., lethargy, diarrhea, and dizziness) were mild in severity. Suan Zao Ren Tang is effective for improving sleep quality and sleep efficiency among methadone-maintained patients with sleep complaints.

Acute myeloid leukemia (AML) is the most common acute leukemia diagnosed in adults. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that plays a significant role in pathogenesis and autoimmune diseases. The major function of MIF is to promote the cell proliferation, migration, and invasion. The aim of the present study is to identify the association between MIF-173 (rs755662) single nucleotide polymorphism (SNP) and AML in Taiwanese population. DNA samples extracted from 256 AML patients and 256 healthy controls were investigated using polymerase chain reaction followed by restriction fragment length polymorphism analysis. The association between MIF-173 SNP genotype and AML patients were assessed with SPSS software. The results show that the GC genotype of MIF-173 SNP is significantly higher in AML patients than in the healthy controls (OR 1.58, 95 % CI 1.06, P = 0.034). Carrier genotypes GC and CC may be a causative factor for AML cancer (OR 1.39, 95 % CI 0.95, P = 0.085). White blood cell count (10(3)/µl) were significantly associated with AML MIF-173 polymorphism patients (P = 0.002). Our results in this study provide the first evidence that the MIF-173 polymorphism is associated with AML. MIF is a potential biomarker for development of AML cancer in male adult in Taiwanese population. Further validations in other populations are warranted.

Diabetes is associated with hyperglycemia and increased thrombin production. However, it is unknown whether a combination of high glucose and thrombin can modulate the expression of NAPDH oxidase (Nox) subtypes in human aortic endothelial cells (HAECs). Moreover, we investigated the role of a diabetes-associated microRNA (miR-146a) in a diabetic atherothrombosis model. We showed that high glucose (HG) exerted a synergistic effect with thrombin to induce a 10.69-fold increase in Nox4 mRNA level in HAECs. Increased Nox4 mRNA expression was associated with increased Nox4 protein expression and ROS production. Inflammatory cytokine kit identified that the treatment increased IL-8 and IL-6 levels. Moreover, HG/thrombin treatment caused an 11.43-fold increase of THP-1 adhesion to HAECs.In silicoanalysis identified the homology between miR-146a and the 3′-untranslated region of the Nox4 mRNA, and a luciferase reporter assay confirmed that the miR-146a mimic bound to this Nox4 regulatory region. Additionally, miR-146a expression was decreased to 58% of that in the control, indicating impaired feedback restraint of HG/thrombin-induced endothelial inflammation. In contrast, miR-146a mimic transfection attenuated HG/thrombin-induced upregulation of Nox4 expression, ROS generation, and inflammatory phenotypes. In conclusion, miR-146a is involved in the regulation of endothelial inflammation via modulation of Nox4 expression in a diabetic atherothrombosis model.

Oral cancer is the fourth most common cause of death from cancer in Taiwanese men. Indirubin-3′-monoxime (I3M), a potent cyclin-dependent kinase inhibitor, has therapeutic effects in other cancer cells. In this study, we carried out in vitro assays to test cell viability, cell cycle progression, apoptosis, cell migration and invasion in this cancer type. In addition, using an oral tumorigenic animal model, we examined target gene and protein expression using real time qPCR, immunoblotting and immunohistochemical staining. Our results demonstrate that I3M has an anti-proliferative effect in both Cal-27 and HSC-3 oral cancer cell lines and that treatment of Cal-27 and HSC-3 cells with I3M results in apoptosis through the activation of cytochrome c. In addition, I3M interrupts the cell cycle in Cal-27 cells in a dose-dependent manner by arresting cells in the G2/M phase. We also found that I3M suppresses migration and invasion in Cal-27 cells by inhibiting the expression of focal adhesion kinase, urokinase-type plasminogen inhibitor, and matrix metalloproteinase 9. Moreover, we identified survivin as a target protein in I3M-treated oral cancer cells. Using an oral cancer mouse model, we demonstrate that topical application of an adhesive gel composed of I3M and poly(vinyl alcohol) (I3M/PVA) has dose-dependent anti-tumorigenic effects. Following treatment, the expression of survivin protein and mRNA was downregulated in cancerous tissues. Furthermore, plasma survivin levels were also reduced in the I3M-treated mice. These results suggest that topical application of I3M, a drug synthesized from indirubin, which is found in Qing-Dai – has therapeutic potential for treating oral cancer.

BackgroundProsthetic joint infection (PJI) after total knee or hip replacement is a devastating complication associated with substantial morbidity and economic cost. The incidence of prosthetic joint infection is increasing as the use of mechanical joint replacement increases. The treatment approach to prosthetic joint infection is based on different clinical situations such as a patient's comorbidities, epidemic microbiology data, and surgical procedures. The aim of our study was to understand clinical characteristics of prosthetic joint infection, the microbiology of the prosthetic joint infection, and the outcomes of different treatment strategies during 2006–2011.MethodsWe retrospectively collected cases of prosthetic joint infection in the National Taiwan University Hospital between January 1, 2006 and December 31, 2011. The patients' characteristics, microbiology, outcomes, and factors associated with treatment success were recorded.ResultsOne hundred and forty-four patients were identified as having PJI. Of these, 92 patients were entered into per-protocol analysis. Staphylococcus aureus was the most common causative organism (29.9%), followed by coagulase-negative Staphylococci (16.7%), and Enterococci (9.7%). The overall treatment success rate was 50%. Patients who received a two-stage revision had a better outcome, compared to patients who underwent other types of surgeries (70% vs. 32.7%, respectively; p

The presence of glycated albumin (GA) is associated with increased diabetic complications. This study investigated the effect of angiotensin-(1–7) on the expression of GA-induced endothelial interleukin-6 (IL-6) in human aortic endothelial cells (HAECs). We also evaluated whether miR-146a is involved in the post-transcriptional regulation of angiotensin-(1–7). HAECs were stimulated with GA with or without angiotensin-(1–7) pretreatment. Inflammatory cytokine screening approach identified that angiotensin-(1–7) (10 −7 M) potently inhibited GA (200 μg/mL)-stimulated endothelial IL-6 expression in conditioned medium. ELISA confirmed this finding. Real-time PCR showed that angiotensin-(1–7) decreased GA-induced intracellular IL-6 mRNA expression and western blotting showed that angiotensin-(1–7) decreased GA-induced intracellular IL-6 protein expression. Bioinformatics’ miR target analysis identified homology between miR-146a and the 3′-UTR of the human IL-6 mRNA, suggesting a potential regulation of IL-6 by miR-146a. Treatment with GA decreased endothelial miR-146a expression to 37.2% of the albumin control, while angiotensin-(1–7) increased endothelial miR-146a expression to 1.9-times that of the medium control. Pretreatment with angiotensin-(1–7) inhibited the GA-mediated downregulation of miR-146a to 78.9% of the albumin control levels. Furthermore, the inhibitory effect of angiotensin-(1–7) on IL-6 expression was abolished in GA-treated, miR-146a inhibitor-transfected HAECs. In conclusion, these results suggest that angiotensin-(1–7) exerted an endothelial protective effect through IL-6 downregulation, and miR-146a modulation is involved in this protective effect.