[Quantitative proteomics of human blood exosomes]. / Kolichestvennyi proteomnyi analiz ékzosom krovi cheloveka.

Exosomes are extracellular membrane vesicles secreted by cells into biological fluids. The outer membrane of exosomes protects their content from degradation and contains markers of the parent cell. Almost all cells of the body produce exosomes, however, tumor cells secrete them more intensively. Due to fact that exosomes contain proteins of cells secreting them, these vesicles could be a valuable source for biomarkers discovery. Currently, a number of studies prove the participation of exosomes in carcinogenesis. However, there is a problem of isolating pure and characterized exosomes for further use in investigation of functions or identification of tumor protein biomarkers. In this work, we have performed experiments on exosomes isolation from human plasma by three methods: differential ultracentrifugation, ultracentrifugation in sucrose cushion, sedimentation of the exosomal fraction from serum by using a commercial kit. The protein composition of the obtained samples was determined by mass spectrometric methods of selected reactions monitoring (SRM) and shotgun proteomic analysis. The obtained exosomal samples were searched for the presence of exosomal markers (CD9, CD82, HSPA8, CD63). In the samples of exosomes isolated by ultracentrifugation with the sucrose cushion, the content of the above markers was determined as 32.85, 15.59, 6.07 fmol/mg of total protein, correspondently. It was shown that the centrifugation method with the sucrose cushion was optimal for the isolation of exosomes.

MMP7-mediated cleavage of nucleolin at Asp255 induces MMP9 expression to promote tumor malignancy.

Nucleolin (NCL) participates in DNA transcription, ribosomal biogenesis and the regulation of RNA stability. However, the contribution of NCL to tumor development is still not clear. Herein, we found that NCL expression correlated with poor prognosis in lung cancer patients. Overexpressed NCL was predominantly cleaved to C-terminal truncated NCL (TNCL). In lung cancer formation, activation of the epidermal growth factor receptor pathway induced NCL expression, and also the expression of matrix metalloproteinase (MMP) 7, which then cleaved NCL at Asp255 to generate TNCL of 55 kDa. TNCL increased the expression of several oncogenes, including MMP9, anaplastic lymphoma kinase (ALK), HIF1a and CBLB, and decreased the expression of tumor suppressors including BRD4, PCM1, TFG and KLF6 by modulating mRNA stability through binding to the 3'-untranslated regions of their transcripts, thus ultimately enhancing metastasis activity. In conclusion, this study identified a novel role of the cleavage form of NCL generated by MMP7 in stabilizing MMP9 mRNA. We also provide a new insight that MMP7 not only cleaves the extracellular matrix to promote tumor invasion but also cleaves NCL, which augment oncogenesis. Blocking NCL cleavage may provide a useful new strategy for lung cancer therapy.

Biochemical study of retired pentachlorophenol workers with and without following dietary exposure to PCDD/Fs.

PCDD/Fs are found as impurities in commercial pesticide sodium pentachlorophenol (Na-PCP) salt. We compared, using multivariate logistic regression analysis adjusted for confounding factors, serum PCDD/F levels and biochemical examinations of retired Na-PCP workers and other inhabitants living near a closed Na-PCP plant that discharged PCP-contaminated wastewater into a nearby pond in Tainan, Taiwan. In this cross-sectional study from October 2006 through May 2009, 1167 participants were divided into groups according to their occupational (retired Na-PCP plant workers versus other residents) and dietary exposure (eating polluted fish versus not eating polluted fish) to PCDD/Fs, and a general population from a large-scale survey. Serum PCDD/F levels were significantly different between these groups (range: from 22.9±10.0pg WHO(98)-TEQ(DF) g(-1) lipid in the general population to 109.6±94.5pg WHO(98)-TEQ(DF) g(-1) lipid in retired Na-PCP workers eating polluted seafood; P(trend)<0.001). Distinct patterns of PCDD/Fs congener profiles, showing a significantly higher proportion of 1,2,3,7,8-PeCDD, 1,2,3,6,7,8-HxDD, and less 2,3,4,6,7,8-HpCDF, OCDF, were also found among workers and residents with different serum PCDD/F levels versus the general population. After adjusting for confounding factors, glucose (adjusted odds ratio [AOR] 7.22 [95% CI: 4.04-12.90]), triglycerides (AOR 4.31 [95% CI: 2.57-7.22]), blood urea nitrogen (AOR 2.90 [95% CI: 1.58-5.33]), creatine (AOR 5.83 [95% CI: 1.12-30.30]) and total protein (AOR 3.74 [95% CI: 1.91-7.31]) levels in retired workers were significantly higher than in the reference group. Occupational exposure to PCDD/Fs is associated with biochemical abnormalities that may persist for years after serum PCDD/F levels have declined.

Interrelationship between exposure to PCDD/Fs and hypertension in metabolic syndrome in Taiwanese living near a highly contaminated area.

Metabolic syndrome (MetS) consists of a constellation of metabolic abnormalities that confer increased risk of cardiovascular disease. There is a positive correlation between exposure to persistent organic pollutants and MetS. We examine the association between PCDD/Fs and MetS components in 1490 non-diabetic persons living near a highly dioxin-contaminated area. We used factor analysis, with a set of core variables considered central features of MetS and PCDD/Fs, to group similar risk factors. Serum PCDD/Fs were positively and significantly correlated with the number of MetS components. Four risk factors-lipidemia, blood pressure, body size, and glycemia-accounted for 72.6% of the variance in the 10 core factors, and PCDD/Fs were linked to MetS through shared correlations with high blood pressure. After adjusting for confounding factors, we found that diastolic blood pressure (ß=0.018; p=0.006), glucose (ß=0.013; p=0.046), and waist circumference (ß=0.721; p=0.042) significantly increased with increasing serum PCDD/F levels. We found significant trends for associations between metabolic syndrome and serum low-chlorinated PCDD/Fs. The highest quintiles of 2,3,4,7,8-PeCDF, 1,2,3,6,7,8-HxCDF and 2,3,7,8-TCDD had the top three adjusted ORs (95% CI) of 3.5 (1.9-6.3), 2.9 (1.7-4.9) and 2.8 (1.6-4.9), respectively. We also found a slight monotonic relationship between serum PCDD/Fs and the prevalence of MetS, especially when the serum dioxin level was higher than 25.4pg WHO(98)-TEQ(DF)g(-1) lipid (the fourth Quintile). We hypothesize that high-dose exposure to PCDD/Fs is a blood pressure-related factor that raises MetS risk.

Multidimensional self reports as a measure of characteristics in people with eating disorders.

This study used multidimensional self report assessments to measure perfectionism, impulsivity and obsessive compulsive characteristics in females with anorexia nervosa (AN), bulimia nervosa (BN) and in matched healthy controls (HC). The Frost Multidimensional Perfectionism Scale (FMPS), Barrett Impulsivity Scale (BIS) and Obsessive Compulsive Inventory-Revised (OCI-R) scale were completed by 107 participants (AN=30, BN=26, HC=51), in parallel with clinical measures. Results show that people with AN have the highest scores on the dimensions of the FMPS as well as on the overall score; the AN and BN groups have the highest scores on the dimensions and on the overall score of the OCI-R; on the BIS, the AN and BN groups have the highest scores on the attention subscale, but there are no group differences on the overall BIS scores. In relation to the FMPS, the global score, and the subscales 'concern over mistakes' and 'doubts about actions' are all highly correlated with both eating pathology (Eating Disorder Examination Questionnaire, EDE-Q) and low global functioning (Structured Clinical Interview for DSM IV, SCID). The subscale 'obsessing' on the OCI-R shows a strong correlation with eating pathology. The overall score and also the subscales of the BIS do not show strong correlations with eating pathology or poor global functioning. In conclusion, therapies should seek to address these specific areas which are highly correlated with eating disorder pathology.

Efficacy of an intravenous proton pump inhibitor after endoscopic therapy with epinephrine injection for peptic ulcer bleeding in patients with uraemia: a case-control study.

BACKGROUND: Patients with peptic ulcer bleeding and uraemia are prone to re-bleeding. AIM: To compare the efficacy of an intravenous proton pump inhibitor in treating peptic ulcer bleeding in patients with uraemia and those without uraemia. METHODS: High-risk peptic ulcer bleeding patients received endoscopic therapy with epinephrine (adrenaline) injection plus intravenous omeprazole (40 mg bolus followed by 40 mg infusion every 12 h) for 3 days. Re-bleeding, volume of blood transfusion, hospital stay, need for surgery, and mortality were analysed. RESULTS: The uraemic group had similar 7-day re-bleeding rate (6/42, 14.29% vs. 6/46, 13.04%, P = 0.865) to that of non-uraemic patients, but more re-bleeding episodes beyond 7 days (4/42, 9.52% vs. 0/46, 0%, P = 0.032, OR [95% CI] = 1.105 [1.002-1.219]) and all-cause mortality (4/42 vs. 0/46 P = 0.032, OR [95% CI] = 1.105 [1.002-1.219]). The uraemic group also had more units of blood transfusion after endoscopic therapy (mean +/- s.d. 4.33 +/- 3.35 units vs. 2.15 +/- 1.65 units, P < 0.001), longer hospital stay (mean +/- s.d. 8.55 +/- 8.12 days vs. 4.11 +/- 1.60 days, P < 0.001) and complications during hospitalization (9/42 vs. 0/46, P = 0.001, OR [95% CI] = 1.273 [1.087-1.490]). CONCLUSION: Endoscopic therapy with epinephrine injection plus an intravenous proton pump inhibitor can offer protection against early re-bleeding in uraemic patients with peptic ulcer bleeding, but has a limited role beyond 7 days.

Comparison of antimicrobial resistance patterns between clinical and sewage isolates in a regional hospital in Taiwan.

AIMS: To compare bacterial populations and antimicrobial resistance patterns between clinical and sewage isolates from a regional hospital in northern Taiwan. The dissemination of antibiotic-resistant bacteria from hospital compartments to the hospital sewage treatment plant was examined. METHODS AND RESULTS: A total of 1020 clinical isolates and 435 sewage isolates were collected between July and September 2005. The percentages of Gram-negative bacteria from the clinical and sewage isolates were 87.2% and 91.0%, respectively (P = 0.033). Escherichia coli were the leading bacterial isolates in both groups. Antimicrobial susceptibility testing showed a significant difference (P < 0.001) in resistance to ampicillin (85.6% vs 94.1%), ampicillin/sulbactam (31.7% vs 55.4%), cefazolin (29.2% vs 71.5%) and cefuroxime (20.7% vs 61.9%) between clinical and sewage coliform isolates, respectively. CONCLUSIONS: The sewage isolates had higher antimicrobial resistance rates than the clinical isolates from the same hospital. SIGNIFICANCE AND IMPACT OF THE STUDY: The low efficacy of the hospital sewage treatment may contribute to the dissemination of multidrug resistant bacteria from this hospital compartments to the environment. Practices which limit the disposal of antimicrobial agents into the wastewater system may be the possible measure to prevent the selection of multidrug-resistant bacteria from sewage treatment plants.

High variation of PCDDs, PCDFs, and dioxin-like PCBs ratio in cooked food from the first total diet survey in Taiwan.

This study determined the levels of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs), and dioxin-like polychlorinated biphenyls (dl-PCBs) in 240 individual food samples, belonging to 37 different foodstuffs in first total diet study (TDS) in Taiwan. The foods were collected from markets located in eight cities or counties around Taiwan. The food was cooked in a laboratory according to recipes typically used in Taiwan. In this study, PCDD/Fs were lower than the limits proposed by the European Union (EU) regulation for commercialized food, except for a notable PCDD/Fs level in ducks (3.660 pg WHO-TEQ/g, fat) obtained from central Taiwan. We hypothesize the duck meat might be probably polluted via emission of a fly ash recycling plant located near the duck farms. In addition to fish, most foods had high PCDD/Fs to dl-PCBs ratio. Needle fish and sea perch had relatively lower PCDD/Fs and dl-PCBs levels compared with those in other fish. Data from this study can be utilized for further consideration about dietary intake.

High average daily intake of PCDD/Fs and serum levels in residents living near a deserted factory producing pentachlorophenol (PCP) in Taiwan: influence of contaminated fish consumption.

An abandoned pentachlorophenol plant and nearby area in southern Taiwan was heavily contaminated by dioxins, impurities formed in the PCP production process. The investigation showed that the average serum PCDD/Fs of residents living nearby area (62.5 pg WHO-TEQ/g lipid) was higher than those living in the non-polluted area (22.5 and 18.2 pg WHO-TEQ/g lipid) (P<0.05). In biota samples, average PCDD/F of milkfish in sea reservoir (28.3 pg WHO-TEQ/g) was higher than those in the nearby fish farm (0.15 pg WHO-TEQ/g), and Tilapia and shrimp showed the similar trend. The average daily PCDD/Fs intake of 38% participants was higher than 4 pg WHO-TEQ/kg/day suggested by the world health organization. Serum PCDD/F was positively associated with average daily intake (ADI) after adjustment for age, sex, BMI, and smoking status. In addition, a prospective cohort study is suggested to determine the long-term health effects on the people living near factory.

Antibody to severe acute respiratory syndrome (SARS)-associated coronavirus spike protein domain 2 cross-reacts with lung epithelial cells and causes cytotoxicity.

Both viral effect and immune-mediated mechanism are involved in the pathogenesis of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection. In this study, we showed that in SARS patient sera there were autoantibodies (autoAbs) that reacted with A549 cells, the type-2 pneumocytes, and that these autoAbs were mainly IgG. The autoAbs were detectable 20 days after fever onset. Tests of non-SARS-pneumonia patients did not show the same autoAb production as in SARS patients. After sera IgG bound to A549 cells, cytotoxicity was induced. Cell cytotoxicity and the anti-epithelial cell IgG level were positively correlated. Preabsorption and binding assays indicated the existence of cross-reactive epitopes on SARS-CoV spike protein domain 2 (S2). Furthermore, treatment of A549 cells with anti-S2 Abs and IFN-gamma resulted in an increase in the adherence of human peripheral blood mononuclear cells to these epithelial cells. Taken together, we have demonstrated that the anti-S2 Abs in SARS patient sera cause cytotoxic injury as well as enhance immune cell adhesion to epithelial cells. The onset of autoimmune responses in SARS-CoV infection may be implicated in SARS pathogenesis.

Serum PCDD/F concentration distribution in residents living in the vicinity of an incinerator and its association with predicted ambient dioxin exposure.

The aim of this study was to evaluate the serum polychlorinated dibenzo-p-dioxin (PCDD) and dibenzofuran (PCDF) concentration distribution in residents living in the vicinity of an incinerator and its association with annual ambient dioxin exposure predicted by an atmospheric dispersion model. A municipal waste incinerator in Northern Taiwan was chosen for this study. This incinerator had been in operation for 6 years at the time of this study. Using the incinerator site as the center, based on the simulated ambient annual average PCDD/F concentrations. Ninety-five volunteers, all live within a radius of 5 km from the incinerator for at least 5 years, who had no occupational exposure potential, were selected based on the population distribution in each district. The average serum PCDD/F concentration for these subjects living within four zones was about 14 pg I-TEQ/g lipid. The serum distribution levels of people of the four study zones, however, were not consistent with the predicted ambient levels. Results also suggest that ambient exposure might not be the most important contributor to serum concentrations when compared to other exposure sources, such as dietary intake.

Brain region-dependent increases in beta-amyloid and apolipoprotein E levels in hypercholesterolemic rabbits.

Recent studies indicate a possible link between serum cholesterol level, beta-amyloid (Abeta) peptide concentrations, and the incidence of Alzheimer's disease (AD). In the present report, the effects of dietary cholesterol on Abeta and apolipoprotein E (APOE) levels in several brain regions were examined using diet-induced hypercholesterolemic rabbits as the animal model. Increased dietary cholesterol levels increased Abeta concentrations in temporal cortex (p = 0.02). A similar trend was observed in the frontal cortex (p = 0.06), yet not in the cerebellum. Interestingly, the regional levels of Abeta in the hypercholesterolemic rabbit paralleled the amyloid pathology observed in AD brain. Elevated APOE levels were also noticed in temporal (p < 0.01) and frontal (p < 0.01) cortices, but not in cerebellum, in the rabbit fed with cholesterol-abundant diet. These results suggest that high serum cholesterol levels could induce the elevation of brain APOE, which may play a role in aggravating the Abeta accumulation.

Striatal formation of 6-hydroxydopamine in mice treated with pargyline, pyrogallol and methamphetamine.

Formation of 6-hydroxydopamine (6-OHDA) has been posited in the striatum following methamphetamine treatment and plays a critical role in methamphetamine-induced nigrostriatal dopaminergic toxicity. We used high performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) to determine the formation of 6-OHDA by the treatments of methamphetamine combined with pargyline, a monoamine oxidase inhibitor, and pyrogallol, a catechol-O-methyl-transferase inhibitor, in female C57BL/6J mouse striatum. A substantial amount of 6-OHDA (9.9 +/- 0.7 pg/mg wet tissue) was detected in mice treated with pargyline (100 mg/kg) and pyrogallol (25 mg/kg) in combination. Greater striatal 6-OHDA levels were observed in mice treated with combined pargyline, pyrogallol and methamphetamine (50 mg/kg) as compared to mice treated with combined pargyline and pyrogallol. However, mice treated with pargyline and pyragollol in combination followed by one and two doses of methamphetamine exhibited comparable striatal 6-OHDA levels (23.2 +/- 4.3, 27.3 +/- 1.3 pg/mg wet tissue) in our protocol. We conclude that blockade of the primary metabolic pathways of dopamine by inhibiting both monoamine oxidase and catechol-O-methyl-transferase activities is sufficient to induce 6-OHDA formation in the striatum. Acute 6-OHDA accumulation in the striatum can be potentiated by methamphetamine, a potent dopamine releaser, administration following such metabolic inhibitions.

Efficacy of foot-and-mouth disease vaccine in pigs with single dose immunization.

Two experiments were conducted to demonstrate the efficacy of a commercial foot-and-mouth disease (FMD) vaccine in pigs born to well-vaccinated sows at various ages with a single injection under field conditions. The first experiment showed that single dose vaccination of pigs could be conducted at an age younger than 10 weeks. Second experiment demonstrated that pigs vaccinated once at the age of 8 weeks had mean serum neutralization (SN) titer of 1.89+/-0.95 log(10)SN(50) with full protection by challenge test at the age of 24 weeks. Results indicate that the most appropriate age for single dose FMD vaccination in pigs born to well-vaccinated sows would be at 8 weeks.

Optimization of foot-and-mouth disease vaccination protocols by surveillance of neutralization antibodies.

An appropriate immunization program for pigs in a foot-and-mouth disease (FMD) endemic area was proposed based on data analysis obtained from serological surveillance in Taiwan, after an intensive vaccination program. To provide an adequate passive immunity for piglets, gilts that have completed two basic vaccinations must be boosted once before breeding. To achieve an efficient response to the FMD vaccine for piglets born to well vaccinated sows, vaccination need to be delayed until 10-12 weeks of ages for the first immunization, followed by a boost 4 weeks later.

A disposable poly(methylmethacrylate)-based microfluidic module for protein identification by nanoelectrospray ionization-tandem mass spectrometry.

The design, fabrication, and analytical use of a poly(methylmethacrylate) (PMMA)-based microfluidic module for nanoelectrospray ionization-tandem mass spectrometry (nano-ESI-MS/MS) were described. The microfluidic module can be mass-produced at low costs and used as a disposable device to generate nano-ESI-MS/MS signals for protein identification from low amounts of protein samples. Compared with commercially available nanospray capillary tips, the module gave comparable signal quality and also offered advantages in convenience and easiness of operation, permitting repeated usage, and disposability.

Quantitative detection of N(7)-(2-hydroxyethyl)guanine adducts in DNA using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry.

High-performance liquid chromatography (HPLC) was combined with electrospray ionization tandem mass spectrometry (ESI-MS/MS) to develop a sensitive and selective method for the quantitative measurement of N(7)-(2-hydroxyethyl)guanine (N(7)-HEG) adducts in DNA obtained from ethylene oxide-exposed biological samples. Selected reaction monitoring (SRM) was used as the detection mode while the fragmentation product ion at m/z 152 generated from the precursor protonated N(7)-HEG (m/z 196) was monitored. The detection limits for N(7)-HEG were estimated by twofold serial dilution and determined to be 4 fmol in neat standard solution and 16 fmol when a matrix effect is considered. When the mass spectrometer was operated in the selected ion monitoring mode using only the first quadrupole (without MS/MS function), the detection limits increased to 128 fmol and 1 pmol (when matrix effect is considered), respectively. A good linear correlation (R(2) = 0.999) was observed for signal intensities obtained by injecting 16 fmol--33 pmol of N(7)-HEG into the HPLC/ESI-MS/MS system. Hep G2 cells were incubated for 8 h with medium containing various concentrations of ethylene oxide (ranging from 0.05 to 5.0 mM). A dose-response relationship was established, indicating that the adduct formation increases with the exposure level. The method shows potential, although the detection limit needs to be lowered for practical applications, for use in monitoring N(7)-HEG formation in other biological systems.