Identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors

BACKGROUND: The tear film is a thin layer of fluid that covers the ocular surface and is involved in lubrication and protection of the eye. Little is known about the protein composition of tear fluid but its deregulation is associated with disease states, such as diabetic dry eyes. This makes this body fluid an interesting candidate for in-depth proteomic analysis. RESULTS: In this study, we employ state-of-the-art mass spectrometric identification, using both a hybrid linear ion trap-Fourier transform (LTQ-FT) and a linear ion trap-Orbitrap (LTQ-Orbitrap) mass spectrometer, and high confidence identification by two consecutive stages of peptide fragmentation (MS/MS/MS or MS(3)), to characterize the protein content of the tear fluid. Low microliter amounts of tear fluid samples were either pre-fractionated with one-dimensional SDS-PAGE and digested in situ with trypsin, or digested in solution. Five times more proteins were detected after gel electrophoresis compared to in solution digestion (320 versus 63 proteins). Ontology classification revealed that 64 of the identified proteins are proteases or protease inhibitors. Of these, only 24 have previously been described as components of the tear fluid. We also identified 18 anti-oxidant enzymes, which protect the eye from harmful consequences of its exposure to oxygen. Only two proteins with this activity have been previously described in the literature. CONCLUSION: Interplay between proteases and protease inhibitors, and between oxidative reactions, is an important feature of the ocular environment. Identification of a large set of proteins participating in these reactions may allow discovery of molecular markers of disease conditions of the eye

Status of complete proteome analysis by mass spectrometry: SILAC labeled yeast as a model system

BACKGROUND: Mass spectrometry has become a powerful tool for the analysis of large numbers of proteins in complex samples, enabling much of proteomics. Due to various analytical challenges, so far no proteome has been sequenced completely. O'Shea, Weissman and co-workers have recently determined the copy number of yeast proteins, making this proteome an excellent model system to study factors affecting coverage. RESULTS: To probe the yeast proteome in depth and determine factors currently preventing complete analysis, we grew yeast cells, extracted proteins and separated them by one-dimensional gel electrophoresis. Peptides resulting from trypsin digestion were analyzed by liquid chromatography mass spectrometry on a linear ion trap-Fourier transform mass spectrometer with very high mass accuracy and sequencing speed. We achieved unambiguous identification of more than 2,000 proteins, including very low abundant ones. Effective dynamic range was limited to about 1,000 and effective sensitivity to about 500 femtomoles, far from the subfemtomole sensitivity possible with single proteins. We used SILAC (stable isotope labeling by amino acids in cell culture) to generate one-to-one pairs of true peptide signals and investigated if sensitivity, sequencing speed or dynamic range were limiting the analysis. CONCLUSION: Advanced mass spectrometry methods can unambiguously identify more than 2,000 proteins in a single proteome. Complex mixture analysis is not limited by sensitivity but by a combination of dynamic range (high abundance peptides preventing sequencing of low abundance ones) and by effective sequencing speed. Substantially increased coverage of the yeast proteome appears feasible with further development in software and instrumentation

Content of Lipids, Fatty Acids, Carbohydrates, and Proteins in Continental Cyanobacteria: A Systematic Analysis and Database Application

The lipid, fatty acid, protein, and carbohydrate contents in cyanobacterial strains and biomass can vary by orders of magnitude. Many publications (thousands of peer-reviewed articles) require more work to extract their precise concentration values (i.e., different units, inaccurate data), which makes them not easily exploitable. For this purpose, tables have been compiled from the literature data, including lipids, fatty acids, proteins, and carbohydrates composition and quantities in cyanobacteria. A lot of data (323) were collected after careful a literature search, according to selected criteria in order to distinguish separately cyanobacteria, and according to categories of genus and species and generate average values of the contents of these cell components. These data are exploited in a first systematic analysis of the content in types of strains. Our database can be a powerful tool for biologists, chemists, and environmental agencies to determine the potential concentration of high-value chemical building blocks directly from low-value bloom biomass, cell cultures, or debris in the sediment, offering the potential to minimize environmental waste and add value to the agro-industrial residues. The database can also support strategies for food manufacturers to develop new products with optimized properties for veterinarian applications

Addressing drinking water salinity due to sea water intrusion in Praia de Leste, Parana, by a brackish water desalination pilot plant

Seawater intrusion into the Pombas River, source of freshwater to Praia de Leste on the coast of Parana in Brazil presents a problem to the water utility as most water treatment plants in Brazil are conventional. To find a solution to this problem, a pilot plant (1 m3 /h) consisting of ultrafiltration (UF) followed by reverse osmosis (RO) was developed and evaluated. For testing, brackish water was produced with a concentration of 1,500 ± 100 mg/L of total dissolved solids (TDS), mixing seawater and fresh water. To evaluate the water quality, TDS, electrical conductivity, pH, temperature, apparent color, turbidity, alkalinity, total hardness, calcium, chloride and sulfate were monitored. For operational performance, flowrates, osmotic pressure, filtration rate, recovery rate and mass balance were analyzed. On average, the UF system removed 96.4% of turbidity and 98.6% of apparent color; whereas the RO system removed 99.4% of TDS. The overall average recovery (UF and RO) was 45.81% with average osmotic pressure of 8.21 bar, filtration rate of 30.7 L/h/m2 in the UF system and 21.7 L/h/m2 in the RO system. From a water quality point of view, the system was effective in processing brackish into fresh water of high quality

Reversion of Steatosis by SREBP-1c Antisense Oligonucleotide did not Improve Hepatic Insulin Action in Diet-induced Obesity Mice

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)The literature has associated hepatic insulin action with NAFLD. In this sense, treatments to revert steatosis and improve hepatic insulin action become important. Our group has demonstrated that inhibition of Sterol Regulatory Element Binding Proteins-1c (SREBP-1c) reverses hepatic steatosis. However, insulin signals after NAFLD reversion require better investigation. Thus, in this study, we investigated if the reversal of NAFLD by SREBP-1c inhibitor results in improvement in the hepatic insulin signal in obesity mice. After installation/achievement of diet-induced obesity and insulin resistance, Swiss mice were divided into 3 groups: i) Lean, ii) D-IHS, diet-induced hepatic steatosis [no treatment with antisense oligonucleotide (ASO)], and iii) RD-IHS, reversion of diet-induced hepatic steatosis (treated with ASO). The mice were treated with ASO SREBP-1c as previously described by our group. After ASO treatment, one set of animals was anesthetized and used for in vivo test, and another mice set was anesthetized and used for histology and Western blot analysis. Reversion of diet-induced hepatic steatosis did not change blood glucose, glucose decay constant (k(ITT)), body weight, or serum insulin levels. In addition, results showed that the protocol did not improve insulin pathway signaling, as confirmed by the absence of changes in IR, IRS1, Akt and Foxo1 phosphorylation in hepatic tissue. In parallel, no alterations were observed in proinflammatory molecules. Thus, our results suggest that the inhibition of SREBP-1c reverts steatosis, but without improving insulin hepatic resistance.4412885890Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundacao de Amparo a Pesquisa do Estado de Santa Catarina (FAPESC)Universidade do Extremo Sul Catarinense (UNESC)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Comparative cytogenetic analysis of two grasshopper species of the tribe Abracrini (Ommatolampinae, Acrididae)

The grasshopper species Orthoscapheus rufipes and Eujivarus fusiformis were analyzed using several cytogenetic techniques. The karyotype of O. rufipes, described here for the first time, had a diploid number of 2n = 23, whereas E. fusiformis had a karyotype with 2n = 21. The two species showed the same mechanism of sex determination (XO type) but differed in chromosome morphology. Pericentromeric blocks of constitutive heterochromatin (CH) were detected in the chromosome complement of both species. CMA3/DA/DAPI staining revealed CMA3-positive blocks in CH regions in four autosomal bivalents of O. rufipes and in two of E. fusiformis. The location of active NORs differed between the two species, occurring in bivalents M6 and S9 of O. rufipes and M6 and M7 of E. fusiformsi. The rDNA sites revealed by FISH coincided with the number and position of the active NORs detected by AgNO3 staining. The variability in chromosomal markers accounted for the karyotype differentiation observed in the tribe Abracrini

Physical Exercise Decreases Fasting Hyperglycemia in Diabetic Mice Through AMPK Activation

Introduction: The deficiency in glucose uptake in peripheral tissues and increased hepatic gluconeogenesis are physiopathological phenomena observed in type 2 diabetes patients. Physical exercise plays an important role in the improvement of glycemic profile in diabetic patients; however, the mechanisms involved in these processes have not been fully elucidated. Objective: to assess the role of AMPK protein in the glycemic control of diabetic mice after exercise. Methods: During fasting condition, the insulin tolerance test (ITT) and Western blot technique, were combined to assess the glucose homeostasis in diabetic mice (ob/ob and db/db) after a single swimming session. Results: Fasting hyperglycemia, severe insulin resistance and deficiency in the AMPk/ACC signaling in muscle and liver observed in the diabetic mice were reversed after the exercise session. The restoration of AMPK/ACC signaling reduced the expression of the gluconeogenic enzyme, PEPCk in the liver, and increased the translocation of GLUT4 in the skeletal muscle. These data indicate that the activation of AMPK/ACC pathway induced by physical exercise is important to reduce fasting glucose levels in experimental models of type 2 diabetes. These data open new insights for determination of physical activity control on the glucose homeostasis in diabetic patients.15317918

Pharmacokinetics of phenoxodiol, a novel isoflavone, following intravenous administration to patients with advanced cancer

Background: Phenoxodiol is a novel isoflavone currently being studied in clinical trials for the treatment of cancer. This study reports the pharmacokinetics of phenoxodiol in patients with cancer.Methods: The pharmacokinetics of phenoxodiol was studied following a single intravenous (iv) bolus dose and during a continuous intravenous infusion. Three men with prostate cancer and 3 women with breast cancer received IV bolus phenoxodiol (5 mg/kg) and plasma was sampled for free and total phenoxodiol levels. On a separate occasion 5 of the same patients received a continuous intravenous infusion of phenoxodiol (2 mg/kg/h) and plasma was again sampled for free and total phenoxodiol levels. Phenoxodiol was measured using gradient HPLC with ultraviolet detection.Results: Following bolus injection, free and total phenoxodiol appeared to follow first order pharmacokinetics. The elimination half-lives for free and total phenoxodiol were 0.67 ± 0.53 h and 3.19 ± 1.93 h, respectively, while the total plasma clearance rates were 2.48 ± 2.33 L/h and 0.15 ± 0.08 L/h, respectively. The respective apparent volumes of distribution were 1.55 ± 0.69 L/kg and 0.64 ± 0.51 L/kg. During continuous intravenous infusion, free phenoxodiol accumulated rapidly to reach a mean concentration at steady state of 0.79 ± 0.14 μg/ml after 0.87 ± 0.18 h. The apparent accumulation half-life of free phenoxodiol was 0.17 ± 0.04 h while the plasma clearance during continuous infusion was 1.29 ± 0.23 L/h.Conclusions: Phenoxodiol has a short plasma half-life, particularly in the free form, leading to a rapid attainment of steady state levels during continuous intravenous infusion.Trial registration: Australian New Zealand Clinical Trials Registry (ANZCTR): ACTRN12610000334000