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

ELSI-COVID-19 initiative: methodology of the telephone survey on coronavirus in the Brazilian Longitudinal Study of Aging

The COVID-19 pandemic (caused by the SARS-CoV-2) is a public health emergency of international concern that particularly affects older people. Brazil is one of the countries most affected by the pandemic, ranking second with the highest number of confirmed cases and deaths worldwide as of mid-June 2020. The ELSI-COVID-19 initiative is based on telephone interviews with participants of the Brazilian Longitudinal Study of Aging (ELSI-Brazil), conducted on a nationally representative sample of the population aged 50 or older. This initiative aims to provide information on adherence to preventive measures (social distancing, wearing masks, and handwashing/hygiene); reasons for leaving the house, when that was the case; difficulties obtaining medications, medical diagnosis of COVID-19, and receipt of confirmatory results; use of health-care services (recent care-seeking, care-seeking location, care receipt, among other aspects); and mental health (sleep, depression, and loneliness). The first round of telephone interviews was conducted between May 26 and June 8, 2020. The second and third rounds are expected to occur within the coming months. This article presents this initiative methodology and some sociodemographic characteristics of the 6,149 participants in the survey first round, relative the Brazilian population within the same age group

Diversidade e variação sazonal de moscas-das-frutas (Diptera: Tephritidae, Lonchaeidae) e seus parasitóides (Hymenoptera: Braconidae, Figitidae) em pomares de goiaba, nêspera e pêssego

This work was carried out in orchards of guava progenies, and loquat and peach cultivars, in Monte Alegre do Sul, SP, Brazil, in 2002 and 2003. Guavas and loquats were bagged and unbagged bi-weekly and weekly, respectively, for assessment of the infestation period. Peach was only bagged weekly. The assays started when the fruits were at the beginning of development, but still green. Ripe fruits were taken to the laboratory and placed individually into plastic cups. McPhail plastic traps containing torula yeast were hung from January 2002 to January 2004 to assess the fruit fly population in each orchard, but only the Ceratitis capitata population is here discussed. Five tephritid species were reared from the fruits: Anastrepha bistrigata Bezzi, A. fraterculus (Wiedemann), A. obliqua (Macquart), A. sororcula Zucchi, and C. capitata, in addition to six lonchaeid species: Neosilba certa (Walker), N. glaberrima (Wiedemann), N. pendula (Bezzi), N. zadolicha McAlpine and Steyskal, Neosilba sp. 4, and Neosilba sp. 10 (both species are in the process of being described by P. C. Strikis), as well as some unidentified Neosilba species. Ten parasitoid species were obtained from fruit fly puparia, of which five were braconids: Asobara anastrephae (Muesebeck), Doryctobracon areolatus (Szépligeti), D. brasiliensis (Szépligeti), Opius bellus Gahan, and Utetes anastrephae (Viereck), and five figitids: Aganaspis pelleranoi (Brèthes), Dicerataspis grenadensis Ashmead, Lopheucoila anastrephae (Rhower), Leptopilina boulardi (Barbotin, Carlton and Kelner-Pillaut), and Trybliographa infuscata Diaz, Gallardo and Uchôa. Ceratitis capitata showed a seasonal behavior with population density peaking at the second semester of each year. Anastrepha and Neosilba species remained in the orchards throughout both years.Este trabalho foi realizado em três pomares em Monte Alegre do Sul, SP, em 2002 e 2003, representados por coleção de progênies de goiabeiras, de cultivares de nespereiras e de cultivares de pessegueiros. O período de infestação foi determinado por meio de ensacamento e desensacamento quinzenal e semanal de goiabas e nêsperas, respectivamente, e pelo ensacamento semanal de pêssegos. Os ensaios iniciaram-se com os frutos verdes (princípio de desenvolvimento). Os frutos maduros foram levados ao laboratório e acondicionados individualmente em copos plásticos. A flutuação populacional de Ceratitis capitata (Wiedemann) foi avaliada por meio de armadilhas plásticas modelo McPhail com torula em cada pomar, de janeiro/2002 a janeiro/2004. Dos frutos foram obtidas cinco espécies de tefritídeos: Anastrepha bistrigata Bezzi, A. fraterculus (Wiedemann), A. obliqua (Macquart), A. sororcula Zucchi e C. capitata e seis de lonqueídeos: Neosilba certa (Walker), N. glaberrima (Wiedemann), N. pendula (Bezzi), N. zadolicha McAlpine and Steyskal, Neosilba sp. 4 e Neosilba sp. 10, além de algumas espécies não-identificadas. Foram obtidas 10 espécies de parasitóides, cinco da família Braconidae - Asobara anastrephae (Muesebeck), Doryctobracon areolatus (Szépligeti), D. brasiliensis (Szépligeti), Opius bellus Gahan e Utetes anastrephae (Viereck) - e cinco da família Figitidae - Aganaspis pelleranoi (Brèthes), Dicerataspis grenadensis Ashmead, Lopheucoila anastrephae (Rhower), Leptopilina boulardi (Barbotin, Carlton and Kelner-Pillaut) e Trybliographa infuscata Diaz, Gallardo and Uchôa. Ceratitis capitata apresentou comportamento sazonal com picos populacionais durante o segundo semestre dos dois anos. As espécies de Anastrepha e de Neosilba permaneceram nos pomares durante os dois anos

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

Biomarkers and in vitro strategies for nephrotoxicity and renal disease assessment

Acute kidney injury (AKI) is a global public health concern, impacting nearly 13.3 million patients and resulting in three million deaths per year. Chronic kidney disease has increased by 135% since 1990, representing the pathology with the fastest growth rate worldwide. The annual costs of dialysis and kidney transplants range between US$35,000 and US$100,000 per patient. Despite its great impact, kidney disease has remained mostly asymptomatic for many years. AKI continues to be a major, unmet medical condition for which there are no pharmacological treatments available, while animal models are limited to provide direction for therapeutic translation into humans. Currently, serum creatinine is the standard biomarker to identify nephrotoxicity; however, it is a late stage biomarker. Hence, there is a pressing need to study in vitro biomarkers for the assessment of nephrotoxicity in order to develop new and safer drugs. Understanding of the mechanisms by which molecules produce nephrotoxicity is vital in order to both prevent adversity and treat kidney injury. In this review, we address new technologies and models that may be used to identify earlier biomarkers and pathways involved in nephrotoxicity, such as cell culture, omics, bioinformatics platform, CRISPR/Cas9 genome-editing, in silico, organoids and 3D bioprinting, considering AOP

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