NanoUPLC-MSE proteomic data assessment of soybean seeds using the Uniprot database.

Background: Recombinant DNA technology has been extensively employed to generate a variety of products from genetically modified organisms (GMOs) over the last decade, and the development of technologies capable of analyzing these products is crucial to understanding gene expression patterns. Liquid chromatography coupled with mass spectrometry is a powerful tool for analyzing protein contents and possible expression modifications in GMOs. Specifically, the NanoUPLC-MSE technique provides rapid protein analyses of complex mixtures with supported steps for high sample throughput, identification and quantization using low sample quantities with outstanding repeatability. Here, we present an assessment of the peptide and protein identification and quantification of soybean seed EMBRAPA BR16 cultivar contents using NanoUPLC-MSE and provide a comparison to the theoretical tryptic digestion of soybean sequences from Uniprot database. Results: The NanoUPLC-MSE peptide analysis resulted in 3,400 identified peptides, 58% of which were identified to have no miscleavages. The experiment revealed that 13% of the peptides underwent in-source fragmentation, and 82% of the peptides were identified with a mass measurement accuracy of less than 5 ppm. More than 75% of the identified proteins have at least 10 matched peptides, 88% of the identified proteins have greater than 30% of coverage, and 87% of the identified proteins occur in all four replicates. 78% of the identified proteins correspond to all glycinin and betaconglycinin chains. The theoretical Uniprot peptide database has 723,749 entries, and 548,336 peptides have molecular weights of greater than 500 Da. Seed proteins represent 0.86% of the protein database entries. At the peptide level, trypsin-digested seed proteins represent only 0.3% of the theoretical Uniprot peptide database. A total of 22% of all database peptides have a pI value of less than 5, and 25% of them have a pI value between 5 and 8. Based on the detection range of typical NanoUPLC-MSE experiments, i.e., 500 to 5000 Da, 64 proteins will not be identified. Conclusions: NanoUPLC-MSE experiments provide good protein coverage within a peptide error of 5 ppm and a wide MW detection range from 500 to 5000 Da. A second digestion enzyme should be used depending on the tissue or proteins to be analyzed. In the case of seed tissue, trypsin protein digestion results offer good databank coverage. The Uniprot database has many duplicate entries that may result in false protein homolog associations when using NanoUPLC-MSE analysis. The proteomic profile of the EMBRAPA BR-16 seed lacks certain described proteins relative to the profiles of transgenic soybeans reported in other works

Protein families, natural history and biotechnological aspects of spider silk.

Spiders are exceptionally diverse and abundant organisms in terrestrial ecosystems and their evolutionary success is certainly related to their capacity to produce different types of silks during their life cycle, making a specialized use on each of them. Presenting particularly tandemly arranged amino acid repeats, silk proteins (spidroins) have mechanical properties superior to most synthetic or natural high-performance fibers, which makes them very promising for biotechnology industry, with putative applications in the production of new biomaterials. During the evolution of spider species, complex behaviors of web production and usage have been coupled with anatomical specialization of spinning glands. Spiders retaining ancestral characters, such as the ones belonging to the Mygalomorph group, present simpler sorts of webs used mainly to build burrows and egg sacs, and their silks are produced by globular undifferentiated spinning glands. In contrast, Araneomorphae spiders have a complex spinning apparatus, presenting up to seven morphologically distinct glands, capable to of rigidness and elasticity associated with distinct behaviors. Aiming to provide a discussion involving a number of spider silks? biological aspects, in this review we present descriptions of members from each family of spidroin identified from five spider species of the Brazilian biodiversity, and an evolutionary study of them in correlation with the anatomical specialization of glands and spider?s spinning behaviors. Due to the biotechnological importance of spider silks for the production of new biomaterials, we also discuss about the new possible technical and biomedical applications of spider silks and the current status of it

Epidemiology and outcomes of septic shock in children with complex chronic conditions in a developing country PICU

Objective To investigate the role of Complex Chronic Conditions (CCCs) on the outcomes of pediatric patients with refractory septic shock, as well as the accuracy of PELOD-2 and Vasoactive Inotropic Score (VIS) to predict mortality in this specific population. Methods This is a single-center, retrospective cohort study. All patients diagnosed with septic shock requiring vasoactive drugs admitted to a 13-bed PICU in southern Brazil, between January 2016 and July 2018, were included. Clinical and demographic characteristics, presence of CCCs and VIS, and PELOD-2 scores were accessed by reviewing electronic medical records. The main outcome was considered PICU mortality. Results 218 patients with septic shock requiring vasoactive drugs were identified in the 30-month period and 72% of them had at least one CCC. Overall mortality was 22%. Comparing to patients without previous comorbidities, those with CCCs had a higher mortality (26.7% vs 9.8%; OR = 3.4 [1.3–8.4]) and longer hospital length of stay (29.3 vs 14.8; OR 2.39 [1.1- 5.3]). Among the subgroups of CCCs, “Malignancy” was particularly associated with mortality (OR = 2.3 [1.0–5.1]). VIS and PELOD-2 scores in 24 and 48 hours were associated with mortality and a PELOD-2 in 48 hours > 8 had the best performance in predicting mortality in patients with CCC (AUROC = 0.89). Conclusion Patients with CCCs accounted for the majority of those admitted to the PICU with septic shock and related to poor outcomes. The high prevalence of hospitalizations, use of resources, and significant mortality determine that patients with CCCs should be considered a priority in the healthcare system

DETERMINATION OF ARGININE AND ASCORBIC ACID IN EFFERVESCENT TABLETS BY MULTIDIMENSIONAL CHROMATOGRAPHY

This paper describes a fast and direct method for quantification of arginine and ascorbic acid (vitamin C) in effervescent tablets. HPLC-UV multidimensional chromatography (RP18 and cyanopropyl columns) was developed and validated. Phosphate buffer 10 mMpH 3.4 was used as mobile phase. Satisfactory resolution between the drugs was obtained with analysis time less than 8.0 min. Method was linear in the ranges of 140-320 mg.mL-1 and 240-560 mg.mL-1 for arginine and ascorbic acid, respectively. Precision showed RSD <2.0%. Recovery was 99.5% and 100.0% for arginine and ascorbic acid, respectively. Robustness was confirmed through factorial analysis 22 (pH and mobile phase flow rate as factors).This paper describes a fast and direct method for quantification of arginine and ascorbic acid (vitamin C) in effervescent tablets. HPLC-UV multidimensional chromatography (RP18 and cyanopropyl columns) was developed and validated. Phosphate buffer 10 mMpH 3.4 was used as mobile phase. Satisfactory resolution between the drugs was obtained with analysis time less than 8.0 min. Method was linear in the ranges of 140-320 mg.mL-1 and 240-560 mg.mL-1 for arginine and ascorbic acid, respectively. Precision showed RSD <2.0%. Recovery was 99.5% and 100.0% for arginine and ascorbic acid, respectively. Robustness was confirmed through factorial analysis 22 (pH and mobile phase flow rate as factors)

Recombinant biosynthesis of functional human growth hormone and coagulation factor IX in transgenic soybean seeds.

Edição do Congress of the Brazilian Biotechnology Society, Florianópolis, 2013