Recovery of Proteases and Protease Inhibitors from Ganoderma spp. Cultivated in Amazonian Lignocellulose Wastes.

BACKGROUND: Ganoderma spp. are a great source of bioactive molecules. The production and recovery of bioactive molecules vary according to strain, growth substrate, and extraction solution. Variations in protease and their inhibitors in basidiomata from a commercial strain (G. lingzhi) and an Amazonian isolate (Ganoderma sp.) cultivated in Amazonian lignocellulosic wastes and extracted with different solutions are plausible and were investigated in our study. METHODS: Basidiomata from cultivation in substrates based on açaí seed, guaruba-cedro sawdust and three lots of marupá sawdust were submitted to extraction in water, Tris-HCl, and sodium phosphate. Protein content, proteases, and protease inhibitors were estimated through different assays. The samples were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR). RESULTS: Tris-HCl provided higher protein extraction from Ganoderma sp. and higher caseinolytic, gelatinolytic, and fibrinolytic activity for G. lingzhi cultivated in açaí. Water extracts of Ganoderma sp., in general, exhibited higher trypsin and papain inhibitor activities compared to G. lingzhi. Extracts in Tris-HCl and sodium phosphate showed more intense protein bands in SDS-- PAGE, highlighting bands of molecular weights around 100, 50, and 30 kDa. FTIR spectra showed patterns for proteins in all extracts, with variation in transmittance according to substrate and extractor. CONCLUSION: Water extract from Amazonian Ganoderma sp. cultivated in marupá wastes are promising as a source of protease inhibitors, while the Tris-HCL extract of G. lingzhi from açaí cultivation stands out as a source of proteases with fibrinolytic, caseinolytic, and gelatinolytic activities.

Whey protein dynamics in goat mammary secretions during colostrum and early lactation periods.

The protein composition in goat milk undergoes changes throughout the different lactation periods, displaying distinct characteristics that are influenced by the dynamic nature of protein composition and concentration during the transition from colostrum secretion to mature milk. To evaluate the dynamics of whey proteins of Saanen goats during the colostral phase and the first month of lactation, 110 milk samples from 11 healthy mammary halves of seven Saanen goats were selected through a clinical evaluation. Whey was obtained by rennet coagulation of the mammary secretion. The biuret method determined total protein concentration, and their fractions were identified by 12% dodecyl sulfate-polyacrylamide gel electrophoresis. Maximum concentrations of all protein fractions were observed in the first 12 h of lactation, reducing throughout the study. Modification of the protein predominance was also observed. The transition from colostrum secretion to milk occurred 5 or 7 d postpartum.

Physicochemical and protein profile of goat whey powder / Perfil físico-químico e proteico do soro de leite de cabra

The cheese after processing, generates whey, which is a residue that has potential benefit to human health. However, there are still few studies that seek to evaluate the composition and potential benefits of goat milk whey, even though it is a promising dairy source. Despite this, the large-scale generation of this waste can favor the environmental impact when improperly disposed of. Given this, the use of all content of dairy production can reverse this impact and increase the income of the dairy industry. Thus, this research prepared a powdered product based on goat's milk whey from the spray drying technique and evaluate its physicochemical composition by chemical analytical methods and protein profile through polyacrylamide gel electrophoresis. The elaborated product had a higher carbohydrate content than protein, and the identification of protein fractions showed that the most expressive bands represented beta-lactoglobulin and alpha-lactoalbumin. Therefore, the elaborated product has the potential to generate novel products for human consumption and with potential health benefits.

O queijo após processado gera o soro de leite, sendo este um resíduo que possui potencial benefício à saúde humana. No entanto, ainda são poucos os estudos que buscam avaliar a composição e os potenciais benefícios do soro de leite de cabra, mesmo esta sendo uma fonte láctea promissora. Apesar disso, a geração em larga escala deste resíduo pode favorecer o impacto ambiental quando descartado inadequadamente. Em vista disso, a utilização de todo conteúdo gerado na produção de lácteos pode reverter este impacto e aumentar o rendimento da indústria de laticínios. Diante disso, é importante buscar o desenvolvimento de novos produtos para favorecer a redução do impacto gerado. Assim, este trabalho teve como objetivo elaborar um produto em pó, à base de soro de leite de cabra a partir da secagem pela técnica de spray drying e avaliar sua composição físico-química por métodos químicos analíticos e perfil proteico através da eletroforese em gel de poliacrilamida. O produto elaborado apresentou maior teor de carboidratos do que proteína, e a identificação das frações proteicas mostrou que as bandas mais expressivas representavam beta-lactoglobulina e alfa-lactoalbumina. Portanto, o produto elaborado apresenta potencial para geração de novos produtos para consumo humano e com prováveis benefícios à saúde.

Isolation and Characterization of Protein Fractions for Valorization of Sacha Inchi Oil Press-Cake.

The growing interest in plant-based food protein sources has provided opportunities for the valorization of agri-food by-products, driving the food industry towards more sustainable development. In this study, three extraction procedures (varying the pH value (7.0 and 11.0) and the addition of salt (0 and 5%)) were investigated to obtain seven different protein fractions (SIPF) from Sacha Inchi oil press-cake (SIPC), which were characterized in terms of their protein content, electrophoretic profile, secondary structure, and techno-functional properties. Extractions at pH 11.0 without salt addition produced the highest values of protein content, extraction yield, protein recovery, and protein concentration increase (84.0%, 24.7%, 36.5%, and 1.5-fold, respectively). Under these extraction conditions, the electrophoretic analysis indicated that most of the SIPC proteins were extracted. SIPF displayed an excellent oil absorption capacity (4.3-9.0 w/w), and interesting foam activity (36.4-133.3%). The solubility and emulsifying activity of the albumin fractions were significantly higher than those of the other fractions (~87 vs. <15.8%, and 280-370 vs. <140 m2/g, respectively). Correlation analysis showed that the secondary structure of the SIPF significantly influences their techno-functional properties. These results indicate that SIPC is a by-product of great potential for protein extraction processes, and as a valorization strategy for technical cycle solutions for the Sacha Inchi productive chain in the circular economy context.

Proteinuria and Electrophoretic Pattern in Dogs with Comorbidities Associated with Chronic Kidney Disease.

In animals with chronic pathologies, the detection of proteinuria via the proteinuria: creatininuria ratio (UPC) and urinary protein electrophoresis allows for the early diagnosis of chronic kidney disease (CKD). The objective of this work was to identify and determine the magnitude of proteinuria and its electrophoretic pattern characterization in dogs with chronic diseases pathophysiologically related to proteinuria. With the studied patients, five groups were formed. The control group (CG) contained non-proteinuric cases. The cases with proteinuria were classified into four groups according to the concurrent disease: chronic inflammatory diseases (IG), neoplasms (NG), heart diseases (HG), and endocrine diseases (EG). For the statistical analysis, descriptive statistics and non-parametric tests were used. Data from 264 dogs were obtained; in the disease groups, proteinuria was observed in more than 30% as the only finding of kidney disease, evidencing a greater risk factor for proteinuria in the HG group (OR 4.047, CI 1.894-8.644, p < 0.0001). In the HG, NG, and EG groups, a higher frequency of glomerular pattern (GEP) related to glomerular hypertension was observed; in the IG, a higher frequency of mixed pattern (MEP) was observed. These findings are secondary to the hyperfiltration process that affects the glomerulus and the renal tubule.

Montane Rattlesnakes in México: Venoms of Crotalus tancitarensis and Related Species within the Crotalus intermedius Group.

The Crotalus intermedius group is a clade of rattlesnakes consisting of several species adapted to a high elevation habitat, primarily in México. Crotalus tancitarensis was previously classified as C. intermedius, until individuals occurring on Cerro Tancítaro in Michoacán, México, were reevaluated and classified as a new species (C. tancitarensis) based on scale pattern and geographic location. This study aimed to characterize the venom of C. tancitarensis and compare the venom profile to those of other species within the Crotalus intermedius group using gel electrophoresis, biochemical assays, reverse-phase high performance liquid chromatography, mass spectrometry, and lethal toxicity (LD50) assays. Results show that the venom profiles of species within the Crotalus intermedius group are similar, but with distinct differences in phospholipase A2 (PLA2), metalloproteinase PI (SVMP PI), and kallikrein-like serine proteinase (SVSP) activity and relative abundance. Proteomic analysis indicated that the highland forms produce venoms with 50-60 protein isoforms and a composition typical of type I rattlesnake venoms (abundant SVMPs, lack of presynaptic PLA2-based neurotoxins), as well as a diversity of typical Crotalus venom components such as serine proteinases, PLA2s, C-type lectins, and less abundant toxins (LAAOs, CRiSPs, etc.). The overall venom profile of C. tancitarensis appears most similar to C. transversus, which is consistent with a previous mitochondrial DNA analysis of the Crotalus intermedius group. These rattlesnakes of the Mexican highlands represent a radiation of high elevation specialists, and in spite of divergence of species in these Sky Island habitats, venom composition of species analyzed here has remained relatively conserved. The majority of protein family isoforms are conserved in all members of the clade, and as seen in other more broadly distributed rattlesnake species, differences in their venoms are largely due to relative concentrations of specific components.

Understanding the Anti-Diarrhoeal Properties of Incomptines A and B: Antibacterial Activity against Vibrio cholerae and Its Enterotoxin Inhibition.

Incomptines A (IA) and B (IB) are two sesquiterpene lactones with antiprotozoal, antibacterial, cytotoxic, antitumor, spermicidal, and phytotoxic properties. The antibacterial activity of IA and IB against bacteria causing diarrhoea have been reported; however, no information is available regarding their antibacterial activity on Vibrio cholerae. In this work, both compounds were evaluated for their anti-diarrhoeal potential using the bacterium V. cholerae, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis on cholera toxin, and a cholera toxin-induced diarrhoea model in male Balb/c mice. In addition, a molecular docking study was carried out to understand the interaction of IA and IB with cholera toxin. In terms of antibacterial activity, IB was three times more active than IA on V. cholerae. In the case of SDS-PAGE analysis and the in silico study, IA was most effective, revealing its potential binding mode at a molecular level. In terms of anti-diarrhoeal activity, IA was 10 times more active than IB and racecadotril, an antisecretory drug used as positive control; the anti-diarrheal activity of IB was also closer than racecadotril. The results obtained from in vitro, in vivo, and computational studies on V. cholerae and cholera toxin support the potential of IA and IB as new anti-diarrhoeal compounds.

Mobilization of seed storage proteins is crucial to high vigor in common bean seeds / Mobilização das proteínas de reserva é crucial para o alto vigor em sementes de feijão

Seed germination is a complex process controlled by many factors, in which physical and biochemical mechanisms are involved and the mobilization of reserves is crucial for this process to occur. Although, seed reserve mobilization is usually thought to be a post-germination process, seed reserve proteins mobilization occurs during germination. This study quantified seed proteins of bean genotypes during different hydration times, in order to understand the process of protein mobilization and whether there is relationship of this biochemical component with seed vigor. This study was conducted using seeds with different levels of vigor, genotypes with highest (13, 42, 55 and 81) and lowest (07, 23, 44, 50, IPR-88-Uirapurú and Iapar 81) physiological quality. High vigor genotypes showed greater efficiency in hydrolysis and mobilization of protein component, because they presented low globulins content in cotyledons at radicle protrusion in relation to low vigor genotypes (07, 23 and 50). The protein alpha-amylase inhibitor, observed in all genotypes, is involved with the longer time needed for radicle protrusion, according to the band intensity difference in genotypes 07, 44 and Iapar 81.

A germinação de sementes é um processo complexo controlado por muitos fatores, nos quais mecanismos físicos e bioquímicos estão envolvidos e a mobilização de reservas é decisiva para que esse processo ocorra. Embora a mobilização de reservas de sementes seja considerada um processo pós-germinativo, a mobilização das proteínas de reserva de sementes ocorre durante a germinação. Este estudo teve como objetivo quantificar as proteínas de sementes de genótipos de feijão durante os diferentes tempos de hidratação, a fim de compreender o processo de mobilização proteica e se há relação desse componente bioquímico com o vigor das sementes. Este estudo foi realizado utilizando sementes com diferentes níveis de vigor, genótipos com maior (13, 42, 55 e 81) e menor (07, 23, 44, 50, IPR-88-Uirapurú e Iapar 81) qualidade fisiológica. Os genótipos de alto vigor apresentaram maior eficiência na hidrólise e mobilização do componente proteico, pois apresentaram baixo teor de globulinas nos cotilédones na protrusão radicular em relação aos genótipos de baixo vigor (07, 23 e 50). A proteína inibidora da alfa-amilase, observada em todos os genótipos, está envolvida com o maior tempo necessário para a protrusão da radícula, de acordo com a diferença de intensidade da banda nos genótipos 07, 44 e Iapar 81.

Biochemical characterization for determination of genetic distances among different indigenous chickpea (Cicer arietinum L. ) varieties of North-West Pakistan / Caracterização bioquímica para determinação de distâncias genéticas entre diferentes variedades indígenas de grão-de-bico (Cicer arietinum L. ) do noroeste do Paquistão

Abstract Genetic distances among different chickpea varieties and evaluation of their free amino acid profiles were determined on the basis of Sodium dodecyle sulphate polyacrylamide gels electrophoresis (SDS-PAGE). Total soluble proteins were resolved on 10% SDS Polyacrylamide gel. Low variability in tested varieties was observed. Dendogram based on electrophoretic data clustered the genotypes into 2 groups. The results showed that the average protein content of all the varieties was 26.01% within the range 22.8% for Thal-2006 to 34.06% Sheenghar-2000 of dry seed weight. On the basis of total protein content Bittal-98, Dasht and Sheen Ghar-2000, Karak-3 and CM-98, Paidar -91 and Fakhr-e-Thal, C-44, Balaksar and KK-1showed similar concentrations for protein contents among each other but showed variation from the rest of the varieties. Different proteins were separated on the basis of changes in their molecular weights by means of Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Dasht, CM-98, and Sheen Ghar showed 100% similarity. Balaksar and Fakhr-e- Thal, KK-2 and Chattan and KC-98, KK-1 and Lawaghar were 100% similar among each other but showed variation from the rest of the accessions. The overall dendrogram showed high and low level of variation among the accessions. The concentration of free amino acids varied among the 16 chickpea varieties. A significant difference of both essential and non-essential amino acids was found among the chickpea cultivars. The total concentration of essential amino acid was recorded 40.81 g/100 g protein while non-essential was recorded 59.18343 g/100 g protein in the given cultivars. The highest concentration of essential amino acids was found in C-44 followed by KK-2, KK-1 and Fakhr E Tal while the lowest concentration was recorded in Cm-98, Paidar-91 and Sheen Ghar-2000 respectively. Cultivars TAL-2006, Chattan and Karak-3 showed maximum concentration of both essential and endogenous amino acids. In conclusion; for broadening the genetic pools in breeding programs or to search for exotic characters, for instance new disease resistance alleles, accession with low similarity coefficients (Lawaghar and Battal-98) may be utilized. Furthermore the information acquired from this study could be used to device a proficient breeding approach intended at improving nutritional as well as broadening the genetic base of this essential food crop of Pakistan.

Resumo As distâncias genéticas entre as diferentes variedades de grão-de-bico e a avaliação de seus perfis de aminoácidos livres foram determinadas com base na eletroforese em gel de poliacrilamida com dodecil sulfato de sódio (SDS-PAGE). As proteínas solúveis totais foram resolvidas em SDS-PAGE a 10%. Foi observada baixa variabilidade nas variedades testadas. O dendrograma fundamentado em dados eletroforéticos agrupou os genótipos em dois grupos. Os resultados mostraram que o teor médio de proteínas de todas as variedades foi de 26,01%, na faixa de 22,8% para Thal-2006 a 34,06% para Sheenghar-2000 do peso de sementes secas. Com base no conteúdo total de proteínas, Bittal-98, Dasht, Sheen Ghar-2000, Karak-3, CM-98, Paidar-91, Fakhr-e-Thal, C-44, Balaksar e KK-1 apresentaram concentrações semelhantes para o conteúdo de proteínas entre si, mas tiveram variação quanto ao restante das variedades. Diferentes proteínas foram separadas com base nas alterações de seus pesos moleculares por meio de eletroforese em gel de poliacrilamida com dodecil sulfato de sódio (SDS-PAGE). Dasht, CM-98 e Sheen Ghar mostraram 100% de similaridade. Balaksar, Fakhr-e-Thal, KK-2, Chattan e KC-98, KK-1 e Lawaghar foram 100% semelhantes entre si, mas apresentaram variação em relação ao restante dos acessos. O dendrograma geral mostrou alto e baixo nível de variação entre os acessos. A concentração de aminoácidos livres variou entre as 16 variedades de grão-de-bico. Foi encontrada uma diferença significativa entre os aminoácidos essenciais e não essenciais nas cultivares de grão-de-bico. A concentração total de aminoácidos essenciais foi registrada em 40,81 g / 100 g de proteína, enquanto a não essencial foi registrada em 59,18343 g / 100 g de proteína nas cultivares. A maior concentração de aminoácidos essenciais foi encontrada em C-44, seguida de KK-2, KK-1 e Fakhr-e-Thal, enquanto a menor concentração foi registrada em CM-98, Paidar-91 e Sheen Ghar-2000. As cultivares TAL-2006, Chattan e Karak-3 apresentaram concentração máxima de aminoácidos essenciais e endógenos. Em conclusão, para ampliar os pools genéticos em programas de melhoramento ou procurar caracteres exóticos, por exemplo, novos alelos de resistência a doenças, pode ser utilizada a adesão com baixos coeficientes de similaridade (Lawaghar e Battal-98). Além disso, as informações adquiridas neste estudo poderiam ser usadas para criar uma abordagem de criação eficiente, com o objetivo de melhorar a nutrição e ampliar a base genética dessa cultura alimentar essencial do Paquistão.

Biochemical characterization for determination of genetic distances among different indigenous chickpea (Cicer arietinum L. ) varieties of north-west Pakistan / Caracterização bioquímica para determinação de distâncias genéticas entre diferentes variedades indígenas de grão-de-bico (Cicer arietinum L. ) do noroeste do Paquistão

Genetic distances among different chickpea varieties and evaluation of their free amino acid profiles were determined on the basis of Sodium dodecyle sulphate polyacrylamide gels electrophoresis (SDS-PAGE). Total soluble proteins were resolved on 10% SDS Polyacrylamide gel. Low variability in tested varieties was observed. Dendogram based on electrophoretic data clustered the genotypes into 2 groups. The results showed that the average protein content of all the varieties was 26.01% within the range 22.8% for Thal-2006 to 34.06% Sheenghar-2000 of dry seed weight. On the basis of total protein content Bittal-98, Dasht and Sheen Ghar-2000, Karak-3 and CM-98, Paidar -91 and Fakhr-e-Thal, C-44, Balaksar and KK-1showed similar concentrations for protein contents among each other but showed variation from the rest of the varieties. Different proteins were separated on the basis of changes in their molecular weights by means of Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Dasht, CM-98, and Sheen Ghar showed 100% similarity. Balaksar and Fakhr-e- Thal, KK-2 and Chattan and KC-98, KK-1 and Lawaghar were 100% similar among each other but showed variation from the rest of the accessions. The overall dendrogram showed high and low level of variation among the accessions. The concentration of free amino acids varied among the 16 chickpea varieties. A significant difference of both essential and non-essential amino acids was found among the chickpea cultivars. The total concentration of essential amino acid was recorded 40.81 g/100 g protein while non-essential was recorded 59.18343 g/100 g protein in the given cultivars. The highest concentration of essential amino acids was found in C-44 followed by KK-2, KK-1 and Fakhr E Tal while the lowest concentration was recorded in Cm-98, Paidar-91 and Sheen Ghar-2000 respectively. Cultivars TAL-2006, Chattan and Karak-3 showed maximum concentration of both essential and endogenous amino acids. In conclusion; for broadening the genetic pools in breeding programs or to search for exotic characters, for instance new disease resistance alleles, accession with low similarity coefficients (Lawaghar and Battal-98) may be utilized. Furthermore the information acquired from this study could be used to device a proficient breeding approach intended at improving nutritional as well as broadening the genetic base of this essential food crop of Pakistan.(AU)

As distâncias genéticas entre as diferentes variedades de grão-de-bico e a avaliação de seus perfis de aminoácidos livres foram determinadas com base na eletroforese em gel de poliacrilamida com dodecil sulfato de sódio (SDS-PAGE). As proteínas solúveis totais foram resolvidas em SDS-PAGE a 10%. Foi observada baixa variabilidade nas variedades testadas. O dendrograma fundamentado em dados eletroforéticos agrupou os genótipos em dois grupos. Os resultados mostraram que o teor médio de proteínas de todas as variedades foi de 26,01%, na faixa de 22,8% para Thal-2006 a 34,06% para Sheenghar-2000 do peso de sementes secas. Com base no conteúdo total de proteínas, Bittal-98, Dasht, Sheen Ghar-2000, Karak-3, CM-98, Paidar-91, Fakhr-e-Thal, C-44, Balaksar e KK-1 apresentaram concentrações semelhantes para o conteúdo de proteínas entre si, mas tiveram variação quanto ao restante das variedades. Diferentes proteínas foram separadas com base nas alterações de seus pesos moleculares por meio de eletroforese em gel de poliacrilamida com dodecil sulfato de sódio (SDS-PAGE). Dasht, CM-98 e Sheen Ghar mostraram 100% de similaridade. Balaksar, Fakhr-e-Thal, KK-2, Chattan e KC-98, KK-1 e Lawaghar foram 100% semelhantes entre si, mas apresentaram variação em relação ao restante dos acessos. O dendrograma geral mostrou alto e baixo nível de variação entre os acessos. A concentração de aminoácidos livres variou entre as 16 variedades de grão-de-bico. Foi encontrada uma diferença significativa entre os aminoácidos essenciais e não essenciais nas cultivares de grão-de-bico. A concentração total de aminoácidos essenciais foi registrada em 40,81 g / 100 g de proteína, enquanto a não essencial foi registrada em 59,18343 g / 100 g de proteína nas cultivares. A maior concentração de aminoácidos essenciais foi encontrada em C-44, seguida de KK-2, KK-1 e Fakhr-e-Thal, enquanto a menor concentração foi registrada em CM-98, Paidar-91 e Sheen Ghar-2000. As cultivares TAL-2006, Chattan e Karak-3 apresentaram concentração máxima de aminoácidos essenciais e endógenos. Em conclusão, para ampliar os pools genéticos em programas de melhoramento ou procurar caracteres exóticos, por exemplo, novos alelos de resistência a doenças, pode ser utilizada a adesão com baixos coeficientes de similaridade (Lawaghar e Battal-98). Além disso, as informações adquiridas neste estudo poderiam ser usadas para criar uma abordagem de criação eficiente, com o objetivo de melhorar a nutrição e ampliar a base genética dessa cultura alimentar essencial do Paquistão.(AU)

Evaluation of flour protein for different bread wheat genotypes / Avaliação da proteína de farinha para diferentes genótipos de trigo de pão

Abstract Six different bread wheat genotypes; two Egyptian commercial varieties (control); Giza-168 and Gemmeiza-11, and four promising lines; L84 and L148, resulted via hybridization and M10 and M34 via radiation mutation program) were rheologically evaluated using extensograph and for protein, analysis using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The radiation mutant M10 and M34 had the highest maximum resistance which is a very good indicator of strong gluten. The amount of gluten content was higher in M10, L148, and M34 compared to the control samples Gz168 and Gm11. Sulfide amino acids (CYS and MET) are slightly higher in M10. The electrophoretic results and amino acid analyzers show that the best technological quality was exhibited by M10. Radiation mutants wheat genotypes have a protein with good characteristics, mainly gluten which is significantly higher compared to control samples. The rheological properties measured as extensograph and gel electrophoresis were much better in irradiated lines M10 and M34.

Resumo Seis diferentes genótipos de trigo de pão, duas variedades comerciais egípcias (controle) - Giza-168 e Gemmeiza-11 - e quatro linhas promissoras - L84 e L148, obtidas via hibridação, e M10 e M34, via programa de mutação por radiação - foram avaliados reologicamente por meio de extensógrafo, enquanto, para proteínas, foram feitas análises utilizando eletroforese em gel de poliacrilamida com dodecilsulfato de sódio (SDS-PAGE). Os mutantes de radiação M10 e M34 apresentaram a maior resistência máxima, o que é um indicador muito bom de glúten forte. A quantidade de glúten foi maior em M10, L148 e M34 em comparação com as amostras de controle Gz168 e Gm11. Os aminoácidos sulfurados (CYS e MET) são um pouco mais altos no M10. Os resultados eletroforéticos e analisadores de aminoácidos mostram que a melhor qualidade tecnológica foi exibida pelo M10. Os genótipos de trigo mutantes da radiação possuem uma proteína com boas características, principalmente o glúten, que é significativamente maior em comparação às amostras do grupo controle. As propriedades reológicas medidas, como extensógrafo e eletroforese em gel, foram muito melhores nas linhas irradiadas M10 e M34.

Evaluation of flour protein for different bread wheat genotypes / Avaliação da proteína de farinha para diferentes genótipos de trigo de pão

Six different bread wheat genotypes; two Egyptian commercial varieties (control); Giza-168 and Gemmeiza-11, and four promising lines; L84 and L148, resulted via hybridization and M10 and M34 via radiation mutation program) were rheologically evaluated using extensograph and for protein, analysis using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The radiation mutant M10 and M34 had the highest maximum resistance which is a very good indicator of strong gluten. The amount of gluten content was higher in M10, L148, and M34 compared to the control samples Gz168 and Gm11. Sulfide amino acids (CYS and MET) are slightly higher in M10. The electrophoretic results and amino acid analyzers show that the best technological quality was exhibited by M10. Radiation mutants wheat genotypes have a protein with good characteristics, mainly gluten which is significantly higher compared to control samples. The rheological properties measured as extensograph and gel electrophoresis were much better in irradiated lines M10 and M34.(AU)

Seis diferentes genótipos de trigo de pão, duas variedades comerciais egípcias (controle) Giza-168 e Gemmeiza-11 e quatro linhas promissoras L84 e L148, obtidas via hibridação, e M10 e M34, via programa de mutação por radiação foram avaliados reologicamente por meio de extensógrafo, enquanto, para proteínas, foram feitas análises utilizando eletroforese em gel de poliacrilamida com dodecilsulfato de sódio (SDS-PAGE). Os mutantes de radiação M10 e M34 apresentaram a maior resistência máxima, o que é um indicador muito bom de glúten forte. A quantidade de glúten foi maior em M10, L148 e M34 em comparação com as amostras de controle Gz168 e Gm11. Os aminoácidos sulfurados (CYS e MET) são um pouco mais altos no M10. Os resultados eletroforéticos e analisadores de aminoácidos mostram que a melhor qualidade tecnológica foi exibida pelo M10. Os genótipos de trigo mutantes da radiação possuem uma proteína com boas características, principalmente o glúten, que é significativamente maior em comparação às amostras do grupo controle. As propriedades reológicas medidas, como extensógrafo e eletroforese em gel, foram muito melhores nas linhas irradiadas M10 e M34.(AU)

New Cationic fac-[Re(CO)3(deeb)B2]+ Complex, Where B2 Is a Benzimidazole Derivative, as a Potential New Luminescent Dye for Proteins Separated by SDS-PAGE.

Sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) can be used to separate proteins based mainly on their size such as in denaturing gels. Different staining methods have been reported to observe proteins in the gel matrix, where the most used dyes are generally anionic. Anionic dyes allow for interactions with protonated amino acids, retaining the dye in the proteins. Fluorescent staining is an alternative technique considered to be sensitive, safe, and versatile. Some anionic complexes based on d6 transition metals have been used for this purpose, where cationic dyes have been less explored in this context. In this work, we synthesized and characterized a new monocationic rhenium complex fac-[Re(CO)3(deeb)B2]+ (where deeb is 4,4'-bis(ethoxycarbonyl)-2,2'-bpy and B2 is 2,4-di-tert-butyl-6-(3H-imidazo[4,5-c]pyridine-2-yl)phenol). We carried out a structural characterization of this complex by MS+, FTIR, 1H NMR, D2O exchange, and HHCOSY. Moreover, we carried out UV-Vis, luminescence, and cyclic voltammetry experiments to understand the effect of ligands on the complex's electronic structure. We also performed relativistic theoretical calculations using the B3LYP/TZ2P level of theory and R-TDDFT within a dielectric continuum model (COSMO) to better understand electronic transitions and optical properties. We finally assessed the potential of fac-[Re(CO)3(deeb)B2]+ (as well as the precursor fac-Re(CO)3(deeb)Br and the free ligand B2) to stain proteins separated by SDS-PAGE. We found that only fac-[Re(CO)3(deeb)B2]+ proved viable to be directly used as a luminescent dye for proteins, presumably due to its interaction with negatively charged residues in proteins and by weak interactions provided by B2. In addition, fac-[Re(CO)3(deeb)B2]+ seems to interact preferentially with proteins and not with the gel matrix despite the presence of sodium dodecyl sulfate (SDS). In future applications, these alternative cationic complexes might be used alone or in combination with more traditional anionic compounds to generate counterion dye stains to improve the process.

NanoUPLC-MSE reveals differential abundance of gluten proteins in wheat flours of different technological qualities.

Gluten proteins contribute to the rheological properties of dough. Mass spectrometric techniques help to understand the contribution of these proteins to the quality of the end product. This work aimed to apply modern proteomic techniques to characterize and provide a better understanding of gluten proteins in wheat flours of different technological qualities. Nine Brazilian wheat flours (Triticum aestivum) classified by rheological gluten force were used to extract the proteins. Extracts were pooled together by technological qualities in low (LW), medium (MD), and superior (SP). Peptides were analyzed by nanoUPLC and mass spectrometry multiplex method (MSE). Collectively, 3545 peptides and 1297 proteins were identified, and 116 proteins were found differentially abundant. Low molecular weight glutenin subunits (LMW-GS) were found up-regulated only in SP samples. Proteins related to wheat grain hardness, such as puroindoline-A, were found in significant concentration in LW samples. After domain prediction, LW presented a different pattern with a lower abundance of functional domains, and SP presented chaperones, known to be involved in adequate folding of the storage proteins. NanoUPLC-MSE was efficient in analyzing and distinguishing the proteomic pattern of wheat flours from different qualities, pointing out the differentially abundant gluten proteins and providing a better understanding of wheat flour quality. SIGNIFICANCE: Common wheat is one of the most important staple food sources in the world. The improvement and comprehension of wheat quality has been a major objective of plant breeders and cereal chemists. Our findings highlighted the application of a modern proteomic approach to obtain a better understanding of the impact of gluten proteins on the technological quality of different wheat flours. The obtained data revealed different abundances of wheat quality-related proteins in superior quality flours when compared with samples of low rheological properties. In addition, multivariate statistical analysis clearly distinguished the flours of different qualities. This work contributes to the consolidation of research in the field of wheat technological quality.

Influence of the capping of biogenic silver nanoparticles on their toxicity and mechanism of action towards Sclerotinia sclerotiorum.

BACKGROUND: Biogenic nanoparticles possess a capping of biomolecules derived from the organism employed in the synthesis, which contributes to their stability and biological activity. These nanoparticles have been highlighted for the control of phytopathogens, so there is a need to understand their composition, mechanisms of action, and toxicity. This study aimed to investigate the importance of the capping and compare the effects of capped and uncapped biogenic silver nanoparticles synthesized using the filtrate of Trichoderma harzianum against the phytopathogenic fungus Sclerotinia sclerotiorum. Capping removal, investigation of the composition of the capping and physico-chemical characterization of the capped and uncapped nanoparticles were performed. The effects of the nanoparticles on S. sclerotiorum were evaluated in vitro. Cytotoxicity and genotoxicity of the nanoparticles on different cell lines and its effects on nontarget microorganisms were also investigated. RESULTS: The capped and uncapped nanoparticles showed spherical morphology, with greater diameter of the uncapped ones. Functional groups of biomolecules, protein bands and the hydrolytic enzymes NAGase, ß-1,3-glucanase, chitinase and acid protease from T. harzianum were detected in the capping. The capped nanoparticles showed great inhibitory potential against S. sclerotiorum, while the uncapped nanoparticles were ineffective. There was no difference in cytotoxicity comparing capped and uncapped nanoparticles, however higher genotoxicity of the uncapped nanoparticles was observed towards the cell lines. Regarding the effects on nontarget microorganisms, in the minimal inhibitory concentration assay only the capped nanoparticles inhibited microorganisms of agricultural importance, while in the molecular analysis of the soil microbiota there were major changes in the soils exposed to the uncapped nanoparticles. CONCLUSIONS: The results suggest that the capping played an important role in controlling nanoparticle size and contributed to the biological activity of the nanoparticles against S. sclerotiorum. This study opens perspectives for investigations concerning the application of these nanoparticles for the control of phytopathogens.

Toxicogenomic Analysis.

The biological functions of a cell may change in response to exposure to toxic agents. Toxicogenomics employs the recent developments in genomics, transcriptomics, and proteomics to study how a chemical impacts gene/protein expression and cell functions. We describe a method for transcriptomic analysis by RNA sequencing based on Illumina HiSeq, NextSeq, or NovaSeq Systems followed by real-time qPCR validation. We also depict a method for proteomic analysis by "one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis" (1D SDS-PAGE) and a sample preparation procedure for "liquid chromatography in tandem with mass spectrometry" (LC-MS/MS), and we present some generic points to consider during LC-MS/MS.

Evaluation of salivary protein patterns among a rural population exposed and non-exposed to arsenic-contaminated drinking water in areas of Tucumán (Argentina): a pilot study

Abstract Arsenic contamination in the environment and groundwater is a major global public health problem. Several researchers suggest that the toxicity of arsenic could be related to oral cancer development, usually resulting from potentially malignant lesions. During pathological processes, salivary proteins suffer modifications, which could lead to the discovery of new biomarkers. Objective To analyze the protein profile in human saliva samples from a rural population exposed to high levels of arsenic in drinking water and its association with potentially malignant lesions. Methodology This observational, analytic and cross-sectional design included 121 patients from the state of Graneros (Tucumán, Argentina). Arsenic concentration in drinking water was determined and, according to the values obtained, individuals were divided into 2 groups: exposed group and non-exposed group. Saliva samples were obtained, and total protein concentration was measured by Bradford method. Finally, Laemmli SDS-polyacrylamide gel electrophoresis was conducted to obtain the protein profile. Results Total protein concentration in saliva was lower in the exposed group than in the non-exposed group. Average areas of 20 and 42 KDa bands were significantly lower in exposed group than non-exposed group. Conclusion Chronic intake of high arsenic concentrations in drinking water produces changes in the salivary protein profile, which is associated with the presence of potentially malignant lesions.

Biological and Oxidative Effect of Ellagic Acid on Saccharomyces cerevisiae: A New Way for Culture Developing

Abstract In this study, the effects of Ellagic acid (EA) on protein expression in yeasts and cellular development were investigated. Four groups were formed. Groups: 1) Control group; yeast only cultivated group; 2) Ellagic Acid (EA) group: EA (10%) given group; 3) Hydrogen peroxide (H2O2) Group: The group given H2O2 (15 mM); 4) EA + H2O2 group: EA (10%) + H2O2 (15 mM) group. After sterilization, EA (10%) and H2O2 (15 mM) were added to the Saccharomyces cerevisiae (S. cerevisiae) cultures and the cultures were grown at 30 °C for 1 hour, 3 hours, 5 hours and 24 hours (overnight). S. cerevisiae cell growth, lipid peroxidation MDA (malondialdehyde) analysis and GSH (glutathione) level were analyzed by spectrophotometer. Total protein changes were determined by SDS-PAGE electrophoresis and measured by the Bradford method. According to the obtained results, compared with the H2O2 group, cell development (1, 3, 5 and 24 hours), GSH level and total protein synthesis (24 hours) were increased with EA, while MDA level (24 hours) decreased. These results show that EA reduces oxidative damage, increases cell growth and it has a protective effect to promote protein synthesis in S. cerevisiae culture.

Extraction, Partial Purification and Characterization of Bromelain from Pineapple (Ananas Comosus) Crown, Core and Peel Waste

Abstract Ananas Comosus (also known as pineapple) is a part of Bromeliaceae family and it is consumed as food as well as folk medicine for the treatment of various diseases. It is reported that pineapple is a rich source of bromelain, a cysteine protease and it is considered as an important enzyme in different industries due to its significant therapeutic and industrial applications such as anticancer, anti-inflammatory and meat tenderizing. Bromelain is mostly present in fruit and stem of pineapple, but it is reported that crown, core, and peels, which constitute the waste of the pineapple plant, also contain bromelain but limited data is available. Therefore, the proposed study aimed at utilizing pineapple waste for the extraction and characterization of bromelain. Firstly, crude bromelain was extracted with phosphate buffer (pH 7), then it was subjected to partial purification using different fractions of ammonium sulphate (NH4)2SO4 such as 30, 40, 50 and 60% followed by desalting and concentration. Enzyme activity was calculated by using casein digesting unit (CDU) method. The results demonstrated that the crown bromelain showed highest purification of 4.34-fold at 30% (NH4)2SO4 saturation, whereas core and peel bromelain showed highest purification of 2.75 and 2.59-fold at 40% (NH4)2SO4 saturation. The molecular weight of crude and partially purified bromelain was determined by SDS-PAGE analysis and found to be 26 KDa. The pH and thermal stability of all the parts of pineapple showed maximum stability at pH 7 and at 35oC temperature.

Hematologic response and serum and peritoneal fluid proteinogram of cattle affected by intestinal diseases and traumatic reticuloperitonitis / Resposta hematológica e proteinograma sérico e do líquido peritoneal de bovinos acometidos com enfermidades intestinais e reticuloperitonite traumática

This study aimed to evaluate the hematologic response and the serum and peritoneal fluid (PF) proteinogram of cattle affected by digestive diseases. Twenty-seven animals were distributed in two groups: GI (intestinal diseases) and GII (traumatic reticuloperitonitis, TRP). The animals were previously submitted to a physical exam. Subsequently, blood samples were collected to perform the complete blood count, determine the plasma protein and fibrinogen, and obtain the serum for proteinogram in polyacrylamide gel (SDS-PAGE). Simultaneously, PF was collected to perform physical and chemical evaluation and the electrophoretic profile (SDS-PAGE). ANOVA at the 5% probability level was used to compare the groups. The animals showed signs of apathy, dehydration, and gastrointestinal hypomotility in both groups. However, GI animals showed more significant clinical changes. The blood count of both groups (P > 0.05) showed leukocytosis due to neutrophilia and a regenerative left shift with hyperfibrinogenemia. The proteinogram of both body fluids allowed the identification of proteins albumin (ALB), transferrin (TRF), ceruloplasmin, haptoglobin, α1-acid glycoprotein (α1-AGP), MW 23000 Da, α1-antitrypsin, IgA, and IgG. The [PT] PF/[PT] blood serum ratio of each of the identified proteins increased, showing statistical differences between groups (P < 0.05) regarding PT, ALB, TRF, α1-AGP, and IgG values, with GI animals showing the highest ratio. Intestinal diseases and TRP triggered a systemic and local response characterized by clinical, hematological, and serum and PF proteinogram alterations. The proteins α1-GPA, haptoglobin, and TRF measured in PF were good inflammation biomarkers and useful as an auxiliary tool for the diagnosis and prognosis of digestive diseases in cattle.(AU)

O objetivo deste estudo foi avaliar a resposta hematológica e o proteinograma sérico e do líquido peritoneal (LP) de bovinos acometidos com doenças digestivas. Foram avaliados 27 bovinos distribuídos em dois grupos, GI (enfermidades intestinais) e GII (reticuloperitonite traumática-RPT). Os animais foram previamente submetidos ao exame físico. Posteriormente foram colhidas amostras de sangue para realização do hemograma, determinação plasmática da proteína e do fibrinogênio e obtenção do soro para realização do proteinograma em gel de poliacrilamida (SDS-PAGE). Simultaneamente foi colhido o LP para avaliação física e química, assim como a realização do perfil eletroforético (SDS-PAGE). Empregou-se a análise de variância ao nível de 5% de probabilidade visando à comparação entre os grupos. Em ambos os grupos os animais demonstraram sinais de apatia, desidratação e hipomotilidade gastrointestinal, no entanto, os animais do GI apresentaram alterações clínicas mais expressivas. No hemograma observou-se em ambos os grupos (P > 0,05) leucocitose por neutrofilia e desvio à esquerda regenerativo com hiperfibrinogenia. O proteinograma de ambos os fluidos corpóreos permitiu a identificação das proteínas albumina (ALB), transferrina (TRF), ceruloplasmina, haptoglobina, α-1 glicoproteína ácida (α1-GPA), PM 23.000 Da, α-1 anti-tripsina, IgA e IgG. Os valores da relação [PT] LP / [PT] soro sanguíneo de cada uma das proteínas identificadas demonstrou elevação dos mesmos, bem como diferença estatística entre grupos (P < 0,05) nos valores da PT, ALB, TRF, α1-GPA e IgG, nos quais a relação foi mais elevada nos animais do GI. As enfermidades intestinais e a RPT desencadearam resposta sistêmica e local caracterizada pelas alterações clínicas, hematológicas, e do proteinograma sérico e do LP. A α1-GPA, a haptoglobina e a TRF, mensuradas no LP se mostraram bons biomarcadores de inflamação, sendo úteis como recurso auxiliar de diagnóstico e prognóstico das doenças digestivas dos bovinos.(AU)