A chromatographic network for the purification of detergent-solubilized six-transmembrane epithelial antigen of the prostate 1 from Komagataella pastoris mini-bioreactor lysates

Funding Information: The authors acknowledge the support from FEDER funds through the POCI-COMPETE 2020–Operational Programme Competitiveness and Internationalisation in Axis I–Strengthening Research, Technological Development and Innovation (Project POCI-01-0145-FEDER-007491), Jorge Barroca-Ferreira's and Ana M. Gonçalves's individual PhD Fellowships (SFRH/BD/130068/2017 and SFRH/BD/147519/2019, respectively), and Luís A. Passarinha's sabbatical fellowship (SFRH/BSAB/150376/2019) from FCT–Fundação para a Ciência e Tecnologia. This work was also supported by the Health Sciences Research Centre CICS-UBI (UIDB/00709/2020 and UIDP/00709/2020), the Applied Molecular Biosciences Unit UCIBIO (UIDB/04378/2020 and UIDP/04378/2020) and the Associate Laboratory Institute for Health and Bioeconomy–i4HB (project LA/P/0140/2020) which are financed by National Funds from FCT/MCTES. Publisher Copyright: © 2022The Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) is an integral membrane protein involved in cellular communications, in the stimulation of cell proliferation by increasing Reactive Oxygen Species levels, and in the transmembrane-electron transport and reduction of extracellular metal-ion complexes. The STEAP1 is particularly over-expressed in prostate cancer, in contrast with non-tumoral tissues and vital organs, contributing to tumor progression and aggressiveness. However, the current understanding of STEAP1 lacks experimental data on the respective molecular mechanisms, structural determinants, and chemical modifications. This scenario highlights the relevance of exploring the biosynthesis of STEAP1 and its purification for further bio-interaction and structural characterization studies. In this work, recombinant hexahistidine-tagged human STEAP1 (rhSTEAP1-His6) was expressed in Komagataella pastoris (K. pastoris) mini-bioreactor methanol-induced cultures and successfully solubilized with Nonidet P-40 (NP-40) and n-Decyl-β-D-Maltopyranoside (DM) detergents. The fraction capacity of Phenyl-, Butyl-, and Octyl-Sepharose hydrophobic matrices were evaluated by manipulating the ionic strength of binding and elution steps. Alternatively, immobilized metal affinity chromatography packed with nickel or cobalt were also studied in the isolation of rhSTEAP1-His6 from lysate extracts. Overall, the Phenyl-Sepharose and Nickel-based resins provided the desired selectivity for rhSTEAP1-His6 capture from NP-40 and DM detergent-solubilized K. pastoris extracts, respectively. After a polishing step using the anion-exchanger Q-Sepharose, a highly pure, fully solubilized, and immunoreactive 35 kDa rhSTEAP1-His6 fraction was obtained. Altogether, the established reproducible strategy for the purification of rhSTEAP1-His6 paves the way to gather additional insights on structural, thermal, and environmental stability characterization significantly contributing for the elucidation of the functional role and oncogenic behavior of the STEAP1 in prostate cancer microenvironment.publishersversionpublishe

Physiological and thermographic response to heat stress in zebu cattle

AbstractThe objective of this study was to evaluate the heat tolerance of five zebu breeds using physical, physiological and hematological traits as well as thermographic responses. Forty cows of the Gir, Girolando, Nelore, Sindhi and Indubrasil breeds (eight cows each), approximately three years of age, were evaluated. Body weight, withers and hump heights as well as thoracic circumference were recorded. The density and length of the hair was obtained by collecting one square centimeter in the rump region and skin color using the CIELAB system. Rectal temperature, heart and respiratory rates were evaluated during the morning at 4:30h, and in the afternoon, at 14:30h, with six repetitions. Blood samples were collected for hematological evaluation. The surface temperature was obtained using an infrared camera FLIR® T400. Two images were taken from each animal, one laterally of the whole body and the other of the head region. Air temperature, wind speed, relative humidity were obtained from a mobile weather station. The statistics analysis included an analyzes of variance, principal factors, as well as cluster, discriminant and canonical analyzes, logistic regression and calculation of odds ratio. There were significant differences in the rectal temperature, heart and respiratory rates between breeds. Gir and Indubrasil breeds had the highest rectal temperatures. Breed was significant for surface temperatures and showed that physical and physiological factors affected breeds in different ways. Eye and brain surface temperatures were the most affected by environmental parameters. Also, environmental parameters affected packed cell volume and red cell number. Odds ratio test showed that the Gir breed was three times more likely to have higher rectal temperature compared with Sindhi as confirmed by the logistic regression. When the black globe temperature approached 35°C, the probability of the Gir animals having rectal temperatures above normal was approximately 70%. Gir was the breed least adapted to climate conditions of the experiment while the Sindhi and Girolando breeds showed the best physiological response to thermal stress

COMPACTAÇÃO DE SOLOS EM LABORATÓRIO: EFEITO DO DIÂMETRO E DO NÚMERO DE CAMADAS DO CORPO DE PROVA

RESUMONeste trabalho, estudou-se a influência do diâmetro e do número de camadas do corpo de prova nas curvas de compactação de laboratório e na resistência mecânica de dois solos residuais de gnaisse da Zona da Mata Norte de Minas Gerais, respectivamente, de texturas argilo-areno-siltosa (Solo 1) e areno-silto-argilosa (Solo 2), com vistas à construção de estradas florestais. Além dos resultados dos ensaios de compactação, determinou-se a resistência à compressão não confinada dos solos empregando corpos de prova compactados na umidade ótima (wot) e nos teores de umidade 3% abaixo e 2% acima, considerando como referência a energia de compactação do ensaio Proctor normal e empregando corpos de prova compactados em uma, duas e três camadas, bem como nos diâmetros de 35 mm, 73 mm e 100 mm, com nove repetições. Para fins práticos de engenharia e com base em análise estatística aplicada aos parâmetros massa específica aparente seca e resistência à compressão não confinada, pode-se concluir que: (i) há diferenças significativas entre as compactações realizadas em uma e em três camadas, não ocorrendo o mesmo nas compactações realizadas em duas e em três camadas, para ambos os solos; e (ii) há diferenças significativas entre a compactação de corpos de prova de diâmetro 100 mm e os demais de 73 mm e 35 mm, para o solo 1 (argiloso), bem como há também diferenças apenas no ramo seco da curva de compactação, para o solo 2 (arenoso)

Promising prospective effects of Withania somnifera on broiler performance and carcass characteristics: A comprehensive review

Poultry production contributes markedly to bridging the global food gap. Many nations have limited the use of antibiotics as growth promoters due to increasing bacterial antibiotic tolerance/resistance, as well as the presence of antibiotic residues in edible tissues of the birds. Consequently, the world is turning to use natural alternatives to improve birds' productivity and immunity. Withania somnifera, commonly known as ashwagandha or winter cherry, is abundant in many countries of the world and is considered a potent medicinal herb because of its distinct chemical, medicinal, biological, and physiological properties. This plant exhibits antioxidant, cardioprotective, immunomodulatory, anti-aging, neuroprotective, antidiabetic, antimicrobial, antistress, antitumor, hepatoprotective, and growth-promoting activities. In poultry, dietary inclusion of W. somnifera revealed promising results in improving feed intake, body weight gain, feed efficiency, and feed conversion ratio, as well as reducing mortality, increasing livability, increasing disease resistance, reducing stress impacts, and maintaining health of the birds. This review sheds light on the distribution, chemical structure, and biological effects of W. somnifera and its impacts on poultry productivity, livability, carcass characteristics, meat quality, blood parameters, immune response, and economic efficiency

The role of alveolar type II cells in swine leptospirosis

Abstract: This study aimed to investigate a possible relationship between alveolar type II cells and the inflammatory response to infection with Leptospira spp., and thus comprise a further element that can be involved in the pathogenesis of lung injury in naturally infected pigs. The study group consisted of 73 adult pigs that were extensively reared and slaughtered in Teresina, Piauí state, and Timon, Maranhão state, Brazil. The diagnosis of leptospirosis was made using the microscopic agglutination test (MAT) aided by immunohistochemistry and polymerase chain reaction. The MAT registered the occurrence of anti-Leptospira antibodies in 10.96% (8/73) of the pigs. Immunohistochemistry allowed for the visualization of the Leptospira spp. antigen in the lungs of 87.67% (64/73) of the pigs. There was hyperplasia of bronchus-associated lymphoid tissue and circulatory changes, such as congestion of alveolar septa, parenchymal hemorrhage and edema within the alveoli. Lung inflammation was more intense (p = 0.0312) in infected animals, which also showed increased thickening of the alveolar septa (p = 0.0006). Evaluation of alveolar type II (ATII) cells using an anti-TTF-1 (Thyroid Transcription Factor-1) antibody showed that there were more immunostained cells in the non-infected pigs (53.8%) than in the infected animals (46.2%) and that there was an inverse correlation between TTF-1 positive cells and the inflammatory infiltrate. There was no amplification of Leptospira DNA in the lung samples, but leptospiral DNA amplification was observed in the kidneys. The results of this study showed that a relationship exists between a decrease in alveolar type II cells and a leptospire infection. Thus, this work points to the importance of studying the ATII cells as a potential marker of the level of lung innate immune response during leptospirosis in pigs