DoE to improve supercoiled p53-pDNA purification by O-phospho-L-tyrosine chromatography

P53 is implicated in various cellular functions and several studies have shown that transfection of cancer cells with wild-type p53-expressing plasmids could directly drive cells into growth arrest and/or apoptosis. In the present work, the 6.07 kbp pcDNA3-FLAG-p53 plasmid, which encodes the p53 tumor suppressor, was produced and recovered from a recombinant cell culture of Escherichia coli DH5α. Following plasmid biosynthesis, the O phospho-L-tyrosine chromatographic matrix was explored to purify the supercoiled p53-encoding plasmid. In order to quickly determine the optimal chromatographic performance and to obtain the required purity degree, maximizing the recovery yield of the supercoiled plasmid DNA, the Composite Central Face design was applied. The model revealed to be statistically significant (p-value < 0.05), with coefficient of determination of 0.9434 for the recovery yield and 0.9581 for purity and the central point was successfully validated. After the chro matographic process optimization by using the design of experiments tool, 49.7% of the supercoiled p53-en coding plasmid was recovered with 98.2% of purity, when a decreasing ammonium sulphate gradient was ap plied. The dynamic binding capacity of the O-phospho-L-tyrosine agarose column was 0.35 ± 0.02 mg pDNA/ mL matrix at 50% of the breakthrough. Finally, the purified sample was analysed to assess the content of en dotoxins, proteins and genomic DNA, showing that all these impurity levels were below the recommendations of the regulatory agenciesinfo:eu-repo/semantics/publishedVersio

Effect of Chromatographic Conditions on Supercoiled Plasmid DNA Stability and Bioactivity

Funding: This work was supported by FEDER funds through the POCI—COMPETE 2020 Operational Programme Competitiveness and Internationalization in Axis I—Strengthening research, technological development, and innovation (Project POCI-01-0145-FEDER-007491), and National Funds by FCT Foundation for Science and Technology (Project UID/Multi /00709/2013). This work was also developed within the scope of the project CICECO-Aveiro Institute of Materials, FCT Ref. UID/CTM/50011/2019, financed by national funds through the FCT/MCTES. G.M. Azevedo acknowledges the support and fellowship of Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/203482/2014-0). J.F.A. Valente acknowledges the PhD fellowship (Ref SFRH/BD/96809/2013) from FCT.Acknowledgments: The authors would like to thank Thomas Roberts for providing the pcDNA3–FLAG–p53 construct through Addgene, ref: 10838.The dysfunction of the tumor suppressor gene TP53 has been associated with the pathogenesis of the majority of the cases of cancer reported to date, leading the cell to acquire different features known as the cancer hallmarks. In normal situations, the protein p53 protects the cells against tumorigenesis. By detecting metabolic stress or DNA damage in response to stress, p53 can lead the cell to senescence, autophagy, cell cycle arrest, DNA repair, and apoptosis. Thus, in the case of p53 mutations, it is reasonable to assume that the reestablishment of its function, may restrain the proliferation of cancer cells. The concept of cancer gene therapy can be based on this assumption, and suitable biotechnological approaches must be explored to assure the preparation of gene-based biopharmaceuticals. Although numerous procedures have already been established to purify supercoiled plasmid DNA (sc pDNA), the therapeutic application is highly dependent on the biopharmaceutical’s activity, which can be affected by the chromatographic conditions used. Thus, the present work aims at comparing quality and in vitro activity of the supercoiled (sc) isoform of the p53 encoding plasmid purified by three different amino acids-based chromatographic strategies, involving histidine–agarose, arginine–macroporous, and histidine–monolith supports. The B-DNA topology was maintained in all purified pDNA samples, but their bioactivity, related to the induction of protein p53 expression and apoptosis in cancer cells, was higher with arginine–macroporous support, followed by histidine–monolith and histidine–agarose. Despite the purity degree of 92% and recovery yield of 43% obtained with arginine–macroporous, the sc pDNA sample led to a higher expression level of the therapeutic p53 protein (58%) and, consequently, induced a slightly higher apoptotic effect (27%) compared with sc pDNA samples obtained with histidine–monolithic support (26%) and histidine–agarose support (24%). This behavior can be related to the mild chromatographic conditions used with arginine–macroporous support, which includes the use of low salt concentrations, at neutral pH and lower temperatures, when compared to the high ionic strength of ammonium sulfate and acidic pH used with histidine-based supports. These results can contribute to field of biopharmaceutical preparation, emphasizing the need to control several experimental conditions while adapting and selecting the methodologies that enable the use of milder conditions as this can have a significant impact on pDNA stability and biological activity.info:eu-repo/semantics/publishedVersio

Blood Pressure And Cardiac Autonomic Modulation At Rest, During Exercise And Recovery Time In The Young Overweight

This study aimed to assess the blood pressure (BP), cardiac autonomic modulation at rest, in physical exercise and in the recovery in untrained eutrophic (E) and overweight (O) youth. The body mass index (BMI), waist circumference (WC), systolic BP-SBP (E: 109.80 ± 10.05; O: 121.85 ± 6.98 mmHg) and diastolic BP DBP (E: 65.90 ± 7.28; O: 73.14 ± 12.22 mmHg) were higher in overweight and the heart rate recovery (%HRR) was lower as compared with E volunteers. The BMI was associated with SBP (r= 0.54), DBP (r= 0.65), load on the heart rate variability threshold-HRVT (r=-0.46), %HRR2' (r=-0.48) and %HRR 5′ (r=-0.48), and WC was associated with SBP (r= 0.54), DBP (r= 0.64) and HRR2' (r=-0.49). The %HRR was associated to SBP, DBP and HRVT. In summary, the anthropometric variables, BP and cardiac autonomic modulation in the recovery are altered in overweight youth.221273

Same-sign dileptons as a signature for heavy Majorana neutrinos in hadron-hadron collisions

We discuss the possibility of same-sign dileptons as a signature for Majorana neutrinos. The production mechanism is given by a single heavy neutrino production and decay proton-proton -> l^{\pm} N X -> l^{\pm} l^{\pm} X'. Cross section and distributions are presented for the LHC energies.Comment: 07 pages, LaTeX; to be published in Physics Letters

Dietary supplementation of heat-treated Gracilaria and Ulva seaweeds enhanced acute hypoxia tolerance in gilthead sea bream (Sparus aurata)

Intensive aquaculture practices involve rearing fish at high densities. In these conditions, fish may be exposed to suboptimal dissolved O2 levels with an increased formation of reactive O2 species (ROS) in tissues. Seaweeds (SW) contain biologically active substances with efficient antioxidant capacities. This study evaluated the effects of dietary supplementation of heat-treated SW (5% Gracilaria vermiculophylla or 5% Ulva lactuca) on stress bioindicators in sea bream subjected to a hypoxic challenge. 168 fish (104.5 g average weight) were distributed in 24 tanks, in which eight tanks were fed one of three experimental diets for 34 days: (i) a control diet without SW supplementation, (ii) a control diet supplemented with Ulva, or (iii) a control diet with Gracilaria. Thereafter, fish from 12 tanks (n=4 tanks/dietary treatment) were subjected to 24 h hypoxia (1.3 mg O2 l-1) and subsequent recovery normoxia (8.6 mg O2 l-1). Hypoxic fish showed an increase in hematocrit values regardless of dietary treatment. Dietary modulation of the O2-carrying capacity was conspicuous during recovery, as fish fed SW supplemented diets displayed significantly higher haemoglobin concentration than fish fed the control diet. After the challenge, survival rates in both groups of fish fed SW were higher, which was consistent with a decrease in hepatic lipid peroxidation in these groups. Furthermore, the hepatic antioxidant enzyme activities were modulated differently by changes in environmental O2 condition, particularly in sea bream fed the Gracilaria diet. After being subjected to hypoxia, the gene expression of antioxidant enzymes and molecular chaperones in liver and heart were down regulated in sea bream fed SW diets. This study suggests that the antioxidant properties of heat-treated SW may have a protective role against oxidative stress. The nature of these compounds and possible mechanisms implied are currently being investigated. © 2017. Published by The Company of BiologistsThe authors are grateful to Pedro Eloi, Fernando Magalhães and Carlos Morais (ICBAS-UP) for fish maintenance. We would like to thank Kevin Tromp (WIAS-WU), Luis Pereira, Francisca Silva-Brito, and Filipa Fontinha for technical assistance during sampling and analysis (CIIMAR-UP). The authors would like to express gratitude to Rui Pereira (ALGA plus), Jorge Dias (Sparos Lda.), Jon Svendsen and Maria João Peixoto (CIIMAR-UP) for technical advic

Holographic Description of Heavy-Quark Potentials in an Inflationary Braneworld Scenario

We study heavy-quark potential in an inflationary braneworld scenario. The scenario we consider is an (Euclidean) inflating braneworld $AdS_4$ embedded in a static Euclidean $AdS_5$. Using gauge/gravity duality we obtain a confining phase depending on the ratio between the Hubble parameter $H$ in the braneworld and the brane tension $\sigma$.Comment: 16 pages. Minor modifications. To appear in JHE

Recomendações Para O Tratamento Da Crise Migranosa - Um Consenso Brasileiro

In this article, a group of experts in headache management of the Brazilian Headache Society developed through a consensus strategic measurements to treat a migraine attack in both the child and the adult. Particular emphasis was laid on the treatment of migraine in women, including at pregnancy, lactation and perimenstrual period. © 2016, Associacao Arquivos de Neuro-Psiquiatria. All rights reserved.74326227

Hydrological influence on the evolution of a subtropical mangrove ecosystem during the late Holocene from Babitonga Bay, Brazil

Mangroves are key ecosystems which respond to global changes in tropical and subtropical regions worldwide. We describe late Holocene mangroves that established close to the southernmost limit (28°S) for this type of ecosystem in South America. Our findings are based on a C dated core obtained from Babitonga Bay, Santa Catarina State, Brazil (26°12′S, 48°33′W). Analysis of palynology, sedimentary facies, isotopic and elemental data shows that mangrove establishment took place ~500 yrs. B.C.E., following an increase in humidity, and expanded further during the Roman Warm Period and at the end of Dark Age Cold Period. Mangrove and precipitation proxies records appear to be sensitive to rainfall patterns imposed both by the expansion/retraction of the Intertropical Convergence Zone and also the interaction with the South Atlantic Subtropical Anticyclone which affects coastal region due to sea surface temperature variations.The authors thank the Coastal Dynamic Laboratory (LADIC-UFPA) , C-14 Laboratory of the Center for Nuclear Energy in Agriculture (CENA-USP) , University of Joinville (UNIVILLE) and Radiocarbon Laboratory (LAC-UFF) for all infrastructure and support. We also thank three anonymous Reviewers and Prof. H. Falcon-Lang for their constructive comments. The first and third author thanks Brazilian Council for Technology and Science-CNPq for fellowship (process 131813/2016-1 , 165911/2015-8 and 305074/2017-2 ). This study was financed by CNPq ( 445111/2014-3 , 405060/2013- 0 ) and FAPESP ( 2011/00995-7 , 2017/03304-1, and 2020/13715-1 ). This study also was financed in part by the Coordenação de Aperfeiçoamento de Pessoal Nível Superior – Brazil (CAPES) – Finance Code 001