Caracterização da composição do lisado plaquetário

A cultura de células in vitro utiliza diversas formulações de meios de cultura, os quais contém uma fonte de fatores de crescimento; o padrão ouro atualmente utilizado para essa finalidade é o soro fetal bovino (SFB). Entretanto, existem alternativas melhores que superam sua eficácia e limitações. O uso do suplemento lisado plaquetário (LP) humano como substituto do SFB na cultura de células-tronco mesenquimais (CTM) humanas, por exemplo, é particularmente importante pois elimina o risco de xenorreação em pacientes tratados com essas células, além de evitar a transmissão de zoonoses, como a encefalopatia espongiforme bovina. Diversas agências regulatórias como a Organização Mundial da Saúde solicitaram a não utilização de suplementos de origem animal na manipulação de produtos biológicos que têm como finalidade a terapia em humanos. Produzido a partir de bolsas de plaquetas humanas descartadas pelo banco de sangue devido ao prazo de validade, o lisado plaquetário é uma fonte alternativa de fatores de crescimento atualmente viável, sustentável e segura para cultivo celular. Ele promove crescimento acelerado de CTMs em especial, permitindo a obtenção de uma dose terapêutica em menos tempo, mantendo características celulares imunofenotípicas e diferenciativas. A análise do conteúdo desse suplemento, porém, carece de dados, já que ele é preparado in-house, com variações dependendo do protocolo utilizado, sendo muitos protegidos por patente. Sabe-se que a ativação e rompimento das plaquetas no processo de coagulação sanguínea promove a liberação de mais de trezentas moléculas intracelulares em grânulos heterogêneos para o sangue, orquestrando diversas funções fisiológicas, como regeneração tecidual e angiogênese. No Centro de Processamento Celular Avançado (CPCA) do Hospital de Clínicas de Porto Alegre é utilizada grande quantidade de LP para diversos ensaios clínicos. Este trabalho busca, portanto, caracterizar a composição desse produto por metodologias não convencionalmente descritas na literatura, ao passo em que permite futuras investigações sobre seu modo de ação. Através da dosagem de proteínas totais foi observado que o LP possui 49,57 ± 3,65 mg/mL, concentração significativamente maior que o SFB (30,54 ± 1,76 mg/mL); além disso, a análise proteômica por ontologia gênica e função proteica identificou proteínas relacionadas a proliferação, morfogênese, diferenciação, biossíntese, adesão e metabolismo celular, que estão sendo possivelmente metabolizadas ou recicladas pelas células. Ainda, foram detectadas proteínas responsáveis pela ligação e ativação dos fatores de crescimento TGF-β, HGF e IGF. Na análise de partículas por espalhamento dinâmico de luz observou-se a presença de um perfil heterogêneo e polidisperso de partículas, com dois padrões populacionais em 231 ± 96 d.nm com 61% de intensidade e em 30 ± 8 d.nm com 26% de intensidade; sendo possivelmente agregados proteicos ou distintas populações de vesículas, um importante meio de transporte de proteínas no microambiente celular. A realização de estudos como esse contribui essencialmente para a indústria biomédica, fornecendo dados para a melhor compreensão de diversos mecanismos moleculares envolvidos em terapia celular, impulsionando tecnologias na área da saúde e oferecendo alternativas melhores de tratamento para diversas patologias.In vitro cell culture make use of several culture media formulations, that contain a source of growth factors; the gold standard currently used to this aim is fetal bovine serum (FBS). However, there are better alternatives that outweigh its effectiveness and limitations. The use of human platelet lysate (PL) as a substitute for FBS in human mesenchymal stem cell (MSC) culture is particularly important because it eliminates the xenoreaction risk in patients treated with these cells, as well as avoiding the zoonosis transmission, like bovine spongiform encephalopathy. Several health regulatory agencies such as the World Health Organization requested that animal origin supplements are not to be utilized in the manipulation of biological products purposed as therapy in humans. Made from expired human platelet bags discarded by the blood bank, the platelet lysate is an alternative source of growth factors actually viable, sustainable and safe to cell culture. It promotes MSCs faster growth in particular, allowing to achieve the required therapeutic dose in a shorter period of time, maintaining the immunophenotypic and differential cellular characteristics. The analysis of this supplement content, besides, lack data, since it is prepared in-house, with variations according to the utilized protocol, many being patent protected. It is known that the platelet activation and lysis in the blood coagulation process promote the release of more than three hundred intracellular molecules contained in heterogeneous granules to the bloodstream, which coordinate many physiological functions like tissue regeneration and angiogenesis. At the Advanced Cellular Processing Center (CPCA) from the Hospital de Clínicas de Porto Alegre, a large amount of PL is utilized in several clinical trials. This work aim, therefore, to characterize this product composition by unconventional literature-described methodologies, while allowing further investigations about its mechanism of action. Through total protein quantification, it was observed that PL has 49,57 ± 3,65 mg/mL, a significant higher amount than FBS (30,54 ± 1,76 mg/mL); besides, the proteomics analysis by gene ontology and protein function identified proteins related to cellular proliferation, morphogenesis, differentiation, biosynthesis, adhesion and metabolism, which are possibly being metabolized or having its structure recycled by cells. Also, several proteins responsible for activating and binding of the growth factors TGF-β, HGF and IGF were detected. Particle analysis through dynamic light scattering showed a heterogeneous and polydisperse profile of particles, with two population patterns at 231 ± 96 d.nm with 61% of intensity and at 30 ± 8 d.nm with 26% intensity; possibly being protein aggregates or distinct vesicle populations, an important way of protein transportation through the cellular microenvironment. Making studies like this one contribute essentially to the biomedical industry, with data to better comprehend several molecular mechanisms involved in cellular therapy, pushing healthcare technologies forward and offering patients better treatment options for several pathologies

The impact of intensive high performance training on adult height of female artistic gymnasts: a retrospective study

Resumo O objetivo deste estudo foi identificar o potencial impacto do treinamento de alta intensidade sobre as características antropométricas e maturacionais de ex-atletas da ginástica artística feminina e comparar a estatura adulta com padrões internacionais de crescimento. Uma amostra composta de 23 ex-atletas de elite da ginástica artística feminina brasileira foi avaliada. A partir de uma retrospectiva histórica média de 29,56 anos, foi determinado o tempo médio de formação [9,3 (2,6) anos], horas semanais de treinamento [24,26 (4,2)] e tempo após encerramento da carreira competitiva (13,61 ± 5,12 anos). A partir dos dados antropométricos das ginastas (quando ativas), o alvo parental da estatura das ex-atletas, de suas irmãs mais velhas (1) e mais jovens (2) foi calculado pelo método de Tanner {[(Altura do pai - 13 cm) + Altura da mãe] ÷ 2}. Além disso, o histórico de treino, idade de ocorrência da menarca e classificação do crescimento segundo os padrões internacionais, foram registrados para comparações. A estatura média das ex-atletas apresentou normalidade esperada para estatura adulta (NCHS), e em alguns casos ultrapassaram os valores dos percentis 75%, 90% e 95%. As ex-atletas foram maiores do que suas mães (p = 0,039), mas não diferiram da irmã 1 (0,952) ou irmã 2 (p = 0,998). A idade da menarca daquelas foi significantemente mais tardia do que de suas irmãs 1 (p = 0,008) e 2 ( p = 0,017). Em relação às referências brasileiras (IBGE), a altura final das ex-ginastas foi sempre maior do que o padrão nacional, ligeiramente menor do que suas irmãs 2 em alguns momentos (centis 5 e 10), mas eram mais altas do que suas mães e irmãs 1. Em conclusão, não há evidências de que o treinamento de alto impacto da ginástica artística resulte em efeitos adversos sobre a estatura adulta final das atletas.Abstract The purpose of this study was to identify the potential impact of the high performance training on the anthropometric and maturational characteristics of female artistic gymnasts and to compare the adult target height with international standards of growth. A sample consisted of 23 elite Brazilian female former gymnasts was evaluated. From a historic retrospective, about 29.56 yrs. ago (average of all) they devoted themselves to the training for 9.3 (2.6) years, with a weekly 24.26 (4.2) training hours, and had been retired from competition around at 13.61 (5.12) years old. From the anthropometric data of the gymnasts (while still competing), their parents and their older sister (1) and younger sister (2), the target height was calculated by Tanner method {[(father’s height -13 cm) + mother’s height] ÷ 2}. Additionally, training history, age of menarche occurrence, and growth classifying in accordance with the international standards was recorded, aimed at comparisons. The average stature shown normal adult stature (NCHS), and some cases surpassed the standard 75th, 90th and 95th percentile. They are higher than their mother (p = 0.039), but not their sister 1 (0.952) or sister 2 (p = 0.998), but the age of menarche was significantly later only than their sisters 1 (p = 0.008) and 2 (p = 0.017). According to the Brazilian percentiles reference (IBGE), the final height of the former gymnasts was always higher of the standard, slightly smaller than their sisters 2 (5th and 10th), but was taller than their mothers and sisters 1. In conclusion, there is no evidence of artistic gymnastics high performance training having adverse effects on the final adult height

Biological saline produced water treatment, a sustainable process towards lipids production

Oil and gas industry is responsible for the generation of large volumes of oil-contaminated wastewaters, such as saline produced water (PW), that without proper treatment can cause environmental contamination. Alcanivorax borkumensis SK2 is a biosurfactant producer capable of degrading and converting hydrocarbons into bacterial lipids under nutrient limiting conditions (e.g. nitrogen and/or oxygen). Recently, the industrial interest in biosurfactants and bacterial lipids for biotechnological applications (e.g. bioremediation or biofuels production) is increasing. Pursuing the interest of treating and simultaneously valorizing PW, a sequencing batch airlift reactor (SBAR) strategy consisting of sequential cycles of feast and famine stages was developed. A. borkumensis SK2 was used as bioreactor inoculum and the effect of cycle duration, total petroleum hydrocarbon to nitrogen ratio (TPH/N) and dissolved oxygen (DO) concentration (7-8 mg L-1 and 2-3 mg L-1 (famine and feast stage); and 7-8/1-2 mg L-1 (famine/feast stage)) were investigated. The system provided an efficient PW treatment, achieving TPH removal efficiencies in a narrow range from 90 ± 2.1 to 96 ± 1.8 %. Intracellular lipid production increased from 0.48 to 0.74 g g-1 of cellular dry weight (CDW) with the application of higher feast stage duration and lower TPH/N ratios suggesting that nitrogen availability is the most relevant factor to promote accumulation. Under 2-3 mg L-1 and 7-8/1-2 mg L-1 of oxygen, lipid accumulation dropped to 0.50 g g-1 of CDW. Intracellular lipid profile changed according to the DO concentration. Triacylglycerols (TAG) and wax esters (WE) were accumulated under maximum and limiting DO concentrations, while polyhydroxyalkanoates (PHA) accumulation was triggered by the application of alternated DO conditions, indicating that the type of intracellular compound can be selected by manipulating the oxygen concentration in the SBAR. Extracellular lipid production (TAG and WE) was not significantly affected by none of the operational conditions applied (0.06 g L-1). Moreover, the production of a cell-bound and an extracellular glycolipid biosurfactant capable of reducing the medium surface tension from 65 to approximately 41 mN m-1 was observed. The proposed strategy showed that biological PW treatment in a SBAR can be a sustainable process through the production of added-value compounds contributing to a circular economy model.This research was supported by the Portuguese Foundation for Science and Technology (FCT I.P.) and European Regional Development Fund (ERDF) under the scope of project SaltOil+ (POCI-01- 0145- FEDER-030180) (Portugal 2020, COMPETE 2020); Rita M. Silva PhD grant (SFRH/BD/116154/2016) was funded by FCT.info:eu-repo/semantics/publishedVersio

Relationship between functional fitness, medication costs and mood in elderly people

Objective: to verify if functional fitness (FF) is associated with the annual cost of medication consumption and mood states (MSt) in elderly people. Methods: a cross-sectional study with 229 elderly people aged 65 years or more at Santa Casa de Misericórdia de Coimbra, Portugal. Seniors with physical and psychological limitations were excluded, as well as those using medication that limits performance on the tests. The Senior Fitness Test was used to evaluate FF, and the Profile of Mood States - Short Form to evaluate the MSt. The statistical analysis was based on Mancova, with adjustment for age, for comparison between men and women, and adjustment for sex, for comparison between cardiorespiratory fitness quintiles. The association between the variables under study was made with partial correlation, controlling for the effects of age, sex and body mass index. Results: an inverse correlation between cardiorespiratory fitness and the annual cost of medication consumption was found (p < 0.01). FF is also inversely associated with MSt (p < 0.05). Comparisons between cardiorespiratory fitness quintiles showed higher medication consumption costs in seniors with lower aerobic endurance, as well as higher deterioration in MSt (p < 0.01). Conclusion: elderly people with better FF and, specifically, better cardiorespiratory fitness present lower medication consumption costs and a more positive MSt

Nirmatrelvir/ritonavir in COVID-19 patients with haematological malignancies: a report from the EPICOVIDEHA registry

Background: Nirmatrelvir/ritonavir treatment decreases the hospitalisation rate in immunocompetent patients with COVID-19, but data on efficacy in patients with haematological malignancy are scarce. Here, we describe the outcome of nirmatrelvir/ritonavir treatment in a large cohort of the latter patients. Methods: This is a retrospective cohort study from the multicentre EPICOVIDEHA registry (NCT04733729) on patients with haematological malignancy, who were diagnosed with COVID-19 between January and September 2022. Patients receiving nirmatrelvir/ritonavir were compared to those who did not. A logistic regression was run to determine factors associated with nirmatrelvir/ritonavir administration in our sample. Mortality between treatment groups was assessed with Kaplan-Meier survival plots after matching all the patients with a propensity score. Additionally, a Cox regression was modelled to detect factors associated with mortality in patients receiving nirmatrelvir/ritonavir. Findings: A total of 1859 patients were analysed, 117 (6%) were treated with nirmatrelvir/ritonavir, 1742 (94%) were treated otherwise. Of 117 patients receiving nirmatrelvir/ritonavir, 80% had received ≥1 anti-SARS-CoV-2 vaccine dose before COVID-19 onset, 13% of which received a 2nd vaccine booster. 5% were admitted to ICU. Nirmatrelvir/ritonavir treatment was associated with the presence of extrapulmonary symptoms at COVID-19 onset, for example anosmia, fever, rhinitis, or sinusitis (aOR 2.509, 95%CI 1.448-4.347) and 2nd vaccine booster (aOR 3.624, 95%CI 1.619-8.109). Chronic pulmonary disease (aOR 0.261, 95%CI 0.093-0.732) and obesity (aOR 0.105, 95%CI 0.014-0.776) were not associated with nirmatrelvir/ritonavir use. After propensity score matching, day-30 mortality rate in patients treated with nirmatrelvir/ritonavir was 2%, significantly lower than in patients with SARS-CoV-2 directed treatment other than nirmatrelvir/ritonavir (11%, p&nbsp;=&nbsp;0.036). No factor was observed explaining the mortality difference in patients after nirmatrelvir/ritonavir administration. Interpretation: Haematological malignancy patients were more likely to receive nirmatrelvir/ritonavir when reporting extrapulmonary symptoms or 2nd vaccine booster at COVID-19 onset, as opposed to chronic pulmonary disease and obesity. The mortality rate in patients treated with nirmatrelvir/ritonavir was lower than in patients with targeted drugs other than nirmatrelvir/ritonavir. Funding: EPICOVIDEHA has received funds from Optics COMMIT (COVID-19 Unmet Medical Needs and Associated Research Extension) COVID-19 RFP program by GILEAD Science, United States (Project 2020-8223)

Interactions lake-atmosphere: The ALEX 2014 field campaign and numerical simulations

The ALqueva hydro-meteorological EXperiment, ALEX 2014, was an integrated field campaign with measurements of chemical, physical and biological parameters in water and air at different experimental sites in the region of the Alqueva reservoir, a 250 km2 man made lake, in the southeast of Portugal. The Field campaign took place from June 1 to September 30, 2014 and comprises an Intensive Observation Period (IOP) of three days (22 to 24 July). During the four months, the over water fluxes of momentum, heat and mass (H2O and CO2) were obtained with an integrated Open-Path CO2 /H2O Gas Analyser and 3D Sonic Anemometer, mounted on a floating platform, where radiative fluxes were also measured, as well as the water temperature profile. Eight near surface weather stations were operating in the area and air quality, atmospheric electrical field (Potential Gradient) and radon (222Rn) concentration were continuous monitored. Along this period, in situ measurements, water samples and biological elements were monthly collected from three fixed platforms placed in the lacustrine zone and from selected sites in the margins. During the IOP, radiosondes were launched every tree hours, allowing a characterization of the atmospheric boundary layer and its evolution. In 10 occasions Geigersondes were coupled to the radiosondes in order to obtain the atmospheric ionization profile. The boundary layer was characterized with a Ceilometer and the vertical distribution of O3 and NO2 were obtained from a Spectrometer. A GPS network of 15 GNSS stations was installed in order to map the water vapour. The sky brightnesson the nights of July 24 and 25, was measured using a Sky Quality Meter. The lake-atmosphere interactions and its impact in the boundary layer structure and in the local circulations are studied using data collected during the ALEX 2014 POI together with results from numerical simulations performed with the non-hydrostatic Meso-NH french model