Role of human umbilical cord mesenchymal progenitors conditioned media in neuronal/glial cell densities, viability, and proliferation

It has been recently reported that mesenchymal progenitor/stem cells isolated from the Wharton’s Jelly (WJ) of umbilical cords (UC) ameliorate the condition of animals suffering from central nervous system (CNS)-related conditions. However, little is known on the mechanisms that regulate these actions. Therefore, the objective of the present work was to determine how the conditioned media (CM) of a population of mesenchymal progenitors present in the UC WJ, known as human umbilical cord perivascular cells (HUCPVCs), regulate processes such as cell viability, survival, and proliferation of postnatal hippocampal neurons and glial cells. For this purpose primary hippocampal and cortical cultures of neurons and glial cells, respectively, were incubated with CM from HUCPVCs. Results revealed that HUCPVCs CM increase glial cell viability and proliferation. Furthermore, it was observed that glial cell cultures exhibited higher numbers of GFAP-positive cells (astrocytes) and O4-positive cells (oligodendrocytes) when incubated with the CM. Additionally, it was also observed that the growth factors presents in the CM did not induce an increase on the microglial cells number. For hippocampal neurons similar results were obtained, as cultures exposed to HUCPVCs CM disclosed higher numbers of MAP-2–positive cells. Moreover it was also observed that the cell viability and proliferation in this primary hippocampal cell culture system was also higher, when compared to control cultures. From these results it was possible to conclude that HUCPVCs release neuroregulatory factors that have a direct impact on the densities, viability, and proliferation of glial cells and hippocampal primary cultures.Portuguese Foundation for Science and Technology through funds from POCTI and/or FEDER programs (funding to ICVS, 3B's Research Group and post doctoral fellowship to A.J. Salgado-SFRH/BPD/17595/2004); funding from Funda ao Calouste de Gulbenkian. The authors would also like to acknowledge Prof. J.E. Davies from the Institute of Biomaterials and Biomedical Engineering at the University of Toronto, Canada, for kindly providing some of the HUCPVCs lots used in the present work. This work was performed following the terms of the cooperation agreement signed between the 3B's Research Group of the University of Minho and the Hospital de Sao Marcos in Braga and approved by its ethical committee

Depression Among Portuguese Pregnant Women During Covid-19 Lockdown: A Cross Sectional Study

Introduction: Coronavirus disease 2019 was declared as a pandemic on March 2020. Research on its psychological effects is still lacking. Perinatal depression is a medical complication of pregnancy, especially in situations of stress. In this study, we aimed to investigate the presence of symptoms of depression in pregnant women during the lockdown period in Portugal. Methods: This study consisted in a cross-sectional study among Portuguese pregnant women, who completed an online self-report questionnaire between 25th April and 30th April 2020. An anonymous online questionnaire was developed to assess depression and concerns related to COVID-19. This study was approved by the IRB of Hospital Dona Estefânia and performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. Eligibility criteria included pregnant women, ≥ 18 years and living in Portugal. The primary outcome was to evaluate the presence of depressive symptoms and its association to socio-demographic characteristics and to concerns related to COVID-19. Results: A total of 1698 pregnant women were enrolled. The mean age was 31.9 years. 82.4% felt a negative impact of the pandemic in the surveillance of pregnancy and 43% felt insufficient support. 26.3% showed "possible depression" according to the EPDS. A regression analysis revealed the possibility of depression increased as the concerns about COVID increased and was lower for women with support. The possibility of depression was higher for women with psychiatric medical history. Conclusion: This study demonstrated a significant increase in clinically significant depressive symptoms in pregnant women during the lockdown. It also revealed some of the socio-demographic characteristics of women at risk for depression. If left untreated, depression tends to persist, affecting the woman and also the child. Our findings suggest that COVID-19 represents a serious challenge for this population and reinforce the urgent need for early detection and intervention on mental health issues during pregnancy, especially during the pandemic.info:eu-repo/semantics/publishedVersio

Prediction of intracellular storage polymers using quantitative image analysis in enhanced biological phosphorus removal systems

Intracellular polymers, such as polyhydroxyalkanoates (PHA) synthesized by microorganisms for energy and carbon storage, can be commercially used as biodegradable plastics in a wide range of applications. The possibility of obtaining high PHA contents (where the most common monomers found are poly-hydroxybutyrate (PHB) and poly-hydroxyvalerate (PHV)) from inexpensive inocula and raw materials emerges as a promising and commercially interesting alternative. For this purpose, mixed cultures operated under feast/famine cycles are most frequently used. PHA is an important storage polymer in the metabolism of microorganisms involved in enhanced biological phosphorus removal (EBPR) systems. It is stored by polyphosphate accumulating organisms (PAO) and glycogen accumulating organisms (GAO), as described in previous publications [1,2]. Monitoring intracellular storage polymers in bacteria is usually performed through laborious and time consuming off-line chemical analyses. Thus, there is clearly a need to develop new techniques in order to promptly monitor these processes. Image analysis techniques have the potential to be a non-invasive and rapid means of assessing the amount of different storage polymers inside microbial cells, providing the evaluation of these important biotechnological processes. The present study focuses on predicting intracellular storage polymers in EBPR systems. For that purpose, quantitative image analysis techniques were developed and partial least squares (PLS) were used to model PHA results. An EBPR fed with synthetic wastewater containing volatile fatty acids (VFAs) and orthophosphate was used. Biomass samples were collected at the end of the anaerobic and aerobic phases. Analytical PHA quantification was performed by biomass digestion and gas chromatography analysis. In the concurring image analysis methodology Nile blue was used as a fluorescence staining method for PHA granules identification. The results from image analysis allowed establishing a PHB prediction ability presenting a regression value (R2) of 0.854, a PHA prediction regression value (R2) of 0.843 and a PHV prediction regression value (R2) of 0.779. The lower prediction ability for PHV could be explained since this parameter presented only a small contribution to the overall PHA. The analysis performed based on the variable importance in the projection (VIP) established a core of three image analysis parameters (granules total area, granules total intensity and image intensity) as the most important regarding PHA, PHB and PHV prediction

Development of image analysis methodologies to quantify intracellular PHA, polyphosphate and glycogen within wastewater treatment plants

In wastewater treatment plants (WWTP), enhanced biological phosphorus removal (EBPR) processes are performed by mixed cultures containing polyphosphate (PAO) and glycogen accumulating organisms (GAO). In these processes, it is of crucial importance to monitor the intracellular metabolism, namely glycogen, polyhydroxyalkanoates (PHA) and polyphosphate (polyP) inclusions, to determine its efficiency. However, traditional monitoring, carried out through off-line chemical analyses, is laborious and time-consuming. Therefore, there is a clear need to develop new techniques to promptly quantify these intracellular polymers, with image analysis emerging as a promising tool. The use of staining methodologies with specific fluorescent dyes is widespread in EBPR research, including Nile blue for PHA and DAPI for polyP. Although rarely applied in EBPR studies, Aniline blue is a fluorescent stain that can be used for glycogen determination. Furthermore, these fluorescent stains have generally been employed for qualitative rather than quantitative analysis. Therefore, this study aim focused on developing fluorescence-based staining methodologies for glycogen, and on acquisition, processing and image analysis procedures for PHA, polyP and glycogen. Image analysis data was then correlated with traditional analytical data by multivariable statistics. Regarding the determination of the glycogen intracellular concentration, results have been promising, presenting a good correlation (R2 of 0.915) between analytical and image analysis data. The staining and image analysis procedures for the determination of the intracellular concentration of PHA and polyP are currently being optimized. This study will provide a quantitative means to assess PAO and GAO metabolic activity in-situ in WWTP, facilitating the optimisation of these processes

Desenvolvimento de metodologias de análise de imagem para quantificar PHA, polifosfatos e glicogénio intracelular em estações de tratamento de águas residuais

O processo de remoção biológica de fósforo, em estações de tratamento de águas residuais, é um processo efetuado por culturas mistas contendo organismos acumuladores de polifosfatos (PAO) e de glicogénio (GAO). No decurso deste processo os microrganismos podem formar inclusões de glicogénio, polihidroxialcanoatos (PHA) e polifosfatos (poli-P). Neste processo, é fulcral monitorizar o metabolismo intracelular para determinar a sua eficiência. Contudo, a sua monitorização, realizada através de análise químicas em diferido, é laboriosa e morosa. Deste modo, existe uma clara necessidade do desenvolvimento de métodos mais expeditos, como metodologias de análise de imagens, para a monitorização destes polímeros intracelulares. Estas técnicas foram implementadas neste estudo, encontrando-se, no caso da determinação da concentração intracelular de poli-P, em fase de desenvolvimento dos protocolos de coloração e aquisição de imagens. Para a determinação da concentração intracelular de glicogénio, foi obtida uma boa correlação inicial. Na determinação da concentração intracelular de PHA, este estudo foca-se na otimização dos protocolos de coloração e no desenvolvimento do programa de análise de imagem

Caracterização dos Hábitos de Sono na Primeira Infância numa População Clínica

Introdução: Os problemas de sono fazem parte das queixas mais referidas, nomeadamente as dificuldades para as crianças se tranquilizarem e adormecerem na hora de dormir e os despertares noturnos frequentes. O objectivo deste estudo foi caracterizar os hábitos de sono na primeira infância. Materiais e Métodos: Construção de questionário sobre os hábitos de sono na primeira infância, aplicando-o a uma população de crianças atendidas em primeira consulta de pedopsiquiatria no Centro de Estudos do Bebé e da Criança / Unidade da Primeira Infância (n=93), dos 0 aos 3 anos de idade, durante o ano de 2014. Resultados: A média de idades é de 27 meses, variando entre 1 e 36 meses. Não existem diferenças estatisticamente significativas entre sexos e idades relativamente ao ambiente em que dormem. 77% das crianças têm rotinas antes de adormecer. As crianças que têm rotinas, têm menor dificuldade em adormecer (p=0,028). 38,04% dos pais considera que existem problemas de sono. A média do número total de horas de sono entre os 3 e os 11 meses foi de 12h e entre os 12 e os 35 meses de 11h. Discussão: Concordante com a literatura, a maioria das crianças acorda durante a noite (61,3%), o número e a duração de sestas diminui ao longo da vida e os problemas de sono são frequentes (38,04%), apesar de neste estudo terem uma prevalência ligeiramente superior. A duração de sono das crianças é inferior ao recomendado. Conclusão: Verifica-se uma associação significativa entre a inexistência de rotinas e a dificuldade em adormecer e uma associação significativa entre crianças que dormem sozinhas e ausência de problemas de sono.info:eu-repo/semantics/publishedVersio

Novel method to quantify intracellular accumulation of polyphosphate in EBPR systems

A new method for intracellular storage polyphosphate (poly-P)identification and quantification in enhanced biological phosphorus removal (EBPR) systems is proposed based on image analysis. In EBPR systems, 4',6-diamidino-2-phenylindole (DAPI) is usually combined with fluororescent in situ hybridization (FISH) to evaluate the microbial community. The proposed technique is based on an image analysis procedure specifically developed for determining poly-P inclusions within biomass suspension using solely DAPI by epifluorescence microscopy. Due to contradictory literature DAPI concentrations used for poly-P detection, the present work assessed the optimal DAPI concentration for samples acquired at the end of the EBPR aerobic stage when the accumulation is performed. Digital images were then acquired and processed by means of image processing and analysis. Regarding image analysis results and considering the current operational conditions, a promising correlation could be found between average poly-P intensity values and the analytical determination, although presenting a correlation coefficient somewhat far from the ideal. The proposed methodology can be seen as a promising alternative procedure to quantify intracellular poly-P accumulation in a faster and less labor intensive way

Biopolymer monitoring using quantitative image analysis techniques

Polyhydroxyalkanoates (PHAs) are intracellular biopolymers with many applications, particularly as substitutes of polypropylene and polyethylene, due to their thermoplastic properties and biocompatible nature. Furthermore, glycogen is a polysaccharide of glucose with high importance in the metabolism of microbial communities and polyphosphate is a microbial storage compound that should be recovered in order to offset the worldwide depletion of phosphorus sources. The determination of these biopolymers by chemical analysis is a laborious task, often involving digestion processes prior to gas and high-performance liquid chromatography, which are time consuming and difficult to apply in industry. Currently, it is important to develop new, rapid and simple techniques to monitor these polymers. Image analysis is a non-invasive and rapid technique that has the potential to be used to quantify these intracellular polymers quickly, in real-time. Mesquita et al. (2013) showed that it is possible to predict the concentration of glycogen and PHAs by quantitative image analysis, using aniline blue and nile blue staining, respectively. Polyphosphate can also be predicted by this technique through DAPI staining, which is currently under development. These biopolymers are produced by several different microorganisms, and combining their quantification with fluorescence in situ hybridization (FISH) techniques for microbial identification can enable the determination of organisms that store high quantities of each biopolymer. In this work, an advanced quantitative technique is developed to perform real time monitoring of these three biopolymers in a bioreactor performing biological phosphorus removal. Image analysis of the biopolymers was combined with FISH to determine the storage level of each compound within the different microbial populations. This technique will further enable the assessment of biopolymer levels within microbial communities, which can be applied in the biopolymer production industry