O impacto da sinalização astrocítica nos efeitos terapêuticos do secretoma de células mesenquimais estaminais no sistema nervoso central

The central nervous system (CNS) has a limited auto-regeneration capacity, which makes it challenging for the development of new therapies. Previous studies from our lab have demonstrated the applicability of mesenchymal stem cells (MSCs) as a possible therapeutic too for CNS, in which their secretome (e.g. the secretion of trophic factors and vesicles) has been described as the most probable mechanism of their therapeutic action, due to its ability to stimulate/modulate neurogenesis. Glial cells as astrocytes are important players in neural activity and in the modulation of neurotransmission, thereby being crucial elements in neurogenesis. Thus, in the present project, we aimed to evaluate the impact of astrocytes on the effects promoted by the application of MSCs secretome as a therapeutic tool for the modulation and generation of new neurons. For this, MSCs secretome was injected into the dentate gyrus (DG) of the hippocampus of a transgenic animal model (with the SNARE complex blocked) without astrocytic transmission, and therefore with their function partially depleted. Additionally, morphological features of astrocytes were assessed. As controls wild type Black 6 mice, in which the astrocytic function has not been depleted were used. One week after secretome treatment, animals were sacrificed, and brains collected for molecular and histological analysis. Pre-frontal cortex tissue was used to perform a transgene analysis in order to divide the experimental groups in wild type (Wt), Snare high and Snare low (animals presenting different patterns of gene expression). Brains were immuno-stained for GFAP and Ki-67 and assessed under a confocal microscope for proliferations levels at the SGZ of the hippocampus, at the hilus, for counting of radial glial cells at the SGZ and for GFAP+ densities at the DG. Confocal images were also employed for morphological analysis. Results demonstrated increased levels of proliferation for Wt and Snare high animals at the SGZ when compared to Snare low when treated with secretome, furthermore, secretome increased levels of proliferating radial glial cell in Snare high animals. Morphological assessments revealed increased process hypertrophy and complexity in snare animals treated with secretome. The results could be attributed to trophic factors present in the secretome, previously shown to increase proliferation at the DG and also to alter astrocyte morphology. The impact of transgene expression is harder to explain, nevertheless, impaired exocytosis from astrocytes could have implications for the response to a proliferative stimulus given the established autocrine signaling trough this mechanism.A capacidade de regenereação do sistema nervoso (SNC) central é limitada, gerando um desafio para a produção de novas terapias. Estudos recentes do nosso laboratório evidenciaram um potencial interessante para a aplicação de células estaminais mesenquimatosas (MSC) como um potencial agente terapêutico para o SNC no qual o seu secretoma (secreção de fatores tróficos e vesiculas) tem sido descrito como o principal mecanismo de ação, capaz de estimular neurogénese e a sobrevivência de células gliais. Neste contexto, astrócitos tem grande importância na homeostase do SNC modulando importantes funcões como neurotransmissão, fluxo sanguíneo e neurogénesis. Portanto, este projeto teve o objetivo de investigar o papel dos astroócitos nos efeitos modulatórios promovidos pelo secretoma de MSC’s nomeadamente nos seus níveis de proliferação e nas densidades gliais. Para isso, foi utilizado um modelo animal transgénico que possui o complexo SNARE bloqueado, reduzindo desta forma a transmissão astrocítica. Adicionalmente, a morfologia destes astrócitos foi estudada. Como controlos, foram usados murganhos C57BL/6. Uma semana após a injeção com o secretoma, procedeu-se a avaliações histológicas, os tecidos foram marcados para Ki-67 e GFAP e contramarcados para DAPI. O secretoma apresentou um papel estimulatório sob os níveis de proliferação evidenicados pelo maior número de células positivas para Ki-67 na zona sub-granular do hipocampo nos animais wild type e nos SNARE high, sendo a transmissão astrocitica importante para tal processo tendo em vista que, em comparação com animais SNARE low, os níveis de proliferação foram reduzidos, adicionalmente, o secretoma elevou a proliferação de celulas gliais radiais (RGC) apenas nos animais Snare High, mostrando um possível efeito compansatório sob a redução da transmissão glial. Adicionalmente, os animais tratados com secretoma que possuiam expressão do transgene, apresentaram morfologia hipertrófica e mais complexa quando comparados com animais wild type. Os resultados encontrados devem ser considerados com parcimónia pela natureza exploratória do presente estudo, vale a pena reasaltar que é a primeira vez que este tipo de abordagns experimentais e análises é desenvolvido neste modelo animal

New Menger-like dualities in digraphs and applications to half-integral linkages

We present new min-max relations in digraphs between the number of paths satisfying certain conditions and the order of the corresponding cuts. We define these objects in order to capture, in the context of solving the half-integral linkage problem, the essential properties needed for reaching a large bramble of congestion two (or any other constant) from the terminal set. This strategy has been used ad-hoc in several articles, usually with lengthy technical proofs, and our objective is to abstract it to make it applicable in a simpler and unified way. We provide two proofs of the min-max relations, one consisting in applying Menger's Theorem on appropriately defined auxiliary digraphs, and an alternative simpler one using matroids, however with worse polynomial running time. As an application, we manage to simplify and improve several results of Edwards et al. [ESA 2017] and of Giannopoulou et al. [SODA 2022] about finding half-integral linkages in digraphs. Concerning the former, besides being simpler, our proof provides an almost optimal bound on the strong connectivity of a digraph for it to be half-integrally feasible under the presence of a large bramble of congestion two (or equivalently, if the directed tree-width is large, which is the hard case). Concerning the latter, our proof uses brambles as rerouting objects instead of cylindrical grids, hence yielding much better bounds and being somehow independent of a particular topology. We hope that our min-max relations will find further applications as, in our opinion, they are simple, robust, and versatile to be easily applicable to different types of routing problems in digraphs

In situ and in vitro comparison of laser fluorescence with visual inspection in detecting occlusal caries lesions

The aim of this study was to compare the in situ and in vitro performances of a laser fluorescence (LF) device (DIAGNOdent 2095) with visual inspection for the detection of occlusal caries in permanent teeth. Sixty-four sites were selected, and visual inspection and LF assessments were carried out, in vitro, three times by two independent examiners, with a 1-week interval between evaluations. Afterwards, the occlusal surfaces were mounted on the palatal portion of removable acrylic orthodontic appliances and placed in six volunteers. Assessments were repeated and validated by histological analysis of the tooth sections under a stereomicroscope. For both examiners, the highest intra-examiner values were observed for the visual inspection when in vitro and in situ evaluations were compared. The inter-examiner reproducibility varied from 0.61 to 0.64, except for the in vitro assessment using LF, which presented a lower value (0.43). The methods showed high specificity at the D1 threshold (considering enamel and dentin caries as disease). In vitro evaluations showed the highest values of sensitivity for both methods when compared to the in situ evaluations at D1 and D2 (considering only dentinal caries as the disease) thresholds. For both methods, the results of sensitivity (at D1 and D2) and accuracy (at D1) showed significant differences between in vitro and in situ conditions. However, the sensitivity (at D1 and D2), specificity and accuracy (both at D1) of the methods were not significantly different when the same condition was considered. It can be concluded that visual inspection and LF showed better performance in vitro than in sit

Characterization of lower urinary tract symptoms in patients with idiopathic normal pressure hydrocephalus

AIMS: The purpose of this study was to evaluate lower urinary tract symptoms (LUTS) in idiopathic normal pressure hydrocephalus (iNPH). METHODS: Patients with new-onset iNPH were prospectively evaluated for LUTS via detailed history and physical, and administration of questionnaires from the International Consultation on Incontinence to assess incontinence (ICIq-UI), overactive bladder (ICIq-OAB), and quality of life (ICIq-LUTqol), as well as the American Urological Association Symptom Score bother scale. All patients with moderate-to-severe LUTS were offered urodynamic testing. Sub-analysis was performed based on gender, medical comorbidities, and age. RESULTS: Fifty-five consecutive patients with iNPH completed the initial evaluation and surveys. Total urinary incontinence score was mild to moderate (8.710.64: 0-21 scale) with 90.9% experiencing leakage and 74.5% reporting urge incontinence. The most common OAB symptom was nocturia (2.2 +/- 0.14: 0-4 scale) with urge incontinence the most bothersome (3.71 +/- 0.44: 0-10 scale). Quality-of-life impact was moderate (4.47 +/- 0.4: 0-10 scale) and American Urological Association Symptom Score bother scale was 2.89 +/- 0.22 (0-6 scale). Urodynamics testing revealed 100% detrusor overactivity and mean bladder capacity of 200mL. Several differences were identified based on gender, medical comorbidities, and age. CONCLUSIONS: Patients with iNPH present with mild-moderate incontinence of which nocturia is the most common symptom, urge incontinence the most bothersome, with 100% of patients having detrusor overactivity. Younger patients experienced greater bother related to LUTS. To our knowledge, this is the only prospective evaluation of urinary symptoms in patients with new-onset iNPH

Preclinical comparison of stem cells secretome and levodopa application in a 6-hydroxydopamine rat model of Parkinson’s Disease

Parkinson’s Disease (PD) is characterized by the massive loss of dopaminergic neurons, leading to the appearance of several motor impairments. Current pharmacological treatments, such as the use of levodopa, are yet unable to cure the disease. Therefore, there is a need for novel strategies, particularly those that can combine in an integrated manner neuroprotection and neuroregeneration properties. In vitro and in vivo models have recently revealed that the secretome of mesenchymal stem cells (MSCs) holds a promising potential for treating PD, given its effects on neural survival, proliferation, differentiation. In the present study, we aimed to access the impact of human bone marrow MSCs (hBM-MSCs) secretome in 6-hydroxydopamine (6-OHDA) PD model when compared to levodopa administration, by addressing animals’ motor performance, and substantia nigra (SN), and striatum (STR) histological parameters by tyrosine hydroxylase (TH) expression. Results revealed that hBM-MSCs secretome per se appears to be a modulator of the dopaminergic system, enhancing TH-positive cells expression (e.g., dopaminergic neurons) and terminals both in the SN and STR when compared to the untreated group 6-OHDA. Such finding was positively correlated with a significant amelioration of the motor outcomes of 6-OHDA PD animals (assessed by the staircase test). Thus, the present findings support hBM-MSCs secretome administration as a potential therapeutic tool in treating PD, and although we suggest candidate molecules (Trx1, SEMA7A, UCHL1, PEDF, BDNF, Clusterin, SDF-1, CypA, CypB, Cys C, VEGF, DJ-1, Gal-1, GDNF, CDH2, IL-6, HSP27, PRDX1, UBE3A, MMP-2, and GDN) and possible mechanisms of hBM-MSCs secretome-mediated effects, further detailed studies are needed to carefully and clearly define which players may be responsible for its therapeutic actions. By doing so, it will be reasonable to presume that potential treatments that can, per se, or in combination modulate or slow PD may lead to a rational design of new therapeutic or adjuvant strategies for its functional modeling and repair.This work was supported by the European Regional Development Fund (FEDER), through the Competitiveness Internationalization Operational Programme (POCI), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the projects POCI-01-0145-FEDER-029751 and POCI-01-0145-FEDER-007038. This article has also been developed under the scope of the project NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER)

ANÁLISE COMPARATIVA DE PARÂMETROS DE BALNEABILIDADE EM FAZENDINHA, MACAPÁ-AP

The recreational water quality criteriais are usefull parameters to determine the water quality of the beach when it is used for primary contact. The objective of this investigation is to verify the recreational water quality and the environmental conditions on the Fazendinha beach – Macapá/AP. The methodology included a descriptive study, with analysis of primary and secondary data of water quality considering a period of two years. Information on the monitoring were carried out surveyed by the Institute for the Environment and Land Management of the Amapá State - IMAP. The results showed concentration of E. coli and pH level suitable for recreational water quality. A theoretical study was extended to other beach in order to compare these parameters, and results showed values in non-compliance with current legislation: pH = 4.4 for Curiaú, pH = 5.82 for Perpétuo Socorro, pH = 5.89 for Santa Inês and pH = 5.5 for Ariri proximities. These differences showed the need to intensify monitoring frequency because that it is carried out just once a year. In Fazendinha District, 42% of respondents identified that the beach has been used in other months not monitored by IMAP. We conclud that is necessary to reinforces the importance of monitoring and communication by IMAP about the recreational water quality to greater the health safety to users.Keywords: Water quality; resolution CONAMA 274/2000; environmental quality.A balneabilidade é um instrumento de verificação de parâmetros que determinam se a água da praia tem qualidade para a recreação de contato primário. O objetivo da pesquisa foi verificar a balneabilidade e as condições da qualidade ambiental do balneário da Fazendinha em Macapá. A metodologia contou com uma pesquisa descritiva, com análise de dados primários e secundários de qualidade da água considerando um período de 2 anos. As informações sobre o monitoramento realizado foram pesquisadas junto ao Instituto do Meio Ambiente e de Ordenamento Territorial do Estado do Amapá – IMAP. Os resultados mostraram valores de E. coli e pH satisfatórios para o balneário da Fazendinha. O estudo foi ampliado para outros balneários no sentido de comparar esses parâmetros. Os demais balneários mostraram resultados alterados para o pH, em não conformidade com a legislação. Foram encontrados valores de pH = 4,4 no Curiaú, pH = 5,82 do Perpétuo Socorro, pH = 5,89 no Santa Inês e pH = 5,5 no Ariri. Essas alterações reforçam a necessidade de se intensificar períodos de monitoramento que é feito uma vez ao ano. Na Fazendinha, 42% dos entrevistados identificaram que o balneário é utilizado em outros meses não monitorados. O estudo reforça a importância da comunicação por parte do órgão responsável quanto à qualidade da água para maior satisfação e segurança aos usuários. Palavras-chave: Qualidade da água, resolução CONAMA 274/2000, qualidade ambiental

Microglial depletion has no impact on disease progression in a mouse model of machado–joseph disease

Machado–Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is an autosomal dominant neurodegenerative disorder (ND). While most research in NDs has been following a neuron-centric point of view, microglia are now recognized as crucial in the brain. Previous work revealed alterations that point to an increased activation state of microglia in the brain of CMVMJD135 mice, a MJD mouse model that replicates the motor symptoms and neuropathology of the human condition. Here, we investigated the extent to which microglia are actively contributing to MJD pathogenesis and symptom progression. For this, we used PLX3397 to reduce the number of microglia in the brain of CMVMJD135 mice. In addition, a set of statistical and machine learning models were further implemented to analyze the impact of PLX3397 on the morphology of the surviving microglia. Then, a battery of behavioral tests was used to evaluate the impact of microglial depletion on the motor phenotype of CMVMJD135 mice. Although PLX3397 treatment substantially reduced microglia density in the affected brain regions, it did not affect the motor deficits seen in CMVMJD135 mice. In addition to reducing the number of microglia, the treatment with PLX3397 induced morphological changes suggestive of activation in the surviving microglia, the microglia of wild-type animals becoming similar to those of CMVMJD135 animals. These results suggest that microglial cells are not key contributors for MJD progression. Furthermore, the impact of PLX3397 on microglial activation should be taken into account in the interpretation of findings of ND modification seen upon treatment with this CSF1R inhibitor.Fundação para a Ciência e a Tecnologia (FCT) (PTDC/NEUNMC/3648/2014) and COMPETE-FEDER (POCI-01-0145-FEDER-016818). It was also supported by Portuguese funds through FCT in the framework of the Project POCI-01-0145-FEDER-031987 (PTDC/MED-OUT/31987/2017). A.B.C. was supported by a doctoral fellowship from FCT (PD/BD/127828/2016). B.C. was also supported by FCT (2020.03898.CEECIND). This work was funded by ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI (Portuguese Platform of Bioimaging) (PPBIPOCI-01-0145-FEDER-022122), and by National funds, through FCT—project UIDB/50026/2020 and UIDP/50026/2020

Santas Marias, rogamos por vós: igreja evangélica pernambucana e violência de gênero

O presente trabalho apresenta uma análise do perfil religioso das mulheres vítimas de violência de gênero atendidas pelos programas de proteção em vigor no âmbito da Secretaria da Mulher do estado de Pernambuco, bem como da percepção das igrejas evangélicas a respeito deste tipo de violência. Os dados foram coletados naquele órgão da administração pública, relativos aos anos de 2017 a 2019. O estudo pretende verificar ainda a importância do discurso no espaço religioso para a implementação de políticas públicas que visem combater a violência de gênero contra as mulheres, partindo do pressuposto de que a espécie de violência em questão é caracterizada por fatores bem peculiares que estão mais próximos do espaço privado, no qual as estruturas eclesiásticas desempenham um papel eficiente na construção de sentidos e valores. Dessa forma, acreditamos que o Estado precisa enxergar o campo religioso, em especial as igrejas evangélicas, como um lugar de execução de políticas públicas de prevenção e até mesmo de intervenção contra a violência de gênero contra as mulheres

A infusão de cascas de romã é efetiva na desinfecção de escovas dentais?

Introdução: Os métodos de descontaminação ou desinfecção de escovas dentais têm sido questionados. Objetivo: Este estudo avaliou a eficácia da infusão de cascas de romã como um desinfetante de escovas dentais contra Streptococcus mutans. Material e método: Uma amostra de 16 escolares com idade entre 7 e 9 anos realizou escovação dentária cuidadosa, uma vez ao dia por 5 dias/semana durante 4 semanas. Após cada dia de escovação, as escovas foram lavadas e pulverizadas com uma solução desinfetante. Este procedimento foi repetido por 4 semanas utilizando uma das diferentes soluções por semana: água destilada (G1; grupo controle), infusão de casca de romã (Punica granatum Linn) (G2), hipoclorito de sódio a 1% (G3) e digluconato de clorexidina a 0,12% (G4). Após o quinto dia, as escovas foram coletadas para análise laboratorial. As cabeças das escovas foram agitadas em solução salina diluída em 10–1, 10–2,10–3, e 25μL de cada diluição foi semeada em meio de cultura agar mitis salivarius para contagem de unidade formadora de colônias (UFC) de S. mutans. Um examinador calibrado (Kappa = 0,91) realizou a contagem de UFC mL–1 × 104 . Os testes de Kruskal-Wallis e de Comparações Múltiplas de Dunn foram usados em um nível de significância de 5%. Resultado: G1 apresentou o maior número de UFC (3,9 ± 8,4), seguido de G2 (3,2 ± 4,0). Não foi observado crescimento de S. mutans em G3 e G4. Não houve diferença estatisticamente significante entre G1 e G2 e entre G3 e G4 (p>0,05). Conclusão: A infusão de romã foi completamente ineficaz para a desinfecção de escovas dentais contra S. mutans quando comparada às soluções de hipoclorito de sódio a 1% e digluconato de clorexidina a 0,12%.Introduction: Methods of decontamination or sanitization of toothbrushes have been questioned. Objective: This study assessed the effectiveness of pomegranate peels infusion as a disinfectant of toothbrushes against Streptococcus mutans. Material and method: A sample of 16 schoolchildren aged between 7 and 9 years performed brushing 5 days/week, with a careful brushing once a day. After each day of brushing, the toothbrushes were washed and sprayed with one disinfectant solution. This procedure was repeated for 4 weeks using one of the different solutions per week: distilled water (G1; negative control), pomegranate (Punica granatum Linn) peels infusion (G2), 1% sodium hypochlorite (G3) and 0.12% chlorhexidine digluconate (G4). After the fifth day, toothbrushes were collected for laboratory analysis. Toothbrushes heads were subjected to agitation in saline dilution of 10–1, 10–2,10–3, and 25 μL of each dilution were seeded in mitis salivarius agar culture medium for S. mutans colony-forming unit (CFU) counting. One calibrated examiner (Kappa = 0.91) performed the CFU (mL–1 × 104 ) counts. Kruskal-Wallis and Dunn Multiple Comparison tests were used at a significance level of 5%. Result: G1 presented the highest number of CFU (3.9 ± 8.4), followed by G2 (3.2 ± 4.0). No S. mutans growth was observed in G3 and G4. There was no statistically significant difference between G1 and G2 and between G3 and G4 (p>0.05). Conclusion: Pomegranate infusion was completely ineffective for the disinfection of toothbrushes against S. mutans when compared with 1% sodium hypochlorite and 0.12% chlorhexidine digluconate solution