New insights into the pathogenicity of Listeria monocytogenes clonal complexes

Listeria monocytogenes is a foodborne pathogen that causes listeriosis in humans. This pathogen mainly affects specific vulnerable groups such as the elderly, newborns, pregnant women, and immunocompromised individuals. The severity of the illness varies based on both intrinsic factors of the affected individuals and the pathogen itself. Therefore, different clinical outcomes can be expected, ranging from simple gastroenteritis to central nervous system, maternal-neonatal, and bacteremia infections. Furthermore, L. monocytogenes is genetically and phenotypically diverse species, resulting in a high variation in stress response and virulence potential among strains. Multilocus sequence typing (MLST) has been widely adopted as a reference method to categorize the clonal structure of bacterial species and to define clonal complexes (CCs) of genetically related isolates, i.e., derived from the same ancestor. In L. monocytogenes, the combination of MLST and epidemiological data allows to distinguish hypervirulent CCs, which are notably more prevalent in clinical cases and typically associated with severe forms of the disease. Contrarily, other CCs, denominated hypovirulent are predominantly isolated from food and food processing environments, and they are linked with the occurrence of listeriosis in highly immunosuppressed individuals. The aim of the present work was to assess and compare the virulence potential of L. monocytogenes strains belonging to both hypervirulent (CC1, CC2, CC4, CC6, CC87 and CC388) and hypovirulent CCs (CC9 and CC121). Sixteen isolates collected from clinical cases occurred in Portugal, distributed among eight distinct CCs, were selected and characterized in terms of: i) in vitro invasion efficiency in Caco-2 cells; ii) presence of premature stop codon (PMSC) mutations in the inlA gene; iii) in vivo infection in Galleria mellonella larvae and health index score characterization, iv) hemolytic activity; and v) ability to survive to the acid barrier of the stomach (pH 2.5 and 3). Five strains were further characterized in terms of immunomodulatory effect in infected G. mellonella larvae and expression of five virulence genes (inlA, inlB, actA, hly, plcA, and prfA). The results obtained demonstrated that there is a clear-cut distinction in the invasion efficiencies in Caco-2 between strains belonging to hyper- and hypovirulent CCs, with CC121 and CC9 isolates showing impaired invasion ability and PMSCs in inlA. Although this CC-related evidence was found in in vitro assays, isolates from CC9 showed an augmented virulent phenotype upon infection of G. mellonella larvae, while some isolates from hypervirulent CCs (CC2 and CC6) yielded reduced larvae mortality rates. Additionally, intra-clonal complex differences were observed among CC6 strains. Comparison of haemolytic activity among CCs, performed using classical blood agar plates and a microplate assay, showed that a CC121 strain presented higher ability to lyse red blood cells. This observation was validated by higher expression levels of hly (the gene encoding the protein listeriolysin O, LLO); this strain also exhibited increased expression of the virulence genes inlA and actA. No significant differences were found between the CCs concerning survival at low pH. In conclusion, the present study emphasizes the importance of incorporating both genotypic and phenotypic data when characterizing the virulence potential of L. monocytogenes. However, further research, such as genome analysis, may shed some light and explain some of the reported phenotypes. Moreover, as the zero-risk infection with L. monocytogenes is still not yet achievable, reliable biomarkers for risk assessment in the food industry need to be identified.Listeria monocytogenes é um agente patogénico de origem alimentar que causa listeriose humana. Este agente patogénico afeta principalmente idosos, recém-nascidos, grávidas e indivíduos imunocomprometidos, com diferentes graus de gravidade, dependendo de fatores intrínsecos dos indivíduos e do próprio agente patogénico. Por conseguinte, são esperados diferentes quadros clínicos, desde uma simples gastroenterite a infeções do sistema nervoso central, materno-neonatais e bacteriémicas. Além disso, L. monocytogenes é uma espécie diversa, tanto do ponto de vista genético como fenotípico, o que resulta numa elevada variação da resposta ao stress e do potencial de virulência entre estirpes. O Multilocus sequence typing (MLST) tem sido amplamente usado como método de referência para categorizar a estrutura clonal das espécies bacterianas e definir complexos clonais (CCs) de isolados geneticamente relacionados, ou seja, descendentes de um mesmo antepassado. Nesta espécie, a combinação de MLST com dados epidemiológicos permitiu distinguir CCs hipervirulentos que são notavelmente mais prevalentes em casos clínicos e associados a casos graves de listeriose. Contrariamente, outros CCs, denominados hipovirulentos, são predominantemente isolados de alimentos e de ambientes de processamento alimentar e associados à ocorrência de listeriose em indivíduos altamente imunocomprometidos. O objetivo do presente trabalho foi avaliar e comparar o potencial de virulência de estirpes de L. monocytogenes pertencentes a CCs hiper- (CC1, CC2, CC4, CC6, CC87 e CC388) e hipovirulentos (CC9 e CC12). Dezasseis isolados recolhidos de casos clínicos ocorridos em Portugal, distribuídas por oito CCs distintos, foram selecionados e caracterizados em termos de: i) eficiência de invasão in vitro em células Caco-2; ii) presença de mutações no códão stop-prematuro (PMSC) no gene inlA; iii) infeção in vivo em larvas de Galleria mellonella e caraterização do seu índice saúde, iv) atividade hemolítica; e, v) sobrevivência à barreira ácida do estômago (pH 2,5 e 3). Foram ainda caracterizadas cinco estirpes em termos do seu efeito imunomodulador em larvas de G. mellonella infetadas e a nível da expressão de cinco genes de virulência (inlA, inlB, actA, hly, plcA e prfA). Os resultados obtidos demonstraram que existe uma distinção clara nas eficiências de invasão em células Caco-2 entre estirpes pertencentes a CCs hiper e hipovirulentos, com os isolados do CC121 e CC9 a apresentarem uma baixa capacidade de invasão e PMSCs no gene inlA. Embora esta evidência relacionada com o CC tenha sido encontrada em ensaios in vitro, os isolados do CC9 apresentaram um fenótipo hipervirulento após a infeção de larvas de G. mellonella, enquanto os isolados de CCs hipervirulentos (CC2 e CC6) causaram taxas de mortalidade das larvas reduzidas. Além disso, observaram-se diferenças intra-complexo clonal entre as estirpes do CC6. A comparação da atividade hemolítica entre CCs, realizada utilizando placas de ágar-sangue e um ensaio em microplacas, mostrou que uma estirpe do CC121 apresentava uma maior capacidade de hemólise de glóbulos vermelhos. Esta observação foi validada por níveis de expressão mais elevados do gene hly (que codifica a proteína listeriolisina O, LLO); esta estirpe também apresentou uma expressão aumentada dos genes de virulência inlA e actA. Não foram encontradas diferenças significativas na sobrevivência a pH baixo entre CCs. Em conclusão, o presente estudo sublinhou a importância de incorporar dados genotípicos e fenotípicos na caraterização do potencial de virulência de L. monocytogenes. No entanto, são necessários mais estudos, por exemplo na análise do genoma, para gerar novos conhecimentos e explicar alguns dos fenótipos registados. Além disso, não existindo risco zero de infeção por L. monocytogenes, é necessário identificar biomarcadores fiáveis a utilizar na avaliação de risco na indústria alimentar

Effects of bihemispheric motor cortex transcranial direct current stimulation (tDCS) on dual-task motor performance in chronic stroke: case study

Stroke is the leading cause of acquired adult disability. It can cause multiple impairments and affect multiple domains. These deficits are usually more evident and impairing when performing dual tasks. Dual task is defined as the performance of two or more concurrent tasks simultaneously that require motor and cognitive skills. Recently, tDCS has acquired a growing interest on stroke rehabilitation given its potential to modulate cortical excitability. Evaluate the effects of 9 bihemispheric tDCS sessions over the primary motor cortex in individuals with chronic stroke on dual-task motor performance. In this study case, subject participated in 9 sessions of 20 minutes of bihemispheric tDCS, followed by his traditional rehabilitation activities. Participant was submitted to 4 evaluation moments. The following assessment tools were used: sociodemographic and clinical questionnaire, Mini Mental State Examination, Fugl-Meyer Assessment of Motor Recovery after Stroke, Verbal Fluency Test, Digit Span, Jamar Hydraulic Hand Dynamometer, Timed Up and Go (TUG), Functional Reach Test (FRT), Box and Block Test (BBT) e Stroke Upper Limb Capacity Scale (SULCS). When compared baseline and final assessment results, the participant showed better results under dual task in the BBT, with an improvement of 100% of blocks transferred with the paretic hand and 125% in the non-paretic hand. In TUG, the participant showed better results under dual task, reducing the time to complete the task in 28,69%. No FRT, results improved in single task, 71,41%, with slight changes in dual task. In the Jamar Hydraulic Hand Dynamometer, a slight reduction was registered in both single and dual task. This reduction was smaller under dual task, -0,42%. Results show that combining bihemispheric tDCS with conventional rehabilitation techniques may have a positive effect in dual-task motor performance

Dynamics of Simultaneous Arboviral Infection in Host and Vector Cells

In this study, simultaneous infection dynamics were investigated in mammalian and insect cells. Mayaro virus (MAYV; genus Alphavirus), West Nile virus (WNV; genus Flavivirus), and Zika virus (ZIKV; genus Flavivirus) were the arboviruses used in the pairings. Arboviruses are transmitted by arthropods, and Aedes sp. mosquitoes are vectors of multiple viruses, including all three used in this study. In addition to sharing some of the same vectors, MAYV, WNV, and ZIKV can all infect humans. To understand whether the presence of another virus affected viral infection dynamics, mammalian-derived Vero and insect-derived C6/36 cells were infected with either MAYV and ZIKV or WNV and ZIKV. In nature, a simultaneous infection can occur through either coinfection or superinfection, and both conditions were studied here. Infection dynamics were assessed by quantifying viral RNA production for 6 days following infection, using RT-qPCR. Simultaneous MAYV + ZIKV and ZIKV + WNV infections impacted infection dynamics. Interestingly, the outcomes seen in simultaneous infections of Vero cells were similar for both pairings. Viral accommodation was seen in coinfection conditions, as no change was seen in RNA levels for either virus in the pairing. The order in which the viruses infected the cells changed the outcome in superinfection conditions in Vero cells – viral accommodation was observed when ZIKV was the first to infect the cells, while superinfection exclusion was seen when ZIKV tried to infect Vero cells already infected with either MAYV or WNV. In C6/36, replication differed between the virus pairings studied but did not differ between coinfection and superinfections. Viral interference was observed for conditions where MAYV and ZIKV simultaneously infected C6/36 cells, while virus accommodation was at play when the virus pairing consisted of WNV and ZIKV

Physiotherapy case reports on three people with progressive supranuclear palsy

Introduction: Progressive supranuclear palsy (PSP) is a neurodegenerative brain disease that affects patient’s functionality and quality of life. Physiotherapy should be recommended at the time of diagnosis to slow the progression of disability and enhance the quality of life of these patients. Clinical presentation: Here, we describe three cases of patients with PSP, outlining their motor and non-motor symptoms and examining their clinical progression with physiotherapy intervention. During the initial intervention years, a reduction in the number of falls was achieved, along with improvements in gait and balance. Conclusion: Exercise and physiotherapy appear to be beneficial for patients with PSP by enhancing their functionality and quality of life. Controlling or reducing the number of falls should be the primary goal of any intervention for patients with PSP.info:eu-repo/semantics/publishedVersio

Evaluation of miR-124 expression in the human hippocampus with focus on Alzheimer’s Disease

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2019A Doença de Alzheimer (DA) é uma doença neurodegenerativa progressiva cujos mecanismos patológicos ainda não estão totalmente estabelecidos. A doença é caraterizada pela deposição de placas de Beta Amilóide (Aß) e tangles de proteína Tau hiperfosforilada, que geram ativação de microglia e neurodegeneração. Uma das áreas mais afetadas é o hipocampo, uma região relacionada com a memória e cognição, levando à demência e perda de memória, sintomas característicos da DA. Na DA registam-se níveis alterados de microRNA-124 (miR-124), tal como em outras doenças neurodegenerativas. Apesar de o miR- 124 ser considerado um microRNA (miRNA) principalmente neuronal, encontra-se também presente em microglia e astrócitos. Contudo, a sua expressão regional em células gliais nunca foi descrita em amostras humanas de DA. Além disso, ainda há muito para ser compreendido acerca do seu papel na DA, existindo relatos controversos no que respeita à alteração dos seus níveis ou do seu efeito na microglia. Neste contexto, estudámos a distribuição de miR- 124 em amostras de hipocampo humano provenientes de autópsias de DA, através de In Situ Hybridization (ISH). A ISH é uma técnica histológica que permite a deteção de ácidos nucleicos em tecidos, através do uso de uma sonda complementar. Neste trabalho, descrevemos em detalhe o protocolo de ISH e discutimos as suas principais vantagens e desvantagens, evidenciando a sua aplicação em amostras de hipocampo, provenientes de doentes com DA e controlos saudáveis. Especificamente, mostramos co-marcações para marcadores celulares e respetiva análise semi-quantitativa. Os resultados elucidam a presença de miR-124 em células microgliais HLA-DR/DP/DQ-positivas à volta de placas Aß num doente com DA num estadio avançado da doença. É visível co-localização em toda a matéria branca e no dentate gyrus, quer na DA como no controlo. Na DA, observámos a sua presença em astrócitos GFAP-positivos, onde parece ter uma localização nuclear preferencial. A ISH apresenta a característica singular de permitir o estudo e semi- quantificação da distribuição celular de um miRNA, sendo uma ferramenta valiosa que pode ser aplicada para elucidar a função de miRNAs na desregulação da comunicação célula-a- célula em contextos patológicos e na DA.Alzheimer’s Disease (AD) is a progressive neurodegenerative disorder whose pathological mechanisms are still not fully elucidated. The disease is characterized by the deposition of Amyloid Beta (Aß) and hyperphosphorylated Tau protein tangles, leading to microglia activation and ultimately neurodegeneration. One of the most affected areas is the hippocampus, an area related to memory and cognition, leading to dementia and memory loss which are characteristic AD symptoms. Abnormal microRNA-124 (miR-124) levels are observed in AD, as well as in other neurodegenerative disorders. Although miR-124 is regarded as a neuron-enriched microRNA (miRNA), it has been found in microglia and astrocytes. However, its regional expression in glial cells has never been identified in human AD samples. Moreover, much remains to be understood about its role in AD, with controversial reports about it being up or down regulated and scarce information on its effects on microglia. In this context, we studied the distribution of miR-124 in the human hippocampus of AD autopsy samples, by In Situ Hybridization (ISH). ISH is a histological technique that allows the detection of nucleic acids in whole tissues by use of a complementary probe. In this work, we fully describe the ISH protocol, discuss its main advantages and disadvantages and give data examples from its application on human AD and control hippocampal samples. Specifically, we show co-stainings for cell-specific markers and semi-quantitative analysis of co-staining intensity. Results elucidate the miR-124 presence in HLA-DR/DP/DQ-positive microglial cells surrounding Aß plaques in an AD patient at a late disease stage. Co-localization throughout white matter and dentate gyrus was visible in both patient and control samples. In AD, we observed its presence in GFAP-positive astrocytes, where it seems to have a strong nuclear localization. This technique has the unique characteristics of allowing the study of cell-type distribution and semi-quantification, while preserving morphological aspects, which is essential in studying complex cell-to-cell communication in pathological contexts. Thus, ISH is a valuable tool that can be used to further elucidate the role of miRNAs in AD and in cell-to-cell communication deregulation.Academisch Medisch Centrum - Universiteit van Amsterda

Activity schedule and foraging in Protopolybia sedula (Hymenoptera, Vespidae)

Protopolybia sedula is a social swarming wasp, widely spread throughout many countries in the Americas, including most of Brazil. Despite its distribution, studies of its behavioral ecology are scarce. This study aimed to describe its foraging activity and relation to climatic variables in the city of Juiz de Fora in southeastern Brazil. Three colonies were under observation between 07:00 and 18:00 during April 2012, January 2013, and March 2013. Every 30 minutes, the number of foragers leaving and returning to the colony was registered along with air temperature and relative humidity. Activity began around 07:30¸ increased between 10:30 and 14:30, and ended around 18:30. A mean of 52.7 exits and 54 returns were measured every 30 minutes. The daily mean values were 1,107 ± 510.6 exits and 1,135 ± 854.8 returns. Only one colony showed a significant correlation between forager exits and temperature (rs = 0.8055; P \u3c 0.0001) and between exits and relative humidity (rs = -0.7441; P = 0.0001). This paper shows that climatic variables are likely to have little control on the foraging rhythm of P. sedula when compared to other species, suggesting the interaction of other external and internal factors as stimuli of species foraging behavio

Neanderthal Diets in Portugal: Small and Large Prey Consumption during the Marine Isotope Stage 5 (MIS-5)

Gruta da Figueira Brava and Gruta da Oliveira are two key-sites within the Middle Palaeolithic research in the Iberian Peninsula. They are located in Central Portugal, the former occupying a coastal position, whereas the latter is about 60 km inland. They were occupied during the MIS-5 and the retrieval of two important faunal collections are now vital to the reconstruction of the palaeoeconomic activities of the Last Interglacial Neanderthals, as well as to understanding their mobility patterns within the landscape. Both caves were within resource-rich landscapes with permanent water sources nearby. Gruta da Figueira Brava also profited from its proximity to the coast with access to an ecotonal environment. This results in the formation of faunal assemblages proliferous in ungulate remains, leporids, birds, tortoises, molluscs and crabs. After detailed taphonomical analyses, it was possible to ascertain that all faunal remains resulted from human activities with some contributions from other agents of accumulation. Neanderthals brought in complete carcasses of small prey, deer and ibex, whereas only the nutrient-rich parts of larger animals were brought home for further processing and consumption. All prey sizes were being evenly targeted, with systematic use of shellfish resources that led to the formation of deposits in Gruta da Figueira Brava comparable to those from nearby Mesolithic sites. Biometric analyses of limpets and tortoises hint at the systematic use and overexploitation of such resources. Quick moving small prey were targeted, with leporids and birds being used for food and maybe for pelts and feathers. The wide range of species exploited demonstrates that Neanderthals had consistent broad spectrum diets, which had implications on the type of site use, with a tendency for year-round occupations, which could have promoted the development of larger Neanderthal groups, and the consequent formation of more complex, more stratified and more organised social structures

Relationship between Polymer Dielectric Constant and Percolation Threshold in Conductive Poly(styrene)-Type Polymer and Carbon Black Composites

Se presenta un criterio fenomenológico para determinar el umbral de percolación en compuestos poliméricos polímero - negro de carbono, en términos de la constante dieléctrica del polímeroSIE

Physiological responses of a halophytic shrub to salt stress by Na2SO4 and NaCl: oxidative damage and the role of polyphenols in antioxidant protection

Salt stress conditions lead to increased production of reactive oxygen species (ROS) in plant cells. Halophytes have the ability to reduce these toxic ROS by means of a powerful antioxidant system that includes enzymatic and non-enzymatic components. In this research, we used the halophytic shrub Prosopis strombulifera to investigate whether the ability of this species to grow under increasing salt concentrations and mixtures was related to the synthesis of polyphenolic compounds and to the maintenance of leaf pigment contents for an adequate photosynthetic activity. Seedlings of P. strombulifera were grown hydroponically in Hoagland's solution, gradually adding Na2SO4 and NaCl separately or in mixtures until reaching final osmotic potentials of −1, −1.9 and −2.6 MPa. Control plants were allowed to develop in Hoagland's solution without salt. Oxidative damage in tissues was determined by H2O2 and malondialdehyde content. Leaf pigment analysis was performed by high-performance liquid chromatography with ultraviolet, and total phenols, total flavonoids, total flavan-3-ols, condensed tannins, tartaric acid esters and flavonols were spectrophotometrically assayed. Treatment with Na2SO4 increased H2O2 production and lipid peroxidation in tissues and induced a sharp increase in flavonoid compounds (mainly flavan-3-ols) and consequently in the antioxidant activity. Also, Na2SO4 treatment induced an increased carotenoid/chlorophyll ratio, which may represent a strategy to protect photosystems against photooxidation. NaCl treatment, however, did not affect H2O2 content, lipid peroxidation, pigments or polyphenols synthesis. The significant accumulation of flavonoids in tissues under Na2SO4 treatment and their powerful antioxidant activity indicates a role for these compounds in counteracting the oxidative damage induced by severe salt stress, particularly, ionic stress. We demonstrate that ionic interactions between different salts in salinized soils modify the biochemical and morpho-physiological responses of P. strombulifera plants to salinity.Fil: Reginato, Mariana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquímicas y Naturales. Cátedra de Fisiología Vegetal; ArgentinaFil: Castagna, Antonella. Università degli Studi di Pisa; ItaliaFil: Furlan, Ana Laura. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; ArgentinaFil: Castro, Stella Maris. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; ArgentinaFil: Ranieri, Annamaria. Università degli Studi di Pisa; ItaliaFil: Luna, Maria Virginia. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquímicas y Naturales. Cátedra de Fisiología Vegetal; Argentin