Estudio del potencial patógeno de cepas de Saccharomyces cerevisiae aisladas de suplementos dietéticos y probióticos

La levadura Saccharomyces cerevisiae es la especie más utilizada desde un punto de vista biotecnológico. Además, se emplea como suplemento dietético y como agente probiótico bajo el nombre de S. cerevisiae var. boulardii. Hasta ahora, su consumo se había considerado beneficioso por sus propiedades biofuncionales y su valor nutritivo intrínseco, y por otra parte, seguro, dado el carácter “GRAS” (de Generally Recognized As Safe) que se atribuye a esta especie de levadura; sin embargo, actualmente se considera a S. cerevisiae dentro del grupo de patógenos oportunistas emergentes de baja virulencia, capaz de causar infecciones principalmente en hospedadores inmunodeprimidos. Con el fin de conocer el posible papel de esta levadura como patógeno emergente, se obtuvieron en este trabajo cepas de S. cerevisiae a partir de 8 productos dietéticos comerciales con levadura de un total de 22 analizados, con el fin de valorar el potencial patógeno de dichas cepas; en primer lugar, una vez aisladas estas levaduras se aplicaron técnicas moleculares de identificación (análisis de restricción de la región ribosomal 5.8S-ITS) y de caracterización (análisis de restricción del DNA mitocondrial y amplificación por PCR de elementos delta); con la identificación se demostró que 6 de estos aislados pertenecían a la especie S. cerevisiae y 2 fueron híbridos interespecíficos (S. cerevisiae x S. kudriavzevii y S. cerevisiae x S. bayanus). Con la caracterización, se demostró que de estos 8 aislados, 4 de ellos eran la misma cepa de levadura, por tanto, finalmente se obtuvieron 5 aislados de levadura identificados como diferentes. En segundo lugar, una vez obtenidos estos 5 aislados, se llevó a cabo un estudio de rasgos fenotípicos in vitro asociados con la virulencia en levaduras patógenas, como fueron el crecimiento a altas temperaturas, secreción de enzimas extracelulares (proteasas y fosfolipasas), crecimiento pseudofilamentoso y crecimiento invasivo. Se observó que los aislados híbridos presentaron negatividad para la mayoría de estos rasgos estudiados, mientras que el resto de aislados analizados, a los cuales denominamos D2, D4 y D14 presentaron estos rasgos fenotípicos con un nivel alto a moderado, indicando que estos aislados podrían poseer capacidad infectiva independientemente del sistema inmune del hospedador. Seguidamente, se llevó a cabo un estudio de infección sistémica in vivo en ratones inmunocompetentes BALB/c estudiando el alojamiento de estas levaduras en cerebro y riñón a los 7, 15 y 30 días postinfección. Se demostró que el aislado híbrido ensayado, denominado D23, mostró incapacidad para colonizar ambos órganos, mientras que los aislados D2, D4 y D14 mostraron alojamientos en cerebro y riñón, siendo el aislado D14 el que mostró mayor potencial patógeno , ya que fue el único que causó mortalidad en ratones. Se estableció un modelo murino de infección gastrointestinal imitando condiciones clínicas habituales de trauma e inmunosupresión para valorar la capacidad de translocación intestinal y diseminación de cepas de S. cerevisiae. Este estudio se llevó a cabo con el aislado virulento D14 y con el aislado avirulento D23. Se demostró que ambos aislados fueron capaces de atravesar la barrera intestinal, mientras que sólo se detectó diseminación consistente en órganos distantes, como hígado, riñones, cerebro y nódulos mesentéricos en el caso del aislado virulento D14. A continuación, se procedió a llevar a cabo un análisis genómico de 4 cepas de S. cerevisiae, en las cuales se incluía el aislado D14, una cepa de laboratorio avirulenta W303 y dos aislados clínicos virulentos (60 y 102) estudiados previamente en nuestro grupo. Se mostró variabilidad en la composición genómica entre las zonas subteloméricas de estos aislados, siendo ésta mayor en los aislados virulentos (D14, 60 y 102). Además, la mayoría de los genes que presentaban variación en el número de copias se situaban en estas regiones. También se observó una aneuploidía en el aislado clínico 60, la cual podría proporcionar una ventaja selectiva sobre ciertas condiciones ambientales. Se vieron también cambios en el número de copias de genes relacionados con la reparación de bases causada por daño oxidativo o de genes del metabolismo del glutation. Por otra parte, se realizó un estudio comparativo de la supervivencia en sangre humana de las cepas virulentas D14 y 60 y la cepa de laboratorio avirulenta W303, donde se demostró que las cepas virulentas fueron capaces de sobrevivir en sangre humana y el porcentaje de supervivencia fue equivalente al de una cepa patógena ensayada de la especie C. albicans. Utilizando el modelo de supervivencia en sangre humana, se llevó a cabo un análisis transcriptómico de los aislados virulentos D14 y 60, la cepa de laboratorio W303 y una cepa vínica avirulenta CECT 10.431. Se observó una respuesta transcripcional específica por parte de los aislados virulentos (D14 y 60) de genes implicados en la biosíntesis y metabolismo de aminoácidos y en genes de respuesta al estrés oxidativo. Además, tras la aplicación de un estrés oxidativo, las cepas virulentas mostraron mayor resistencia que las cepas avirulentas (CECT 10.431 y W303), sugiriendo una correlación entre la resistencia al estrés oxidativo y la patogenicidad. Por último, los cambios observados en el análisis genómico no se pudieron correlacionar con el nivel de expresión de estos genes en el análisis transcriptómico. Por tanto, cabe pensar que una expresión diferencial entre cepas virulentas y avirulentas sea resultado de la adaptación de las primeras al medio sanguíneo. Este estudio podría ser de gran interés para todas aquellas industrias alimentarias que incluyen a S. cerevisiae en sus preparados, con el fin de evitar la administración de dichas cepas de levaduras a individuos inmunodeprimidos. Puesto que el carácter híbrido no parece conferir habilidad infectiva, el uso de este tipo de cepas en la preparación de suplementos dietéticos podría ser garantía de seguridad.The yeast Saccharomyces cerevisiae is the most used from a biotechnological perspective. It is also used as a dietary supplement and probiotic agent under the name of S. cerevisiae var. boulardii. Until now, its use has been considered beneficial for biofunctional properties and intrinsic nutritional value, and on the other hand, sure, given the "GRAS" character (Generally Recognized As Safe for) that is attributed to this species of yeast; however, currently S. cerevisiae is considered within opportunistic pathogens emerging group of low virulence, mainly capable of causing infections in immunocompromised hosts. In order to know the possible role of this yeast as emerging pathogen, in this study were obtained 8 strains of S. cerevisiae from commercial dietary yeast products of a total of 22 analyzed to evaluate the pathogenic potential of said strains; firstly, once these yeasts were isolated, we applied molecular identification techniques (restriction analysis 5.8S ribosomal-ITS region) and characterization (restriction analysis of mitochondrial DNA and amplification of delta sequences by PCR). With the identification we showed that 6 of these isolates belonged to the species S. cerevisiae and 2 were interespecific hybrids (S. cerevisiae x S. kudriavzevii and S. cerevisiae x S. bayanus). With the characterization, it was shown that these 8 isolates, 4 of them were the same yeast strain, thus finally we obtained 5 yeast isolates identified as different. Secondly, once these five isolates were obtained, we carried out a study of in vitro phenotypic traits associated with the virulence of pathogenic yeast, as growth at high temperatures, secretion of extracellular enzymes (proteases and phospholipases), pseudohyphal growth and invasive growth. It was observed that the hybrids strains showed negative for most of these traits studied, whereas the remaining isolates analyzed, to which we refer as D2, D4 and D14, these phenotypic traits showed a moderate to high level, indicating that these isolates may possess infectivity independently of the host immune system. Next, was conducted a systemic infection in vivo in immunocompetent mice BALB/c, studying the accommodation of these yeasts in brain and kidney after 7, 15 and 30 days postinfection. It was shown that the hybrid strain tested, called D23, showed inability to colonize both organs, while isolates D2, D4 and D14 showed accommodation in brain and kidney, however the strain D14 showed greater pathogenic potential, since it was the only causing mortality in mice. We established a murine model of gastrointestinal infection mimicking normal clinical conditions and immunosuppression trauma to assess the ability of gut translocation and spread of strains of S. cerevisiae. This study was carried out with the virulent strain D14 and with the avirulent strain D23. It was shown that both isolates were able to cross the intestinal barrier, while only detected consisting spread to distant organs, such as liver, kidney, brain and mesenteric lymph in the case of virulent strain D14. Then, we proceeded to carry out a genomic analysis of 4 S. cerevisiae strains, which were the virulent strain D14, an avirulent laboratory strain W303, and two virulent clinical strains (60 and 102) previously studied in our group. It showed genomic variability in subtelomeric areas composition between these isolated, being higher in virulent strains (D14, 60 and 102). Furthermore, most of the genes that showed variation in the number of copies are placed in these regions. Aneuploidy was also observed in the clinical strain 60, which could provide a selective advantage on certain environmental conditions. We also observed changes in the copy number of genes related with the repair of bases caused by oxidative damage or glutathione metabolism genes. Moreover, we conducted a comparative study of survival in human blood with virulent strains D14 and 60 and the avirulent laboratory strain W303, which showed that the virulent strains were able to survive in human blood and the survival rate was equivalent to a pathogenic strain of the species C. albicans. Using the model of survival in human blood, was performed a transcriptomic analysis with virulent strains D14 and 60, the laboratory strain W303 and avirulent wine strain CECT 10.431. There was a specific transcriptional response by virulent isolates (D14 and 60) of genes involved in the biosynthesis and metabolism of aminoacids and genes of oxidative stress response. Furthermore, after the application of oxidative stress, virulent strains showed greater resistance than avirulent strains (CECT 10.431 and W303), suggesting a correlation between oxidative stress resistance and pathogenicity. Finally, the observed changes in genomic analysis could not be correlated with the level of expression of these genes in the transcriptome analysis. Therefore, it is conceivable that a differential expression between virulent and avirulent could be a result of adaptation of virulent strains to the blood environment. This study could be of great interest to all those food industries which include S. cerevisiae in their preparations, to avoid administration of such yeast strains to immunocompromised individuals. Since the hybrid character appears to confer no infective ability, the use of such strains in the dietary supplement preparation would be security guarantee

InmunoProteómica de la respuesta a la infección sistémica en ratón con una cepa Sccharomyces cerevisiae procedente de suplementos dietéticos

Comunicaciones a congreso

BlogForever: D3.1 Preservation Strategy Report

This report describes preservation planning approaches and strategies recommended by the BlogForever project as a core component of a weblog repository design. More specifically, we start by discussing why we would want to preserve weblogs in the first place and what it is exactly that we are trying to preserve. We further present a review of past and present work and highlight why current practices in web archiving do not address the needs of weblog preservation adequately. We make three distinctive contributions in this volume: a) we propose transferable practical workflows for applying a combination of established metadata and repository standards in developing a weblog repository, b) we provide an automated approach to identifying significant properties of weblog content that uses the notion of communities and how this affects previous strategies, c) we propose a sustainability plan that draws upon community knowledge through innovative repository design

Toxicological assessment of mesoporous silica particles in the nematode Caenorhabditis elegans

[EN] Here we report the toxicological evaluation of mesoporous silica particles (MSPs) in the nematode C. elegans. Specifically, we have investigated the effect of bare micro- (M0) and nano-sized (N0) MSPs, and their corresponding functionalized particles with a starch derivative (Glu-N) (M1 and N1, respectively) on C. elegans ageing parameters. The toxicity of MSPs, their impact on C. elegans lifespan, movement capacity, progeny and ability to survive upon exposure to acute oxidative stress were assessed. This study demonstrated that both size particles assayed (M0 and N0), labeled with rhodamine and monitored through fluorescence microscopy, are ingested by the nematode. Moreover, toxicity assays indicated that bare nano-sized particles (N0) have a negative impact on the C. elegans lifespan, reducing mobility and progeny production. By contrast, micro-sized particles (M0) proved innocuous for the nematodes. Furthermore, functionalization of nanoparticles with starch derivative reduced their toxicity in C. elegans. Thus, oral intake of N1 comparatively increased the mean lifespan and activity rates as well as resistance to oxidative stress. The overall findings presented here demonstrate the influence of MSP size and surface on their potential toxicity in vivo and indicate the silica-based mesoporous particles to be a potential support for encapsulation in oral delivery applications. Furthermore, the good correlation obtained between healthy aging variables and viability (mean lifespan) validates the use of C. elegans as a multicellular organism for nanotoxicology studies of MSPs.The authors wish to express their gratitude to the Spanish Government (MINECO Projects AGL2012-39597-C02-01, AGL2012-39597-C02-02, AGL2015-70235-C2-1, MAT2012-38429-C04-01 and MAT2015-64139-C4-1), the Generalitat Valenciana (Project PROMETEOII/2014/047) and Colombian Administrative Department of Science, Technology and Research which supported Ms. Acosta Scholarship. We would also like to thank the Institut de Ciencia dels Materials (ICMUV), the Microscopy Service of the Universitat Politecnica de Valencia and the microscopy service of IATA for technical support. We thank Roquette for the Glucidex samples.Acosta-Romero, C.; Barat Baviera, JM.; Martínez-Máñez, R.; Sancenón Galarza, F.; Llopis Llopis, S.; Gonzalez, N.; Genovés, S.... (2018). Toxicological assessment of mesoporous silica particles in the nematode Caenorhabditis elegans. Environmental Research. 166:61-70. https://doi.org/10.1016/j.envres.2018.05.018S617016

Actigraphic sleep and dietary macronutrient intake in children aged 6–9 years old: A pilot study

The objective of this study was to examine the relationship between different sleep parameters and energy and macronutrient intake in school-aged children. A total of 203 children 6 to 9 years of age participated in this cross-sectional study. Anthropometric measurements were taken first. Diet was assessed with 3-day food logs and sleep was measured with a questionnaire on sleep quality and a wrist actigraph worn for at least 7 days. A decrease of 165.45 kcal was observed per each additional hour of sleep during the week (β (95% CI) = −165.45 (−274.01, −56.88); p = 0.003). This relationship was also observed for fat (β (95% CI) = −11.14 (−18.44, −3.84); p = 0.003) and protein (β (95% CI) = −13.27 (−22.52, −4.02); p = 0.005). An increase in weekend sleep efficiencies for those under the recommended threshold of 85% also had a similar association with energy (β (95% CI) = −847.43 (−1566.77, 128.09); p = 0.021) and carbohydrate (β (95% CI) = −83.96 (−161.76, −6.15); p = 0.035)) intake. An increase in habitual sleep variability was related with a slight increase in protein intake (β (95% CI) = 0.32 (0.031, 0.62); p = 0.031). Children who slept less had a higher energy intake, especially from fat and protein and those who presented inefficient sleep had a higher carbohydrate intake. Strategies to enhance sleep quality and quantity combined with dietary recommendations could help to improve energy and macronutrient intake levels in children

Actigraphic Sleep and Dietary Macronutrient Intake in Children Aged 6-9 Years Old: A Pilot Study

The objective of this study was to examine the relationship between different sleep parameters and energy and macronutrient intake in school-aged children. A total of 203 children 6 to 9 years of age participated in this cross-sectional study. Anthropometric measurements were taken first. Diet was assessed with 3-day food logs and sleep was measured with a questionnaire on sleep quality and a wrist actigraph worn for at least 7 days. A decrease of 165.45 kcal was observed per each additional hour of sleep during the week (β (95% CI) = −165.45 (−274.01, −56.88); p = 0.003). This relationship was also observed for fat (β (95% CI) = −11.14 (−18.44, −3.84); p = 0.003) and protein (β (95% CI) = −13.27 (−22.52, −4.02); p = 0.005). An increase in weekend sleep efficiencies for those under the recommended threshold of 85% also had a similar association with energy (β (95% CI) = −847.43 (−1566.77, 128.09); p = 0.021) and carbohydrate (β (95% CI) = −83.96 (−161.76, −6.15); p = 0.035)) intake. An increase in habitual sleep variability was related with a slight increase in protein intake (β (95% CI) = 0.32 (0.031, 0.62); p = 0.031). Children who slept less had a higher energy intake, especially from fat and protein and those who presented inefficient sleep had a higher carbohydrate intake. Strategies to enhance sleep quality and quantity combined with dietary recommendations could help to improve energy and macronutrient intake levels in children

Vigilância da qualidade microbiológica de água de consumo humano e de água destinada ao abastecimento público da província de Valência durante o período 2002-2010

This study focuses on assessing the microbiological quality of drinking water and of water intended for drinking in the province of Valencia (Spain) between 2002 and 2010. Variation was found regarding space, time and source requirements in samples that did not meet the standards of quality established by Royal Decree 140/2003 for the following: total coliforms (TC); faecal coliforms (FC); Escherichia coli (EC); aerobic bacteria at 22 ºC (AB 22 ºC); faecal streptococci (FS); enterococci (EN); sulphite-reducing clostridia (SC); and Clostridium perfringens (CP). The samples were stratified by those meeting the standards (“Compliance”) and those that did not (“Non-Compliance”), as well as by their relationship with the degree of chlorination.A total of 10057 water samples were examined from various sources: springs; surface waters; denitrifying plant waters; wells; and distribution networks. They were grouped into each of the 17 districts of the province of Valencia.The total number of samples that failed to meet the standards of quality, for each criterion, were as follows: 34.0 % for TC; 16.0 % FS-EN; 13.0 % for FC-EC; 5.6 % for SC-CP; and 15.5 % for AB 22 ºC. Regarding spatial variation in samples, the highest percentages of samples in the “Non-Compliance” group were found in the interior part of the province. For time variation, the highest percentages of “Non-Compliance” were for the years: 2002 - 2004, 2008 and 2009. Regarding source variation, origin of the samples with “Non- compliance” was highest for surface waters, followed by springs and wells.For all samples studied, 39.8 % were within the “Non-Compliance” group, of which 18.3 % came from sources that supply the population (distribution networks).Of the samples within the “Compliance” group, 61 % were chlorinated, which confirms that chlorine is a powerful disinfectant and that chlorination is an effective water disinfection treatment.El presente trabajo tiene como objetivo la evaluación de la calidad microbiológica de las muestras de agua analizadas en la provincia de Valencia durante el período 2002-2010. Se observó la variación espacial, temporal y por origen de las muestras que no cumplían los requisitos especificados en el RD 140/2003 para los coliformes totales (CT), coliformes fecales (CF), Escherichia coli (EC), aerobios a 22 ºC (AB 22 ºC), estreptococos fecales (EF), enterococos (EN), Clostridium sulfito reductores (CS), y Clostridium perfringens (CP), estratificando las muestras en aquellas que cumplían la normativa “Conformes” y las que no “No Conformes”, y su relación con el grado de cloración.Se estudian un total de 10057 muestras de agua procedentes de fuentes que no manan de la red, aguas superficiales, aguas provenientes de plantas desnitrificadoras, pozos y redes de distribución. Se han agrupado en las 17 comarcas de la provincia de Valencia.Del total de las muestras, no cumplían los requisitos de calidad para CT el 34,0 %, para EF-EN el 16,0 %, para AB 22 ºC el 15.5 % para CF-EC el 13,0 % y para CS-CP el 5,6 %. Los porcentajes más elevados de muestras “No Conformes” se observaron situados en la zona interior, y en los años 2002-2004, 2008 y 2009. Respecto a la distribución por origen, se observaron en aguas superficiales, seguidas de fuentes y pozos.Del total de muestras estudiadas, el 39,8 % eran “No Conformes”, y de estas un 18,3 % procedían de abastecimientos que proveen a la población (redes de distribución).El 61,0 % de las muestras “Conformes” estaban cloradas, lo que demuestra que el cloro sigue siendo un tratamiento efectivo de desinfección.Este trabalho tem como objetivo avaliar a qualidade microbiológica de amostras de água analisadas na província de Valência durante o período de 2002 a 2010. Observou-se a variação espacial, temporal e da origem das amostras que não cumprem os requisitos estabelecidos no RD 140/2003, para coliformes totais (CT), coliformes fecais (CF), Escherichia coli (EC), germes aeróbios a 22 ºC (AB 22 ºC), estreptococos fecais (EF), enterococos (EN), Clostridium sulfito (CS) e Clostridium perfringens (CP), estratificando as amostras que cumpriam os requisitos em “conformes” e as que não cumpriam em “não conformes” e a relação com o nível de cloro. Foi estudado um total de 10057 amostras de água de diversas origens: fontes, águas superficiais, centrais de desnitrificação, poços e rede de distribuição. Foram agrupadas nos 17 municípios de Valência.Do total das amostras, não cumpriam os requisitos de qualidade para CT (34 %), EF e EN (16 %), AB 22 ºC (15,5 %), CF e EC (13 %) e CS e CP (5,6 %). Quanto à variação espacial registaram-se as maiores percentagens de amostras “não conformes” na zona interior e nos anos 2002 a 2004, 2008 e 2009. No que diz respeito à distribuição por origens foram observadas maiores percentagens de amostras “não conformes” nas águas de superfície, seguido de nascentes e poços.Do total de amostras analisadas, 39,8 % estavam “não conforme”, sendo que 18,3 % destas eram da rede que abastece a população (rede de distribuição).61 % das amostras “conformes” estão cloradas, o que significa que o cloro ainda é um tratamento eficaz de desinfeção

Las unidades del discurso oral. La propuesta Val.Es.Co. de segmentación de la conversación (coloquial)

El presente artículo resume y examina críticamente la propuesta de segmentación del discurso realizada en Briz y Grupo Val.Es.Co (2003). El modelo Val.Es.Co. consta de ocho unidades (discurso, diálogo, intercambio/ alternancia de turnos, intervención/turno, acto y subacto), tres órdenes (social, estructural e informativo) y cuatro posiciones (inicial, media, final e independiente); se caracteriza, además, por ser jerárquico y recursivo. La aplicación del modelo Val.Es.Co. permite la segmentación de una conversación coloquial sin residuos, así como un adecuado tratamiento de diversos fenómenos conversacionales (actos truncados, solapamientos, elementos suprasegmentales o marcadores discursivos)This paper summarizes and critically reviews the model of discourse segmentation made by Briz and Grupo Val.Es.Co (2003). This model is made up of eight units (discourse, dialogue, exchange/ turn taking, intervention/turn, act and subact), three orders (social, structural and informative) and four positions (initial, medial, final and independent). The Val.Es.Co model is also hyerarchical and recursive. By applying the Val.Es.Co model, a conversation can be divided into parts and subparts without any element remaining unanalyzed. Also, some specific features occurring in conversations such as false starts, overlappings, the segmentation value of prosodic features or discourse markers, can be successfully analyze

Regulation of TFEB and V-ATPases by mTORC1

TORC1 is a key regulator of cell growth in response to nutrients and acts at the surface of the late endosome. This study identifies V-ATPase genes as transcriptional targets of TORC1 and implicates the transcription factor TFEB as an important mediator of TORC1-dependent gene expression and TORC1-regulated endocytosis

Prospective individual patient data meta-analysis of two randomized trials on convalescent plasma for COVID-19 outpatients

Data on convalescent plasma (CP) treatment in COVID-19 outpatients are scarce. We aimed to assess whether CP administered during the first week of symptoms reduced the disease progression or risk of hospitalization of outpatients. Two multicenter, double-blind randomized trials (NCT04621123, NCT04589949) were merged with data pooling starting when = 50 years and symptomatic for <= 7days were included. The intervention consisted of 200-300mL of CP with a predefined minimum level of antibodies. Primary endpoints were a 5-point disease severity scale and a composite of hospitalization or death by 28 days. Amongst the 797 patients included, 390 received CP and 392 placebo; they had a median age of 58 years, 1 comorbidity, 5 days symptoms and 93% had negative IgG antibody-test. Seventy-four patients were hospitalized, 6 required mechanical ventilation and 3 died. The odds ratio (OR) of CP for improved disease severity scale was 0.936 (credible interval (CI) 0.667-1.311); OR for hospitalization or death was 0.919 (CI 0.592-1.416). CP effect on hospital admission or death was largest in patients with <= 5 days of symptoms (OR 0.658, 95%CI 0.394-1.085). CP did not decrease the time to full symptom resolution