39,581 research outputs found

    BMC Gebomics

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    Background. Duck treatments and vaccine designs against the opportunistic human pathogen Pseudomonas aeruginosa have multiple issues, all associated with the diverse genetic traits present in this pathogen, ranging from multi-drug resistant genes to the molecular machinery for the biosynthesis of biofilms. Several candidate vaccines against P. aeruginosa have been developed, which target the outer membrane proteins; However, major issues arise when attempting to establish complete protection against this pathogen due to its presumably genotypic variation at the strain level. To shed light on this concern, we propose this study to assess the P. aeruginosa pangenome and its molecular evolution across multiple strains. Results the P. aeruginosa pangenome was estimated to contain almost 17,000 non-redundant genes, and approximately 15% of these constituted the core genome. Functional analyzes of the accessory genome indicated a wide presence of genetic elements directly associated with pathogenicity. An in-depth molecular evolution analysis revealed the full landscape of selection forces acting on the P aeruginosa pangenome, in which purifying selection drives evolution in the genome of this human pathogen. We also detected distinctive selection wide variety of outer membrane proteins, with the data supporting the concept of substantial genetic variation in proteins probably recognized antigens. Approaching the evolutionary information of genes under extremely positive selection, we designed a new multi-locus sequencing typing assay for and informative, rapid and cost-effective genotyping of P. Aeruginosa on a large scale.Departamento Administrativo de Ciencia, Tecnología e Innovación [CO] Colciencias5817-569-34856Establecimiento de módulo funcionales de secuencia en familias de metiltransferasas de RNA y su uso en la búsqueda de genes de resistencia a antibióticos en patógenos humanosn

    Aplicações biomédicas de materiais fotoativos à base de amido

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    The centrality that light presents in the human being’s life since antiquity to its use in industrial and biomedical applications makes it a topic of intense research. The knowledge of its properties and the way it affects the environment and living beings allows us to find new applications, namely biomedical. Photodynamic therapy is known since the end of the 1970’s as a promising approach in the treatment of neoplasia. This therapeutic approach takes advantage of light’s interaction with photosensitive compounds, named photosensitizers, which in the presence of molecular oxygen produce reactive oxygen species capable of leading tumoral cells to cellular death. From tumoral cells to microbial cells was only a step, and this photodynamic approach has proved very successful, more specifically using porphyrins as photosensitizers, in inactivating microorganisms, including microbial strains multi-resistant to conventional antimicrobial agents (e.g., antibiotics, antifungals). With the rise of microbial resistance to conventional antimicrobials and increasing emergence of chronical conditions such as diabetes and its complications, it becomes imperative to find new therapies which allow to treat and help meliorate patients’ quality of life. The goal of this study was to evaluate the possibility of using cost-friendly materials, such as starch, to support porphyrinic derivatives and in this way to produce photoactive materials with the ability to act as photosensitizers and inactivate common skin infections such as the ones incited by methicillin resistant Staphylococcus aureus and Pseudomonas aeruginosa, and which arise in diabetic ulcers, and furthermore to evaluate its ability to promote tissue regeneration. The porphyrins selected to incorporate in starch were cationic porphyrin 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin (TPP5F) and neutral porphyrin 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin (TMPyP) which were prepared and later immobilized in the starch-based materials. The photoactive films prepared by incorporation of porphyrin in starch were then tested and their antimicrobial properties evaluated in in vitro and ex vivo studies. The TMPyP incorporated starch-based film revealed promising antimicrobial activity as it was able to inactivate S.aureus (MRSA), both in vitro and ex vivo, after white light exposure with an irradiance of 50 mW cm-2 for 60 min and 24h, respectively. Using porcine skin as skin infection model, this TMPyP film revealed potential in preventing the onset of bacterial infections. To evaluate the biocompatibility and possible stimulating effects in tissue regeneration of the starch/ porphyrin films, in vitro studies were conducted in cell lines of fibroblasts (HDF) and endothelial cells (HMEC), two important components of tissue regeneration and wound healing. Four different parameters were tested: cellular viability, cellular migration, ROS formation and cellular adhesion. For that, the starch/porphyrin-based materials were applied and irradiated with a red light from a LED system with an irradiance of 5 mW cm-2 for 15 min. The films of 7.06 mm2 revealed the best results in viability and at these conditions the cells present high levels of cellular ROS. The application of light seems to have influenced all parameters except ROS formation. Low dose red-light seems to positively influence viability and wound healing in endothelial cells, revealing itself as a potential promoter of vascularization. The results obtained with the starch/porphyrin films allow for a perspective of their potential application in the treatment of diabetics’ wounds/ulcers.A centralidade que a luz apresenta na vida do ser Humano desde a antiguidade até à sua utilização em aplicações industriais e biomédicas faz com que esta continue a ser objeto de intensa investigação. O conhecimento das suas propriedades e da forma como afeta o ambiente e os seres vivos permite encontrar novas aplicações nomeadamente biomédicas. A terapia fotodinâmica é reconhecida desde finais da década de 70 como uma abordagem promissora no tratamento de neoplasias. Esta abordagem terapêutica tira partido da interação da luz com compostos fotossensíveis designados de fotosensibilizadores que na presença de oxigénio molecular produzem espécies reativas de oxigénio capaz de levar à morte celular de células tumorais. Das células tumorais às células microbianas foi um passo, e esta abordagem fotodinâmica tem sido muito bem sucedida nomeadamente utilizando porfirinas como fotosensibilizadores, na inativação de microorganismos incluindo estirpes microbianas multi-resistentes aos agentes antimicrobianos convencionais (eg. antibióticos, antifúngicos, etc). Com o aumento da resistência microbiana aos antimicrobianos convencionais e o surgimento crescente de condições crónicas como diabetes e as suas complicações, torna-se imperativo encontrar novas terapias que permitam tratar e melhorar a qualidade de vida dos pacientes. Este estudo teve como objetivo avaliar a possibilidade de utilizar materiais baratos, como seja o amido, para suportar derivados porfirínicos e desta forma produzir materiais fotoactivos com capacidade de atuar como fotosensibilizadores e inactivarem comuns infeções de pele como sejam as provocadas por Staphylococcus aureus resistente à meticilina (MRSA) e Pseudomonas aeruginosa e que surgem em úlceras de diabéticos, e mais do que isso, avaliar a sua capacidade de promoverem a regeneração de tecidos. As porfirinas selecionadas para incorporar no amido foram a porfirina catiónica 5,10,15,20-tetraquis(1- metilpiridinium-4-il)porfirina (TMPyP) e porfirina neutra 5,10,15,20- tetraquis(pentafluorofenil)porfirina (TPP5F) que foram preparadas e posteriormente imobilizadas nos materiais à base de amido. Os filmes fotoactivos preparados por incorporação de porfirina em amido, foram então testados e as suas propriedades antimicrobianas em estudos in vitro e ex vivo avaliadas. O filme à base de amido com a TMPyP incorporada revelou promissora atividade antimicrobiana sendo que conseguiu inativar a S. aureus (MRSA), tanto in vitro como ex vivo, após exposição a luz branca com uma irradiância de 50 mW cm-2 por 60 min e 24h, respetivamente. Utilizando pele de porco como um modelo de infeção de pele, este filme contendo TMPyP revelou potencial em prevenir a instalação de infeções bacterianas. Para avaliar a biocompatibilidade dos filmes de amido-porfirina preparados e possíveis efeitos estimulantes na regeneração de tecidos, foram realizados estudos in vitro em linhas celulares de fibroblastos (HDF) e células endoteliais (HMEC), dois componentes importantes da regeneração de tecido e cura de feridas. Quatro parâmetros diferentes foram testados: viabilidade celular, migração celular, formação de ROS e adesão celular. Para tal os filmes à base de amido-porfirinas foram aplicados e irradiados com luz vermelha proveniente de um sistema LED com uma irradiância de 5 mW cm-2 por 15 min. Os filmes de dimensões 7.06 mm2 revelaram os melhores resultados nos ensaios de viabilidade celular, e as células nestas condições apresentam elevada quantidade de ROS celular. A aplicação de luz parece ter influenciado todos os parâmetros exceto a formação de ROS. A luz vermelha em baixa dose parece influenciar positivamente a viabilidade e regeneração em células endoteliais, revelando-se um potencial promotor de vascularização. Os resultados obtidos com os filmes amido-porfirina permitem perspetivar a sua potencial aplicação no tratamento de feridas/úlceras de diabéticos.Mestrado em Biomedicina Molecula

    Impacto sobre las poblaciones microbianas y análisis de genes destacados durante las interacciones multitróficas de Pseudomonas chlororaphis PCL1606 en la rizosfera de aguacate

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    El efecto de la aplicación de PcPCL1606 sobre las comunidades procariotas en muestras de suelo y rizosfera no fue detectable, y se observaron cambios menores en comunidades eucariotas. En los suelos infestados con el patógeno, la presencia de R. necatrix impacta fuertemente a las comunidades microbianas del suelo y la rizosfera. Sin embargo, después de la aplicación de PcPCL1606 al suelo infestado con R. necatrix, estabilizó las comunidades eucariotas, reduciendo fuertemente la presencia de R. necatrix en suelo y rizosfera de aguacate, a su vez minimizando su efecto sobre el resto de comunidades microbianas. Se analizó este fenotipo de supresión de R. necatrix, y se comprobó que PcPCL1606 tiene un papel clave en la inducción de supresividad; además, este fenotipo dependía en gran medida de la producción de HPR (Tienda et al., 2020). Por lo que, el siguiente objetivo de nuestro trabajo, fue estudiar el papel de genes regulados por HPR, que participan durante los procesos de interacción de PcPCL1606 con la rizosfera de aguacate y durante la interacción multitrófica en el biocontrol entre la bacteria, la planta y el patógeno. Para ello, se realizó una aproximación de genómica funcional, con la técnica RNAseq, y se observó que el compuesto HPR en PcPCL1606, actúa como regulador, siendo mayoritariamente un inductor de genes en interacción con la rizosfera de aguacate y un represor de genes en la interacción multitrófica. En la interacción con la rizosfera, HPR induce la expresión de los genes PCL1606_14510 y PCL1606_46820 (con alta identidad a los genes kgtP y phaG de Pseudomonas aeruginosa, respectivamente). El gen kgtP de PcPCL1606 está implicado en el transporte y metabolismo del compuesto 2-oxoglutarato (2-OG) y en la quimiotaxis hacia dicho compuesto. El gen phaG de PcPCL1606 es responsable de la producción de gránulos de polihidroxialcanoatos (PHA), y estaría implicado en tolerancia a algunos estréses (temperatura y peróxido de hidrógeno). Además, estos dos genes también están implicados en la capacidad de control biológico de PcPCL1606 en el patosistema aguacate-R. necatrix. Por otro lado, en la interacción multitrófica que se da durante el biocontrol frente a R. necatrix, HPR actúa mayoritariamente como un represor de genes, aunque nos pareció interesante el estudio de un gen que estaba inducido por HPR debido al posible papel en la formación de biopelícula, el gen PCL1606_36720, con alta identidad al gen pgdA del grupo P. chlororaphis, en PcPCL1606 no tiene efecto sobre la producción de biopelícula, pero el mutante en este gen muestra una mayor adhesión, una menor movilidad tipo swimming, y muestra una pérdida de la actividad de biocontrol de PcPCL1606 frente a R. necatrix.Pseudomonas chlororaphis PCL1606 (PcPCL1606) es una bacteria modelo aislada de la rizosfera del aguacate, que muestra una fuerte actividad de antagonismo y control biológico contra diferentes patógenos fúngicos transmitidos por el suelo, incluido el agente Rosellinia necatrix, que causa la podredumbre blanca radicular del aguacate en el área mediterránea (Cazorla et al., 2006; González-Sánchez et al., 2013). Ensayos in vitro de producción de antibióticos revelaron que PcPCL1606 produce los compuestos antifúngicos pirrolnitrina (PRN), ácido cianhídrico (HCN) y 2-hexil, 5-propil resorcinol (HPR). Sin embargo, se ha demostrado que solo HPR está directamente involucrado en el antagonismo y la capacidad de biocontrol de esta cepa (Calderón et al., 2015). Además, se demostró que HPR intervenía en otros fenotipos como la producción de biopelícula (Calderón et al., 2019) y en fenotipos relacionados con las interacciones multitróficas que se dan en la rizosfera del aguacate, teniendo HPR un papel fundamental en la colonización de la superficie radicular del aguacate y ejerciendo un efecto deletéreo sobre las hifas fúngicas (Calderón et al., 2014). En este trabajo se evaluó si la aplicación semicomercial de PcPCL1606 al suelo puede afectar potencialmente a las comunidades microbianas del suelo y rizosfera del aguacate en condiciones naturales o en presencia de R. necatrix. Para ello, se preparó un producto formulado basado en PcPCL1606 y se aplicó al suelo mediante riego a plantas de aguacate que crecían en experimentos de mesocosmos. Se demostró que PcPCL1606 formulado controló la podredumbre blanca radicular en condiciones de campo y que sobrevivía hasta el final de los experimentos. Para probar los efectos de PcPCL1606 en las comunidades microbianas, se analizaron las poblaciones utilizando una aproximación metagenómica, secuenciando fragmentos de los genes 16S/ITS

    Innate lymphoid cells in sepsis and trained immunity

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    Innate immune cells are the first line of defense against infections. A recent breakthrough in immunology has been the description of the acquisition of memory by innate immune cells and their progenitors, a process called “trained immunity”. Trained immunity is induced by microbial or danger signals. Trained innate immune cells show an enhanced response to a secondary hit. Many aspects of trained immunity remain open, particularly in the field of infections. Here, we developed a model of training induced by challenging mice with yeast-derived β-glucans to characterize the impact of trained immunity on infections. Here we first investigated: 1) the breadth and 2) the persistence of protection conferred by trained immunity. Using a large panel of preclinical models, we show that training protected mice from systemic, peritoneal, gastrointestinal and pulmonary infections induced by Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Citrobacter rodentium, Pseudomonas aeruginosa and Streptococcus pneumoniae. Training increased bone marrow myeloid progenitors, blood inflammatory monocytes and neutrophils, peritoneal antimicrobial macrophages, and lung monocytes and neutrophils. Monocytes/macrophages and IL-1 signaling were essential for protecting trained mice from systemic listeriosis. Most of the cellular alterations persisted over time. Accordingly, mice trained 9 weeks earlier were protected from lethal listeriosis. Thus, trained immunity confers broad, long- lasting protection from bacterial infections. Innate lymphoid cells (ILCs) are tissue-resident cells and important sources of cytokines shaping immune responses. Thus, we explored ILC subpopulations (ILC1/2/3) during pneumococcal pneumoniae. In trained mice, the number of ILCs remained stable, but the proportion of ILCs shifted toward ILC2 involved in tissue repair. Thus, training promotes antimicrobial defense mechanisms and possibly resolution/repair mechanisms following bacterial pneumonia. ILCs were identified recently. The expression of ILCs in humans suffering from sepsis is not known. Using two cohorts of patients constituted during this thesis, we questioned whether ILCs circulate at different levels in the blood of patients with ventilator-associated pneumoniae (VAP) and Covid-19 pneumonia. ILCs blood counts were not affected upon VAP. On the contrary, ILCs numbers decreased in patients hospitalized with Covid-19, an effect associated with disease severity. Therefore, ILCs may represent biomarkers for Covid-19 but apparently not for VAP sepsis. Overall, our studies suggest that trained immunity could be used to enhance antimicrobial host defenses, for example by designing new vaccine adjuvants, or targeted to counter-balance the deleterious effects associated with enhanced inflammatory responses. Moreover, ILCs may represent an attractive biomarker for sepsis patients suffering from Covid-19. -- Les cellules immunitaires innées constituent la première ligne de défense contre les infections. La description de l'acquisition d'une mémoire par les cellules immunitaires innées constitue une percée récente en immunologie. Ce processus est appelé « trained immunity ou immunité entraînée ». L'immunité entraînée est induite par des signaux microbiens ou de danger. Elle fournit des cellules immunitaires innées répondant de manière accrue à un contact ultérieur avec ces mêmes types de signaux. Dans ce travail, nous avons développé un modèle préclinique d’entrainement par injection de paroi de levure à des souris, puis caractérisé l'impact de l'immunité entraînée sur les infections. Dans un premier temps, nous avons étudié le spectre et la persistance de la protection conférée par l'immunité entraînée. L'entraînement protégeait les souris d’infections systémiques, péritonéales, gastro-intestinales et pulmonaires induites par des bactéries telles Staphylocoque, Listeria, Escherichia coli, Pseudomonas et Streptocoque. L'entraînement augmentait le nombre de cellules souches myéloïdes ainsi que les monocytes et neutrophiles du sang, de la cavité péritonéale et des poumons. Les monocytes et la signalisation induite par l'IL-1 étaient essentiels pour protéger les souris entraînées contre la listériose. La plupart des altérations cellulaires persistaient dans le temps, et les souris entraînées neuf semaines plus tôt étaient protégées contre la listériose. Ainsi, l'immunité entraînée confère une protection large et durable contre les infections bactériennes. Les cellules lymphoïdes innées (ILCs) sont des cellules résidant dans les tissus et une source importante de cytokines qui façonnent les réponses immunitaires. Nous avons exploré les sous-populations d’ILCs (ILC1/2/3) au cours de la pneumonie à pneumocoque. Chez les souris entraînées, le nombre d'ILCs restait stable mais la proportion des ILC2, qui promeuvent la réparation tissulaire, augmentait chez les souris survivantes. Ainsi, l'entraînement favorise les mécanismes de défense antimicrobienne et peut- être les mécanismes de cicatrisation lors d’une pneumonie bactérienne. Finalement, nous avons questionné si les ILCs circulent à des niveaux différents dans le sang de patients souffrant de pneumonie associée à la ventilation mécanique (PAVM) et de Covid-19. Le nombre d’ILCs n’était pas affecté par la PAVM. Au contraire, le nombre d'ILCs diminuait drastiquement chez les patients hospitalisés pour Covid-19. Par conséquent, les ILCs peuvent représenter des biomarqueurs pour la septicémie de Covid-19 mais pas pour la PVA. Globalement, notre étude suggère que l'immunité entraînée pourrait être utilisée pour renforcer les défenses antimicrobiennes de l'hôte, par exemple en concevant de nouveaux adjuvants vaccinaux, ou en ciblant des inhibiteurs pour contrebalancer les effets délétères associés à l’inflammation. En outre, les ILCs pourraient servir de biomarqueur pour les patients atteints de Covid-19. Innate lymphoid cells in sepsis and trained immunity -- Le système immunitaire est un système de défense qui nous protège notamment contre les infections. Il est divisé en deux parties: 1) le système immunitaire inné composé de globules blancs qui reconnaissent de manière non-spécifique toutes sortes de microbes, et 2) le système immunitaire adaptatif qui génère des lymphocytes mémoires qui réagissent beaucoup plus vite et de manière plus intense lors d’une réinfection avec un même pathogène. Cette capacité de mémorisation est utilisée dans le principe des vaccins qui nous protègent contre les infections. Le dogme selon lequel la mémoire immunitaire est l’apanage des lymphocytes et du système immunitaire adaptatif a été remis en question récemment. Ainsi, il a été démontré que le système immunitaire innée préalablement exposé à un microbe (le plus souvent un fragment de levure ou le vaccin BCG) fait preuve de mémoire en ce sens qu’il peut augmenter sa réponse lors d’une réinfection par l’intermédiaire de globules blancs appelés monocytes et neutrophiles. Dans ce projet, nous avons cherché à définir si la mémoire du système immunitaire inné: 1) permet de protéger de toute sorte d’infections, et 2) persiste dans le temps. Nous rapportons en utilisant des modèles précliniques utilisant la souris que le développement de mémoire immunitaire innée protège les souris contre un grand nombre d’infections incluant la septicémie, péritonite, gastro-entérite et pneumonie. Par ailleurs la protection est durable au cours du temps. Les cellules lymphoïdes innées (ILCs) sont des cellules découvertes récemment. Elles résident dans les tissus et sont une source importante de molécules solubles qui façonnent les réponses immunitaires (i.e. des cytokines, qui s’apparentent à des hormones du système immunitaire). Vu ces caractéristiques, nous avons exploré l’expression des ILCs au cours de la pneumonie à pneumocoque en relation avec l’instauration de la mémoire du système immunitaire inné. Nos résultats suggèrent que les ILCs ne sont pas affectées de par leur nombre mais possiblement de par leur fonction lors de l’acquisition de la mémoire innée, favorisant les mécanismes de défense antimicrobienne et peut-être les mécanismes de cicatrisation après une pneumonie bactérienne. Pour terminer, nous avons questionné si les ILCs présentes dans le sang sont affectées chez les patients souffrant de pneumonie à bactérie associée à la ventilation mécanique ou de pneumonie à virus, en l’occurrence de Covid-19. Le nombre des ILCs diminuait drastiquement chez les patients hospitalisés pour Covid-19 mais pas chez les patients souffrant de pneumonie bactérienne, suggérant que les ILCs pourraient être utilisées pour estimer la sévérité de la Covid-19. Globalement, ces travaux suggèrent que la mémoire de l'immunité innée pourrait être utilisée pour renforcer les défenses antimicrobiennes de l'hôte, par exemple en concevant de nouveaux types de vaccins. En outre, les ILCs pourraient servir de marqueur pour les patients atteints de Covid-19

    Studio Longitudinale dei volumi statici polmonari nella Discinesia Ciliare Primaria: valutazione di una casistica della Clinica Pediatrica negli ultimi dieci anni

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    La discinesia ciliare primaria (DCP) è una malattia rara, geneticamente eterogenea, abitualmente trasmessa con modalità autosomica recessiva, ma occasionalmente X-linked, caratterizzata da una alterazione del movimento delle ciglia dell’epitelio respiratorio. La compromissione del trasporto muco-ciliare favorisce infezioni batteriche ricorrenti e croniche delle vie aeree, che possono causare danni permanenti a carico dei bronchi e del parenchima polmonare, con un progressivo declino della funzione respiratoria. Tuttavia, è presente una considerevole variabilità nella gravità e progressione della patologia respiratoria ed emergono nuove correlazioni tra il genotipo ed il fenotipo della malattia. Pochi studi hanno correlato l’evoluzione a lungo termine della spirometria al difetto ultrastrutturale o al genotipo che sono alla base della DCP. Nessun studio ha, invece, mai valutato longitudinalmente i volumi polmonari statici, che potrebbero essere un indice più sensibile della compromissione delle vie aeree periferiche. Solo due studi cross-sectional hanno dimostrato in questa malattia la presenza di un incremento della capacità funzionale residua (FRC), del volume residuo (RV) e del rapporto RV/capacità polmonare totale (TLC) e uno soltanto documenta l’associazione tra RV ed infezione da Pseudomonas aeruginosa (P.a.). Anche la nutrizione e l’accrescimento potrebbero essere alterati, dato che sono stati fortemente associati alla funzione respiratoria. Lo scopo di questo studio era, pertanto, di valutare l’evoluzione correlata all’età dei volumi polmonari statici ed identificare la loro associazione con i difetti ultrastrutturali e le mutazioni genetiche responsabili della DCP. Volevamo, inoltre, valutare la relazione di tali parametri con l’indice di massa corporea (BMI) e con il cambiamento nella prevalenza delle infezioni batteriche delle vie aeree, in particolare di quelle sostenute da P.a., allo scopo di ampliare la conoscenza delle correlazioni genotipo-fenotipo. Per poter essere inclusi nello studio i pazienti, presentando una storia clinica compatibile, dovevano aver ricevuto la diagnosi di DCP dopo essere stati sottoposti ad esami ultrastrutturali e/o funzionali delle ciglia dell’epitelio respiratorio, alla ciliogenesi in coltura, alla misurazione dell’ossido nitrico nasale e/o della ricerca di mutazioni bialleliche a carico di uno dei geni responsabili della malattia. Inoltre, venivano inclusi nello studio tutti i pazienti afferenti all’U.O. Pediatria dell’Azienda Ospedaliero-Universitaria Pisana per 2 volte all’anno negli ultimi 10 anni, in grado di eseguire correttamente l’esame pletismografico. Il muco espettorato o raccolto mediante aspirazione faringea nei non collaboranti era utilizzato per le indagini microbiologiche. Nella elaborazione statistica, per valutare l’associazione tra morfologia ciliare, mutazioni genetiche e caratteristiche cliniche venivano utilizzati modelli lineari ad effetti misti. Venivano arruolati nello studio 118 pazienti di età ≥ 5 anni, che avevano eseguito complessivamente 1096 visite, in media 9 per paziente, nel corso di un follow-up medio di 5 anni. All’arruolamento, quasi tutti i parametri spirometrici e, specialmente negli adulti, anche pletismografici erano significativamente peggiori nei pazienti con assenza dei bracci interni di dineina, difetti dell’apparato centrale e disorganizzazione microtubulare (IDA/CA/MTD) rispetto ai pazienti con Normale Microscopia Elettronica (EM). Il trend medio di incremento nel tempo per RV era significativamente più elevato nei pazienti con IDA/CA/MTD rispetto a quelli con difetto dei bracci esterni di dineina (ODA) e con Normale EM (p=0.021 e p=0.022, rispettivamente). Il trend medio di RV/capacità polmonare totale (TLC) nel gruppo di pazienti con IDA/CA/MTD era significativamente peggiore che in tutti gli altri gruppi. L’incremento maggiore dei volumi polmonari veniva osservato nel gruppo con mutazioni bialleliche nel gene CCDC39 & CCDC40, mentre si rilevava un minore incremento dell’iperinsufflazione nei gruppi con mutazioni nei geni DNAH5 e DNAH11. Si registrava, inoltre, un aumento più ripido del RV/TLC nei pazienti con infezione da P.a. rispetto a quelli con altre infezioni batteriche (p=0.007), o senza infezione (p=0.003). Infine, non si osservava alcuna associazione statisticamente significativa tra variazioni dei volumi statici polmonari e BMI. Pertanto, con questo studio abbiamo confermato in maniera robusta la peggiore prognosi per alcuni difetti genetici ed ultrastrutturali (CCDC39 & CCDC40 o IDA/CA/MTD) rispetto ad altri (DNAH5 e DNAH11 o ODA e Normale EM), la cui associazione con una maggiore compromissione della funzione respiratoria finora si basava esclusivamente sulla spirometria. La valutazione dei volumi polmonari dovrebbe essere, quindi, sempre associata alla spirometria nel management clinico dei pazienti con DCP collaboranti. Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, usually autosomal recessive, but occasionally X-linked, disorder, characterized by dysfunction of motile cilia. Impaired mucociliary clearance results in recurrent and chronic bacterial infection of the airways, eventually causing permanent bronchial and parenchymal damage, with progressive decline in lung function. However, there is considerable variability in the severity and progression of lung disease, and new genotype-phenotype relationships are emerging. A few studies have related long-term evolution of spirometry to the underlying ultrastructural defect or genotype. No longitudinal study has evaluated static lung volumes in PCD, which may be a more sensitive index of distal airway disease. A few cross-sectional studies demonstrated hyperinflation, with an increase in functional residual capacity (FRC), residual volume (RV) and RV/total lung capacity (TLC) ratio, in one study, worse hyperinflation was associated with Pseudomonas aeruginosa (P.a.) infection. Furthermore, both growth and nutrition are adversely affected in PCD patients and are both strongly associated with lung function. The aim of this study was to investigate the age-related evolution of static lung volumes and identify their association with ultrastructural defects, and individual gene mutations in a large cohort of adult and paediatric PCD patients. We also assessed the relationships of these parameters with body mass index (BMI) and the change in prevalence of bacterial airway infections, especially P.a., in order further to extend our knowledge of genotype-phenotype correlations. Participants had a confirmed diagnosis of PCD, based on ultrastructural and/or functional ciliary abnormalities, in vitro ciliogenesis and/or biallelic PCD disease-causing mutations, and nasal NO combined with compatible clinical features. Study visits occurred twice a year for up to 10 years. The current analysis was limited to patients who were able successfully to perform plethysmography. Expectorated sputum or pharyngeal aspirates were collected for microbiological analysis, as appropriate. Linear mixed effects models were used to assess associations between ciliary morphology, genetic mutations, and clinical features. One hundred and eighteen PCD patients, aged ≥ 5 years, met the inclusion criteria and were enrolled in the study. There were 1096 study visits, mean 9 per patient. Mean (range) followup was 5 years. At enrolment, almost all spirometric and, especially in adults, plethysmographic parameters were significantly worse in absent inner dynein arms, central apparatus defects, and microtubular disorganization (IDA/CA/MTD) compared with Normal Electron Microscopy (EM) patients. Mean trend increase with time for residual volume (RV) was significantly higher in IDA/CA/MTD compared to outer dynein arms (ODA) defect (p=0.021) and Normal EM (p=0.022) groups. Mean trend of RV/total lung capacity (TLC) in IDA/CA/MTD was significantly worse than in all other groups. The steepest rise in lung volumes was in CCDC39/CCDC40, while hyperinflation increased less in DNAH5 and DNAH11 groups. RV/TLC showed a significantly steeper rise in patients with Pseudomonas aeruginosa compared to other infections (p=0.007), or without infection (p=0.003). Therefore, PCD patients with IDA/CA/MTD defects or CCDC39/CCDC40 mutations had the greatest increase in hyperinflation, while those with ODA defect and Normal EM or DNAH11 and DNAH5 mutations had less severe changes. We have robustly confirmed the worse prognosis for some genetic and ultrastructural defects, which association hitherto rested solely on spirometry. Lung volumes evaluation should be always associated with spirometry in clinical management of collaborating PCD patients

    La matriz extracelular de la biopelícula en la ecología de dos especies de Pseudomonas asociadas a plantas

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    Por un lado, la reducción de la formación de biopelícula en la cepa mutante en Psl de PssUMAF0158 incrementa la virulencia en foliolos de tomate, tal y como se había descrito previamente para el polisacárido celulosa. Por otro lado, la reducción de la formación de biopelícula en la cepa mutante en Psl de PcPCL1606 tiene un impacto negativo sobre la efectividad de la actividad de biocontrol frente a la podredumbre blanca radicular del aguacate causada por el hongo fitopatógeno Rosellinia necatrix. La fibra de tipo amiloide Fap, la cual es producida por PcPCL1606 pero no por PssUMAF0158, no posee un papel relevante en la formación de biopelículas in vitro, pero sí ha mostrado tener un papel en adhesión temprana a superficies de tipo abiótico y biótico y en la eficacia de la actividad de control biológico en el sistema mencionado. Adicionalmente, en esta tesis doctoral se ha demostrado que la temperatura y la luz influyen en la producción de polisacáridos en las cepas de P. syringae pv. syringae y, por lo tanto, que esta bacteria puede modular la formación de biopelículas en respuesta a factores ambientales.La formación de biopelículas ha mostrado ser una característica relevante de la bacteria fitopatógena Pseudomonas syringae pv. syringae y del agente de control biológico Pseudomonas chlororaphis durante la interacción con plantas. Un análisis genómico comparativo reveló que los polisacáridos alginato y Psl, así como la fibra de tipo amiloide Fap, relevantes para la formación de biopelículas en otras Pseudomonas, podrían ser componentes de la matriz extracelular de Pseudomonas syringae pv. syringae UMAF0158 (PssUMAF0158), agente causal de la necrosis apical del mango, y/o del agente de biocontrol Pseudomonas chlororaphis PCL1606 (PcPCL1606). A diferencia del polisacárido alginato, el polisacárido Psl sólo ha sido estudiado en Pseudomonas aeruginosa, a pesar de que la región genómica que lo codifica está presente en varias cepas de pseudomonas asociadas a plantas, incluidas PssUMAF0158 y PcPCL1606. Los resultados obtenidos han mostrado que el polisacárido alginato no desempeña un papel esencial para la formación de biopelículas en estos dos modelos de estudio, como se había descrito previamente en P. aeruginosa. El clúster génico de tipo psl de la cepa PssUMAF0158 está presente en todos los filogrupos del complejo P. syringae que están asociados a plantas. Sin embargo, la presencia del clúster génico de tipo psl de la cepa PcPCL1606 dentro del complejo Pseudomonas fluorescens es más variable, aunque está presente en todas las cepas del filogrupo P. chlororaphis que se han analizado. Estudios de formación de biopelículas y virulencia en PssUMAF0158 y de formación de biopelículas y actividad de biocontrol en PcPCL1606 con las cepas silvestres y mutantes afectados en la producción de Psl han revelado que este polisacárido es un componente de la matriz extracelular de estas cepas con un papel importante para la formación de biopelículas y la ecología bacteriana

    Complete genome sequence of functional probiotic candidate Lactobacillus amylovorus CACC736

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    Lactobacillus amylovorus CACC736 was originated from swine feces in Korea. The complete genome sequences of the strain contained one circular chromosome (2,057,809 base pair [bp]) with 38.2% guanine-cytosine (GC) content and two circular plasmids, namely, pCACC736-1 and pCACC736-2. The predicted protein-coding genes, which are encoding the clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins, biosynthesis of bacteriocin (helveticin J), and the related proteins of the bile, acid tolerance. Notably, the genes related to vitamin B-group biosynthesis (riboflavin and cobalamin) were also found in L. amylovorus CACC736. Collectively, the complete genome sequence of the L. amylovorus CACC736 will aid in the development of functional probiotics in the animal industry

    Targeting the <i>Pseudomonas aeruginosa</i> quorum sensing system to inhibit virulence factors and eradicate biofilm formation using AHL-analogue phytochemicals

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    Quorum sensing plays a major role in the expression of virulence and development of biofilm in the human pathogen Pseudomonas aeruginosa. Natural compounds are well-known for their antibacterial characteristics by blocking various metabolic pathways. The goal of this study is to find natural compounds that mimic AHL (Acyl homoserine lactone) and suppress virulence in P. aeruginosa, which is triggered by quorum sensing-dependent pathways as an alternative drug development strategy. To support this rationale, functional network analysis and in silico investigations were carried out to find natural AHL analogues, followed by molecular docking studies. Out of the 16 top-hit AHL analogues derived from phytochemicals, seven ligands were found to bind to the quorum sensing activator proteins. Cassialactone, an AHL analogue, exhibited the highest binding affinity for RhlI, RhlR, and PqsE of P. aeruginosa, with a docking score of −9.4, −8.9, and −8.7 kcal/mol, respectively. 2(5H)-Furanone, a well-known inhibitor, was also docked to compare the docking score and intermolecular interactions between the ligand and the target protein. Furthermore, molecular dynamics simulations and binding free energy calculations were performed to determine the stability of the docked complexes. Additionally, the ADME properties of the analogues were also analyzed to evaluate the pharmacological parameters. Functional network analysis further showed that the interconnectedness of proteins such as RhlI, RhlR, LasI, and PqsE with the virulence and biofilm phenotype of the pathogen could offer potential as a therapeutic target. Communicated by Ramaswamy H. Sarma</p

    PHYTOCHEMICAL AND ORGANOLEPTIC CHARACTERISTIC OF Sterculia quadrifida R.Br. TREE BARK HERBAL TEA

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    Sterculia quadrifida R.Br. Bark decoction is used to treat hepatitis and consumed as a tonic by Timorese in the East Nusa Tenggara Province, Indonesia. Raw herbal materials are susceptible to fungi contamination, have limitations in transportation, and have low economic value. Processing the bark into powder packed in tea bags is expected to overcome these obstacles. Stevia, ginger, and mint leaves are herbs often added to herbal drink formulas. This study aimed to evaluate the antioxidant capacity, total flavonoid content (TFC), total phenol content (TPC), physicochemical properties, and sensory properties of six formulas, namely: (F1) pure S. quadrifida bark; (F2) bark of S. quadrifida and leaves of stevia; (F3) stem bark of S. quadrifida and ginger; (F4) bark of S. quadrifida, stevia, and ginger; (F5) bark of S. quadrifida and mint leaves; (F6) bark of S. quadrifida, stevia, and mint. Total phenolic content was measured using the Folin-Ciocalteu method, and total flavonoids were determined using the AICI3 method. Antioxidant activity was measured using the 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) method. The TPC of S. quadrifida tea ranged from 14.09±0.7 to 68.20±0.95% Gallic Acid Equivalent (GAE), and the TFC ranged from 0.03±0.005 to 0.09±0.004% QE. The Ascorbic acid Equivalent Antioxidant Capacity (AEAC) of F1, F2, F5, and F6 were insignificantly different. The pure S. quadrifida bark formula (F1) contained the highest levels of antioxidants (1,044.3±28.48 ppm Ascorbic Acid Equivalent (AAE)). Formulas with the addition of stevia, mint, and ginger showed weaker antioxidant activity than pure S. quadrifida. The formula of pure S. quadrifida bark (F1) and S. quadrifida with the addition of stevia (F2) have the potential to be developed as commercial herbal tea

    Exploration of Compost Soil for the Production of Thermo-Stable <i>Bacillus</i> Protease to Synthesize Bioactive Compounds through Soy Protein Hydrolysis

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    Application of bioactive peptides (BAPs) is promising due to their potential antimicrobial, antioxidant, agonistic, and ACE inhibition properties. To achieve a stable and active peptide at relatively high pH and temperatures by microbial fermentation, a wide variety of microorganisms need to be explored from diverse habitats, and compost is the excellent source. In an attempt to isolate potent protease-producing bacteria, gelatin-supplemented DM agar medium was used. Out of 140 pure cultures, initial protease production selects isolate D3L/1 (26 U/mL), and 16S rDNA sequencing confirmed it as Bacillus subtilis. Protease production was increased to 55.55 U/mL, with pH 7.5, 1% glucose, 1% casein, 1% ammonium sulfate, for 96 h of fermentation, at 37 °C under 140 rpm of shaking. Ion-exchange, and size-exclusion chromatography, 30 KDa protease was purified up to 4.1-fold (specific activity 3448.62 U/mL; 67.66% yield). The enzyme was active under broad temperatures (60 °C optimum), organic solvents, and pH variations. A total of 5% H2O2 can only reduce 40% of enzyme activity. However, 1 mM, Fe2+, and Cu2+ increased enzyme activity by five times. Soy hydrolysis (SPI) byD3L/1 protease produces bioactive compound (Serratia marcescens but active against Escherechia coli (47%), Staphylococcus aureus (28%), and Pseudomonas aeruginosa (12%)
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