Promoter trapping in microalgae using the antibiotic paromomycin as selective agent

The lack of highly active endogenous promoters to drive the expression of transgenes is one of the main drawbacks to achieving efficient transformation of many microalgal species. Using the model chlorophyte Chlamydomonas reinhardtii and the paromomycin resistance APHVIII gene from Streptomyces rimosus as a marker, we have demonstrated that random insertion of the promoterless marker gene and subsequent isolation of the most robust transformants allows for the identification of novel strong promoter sequences in microalgae. Digestion of the genomic DNA with an enzyme that has a unique restriction site inside the marker gene and a high number of target sites in the genome of the microalga, followed by inverse PCR, allows for easy determination of the genomic region, which precedes the APHVIII marker gene. In most of the transformants analyzed, the marker gene is inserted in intragenic regions and its expression relies on its adequate insertion in frame with native genes. As an example, one of the new promoters identified was used to direct the expression of the APHVIII marker gene in C. reinhardtii, showing high transformation efficiencies.Junta de Andalucía (P09-CVI-5053)Junta de Andalucía (BIO-214

Using agro-industrial wastes for mixotrophic growth and lipids production by the green microalga Chlorella sorokiniana

There has been growing interest in the use of microalgae for the production of biofuels, but production costs continue to be too high to compete with fossil fuel prices. One of the main limitations for photobioreactor productivity is light shielding, especially at high cell densities. The growth of the green microalga Chlorella sorokiniana, a robust industrial species, has been evaluated under different trophic conditions with traditional carbon sources, such as glucose and sucrose, and alternative low cost carbon sources, such as carob pod extract, industrial glycerol and acetate-rich oxidized wine waste lees. The mixotrophic cultivation of this microalga with wine waste lees alleviated the problems of light shielding observed in photoautotrophic cultures, improving specific growth rate (0.052 h-1) compared with the other organic sources. The fed-batch mixotrophic culture of Chlorella sorokiniana in a 2 L stirred tank reactor, with optimized nutritional conditions, 100 mM of acetate coming from the oxidized wine waste lees and 30 mM of ammonium, produced an algal biomass concentration of 11 g L-1 with a lipid content of 38 % (w/w). This fed-batch strategy has been found to be a very effective means to enhance the biomass and neutral lipid productivity.Research grants from theSpanish (AGL2016-74866-C32R-AEI/FEDER) and European governments(INTERREG VA-POCTEP- 2014-2020; 0055_ALGARED_PLUS_5_E) and the Portuguese Science Foundation (FCT) through the grant UID/MAR/00350/2013 to the CIMA of the University of Algarve. We thank Dr.Molinari from the University of Milan, for kindly providing the Acetobacteracetiistrain. The help of CEIMAR University Excellence Campus is also acknowledged.info:eu-repo/semantics/publishedVersio

Certificação de produtos orgânicos: obstáculos à implantação de um sistema participativo de garantia na Andaluzia, Espanha.

O trabalho analisa o processo de organização de produtores orgânicos da Andaluzia que estiveram envolvidos em uma tentativa de implantação de um sistema participativo de garantia. Esta iniciativa foi liderada pela administração dessa comunidade autônoma espanhola entre 2006 e 2008. O estudo baseia-se em entrevistas realizadas com atores sociais que estiveram implicados nesse processo, identificando os obstáculos políticos e organizativos que impediram que essa proposta pudesse avançar

RICORS2040 : The need for collaborative research in chronic kidney disease

Chronic kidney disease (CKD) is a silent and poorly known killer. The current concept of CKD is relatively young and uptake by the public, physicians and health authorities is not widespread. Physicians still confuse CKD with chronic kidney insufficiency or failure. For the wider public and health authorities, CKD evokes kidney replacement therapy (KRT). In Spain, the prevalence of KRT is 0.13%. Thus health authorities may consider CKD a non-issue: very few persons eventually need KRT and, for those in whom kidneys fail, the problem is 'solved' by dialysis or kidney transplantation. However, KRT is the tip of the iceberg in the burden of CKD. The main burden of CKD is accelerated ageing and premature death. The cut-off points for kidney function and kidney damage indexes that define CKD also mark an increased risk for all-cause premature death. CKD is the most prevalent risk factor for lethal coronavirus disease 2019 (COVID-19) and the factor that most increases the risk of death in COVID-19, after old age. Men and women undergoing KRT still have an annual mortality that is 10- to 100-fold higher than similar-age peers, and life expectancy is shortened by ~40 years for young persons on dialysis and by 15 years for young persons with a functioning kidney graft. CKD is expected to become the fifth greatest global cause of death by 2040 and the second greatest cause of death in Spain before the end of the century, a time when one in four Spaniards will have CKD. However, by 2022, CKD will become the only top-15 global predicted cause of death that is not supported by a dedicated well-funded Centres for Biomedical Research (CIBER) network structure in Spain. Realizing the underestimation of the CKD burden of disease by health authorities, the Decade of the Kidney initiative for 2020-2030 was launched by the American Association of Kidney Patients and the European Kidney Health Alliance. Leading Spanish kidney researchers grouped in the kidney collaborative research network Red de Investigación Renal have now applied for the Redes de Investigación Cooperativa Orientadas a Resultados en Salud (RICORS) call for collaborative research in Spain with the support of the Spanish Society of Nephrology, Federación Nacional de Asociaciones para la Lucha Contra las Enfermedades del Riñón and ONT: RICORS2040 aims to prevent the dire predictions for the global 2040 burden of CKD from becoming true

Clínica jurídica de acción social

Memoria ID-0032. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2017-2018

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

A Large-Scale Genetic Analysis Reveals a Strong Contribution of the HLA Class II Region to Giant Cell Arteritis Susceptibility

We conducted a large-scale genetic analysis on giant cell arteritis (GCA), a polygenic immune-mediated vasculitis. A case-control cohort, comprising 1,651 case subjects with GCA and 15,306 unrelated control subjects from six different countries of European ancestry, was genotyped by the Immunochip array. We also imputed HLA data with a previously validated imputation method to perform a more comprehensive analysis of this genomic region. The strongest association signals were observed in the HLA region, with rs477515 representing the highest peak (p = 4.05 × 10−40, OR = 1.73). A multivariate model including class II amino acids of HLA-DRβ1 and HLA-DQα1 and one class I amino acid of HLA-B explained most of the HLA association with GCA, consistent with previously reported associations of classical HLA alleles like HLA-DRB1∗04. An omnibus test on polymorphic amino acid positions highlighted DRβ1 13 (p = 4.08 × 10−43) and HLA-DQα1 47 (p = 4.02 × 10−46), 56, and 76 (both p = 1.84 × 10−45) as relevant positions for disease susceptibility. Outside the HLA region, the most significant loci included PTPN22 (rs2476601, p = 1.73 × 10−6, OR = 1.38), LRRC32 (rs10160518, p = 4.39 × 10−6, OR = 1.20), and REL (rs115674477, p = 1.10 × 10−5, OR = 1.63). Our study provides evidence of a strong contribution of HLA class I and II molecules to susceptibility to GCA. In the non-HLA region, we confirmed a key role for the functional PTPN22 rs2476601 variant and proposed other putative risk loci for GCA involved in Th1, Th17, and Treg cell function

Optimización de herramientas moleculares para la manipulación genética de microalgas y su aplicación en procesos de biofloculación

Las microalgas son un grupo muy diverso de microorganismos foto sintéticos y eucariotas. Poseen un alto potencial biotecnológico debido a que son productores de numerosos compuestos bioactivos de interés industrial. La recogida de biomasa de microalgas se vuelve particularmente importante debido a que es un paso crítico en el proceso de producción, representando alrededor del 20-30% del coste total y por tanto, su mejora es necesaria. En la presente tesis doctoral, se lleva a cabo el desarrollo y la optimización de herramientas moleculares para la manipulación genética de microalgas y su posterior aplicación en la mejora de los métodos de recogida de biomasa microalgal, a través de procesos de biofloculación. En el Capítulo 2, en Chlamydomonas reinhardtii, se evaluó la eficiencia de dos promotores heterólogos: el promotor CaMV35S del virus del mosaico de la coliflor y el promotor NOS, de la nopalina sintasa de Agrobactereium tumefaciens. El gen de resistencia a paramomicina {APHVIII) fue seleccionado como gen marcador. La eficiencia de transformación y los niveles de transcrito y proteína APHVIII, se analizaron en una serie de transformantes seleccionados. Los mayores valores se encontraron para el promotor NOS, seguido del promotor CaMV 35S. Aunque el uso de promotores heterólogos es una herramienta eficiente, la búsqueda de promotores endógenos podría ser una alternativa viable para encontrar un promotor universal y fuerte, propio de microalgas. En el Capítulo 3, se transformó genéticamente Chlamydomonas reinhardtii, utilizando el APHVIII como gen marcador, sin promotor. Los transformantes que exhibieron un fenotipo más fuerte se analizaron mediante PCR inversa y se identificó la región genómica precedente al gen marcador. En la mayoría de los transformantes analizados, el gen marcador se encontraba inserto en regiones intragénicas y su expresión se debió a una adecuada inserción en fase de lectura con los genes endógenos. En el Capítulo 4, los promotores heterólogos y el APHVIII promotorless utilizados en el capítulo 2 y 3, se emplearon para la transformación genética de Chlorella sorokiniana, una de las microalgas actualmente con mayor potencial y relevancia a nivel industrial. Se puso a punto el método de transformación génica mediante electroporación, definiéndose los parámetros óptimos como 2,5 kV de intensidad eléctrica y 3 pulsos. Una vez obtenidos los transformantes, se analizaron. Los resultados obtenidos mostraron los mejores valores de eficiencia utilizando el promotor CaMV 35S, en contraste con los resultados obtenidos para C reinhardtii, confirmando de este modo, que ios promotores heterólogos que se utilizan actualmente son fuertemente dependientes de cada especie. En los siguientes capítulos, tanto herramientas fisiológicas como moleculares son desarrolladas y optimizadas para la mejora de los métodos de biofloculación implicados en la recogida de biomasa de microalgas. En el Capítulo 5, la levadura altamente floculante Saccharomyces bayamis var. iivarum y sus factores floculantes son utilizados para inducir floculación en dos microalgas C. reinhardtii y Picochlorum sp, HM1. La adición de Saccharomyces y dichos factores, indujo agregación en las células de ambas especies, resultando en valores máximos de eficiencia de recuperación del 95% y 75% para Chlamydomonas y Picochlorum respectivamente. Con el fin de inducir fenotipos auto-floculantes en células de C reinhardtii, en el Capítulo 6, una cepa silvestre de esta clorofita, fue transformada genéticamente con uno de los genes dominantes responsables de la floculación en la levadura Saccharomyces bayanus var. uvarum, el gen FL05. De entre los muchos transformantes de C. reinhardtii analizados, tres de ellos: CrFL0511, CrFL0513 y CrFL0520, fueron identificados habiendo integrado establemente el gen de floculación en su genoma y exhibiendo fenotipos de auto-floculación.Microalgae are a highly diversified group of eukaryotic and photosynthetic microorganisms with a relevant biotechnological potential because of they are producers of numerous bioproducts used in feed, cosmetic, pharmaceutical and bioenergy industry. The harvesting of the microalgal biomass becomes particularly important because it is a critical step which accounts for about 20-30% of the total production cost and its improvement is necessary. In this thesis, the optimization and the development of molecular tools are performed for genetic manipulation of microalgae and their further application for the improvement of bioflocculation methods to harvest microalgal biomass. In Chapter 2, using the genetic transformation developed for Chlamydomonas reinhardtii, the efficiency of the heterologous promoters of cauliflower mosaic virus 35S (CaMV 35S) and Agrobacterium nopaline synthase (NOS) genes was evaluated. The paromomycin resistance gene (APHV1II) was selected as marker gene. The transformation efficiency and the APHVIII transcript and protein levels were evaluated in a series of transformants for each promoter. Among the two heterologous promoters used, CaMVSSS and NOS, the highest transformation efficiencies and levels of APHVIII expression were found using the NOS promoter. Although the use of heterologous promoters has been a suitable technique for the expression of exogenous genes in microalgae, the search of endogenous promoters could be a feasible alternative to find a strong universal microalgal promoter. In Chapter 3, a nuclear genetic transformation in Chlamydomonas reinhardtii cells was carried out, using the APHVIII as a promoterless marker gene. The random insertions of the promoterless in the most robust selected transformants was demonstrated, allowing the identification of novel strong promoter sequences in microalgae. The inverse PCR technique allowed an easy determination of the genomic region, which precedes the marker gene. In most of the transformants analysed, the marker gene was inserted in intragenic regions and its expression relied on its adequate insertion in frame with native genes. In Chapter 4, the efficiency of the two heterologous promoters and APHVIII promoterless checked in Chapter 2 and 3, were also used for genetic transformation of the industrially important microalga Chlorella sorokinianci. Firstly, a cheap, simple and feasible method of electroporation was developed for this microalga, defining 2.5 kV of electric field strength and 3 electric pulses as the suitable parameters. And then, the efficiencies of the chosen promoters was analysed. The results showed the best efficiency values using the CaMV 35S promoter, in contrast with the results obtained for C. reinhardtii, confirming thus, that the heterologous promoters currently used are strongly specie-dependent. In the following chapters, physiological and molecular tools are developed and optimized to the improvement of bioflocculation methods for the microalgal biomass harvesting. In Chapter 5, the highly self-flocculating yeast Saccharomyces bayamis var. uvarum and its flocculating factors are used to induce flocculation in the two microalgae C. reinhardtii and Picochlorum sp. HM1. The addition of Saccharomyces and flocculating factors induced cell aggregation in both microalgal species studied, resulting in maximum recovery efficiency values of 95% and 75% for Chlamydomonas and Picochlorum respectively. In order to induce self-flocculating phenotypes in C. reinhardtii cells, in Chapter 6, a wild type strain of this chlorophyte was genetically transformed with a flocculin gene (FLOS) from the self-flocculating yeast Saccharomyces bayamis var. uvarum and, three Chlamydomonas transformants, CrFLOSll, CrFL0513 and CrFL0520, were identified having integrated the flocculin gene into their genome and exhibiting self-flocculating phenotypes