Lipase assay in yarrowia lipolytica

Lipases (triacylglycerol acyl hydrolases, EC 3. 1.1.3) are key enzymes in fat metabolism produced by microorganisms, plants, and animals that catalyze the breakdown of triacylglycerols to free fatty adds and glycerol. In Y. lipolytica severaJ genes encoding lipases have been isoiated,LlP2 coding for a secreted lipase (Pignede et al. 1000) and LlPl and LIP3 encoding two lipase genes of the carboxylesrerase family (Choupina et al. 1999). Lipase expression was repressed by glucose and induced by fatty acids and olive oil

Producción de lipasas por la levadura Yarrowia lipolytica

Yarrowia lipolytica es una levadura dimórfica que secreta gran cantidad de proteínas al medio de cultivo, entre ellas: proteasas - alcalinas, neutras y ácidas -, ribonucleasas, fosfatasas y lipasas. Las lipasas son enzimas importantes en el metabolismo de ácidos grasos, catalizando la hidrolisis de triglicéridos a ácidos grasos y glicerol

Non-conventional yeasts as hosts for heterologous protein production

Yeasts are an attractive group of lower eukaryotic microorganisms, some of which are used in several industrial processes that include brewing, baking and the production of a variety of biochemical compounds. More recently, yeasts have been developed as host organisms for the production of foreign (heterologous) proteins. Saccharomyces cerevisiae has usually been the yeast of choice, but an increasing number of alternative non-Saccharomyces yeasts has now become accessible for modern molecular genetics techniques. Some of them exhibit certain favourable traits such as high-level secretion or very strong and tightly regulated promoters, offering significant advantages over traditional bakers’ yeast. In the present work, the current status of Kluyveromyces lactis, Yarrowia lipolytica, Hansenula polymorpha and Pichia pastoris (the best-known alternative yeast systems) is reviewed. The advantages and limitations of these systems are discussed in relation to S. cerevisiae

Non-conventional yeasts as hosts for heterologous protein production.

Yeasts are an attractive group of lower eukaryotic microorganisms, some of which are used in several industrial processes that include brewing, baking and the production of a variety of biochemical compounds. More recently, yeasts have been developed as host organisms for the production of foreign (heterologous) proteins. Saccharomyces cerevisiae has usually been the yeast of choice, but an increasing number of alternative non-Saccharomyces yeasts has now become accessible for modern molecular genetics techniques. Some of them exhibit certain favourable traits such as high-level secretion or very strong and tightly regulated promoters, offering significant advantages over traditional bakers' yeast. In the present work, the current status of Kluyveromyces lactis, Yarrowia lipolytica, Hansenula polymorpha and Pichia pastoris (the best-known alternative yeast systems) is reviewed. The advantages and limitations of these systems are discussed in relation to S. cerevisiae.Spanish Society for Microbiolog

Seroprevalence of SARS-CoV-2 Antibodies and Factors Associated with Seropositivity at the University of Salamanca: The DIANCUSAL Study

© 2021 by the authors[Background]: Systematic screening for antibodies against SARS-CoV-2 is a crucial tool for surveillance of the COVID-19 pandemic. The University of Salamanca (USAL) in Spain designed a project called “DIANCUSAL” (Diagnosis of New Coronavirus, COVID-19, in University of Salamanca) to measure antibodies against SARS-CoV-2 among its ~34,000 students and academic staff, as the influence of the university community in the spread of the SARS-CoV-2 pandemic in the city of Salamanca and neighboring towns hosting USAL campuses could be substantial.[Objective]: The aim of this study was to estimate the prevalence of SARS-CoV-2 antibodies among USAL students, professors and staff and to evaluate the demographic, academic, clinical and lifestyle and behavioral factors related to seropositivity.[Methodology]: The DIANCUSAL study is an ongoing university population-based cross-sectional study, with the work described herein conducted from July–October 2020. All USAL students, professors and staff were invited to complete an anonymized questionnaire. Seroprevalence of anti-SARS-CoV-2 antibodies was detected and quantified by using chemiluminescent assays for IgG and IgM. Principal findings: A total of 8197 (24.71%) participants were included. The mean age was 31.4 (14.5 SD) years, and 66.0% of the participants were female. The seroprevalence was 8.25% overall and was highest for students from the education campus (12.5%) and professors from the biomedical campus (12.6%), with significant differences among faculties (p = 0.006). Based on the questionnaire, loss of smell and fever were the symptoms most strongly associated with seropositivity, and 22.6% of seropositive participants were asymptomatic. Social distancing was the most effective hygiene measure (p = 0.0007). There were significant differences in seroprevalence between participants with and without household exposure to SARS-CoV-2 (p = 0.0000), but not between students who lived in private homes and those who lived in dormitories. IgG antibodies decreased over time in the participants with confirmed self-reported COVID-19 diagnoses.[Conclusions]: The analysis revealed an overall 8.25% seroprevalence at the end of October 2020, with a higher seroprevalence in students than in staff. Thus, there is no need for tailored measures for the USAL community as the official average seroprevalence in the area was similar (7.8% at 22 June and 12.4 at 15 November of 2020). Instead, USAL members should comply with public health measures.The DIANCUSAL (Diagnosis of New Coronavirus, COVID-19, in University of Salamanca) study was funded by a grant from the University of Salamanca (USAL)

Isolation, characterization and expression of genes encoding enzymes with lipase activity in Yarrowia lipolytica

Yarrowia lipolytica is a dimophic yeast with very high secretory capacities and able to grow in alkanes and fatty acids as the sole carbon source. Using PCR, we have isolated two DNA fragments that after sequencing show homology with fungal lipases. By genomic DNA subcloning, we have isolated a gene nominated YlLIP1 located on Y. lipolytica chromosome IV. The gene presents an ORF of 1458 bp which encodes a putative protein of 486 amino acids with an apparent molecular weight of 55.4 kDa. The nucleotide sequence of the 5' and 3' ends shows the consensus signals for translation initiation and transcription termination motifs in the yeast. A disruption of YlLIP1 has been performed. The deleted ΔYllip1 strain shows lower lipase activity in comparison to the wild type. The DNA encoding YlLip1p was expressed in Saccharomyces cerevisiae and Kluyveromyces lactis. Both yeast species are able to synthesize a functional enzyme under the control of GAL1 promoter. We are currently isolating the second gene, both in the wild type and in the ΔYllip1 strain

The lipase system of Yarrowia lipolytica

Among yeast species, Yarrowia lipolytica is one of the highest producers of extracellular proteins ( acid, neutral and alkaline proteases, ácid phosphatase, ribonucleases and lipases). Lipases ( triacylglycerol hydrolases) are important enzymes in fat metabolism, catalyzing the breakdown of triacilglycerols to free fatty acids and glycerol. Owing to the very low solubility of ther natural substrats, this hydrolysis is catalysed at the interfase beteween an insoluble substrat and the aqueous phase in which the enzyme is solubilized. This feature distinguishes them from esterases, which preferentially catalyze the hydrolisys of soluble esters in water.EU (BIO4- CT96-0003

Systematic Review and Meta-Analysis of Artemisinin Based Therapies for the Treatment and Prevention of Schistosomiasis

<div><h3>Background</h3><p>Chemotherapy based on repeated doses of praziquantel is still the most effective control strategy against Schistosomiasis, however artemisinin derivatives emerged as a family of compounds with schistomicide activity. The aim of the present work is to compare the efficacy of artemisinin-based therapies in the treatment and prophylaxis of human schistosomiasis. The design of this work involved a quantitative systematic review and meta-analysis.</p> <h3>Methodology/Principal Findings</h3><p>Retrieval of published studies was carried out through an electronic search of the PubMed (MEDLINE), EMBASE, Cochrane Library and CINAHL databases. This included reports comparing the therapeutic efficacy of artesunate alone, artesunate <em>plus</em> sulfadoxine-pyrimethamine and a combination of artemisinin derivatives <em>plus</em> praziquantel against praziquantel alone on different types of schistosomiasis. Moreover, studies on artesunate and artemether used as preventive drugs were also analyzed against placebo. The primary outcome measure for schistosomiasis treatment was “parasitological cure”, whereas for the prophylaxis the outcome evaluated was “infection rate”. Our results show that patients treated with artesunate alone have significantly lower cure rates than those treated with praziquantel (OR = 0.27 (95% C.I. 0.13–0.53; p<0.001)) and that the combined therapy of artesunate <em>plus</em> sulfadoxine-pyrimethamine is also significantly less effective than praziquantel treatment (OR = 0.14 (95% C.I. 0.02–0.92; p = 0.04)). However, the combination of an artemisinin derivatives <em>plus</em> praziquantel showed a higher cure rate than praziquantel monotherapy with OR = 2.07 (95% C.I. 1.27–3.36; p = 0.003). Finally, chemoprophylaxis with either artesunate (RR = 0.11 (95% C.I. 0.06–0.22; p<0.001)) or artemether (RR = 0.25 (95% C.I. 0.16–0.40; p<0.001)) was significantly better than a placebo in both cases.</p> <h3>Conclusions/Significance</h3><p>This meta-analysis confirms that artemisinin derivatives used in combination with praziquantel have the potential to increase the cure rates in schistosomiasis treatment, but not artesunate alone. It is also confirmed that repeated doses of artemisinin derivatives play a prophylactic role, significantly reducing the incidence of <em>Schistosoma japonicum</em> infections compared with placebo.</p> </div

Identification of genomic regions implicated in susceptibility to "Schistosoma mansoni" infection in a murine genetic model (backcross)

This dataset was intended to describe schistosomiasis severity in a backcross cohort and to study the genetic linkage analysis with parasitological, pathological and immunological variables.[Description of methods used for collection/generation of data] F1BX mice were infected with 150 ± 5 S. mansoni cercariae each mouse and nine weeks post-infection were euthanized. We considered 20 variables: granulomas; affected liver surface (mm2/cm2); the number of adult male and female worms; eggs per gram of liver and small intestine; eggs in liver and small intestine per female; CD4, CD8, CD45, CD220 in blood or spleen; IgG, IgG1, IgG2a, IgM antibodies. Multivariate models (cluster and principal component analyses and K-means) identified four levels of infection intensity in the cohort. The genetic regions associated with severity were assessed by massive genotyping and genetic linkage analysis using 961 informative SNPs.[Methods for processing the data] Mean and standard error in each variable, Kolmogorov-Smirnov test. ANOVA and Tukey’ test or Student t-test. The Pearson correlation coefficient (r) and Student t-test for the statistical significance. Multivariant models considering sex influence in worm recovery, eggs in the liver and intestine, fecundity, granulomas and the affected liver surface. All the variables were standardized to 0 mean and standard deviation to 1. Cluster analysis, dendrogram, Principal components analysis and conglomerates by k-means were used to generate clusters. Median proportions were performed. SIMFIT statistical package for Windows version 7.3.7 were used Massive genotyping and geneticlinkage analysis using 961 informative SNPs: The genetic distance based on the recombination frequencies between markers in the F1BX cohort was compared with http://cgd.jax.org/mousemapconverter using maximum-likelihood mapping with HM algorithm. The Haldane function was used with a step size of 2.5 cM and a genotyping error of 0.001. We used the LOD-score to calculate the statistical significance of the linkage of the QTLs found. LOD-score higher than 1.4 suggested linkage. The Ensembl bioinformatics tool (https://www.ensembl.org/index.html) was used to identify syntenic regions between mouse and human.Here we present the dataset used in our study entitled "Identification of genomic regions implicated in susceptibility to Schistosoma mansoni infection in a murine genetic model (backcross)". Thus, we crossed the C57BL/6 mouse strain with the CBA one and then the F1B6CBA females (C57 x CBA) were backcrossed with CBA males generating the F1BX cohort of the study. The study consists of the identification of genetic markers of schistosomiasis. High infection levels and severe liver fibrosis in schistosomiasis appear only in a few highly susceptible infected people. Schistosomiasis could be a complex trait disease and it could be possible to identify genetic markers associated with severity. This study uses a genetically heterogeneous back-cross cohort with genetically unique mice. A backcross (F1BX) mouse cohort was generated after two stages; firstly, we crossed a mouse strain (CBA/2J) susceptible to schistosomiasis with a resistant one (C57BL/6J) to generate the F1B6CBA mice; secondly, the F1BX mice were generated by backcrossing. F1B6CBA female mice with CBA/2J males. F1BX mice were infected with 150 ± 5 S. mansoni cercariae each mouse and nine weeks post-infection were euthanized. We considered 20 variables: granulomas; affected liver surface (mm2/cm2); the number of adult male and female worms; eggs per gram of liver and small intestine; eggs in liver and small intestine per female; CD4, CD8, CD45, CD220 in blood or spleen; IgG, IgG1, IgG2a, IgM antibodies. Multivariate models (cluster and principal component analyses and K-means) identified four levels of infection intensity in the cohort. The genetic regions associated with severity were assessed by massive genotyping and genetic linkage analysis using 961 informative SNPs.The main research project is: Red de Investigación Colaborativa en Enfermedades Tropicales (RICET) Ref.: RD16/0027/0018.Peer reviewe