Polymorphism and genetic diversity of Isospora parnaitatiaiensis Silva,Rodrigues, Lopes,Berto, Luz,Ferreira & Lopes, 2015 (Eimeriidae) from antbirds (Thamnophilidae) in Brazil

Systematic Parasitology - Isospora parnaitatiaiensis Silva, Rodrigues, Lopes, Berto, Luz, Ferreira & Lopes, 2015 was identified from a new host, the plain antvireo Dysithamnus mentalis...     

Efficient homologous recombination with short length flanking fragments in Ku70 deficient Yarrowia lipolytica strains

In Yarrowia lipolytica, targeted gene replacement occurs only with long length (1 kb) homologous flanking fragments, as this yeast preferentially uses the non-homologous end-joining mechanism (NHEJ) for DNA repair over homologous recombination (HR). To improve the frequency of HR, we identified and disrupted the KU70 and KU80 genes responsible for double strand break repair in the NHEJ pathway in Y. lipolytica. In ku70? HR of URA3 marker at the ADE2 locus occurred with 43 % frequency with as little as 50 bp long flanking regions. The number of Ura⁺ transformants was reduced to 1 % of the Po1d (ura3-302) wild type-like strain level, regardless of the flanking fragment length. On the contrary, even though HR was not improved in ku80?, Ura⁺ transformants was 60 % lower compared to the wild type.     

Importance of the methyl-citrate cycle on glycerol metabolism in the yeast Yarrowia lipolytica

A novel approach to trigger lipid accumulation and/or citrate production in vivo through the inactivation of the 2-methyl-citrate dehydratase in Yarrowia lipolytica was developed. In nitrogen-limited cultures with biodiesel-derived glycerol utilized as substrate, the Δphd1 mutant (JMY1203) produced 57.7 g/L of total citrate, 1.6-fold more than the wild-type strain, with a concomitant glycerol to citrate yield of 0.91 g/g. Storage lipid in cells increased at the early growth stages, suggesting that inactivation of the 2-methyl-citrate dehydratase would mimic nitrogen limitation. Thus, a trial of JMY1203 strain was performed with glycerol under nitrogen-excess conditions. Compared with the equivalent nitrogen-limited culture, significant quantities of lipid (up to ∼31% w/w in dry weight, 1.6-fold higher than the nitrogen-limited experiment) were produced. Also, non-negligible quantities of citric acid (up to ∼26 g/L, though 0.57-fold lower than the nitrogen-limited experiment) were produced, despite remarkable nitrogen presence into the medium, indicating the construction of phenotype that constitutively accumulated lipid and secreted citrate in Y. lipolytica during growth on waste glycerol utilized as substrate.     

A constitutive expression system for <Emphasis Type="Italic">Pichia pastoris</Emphasis> based on the <Emphasis Type="Italic">PGK1</Emphasis> promoter

Objectives

To develop a new vector for constitutive expression in Pichia pastoris based on the endogenous glycolytic PGK1 promoter.

Results

P. pastoris plasmids bearing at least 415 bp of PGK1 promoter sequences can be used to drive plasmid integration by addition at this locus without affecting cell growth. Based on this result, a new P. pastoris integrative vector, pPICK2, was constructed bearing some features that facilitate protein production in this yeast: a ~620 bp PGK1 promoter fragment with three options of restriction sites for plasmid linearization prior to yeast transformation: a codon-optimized α-factor secretion signal, a new polylinker, and the kan marker for vector propagation in bacteria and selection of yeast transformants.

Conclusions

A new constitutive vector for P. pastoris represents an alternative platform for recombinant protein production and metabolic engineering purposes.

     

Effect of polymorphisms in the CD36 and STAT3 genes on different dietary interventions among patients with coronary artery disease: study protocol for a randomized controlled trial

BackgroundCardiovascular disease has become a major health problem, and it has been associated with both environmental and genetic factors. Studies have shown that the Mediterranean Diet (MeDiet), or its components such as nuts and olive oil, may be strongly associated with the improvement of cardiovascular risk factors in specific populations. The purpose of the GENUTRI study is to investigate the interaction of genetics with cardiovascular risk factors in a non-Mediterranean population with coronary artery disease (CAD) according to three different diets: rich in pecan nuts, in extra-virgin olive oil or a control diet.Methods/designThe GENUTRI study is a single-center, randomized, open-label, parallel-group, 12-week pragmatic clinical trial conducted in patients aged 40 to 80 years and diagnosed with CAD. A standardized questionnaire will be applied to data collection and a blood sample will be obtained for lipid, glycemic and inflammatory profile evaluation. Polymorphisms in the CD36 and STAT3 genes will be detected using the TaqMan® SNP Genotyping Assay. Patients will be allocated in three groups: group 1: 30 g/day of pecan nuts; group 2: 30 ml/day of olive oil; and group 3: control diet. The primary outcome will consist of changes in LDL-cholesterol (in mg/dl) after 12 weeks of intervention.DiscussionStudies have shown the beneficial effects of diets rich in nuts and olive oil mainly in the Mediterranean population. GENUTRI is a clinical trial focusing on the effects of nuts or olive oil supplementation in Brazilian individuals. Additionally, we will try to demonstrate that genetic polymorphisms linked to cardiovascular disease may modulate the effects of different diets on biochemical and inflammatory markers among these subjects.

Trial registration

ClinicalTrials.gov Identifier: {"type":"clinical-trial","attrs":{"text":"NCT02202265","term_id":"NCT02202265"}}NCT02202265 (registered on 18 July 2014: first version).     

Knockout of receptor for advanced glycation end‐products attenuates age‐related renal lesions

Pro‐aging effects of endogenous advanced glycation end‐products (AGEs) have been reported, and there is increasing interest in the pro‐inflammatory and ‐fibrotic effects of their binding to RAGE (the main AGE receptor). The role of dietary AGEs in aging remains ill‐defined, but the predominantly renal accumulation of dietary carboxymethyllysine (CML) suggests the kidneys may be particularly affected. We studied the impact of RAGE invalidation and a CML‐enriched diet on renal aging. Two‐month‐old male, wild‐type (WT) and RAGE^?/? C57Bl/6 mice were fed a control or a CML‐enriched diet (200 μg CML/g_food) for 18 months. Compared to controls, we observed higher CML levels in the kidneys of both CML WT and CML RAGE^?/? mice, with a predominantly tubular localization. The CML‐rich diet had no significant impact on the studied renal parameters, whereby only a trend to worsening glomerular sclerosis was detected. Irrespective of diet, RAGE^?/? mice were significantly protected against nephrosclerosis lesions (hyalinosis, tubular atrophy, fibrosis and glomerular sclerosis) and renal senile apolipoprotein A‐II (ApoA‐II) amyloidosis (p < 0.001). A positive linear correlation between sclerosis score and ApoA‐II amyloidosis score (r = 0.92) was observed. Compared with old WT mice, old RAGE^?/? mice exhibited lower expression of inflammation markers and activation of AKT, and greater expression of Sod2 and SIRT1. Overall, nephrosclerosis lesions and senile amyloidosis were significantly reduced in RAGE^?/? mice, indicating a protective effect of RAGE deletion with respect to renal aging. This could be due to reduced inflammation and oxidative stress in RAGE^?/? mice, suggesting RAGE is an important receptor in so‐called inflamm‐aging.     

Effect of acyl-CoA oxidase activity on the accumulation of gamma-decalactone by the yeast Yarrowia lipolytica: a factorial approach

beta-Oxidation is a cyclic pathway involved in the degradation of lipids. In yeast, it occurs in peroxisomes and the first step is catalyzed by an acyl-CoA oxidase (Aoxp). The yeast Yarrowia lipolytica possesses several genes (POX) coding for Aoxps. This study is based on the factorial analysis of results obtained with the many POX derivative strains that have been constructed previously. The effect of interactions between Aoxps on the acyl-CoA oxidase (Aox) activity was important even at the second order. We then investigated the effect of Aox activity on growth and lactone production. Aox activity was correlated with acidification of the medium by cells and with cellular growth but not with lactone production, although Aox activity on short chains was inversely correlated with lactone accumulation. Due to the poor correlation between Aox activity and lactone production, the modeling of this parameter gave no satisfactory results but growth depending on Aox activity was modeled.     

Rapid identification of malaria vaccine candidates based on alpha-helical coiled coil protein motif

To identify malaria antigens for vaccine development, we selected alpha-helical coiled coil domains of proteins predicted to be present in the parasite erythrocytic stage. The corresponding synthetic peptides are expected to mimic structurally "native" epitopes. Indeed the 95 chemically synthesized peptides were all specifically recognized by human immune sera, though at various prevalence. Peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice. These antibodies did not show significant cross reactions, i.e., they were specific for the original peptide, reacted with native parasite proteins in infected erythrocytes and several were active in inhibiting in vitro parasite growth. Circular dichroism studies indicated that the selected peptides assumed partial or high alpha-helical content. Thus, we demonstrate that the bioinformatics/chemical synthesis approach described here can lead to the rapid identification of molecules which target biologically active antibodies, thus identifying suitable vaccine candidates. This strategy can be, in principle, extended to vaccine discovery in a wide range of other pathogens.