Genetic dissection of vitamin E biosynthesis in tomato

Vegetables are critical for human health as they are a source of multiple vitamins including vitamin E (VTE). In plants, the synthesis of VTE compounds, tocopherol and tocotrienol, derives from precursors of the shikimate and methylerythritol phosphate pathways. Quantitative trait loci (QTL) for α-tocopherol content in ripe fruit have previously been determined in an Solanum pennellii tomato introgression line population. In this work, variations of tocopherol isoforms (α, β, γ, and δ) in ripe fruits of these lines were studied. In parallel all tomato genes structurally associated with VTE biosynthesis were identified and mapped. Previously identified VTE QTL on chromosomes 6 and 9 were confirmed whilst novel ones were identified on chromosomes 7 and 8. Integrated analysis at the metabolic, genetic and genomic levels allowed us to propose 16 candidate loci putatively affecting tocopherol content in tomato. A comparative analysis revealed polymorphisms at nucleotide and amino acid levels between Solanum lycopersicum and S. pennellii candidate alleles. Moreover, evolutionary analyses showed the presence of codons evolving under both neutral and positive selection, which may explain the phenotypic differences between species. These data represent an important step in understanding the genetic determinants of VTE natural variation in tomato fruit and as such in the ability to improve the content of this important nutriceutical.     

Alternative Prey and Abundance Covariance Switches an Intraguild Predator’s Functional Response

Positive or negative prey abundance covariances play an important role in determining prey preference of predators. The goal here was to understand how variations in abundance of two blowfly prey species, a native and a non-native species, influence the switching behavior and functional response of Chrysomya albiceps, an intraguild predatory blowfly, under laboratory conditions. The results suggest C. albiceps prefers to consume a native prey species rather than a non-native prey species. However, when prey densities covariate negatively, both species were consumed at the same rate, changing predator’s functional response from type II to type III. The conditions that trigger the switching behavior in blowfly communities are discussed in detail in this study.     

β-Glucan Induces Reactive Oxygen Species Production in Human Neutrophils to Improve the Killing of Candida albicans and Candida glabrata Isolates from Vulvovaginal Candidiasis

Vulvovaginal candidiasis (VVC) is among the most prevalent vaginal diseases. Candida albicans is still the most prevalent species associated with this pathology, however, the prevalence of other Candida species, such as C. glabrata, is increasing. The pathogenesis of these infections has been intensely studied, nevertheless, no consensus has been reached on the pathogenicity of VVC. In addition, inappropriate treatment or the presence of resistant strains can lead to RVVC (vulvovaginal candidiasis recurrent). Immunomodulation therapy studies have become increasingly promising, including with the β-glucans. Thus, in the present study, we evaluated microbicidal activity, phagocytosis, intracellular oxidant species production, oxygen consumption, myeloperoxidase (MPO) activity, and the release of tumor necrosis factor α (TNF-α), interleukin-8 (IL-8), IL-1β, and IL-1Ra in neutrophils previously treated or not with β-glucan. In all of the assays, human neutrophils were challenged with C. albicans and C. glabrata isolated from vulvovaginal candidiasis. β-glucan significantly increased oxidant species production, suggesting that β-glucan may be an efficient immunomodulator that triggers an increase in the microbicidal response of neutrophils for both of the species isolated from vulvovaginal candidiasis. The effects of β-glucan appeared to be mainly related to the activation of reactive oxygen species and modulation of cytokine release.     

Immobilization–stabilization of the lipase from Thermomyces lanuginosus: Critical role of chemical amination

This paper describes the immobilization and stabilization of the lipase from Thermomyces lanuginosus (TLL) on glyoxyl agarose. Enzymes attach to this support only by the reaction between several aldehyde groups of the support and several Lys residues on the external surface of the enzyme molecules at pH 10. However, this standard immobilization procedure is unsuitable for TLL lipase due to the low stability of TLL at pH 10 and its low content on Lys groups that makes that the immobilization process was quite slow. The chemical amination of TLL, after reversible immobilization on hydrophobic supports, has been shown to be a simple and efficient way to improve the multipoint covalent attachment of this enzyme. The modification enriches the enzyme surface in primary amino groups with low pKb, thus allowing the immobilization of the enzyme at lower pH values. The aminated enzyme was rapidly immobilized at pH 9 and 10, with activities recovery of approximately 70%. The immobilization of the chemically modified enzyme improved its stability by 5-fold when compared to the non-modified enzyme during thermal inactivation and by hundreds of times when the enzyme was inactivated in the presence of organic solvents, being both glyoxyl preparations more stable than the enzyme immobilized on bromocyanogen.     

Phenotype and growth behavior of residual β-catenin-positive hepatocytes in livers of β-catenin-deficient mice

Signaling through the Wnt/β-catenin pathway is a crucial determinant of hepatic zonal gene expression, liver development, regeneration, and tumorigenesis. Transgenic mice with hepatocyte-specific knockout of Ctnnb1 (encoding β-catenin) have proven their usefulness in elucidating these processes. We now found that a small number of hepatocytes escape the Cre-mediated gene knockout in that mouse model. The remaining β-catenin-positive hepatocytes showed approximately 25% higher cell volumes compared to the β-catenin-negative cells and exhibited a marker protein expression profile similar to that of normal perivenous hepatocytes or hepatoma cells with mutationally activated β-catenin. Surprisingly, the expression pattern was observed independent of the cell's position within the liver lobule, suggesting a malfunction of physiological periportal repression of perivenously expressed genes in β-catenin-deficient liver. Clusters of β-catenin-expressing hepatocytes lacked expression of the gap junction proteins Connexin 26 and 32. Nonetheless, β-catenin-positive hepatocytes had no striking proliferative advantage, but started to grow out on treatment with phenobarbital, a tumor-promoting agent known to facilitate the formation of mouse liver adenoma with activating mutations of Ctnnb1. Progressive re-population of Ctnnb1 knockout livers with wild-type hepatocytes was seen in aged mice with a pre-cirrhotic phenotype. In these large clusters of β-catenin-expressing hepatocytes, perivenous-specific gene expression was re-established. In summary, our data demonstrate that the zone-specificity of a hepatocyte's gene expression profile is dependent on the presence of β-catenin, and that β-catenin provides a proliferative advantage to hepatocytes when promoted with phenobarbital, or in a pre-cirrhotic environment.     

Pseudomonas putida mutants in the exbBexbDtonB gene cluster are hypersensitive to environmental and chemical stressors

The genes in the exbBexbDtonB cluster of Pseudomonas putida DOT-T1E are co-transcribed. We have generated non-polar mutants in each of the genes by inserting an aphA3 cassette encoding kanamycin resistance. All three mutants show similar phenotypes: the mutants are unable to grow on minimal medium under iron deficiency conditions. Furthermore, regardless of iron conditions, all mutants are hypersensitive to antibiotics, p-hydroxybenzoate and toluene, chemicals that are extruded from the cell by efflux pumps. These findings are discussed in terms of the involvement of the TonB system in the energization of outer membrane functions necessary for the import or export of different compounds in P. putida.     

The phlebotomine fauna (Diptera: Psychodidae) of Guaraí, state of Tocantins,with an emphasis on the putative vectors of American cutaneous leishmaniasis in rural settlement and periurban areas

Phlebotomine sandflies were captured in rural settlement and periurbanareas of the municipality of Guaraí in the state of Tocantins (TO), an endemicarea of American cutaneous leishmaniasis (ACL). Forty-three phlebotomine specieswere identified, nine of which have already been recognised as ACL vectors.Eleven species were recorded for the first time in TO. Nyssomyiawhitmani was the most abundant species, followed byEvandromyia bourrouli, Nyssomyia antunesiand Psychodopygus complexus. The Shannon-Wiener diversity indexand the evenness index were higher in the rural settlement area than in theperiurban area. The evaluation of different ecotopes within the rural areashowed the highest frequencies of Ev. bourrouli and Ny.antunesi in chicken coops, whereas Ny. whitmanipredominated in this ecotope in the periurban area. In the rural settlementarea, Ev. bourrouli was the most frequently captured species inautomatic light traps and Ps. complexus was the most prevalentin Shannon trap captures. The rural settlement environment exhibited greaterphlebotomine biodiversity than the periurban area. Ps.complexus and Psychodopygus ayrozai naturallyinfected with Leishmania (Viannia) braziliensis wereidentified. The data identified Ny. whitmani as a potential ACLvector in the periurban area, whereas Ps. complexus was moreprevalent in the rural environment associated with settlements.     

Faecal genetic analysis to determine the presence and distribution of elusive carnivores: design and feasibility for the Iberian lynx

Noninvasive methods using genetic markers have been suggested as ways to overcome difficulties associated with documenting the presence of elusive species. We present and assess a novel, reliable and effective molecular genetic technique for the unequivocal genetic identification of faeces from the endangered Iberian lynx (Lynx pardinus). From mitochondrial DNA (mtDNA) cytochrome b and D-loop region sequences, we designed four species-specific primers (for products 130-161 bp long) that were considered to be likely to amplify degraded DNA. We compared two DNA extraction methods, various DNA amplification conditions and the robustness and specificity of the primer pairs with 87 lynx samples from 5 potentially different lynx populations and with 328 samples of other carnivore species. The utility of the identification technique was tested with faeces of different ages, with faeces from controlled field experiments, and with faeces collected from locales with possible lynx populations from throughout the state of Andalusia, Spain (8052 km2). Faecal mtDNA extraction was more efficient using PBS wash of the faeces instead of a faeces homogenate. Our assay increased from 92.6 to 99% efficiency with a second amplification and a reduction in template concentration to overcome polymerase chain reaction (PCR) inhibition. Our assay never produced false positives, and correctly identified all lynx faeces. Of 252 faeces samples of unknown species collected throughout Andalusia, 26.6% (from three different areas) were classified as Iberian lynx, 1.4% showed evidence of PCR inhibition and 1.2% were of uncertain origin. This method has proven to be a reliable technique that can be incorporated into large-scale surveys of Iberian lynx populations and exemplifies an approach that can easily be extended to other species.     

Using a logarithmic regression to identify the heart-rate threshold in cyclists

The purpose of this study was to establish an objective method for identifying the heart-rate threshold (HRT) in cyclists. Fifty-six male cyclists were tested on a cycle ergometer to volitional fatigue. Identification of the HRT used a heart-rate increase above a logarithmic regression line of best fit, coupled with the crossover of a linear regression line of best fit. The measures of Vco(2) and blood lactate for ventilatory threshold (VT) and lactate threshold (HLaT), respectively, were used as criterion measures to validate the HRT. Comparison of HRT with VT and HLaT showed significant associations (r = 0.98). Statistical variance between HRT, VT, and HLaT indicated no difference. From these findings, the logarithmic regression method provides an objective means to determine the HRT. Through this method, cyclists may obtain information for establishing accurate training levels and protocols.