A Novel Auxiliary Agarolytic Pathway Expands Metabolic Versatility in the Agar-Degrading Marine Bacterium Colwellia echini A3(T)

Marine microorganisms encode a complex repertoire of carbohydrate-active enzymes (CAZymes) for the catabolism of algal cell wall polysaccharides. While the core enzyme cascade for degrading agar is conserved across agarolytic marine bacteria, gain of novel metabolic functions can lead to the evolutionary expansion of the gene repertoire. Here, we describe how two less-abundant GH96 a-agarases harbored in the agar-specific polysaccharide utilization locus (PUL) of Colwellia echini strain A3(T) facilitate the versatility of the agarolytic pathway. The cellular and molecular functions of the a-agarases examined by genomic, transcriptomic, and biochemical analyses revealed that alpha-agarases of C. echini A3(T) create a novel auxiliary pathway. alpha-Agarases convert even-numbered neoagarooligo-saccharides to odd-numbered agaro- and neoagarooligosaccharides, providing an alternative route for the depolymerization process in the agarolytic pathway. Comparative genomic analysis of agarolytic bacteria implied that the agarolytic gene repertoire in marine bacteria has been diversified during evolution, while the essential core agarolytic gene set has been conserved. The expansion of the agarolytic gene repertoire and novel hydrolytic functions, including the elucidated molecular functionality of alpha-agarase, promote metabolic versatility by channeling agar metabolism through different routes. IMPORTANCE Colwellia echini A3(T) is an example of how the gain of gene(s) can lead to the evolutionary expansion of agar-specific polysaccharide utilization loci (PUL). C. echini A3(T) encodes two a-agarases in addition to the core beta-agarolytic enzymes in its agarolytic PUL. Among the agar-degrading CAZymes identified so far, only a few alpha-agarases have been biochemically characterized. The molecular and biological functions of two alpha-agarases revealed that their unique hydrolytic pattern leads to the emergence of auxiliary agarolytic pathways. Through the combination of transcriptomic, genomic, and biochemical evidence, we elucidate the complete alpha-agarolytic pathway in C. echini A3(T). The addition of alpha-agarases to the agarolytic enzyme repertoire might allow marine agarolytic bacteria to increase competitive abilities through metabolic versatility

Induction of Mutants with Ectopic Expression of Condensed Tannins

Leaves of 47,000 Lotus japonicus plants were screened using a butanol:HCl histochemical test to select “gain of function” mutants. These plants were progeny from L. japonicus lines which were transformed with T-DNA constructs containing either the maize Ds or Ac transposon (Thykjaer et al., 1995). Among 21 putative leaf tannin mutants, five (tan1-5) were characterized for synthesis of condensed tannins, leucocyanidin reductase activity and the presence of Ac and the selectable marker gene, nptII . A range of leaf tannin content among other Lotus species was also characterized

The Healthy Start project: a randomized, controlled intervention to prevent overweight among normal weight, preschool children at high risk of future overweight

BACKGROUND: Research shows that obesity prevention has to start early. Targeting interventions towards subgroups of individuals who are predisposed, but yet normal weight, may prove more effective in preventing overweight than interventions towards unselected normal weight subsets. Finally, interventions focused on other factors than diet and activity are lacking. The objectives were to perform a randomized, controlled intervention aiming at preventing overweight in children aged 2–6 years, who are yet normal weight, but have high predisposition for future overweight, and to intervene not only by improving diet and physical activity, but also reduce stress and improve sleep quality and quantity. METHODS/DESIGN: Based on information from the Danish National Birth Registry and administrative birth forms, children were selected based on having either a high birth weight, a mother who was overweight prior to pregnancy, or a familial low socioeconomic status. Selected children (n = 5,902) were randomized into three groups; an intervention group, a shadow control group followed in registers exclusively, and a control group examined at the beginning and at the end of the intervention. Approximately 21% agreed to participate. Children who presented as overweight prior to the intervention were excluded from this study (n = 92). In the intervention group, 271 children were included, and in the control group 272 were included. Information obtained from the shadow control group is on-going, but it is estimated that 394 children will be included. The intervention took place over on average 1½ year between 2009 and 2011, and consisted of optional individual guidance in optimizing diet and physical activity habits, reducing chronic stress and stressful events and improving sleep quality and quantity. The intervention also included participation in cooking classes and play arrangements. Information on dietary intake, meal habits, physical activity, sleep habits, and overall stress level was obtained by 4–7 day questionnaire diaries and objective measurements. DISCUSSION: If the Healthy Start project is effective in preventing excessive weight gain, it will provide valuable information on new determinants of obesity which should be considered in future interventions, and on new strategies to prevent development of overweight and obesity at an early age. TRIAL REGISTRATION: ClinicalTrials.gov, ID NCT01583335

Can legume synteny be useful in guiding the introgression of wild genes into cultivated peanut?

Edição do International Workshop Integrating Genomics Into Plant Breeding, Porto Alegre, out. 2007

Iron and ferritin accumulate in separate cellular locations in Phaseolus seeds

<p>Abstract</p> <p>Background</p> <p>Iron is an important micronutrient for all living organisms. Almost 25% of the world population is affected by iron deficiency, a leading cause of anemia. In plants, iron deficiency leads to chlorosis and reduced yield. Both animals and plants may suffer from iron deficiency when their diet or environment lacks bioavailable iron. A sustainable way to reduce iron malnutrition in humans is to develop staple crops with increased content of bioavailable iron. Knowledge of where and how iron accumulates in seeds of crop plants will increase the understanding of plant iron metabolism and will assist in the production of staples with increased bioavailable iron.</p> <p>Results</p> <p>Here we reveal the distribution of iron in seeds of three <it>Phaseolus </it>species including thirteen genotypes of <it>P. vulgaris</it>, <it>P. coccineus</it>, and <it>P. lunatus</it>. We showed that high concentrations of iron accumulate in cells surrounding the provascular tissue of <it>P. vulgaris </it>and <it>P. coccineus </it>seeds. Using the Perls' Prussian blue method, we were able to detect iron in the cytoplasm of epidermal cells, cells near the epidermis, and cells surrounding the provascular tissue. In contrast, the protein ferritin that has been suggested as the major iron storage protein in legumes was only detected in the amyloplasts of the seed embryo. Using the non-destructive micro-PIXE (Particle Induced X-ray Emission) technique we show that the tissue in the proximity of the provascular bundles holds up to 500 μg g^-1of iron, depending on the genotype. In contrast to <it>P. vulgaris </it>and <it>P. coccineus</it>, we did not observe iron accumulation in the cells surrounding the provascular tissues of <it>P. lunatus </it>cotyledons. A novel iron-rich genotype, NUA35, with a high concentration of iron both in the seed coat and cotyledons was bred from a cross between an Andean and a Mesoamerican genotype.</p> <p>Conclusions</p> <p>The presented results emphasize the importance of complementing research in model organisms with analysis in crop plants and they suggest that iron distribution criteria should be integrated into selection strategies for bean biofortification.</p

Can legume systeny be useful in guiding the introgression of wild genes into cultivated peanut?

Edição dos resumos do Workshop, Porto Alegre, RS, 2007

Real-time detection of TDP1 activity using a fluorophore-quencher coupled DNA-biosensor

Real-time detection of enzyme activities may present the easiest and most reliable way of obtaining quantitative analyses in biological samples. We present a new DNA-biosensor capable of detecting the activity of the potential anticancer drug target tyrosyl-DNA phosphodiesterase 1 (TDP1) in a very simple, high throughput, and real-time format. The biosensor is specific for Tdp1 even in complex biological samples, such as human cell extracts, and may consequently find future use in fundamental studies as well as a cancer predictive tool allowing fast analyses of diagnostic cell samples such as biopsies. TDP1 removes covalent 3'DNA adducts in DNA single-strand break repair. This enzymatic activity forms the basis of the design of the TDP1-biosensor, which consists of a short hairpin-forming oligonucleotide having a 5'fluorophore and a 3'quencher brought in close proximity by the secondary structure of the biosensor. The specific action of TDP1 removes the quencher, thereby enabling optical detection of the fluorophore. Since the enzymatic action of TDP1 is the only "signal amplification" the increase in fluorescence may easily be followed in real-time and allows quantitative analyses of TDP1 activity in pure enzyme fractions as well as in crude cell extracts. In the present study we demonstrate the specificity of the biosensor, its ability to quantitatively detect up- or down-regulated TDP1 activity, and that it may be used for measuring and for analyzing the mechanism of TDP1 inhibition

Exploring microbial diversity in Greenland Ice Sheet supraglacial habitats through culturing-dependent and -independent approaches

The microbiome of Greenland Ice Sheet supraglacial habitats is still underinvestigated, and as a result there is a lack of representative genomes from these environments. In this study, we investigated the supraglacial microbiome through a combination of culturing-dependent and -independent approaches. We explored ice, cryoconite, biofilm, and snow biodiversity to answer: (1) how microbial diversity differs between supraglacial habitats, (2) if obtained bacterial genomes reflect dominant community members, and (3) how culturing versus high throughput sequencing changes our observations of microbial diversity in supraglacial habitats. Genomes acquired through metagenomic sequencing (133 high-quality MAGs) and whole genome sequencing (73 bacterial isolates) were compared to the metagenome assemblies to investigate abundance within the total environmental DNA. Isolates obtained in this study were not dominant taxa in the habitat they were sampled from, in contrast to the obtained MAGs. We demonstrate here the advantages of using metagenome SSU rRNA genes to reflect whole-community diversity. Additionally, we demonstrate a proof-of-concept of the application of in situ culturing in a supraglacial setting