Assessing the performance and efficiency of environmental DNA/RNA capture methodologies under controlled experimental conditions

Growing interest and affordability of environmental DNA and RNA (eDNA and eRNA) approaches for biodiversity assessments and monitoring of complex ecosystems have led to the emergence of manifold protocols for nucleic acids (NAs) isolation and processing. Although there is no consensus on a standardized workflow, the common practice for water samples is to concentrate NAs via filtration using varying pore size membranes. Using the smallest pore is assumed to be most efficient for NAs capture from a wide range of material (including sub-cellular particles); however, a trade-off must occur between detection of a meaningful molecular signal and cost/time effort when processing samples using fine pore membranes. Comparative studies involving formal efficiency assessments are lacking, which restricts informed decision-making around an optimized sampling approach for applications such as biosurveillance (i.e. detection and monitoring of target taxa—nuisance organisms, endangered and indicator taxa or other species of economic or cultural importance). Here, we present an experimental study using an easily cultured microalgal species (Alexandrium pacificum) to test different filter membranes for capturing NAs in the context of cost/time effort and cell fractions encountered in nature (whole cells, partially lysed and naked NAs). The results showed no statistically significant difference between membrane types for capturing target eDNA signal from intact and partially lysed cell treatments. In terms of time effort and volume processed, higher efficiency ratings were obtained with the larger pore size (5 μm) cellulose membranes. Positively charged nylon demonstrated enhanced capture of naked NAs, and especially eRNA signal, across treatments. Our findings support using coarse pore size filters for adequate capture of target NA signal (from both eDNA and eRNA) with less processing time. The framework presented here can provide a quick and robust feasibility check and comparative assessment of new and existing NA processing technologies, and allows sufficient control over multiple parameters, including physical–chemical water properties, temporal scales, and concentration and type of input material

Inorganic carbon concentrating mechanisms in free-living and symbiotic dinoflagellates and chromerids

Photosynthetic dinoflagellates are ecologically and biogeochemically important in marine and freshwater environments. However, surprisingly little is known of how this group acquires inorganic carbon or how these diverse processes evolved. Consequently, how CO2 availability ultimately influences the success of dinoflagellates over space and time remains poorly resolved compared to other microalgal groups. Here we review the evidence. Photosynthetic core dinoflagellates have a Form II RuBisCO (replaced by Form IB or Form ID in derived dinoflagellates). The in vitro kinetics of the Form II RuBisCO from dinoflagellates are largely unknown, but dinoflagellates with Form II (and other) RuBisCOs have inorganic carbon concentrating mechanisms (CCMs), as indicated by in vivo internal inorganic C accumulation and affinity for external inorganic C. However, the location of the membrane(s) at which the essential active transport component(s) of the CCM occur(s) is (are) unresolved; isolation and characterization of functionally competent chloroplasts would help in this respect. Endosymbiotic Symbiodiniaceae (in Foraminifera, Acantharia, Radiolaria, Ciliata, Porifera, Acoela, Cnidaria, and Mollusca) obtain inorganic C by transport from seawater through host tissue. In corals this transport apparently provides an inorganic C concentration around the photobiont that obviates the need for photobiont CCM. This is not the case for tridacnid bivalves, medusae, or, possibly, Foraminifera. Overcoming these long-standing knowledge gaps relies on technical advances (e.g., the in vitro kinetics of Form II RuBisCO) that can functionally track the fate of inorganic C forms

Influencia de la raza, estación del año y etapa de lactación sobre el contenido mineral de leche de búfalas

El objetivo de este estudio fue determinar la composición mineral de la leche de búfala producida en la Provincia de Corrientes, Argentina, así como investigar variaciones atribuibles al estadio de lactación, época del año y raza. Se trabajó con leche de 25 búfalas de razas Murrah, Mediterránea, Jafarabadi y mestizas ½ Murrah x ½ Mediterránea, de segunda a séptima lactación, durante 19 meses, totalizando 105 muestras. Los animales pertenecían a un tambo localizado en una región de clima subtropical húmedo, con precipitaciones anuales de 1.690 mm y temperatura media anual de 22°C. Se obtuvieron valores medios para Ca (1,12 ± 0,40 g.kg–1), P (0,99 ± 0,32 g.kg–1), Mg (0,08 ± 0,02 g.kg–1), K (0,92 ± 0,25 g.kg–1), Na (0,35 ± 0,11 g.kg–1), Cu (0,35 ± 0,16 mg.kg–1), Mn (0,27 ± 0,10 mg.kg–1), Zn (4,10 ± 1,40 mg.kg–1) y Fe (1,61 ± 0,61 mg.kg–1). Estos parámetros no revelaron diferencias significativas atribuibles a la raza de los animales con excepción del Mg. La época del año afectó la composición de los minerales, con excepción de Mg y Zn. Las etapas de lactación influenciaron sobre los valores de Ca, P, K y Cu. Los resultados obtenidos indican que el contenido mineral en leche de búfalas resulta considerablemente influenciado por factores regionales

Identifying and Characterizing Alternative Molecular Markers for the Symbiotic and Free-Living Dinoflagellate Genus Symbiodinium

Dinoflagellates in the genus Symbiodinium are best known as endosymbionts of corals and other invertebrate as well as protist hosts, but also exist free-living in coastal environments. Despite their importance in marine ecosystems, less than 10 loci have been used to explore phylogenetic relationships in this group, and only the multi-copy nuclear ribosomal Internal Transcribed Spacer (ITS) regions 1 and 2 have been used to characterize fine-scale genetic diversity within the nine clades (A–I) that comprise the genus. Here, we describe a three-step molecular approach focused on 1) identifying new candidate genes for phylogenetic analysis of Symbiodinium spp., 2) characterizing the phylogenetic relationship of these candidate genes from DNA samples spanning eight Symbiodinium clades (A–H), and 3) conducting in-depth phylogenetic analyses of candidate genes displaying genetic divergences equal or higher than those within the ITS-2 of Symbiodinium clade C. To this end, we used bioinformatics tools and reciprocal comparisons to identify homologous genes from 55,551 cDNA sequences representing two Symbiodinium and six additional dinoflagellate EST libraries. Of the 84 candidate genes identified, 7 Symbiodinium genes (elf2, coI, coIII, cob, calmodulin, rad24, and actin) were characterized by sequencing 23 DNA samples spanning eight Symbiodinium clades (A–H). Four genes displaying higher rates of genetic divergences than ITS-2 within clade C were selected for in-depth phylogenetic analyses, which revealed that calmodulin has limited taxonomic utility but that coI, rad24, and actin behave predictably with respect to Symbiodinium lineage C and are potential candidates as new markers for this group. The approach for targeting candidate genes described here can serve as a model for future studies aimed at identifying and testing new phylogenetically informative genes for taxa where transcriptomic and genomics data are available

Influencia del pastizal en la concentración de CLA y Omega 6 y 3 en leche de búfalas de Corrientes, Argentina

El objetivo del estudio fue investigar la influencia del pastizal natural sobre las concentraciones de ácido linoleico conjugado (CLA) y ácidos grasos Omega 6 y 3 en muestras de leche de búfala. Se realizaron dos ensayos durante 35 días con búfalas multíparas de razas Murrah y mestizas Murrah x Mediterránea, mantenidas sobre pastizal natural en primavera y otoño. El primero se realizó durante los meses de octubre y noviembre de 2007 con pastizal de primavera y régimen pluviométrico normal. El segundo entre los meses de mayo y junio de 2009, con pastizal de otoño y régimen pluviométrico menor. En promedio, los mayores valores de CLA (15,74 mg/g de grasa), Omega 6 (7,30 mg/g de grasa) y Omega 3 (4,45 mg/g de grasa), así como la más estrecha relación Omega 6/ Omega 3 (1,64 : 1 mg/g de grasa), fueron obtenidos en primavera, bajo un régimen pluviométrico normal. En el nordeste argentino, donde los animales son mantenidos principalmente sobre campo natural, resulta importante destacar que las condiciones climáticas afectan el crecimiento del pastizal natural, provocando disminución de los valores de los ácidos grasos CLA y Omega 6 y 3.

Lack of Adiponectin Drives Hyperosteoclastogenesis in Lipoatrophic Mice.

Long bones from mammals host blood cell formation and contain multiple cell types, including adipocytes. Physiological functions of bone marrow adipocytes are poorly documented. Herein, we used adipocyte-deficient PPARγ-whole body null mice to investigate the consequence of total adipocyte deficiency on bone homeostasis in mice. We first highlighted the dual bone phenotype of PPARγ null mice: one the one hand, the increased bone formation and subsequent trabecularization extending in the long bone diaphysis, due to the well-known impact of PPARγ deficiency on osteoblasts formation and activity; on the other hand, an increased osteoclastogenesis in the cortical bone. We then further explored the cause of this unexpected increased osteoclastogenesis using two independent models of lipoatrophy, which recapitulated this phenotype. This demonstrates that hyperosteoclastogenesis is not intrinsically linked to PPARγ deficiency, but is a consequence of the total lipodystrophy. We further showed that adiponectin, a cytokine produced by adipocytes and mesenchymal stromal cells is a potent inhibitor of osteoclastogenesis in vitro and in vivo. Moreover, pharmacological activation of adiponectin receptors by the synthetic agonist AdipoRon inhibited mature osteoclast activity both in mouse and human cells by blocking podosome formation through AMPK activation. Finally, we demonstrated that AdipoRon treatment blocks bone erosion in vivo in a murine model of inflammatory bone loss, providing potential new approaches to treat osteoporosis

Effects of temperature and salinity stress on DNA methylation in a highly invasive marine invertebrate, the colonial ascidian Didemnum vexillum

Environmentally induced epigenetic changes may contribute to phenotypic plasticity, increase adaptive potential in changing environments, and play a key role in the establishment and spread of invasive species in new habitats. In this study, we used methylation-sensitive amplified polymorphism (MSAP) to assess environmentally induced DNA methylation changes in a globally invasive clonal ascidian, Didemnum vexillum. We tested the effect of increasing temperature (19, 25 and 27 °C) and decreasing salinity (34, 32, 30, 28 and 26 practical salinity units (PSU)) on global DNA methylation, growth and survival rates. Exposure to 27 °C resulted in significant changes in DNA methylation over time. Growth also decreased in colonies exposed to high temperatures, suggesting they were under thermal stress. In contrast, no differences in growth nor DNA methylation patterns were observed in colonies exposed to a decreasing salinity gradient, potentially due to prior adaptation. The results of this study show that environmental stress can induce significant global DNA methylation changes in an invasive marine invertebrate on very rapid timescales, and that this response varies depending on the type, magnitude, and duration of the stressor. Changes in genomic DNA methylation and the rate of growth may act to ‘buy survival time’ under stressful conditions, expanding the distribution limits of this globally invasive species

Controlling light with light via structural transformations in metallic nanoparticles

Abstract: A nanoparticle undergoing light-induced transformations between structural phases with different optical properties is an inheritably bistable structure and this bistability can be used to create a resonator-free optical memory element, operating at very low power levels. We experimentally demonstrate this memory functionality using a film of gallium nanoparticles, and we present a method for differentially accessing the logic state of the memory using a modulated optical probe beam. Functional nanoparticles are expected to play an important role in highly integrated photonic devices as active elements of waveguiding and switching structures. In recent experiments on reversible light-and electron beam-induced structural transformations in gallium nanoparticles [1, 2] and in particular in the observation of of solid-to-solid light-induced phase transitions in a single nanoparticulate In this paper we report on the experimental demonstration of bistable memory functionality that can be achieved in nanoparticles by engaging transformations between different structural #74509 -$15.00 US