Site-specific chlorophyll reference conditions for lakes in Northern and Western Europe

The Water Framework Directive (WFD) requires EU Member States to assess the “ecological status” of surface waters. As a component of ecological status, many European countries are developing a classification scheme for chlorophyll concentrations as a measure of phytoplankton biomass. The chlorophyll classification must be based on the degree of divergence of a water body from an appropriate baseline or ‘reference condition’. This article describes the development of a series of regression models for predicting reference chlorophyll concentrations on a site-specific basis. For model development, a large dataset of European lakes considered to be in reference condition, 466 lakes in total, was assembled. Data were included from 12 European countries, but lakes from Northern and Western Europe dominated and made up 92% of all reference lakes. Data have been collated on chlorophyll concentration, altitude, mean depth, alkalinity, humic type, surface area and geographical region. Regression models were developed for estimating site-specific reference chlorophyll concentrations from significant predictor ‘typology’ variables. Reference chlorophyll concentrations were found to vary along a number of environmental gradients. Concentrations increased with colour and alkalinity and decreased with lake depth and altitude. Forward selection was used to identify independent explanatory variables in regression models for predicting site-specific reference chlorophyll concentrations. Depth was selected as an explanatory variable in all models. Alkalinity was included in models for low colour and humic lakes and altitude was included in models for low colour and very humic lakes. Uncertainty in the models was quite high and arises from errors in the data used to develop the models (including natural temporal and spatial variability in data) and also from additional explanatory variables not considered in the models, particularly nutrient concentrations, retention time and grazing. Despite these uncertainties, site-specific reference conditions are still recommended in preference to type-specific reference conditions, as they use the individual characteristics of a site known to influence phytoplankton biomass, rather than adopt standards set to generally represent a large population of lakes of a particular type. For this reason, site-specific reference conditions should result in reduced error in ecological status classifications, particularly for lakes close to typology boundaries.     

Salt-induced changes in the plasma membrane proteome of the halotolerant alga Dunaliella salina as revealed by blue native gel electrophoresis and nano-LC-MS/MS analysis

The halotolerant alga Dunaliella salina is a recognized model photosynthetic organism for studying plant adaptation to high salinity. The adaptation mechanisms involve major changes in the proteome composition associated with energy metabolism and carbon and iron acquisition. To clarify the molecular basis for the remarkable resistance to high salt, we performed a comprehensive proteomics analysis of the plasma membrane. Plasma membrane proteins were recognized by tagging intact cells with a membrane-impermeable biotin derivative. Proteins were resolved by two-dimensional blue native/SDS-PAGE and identified by nano-LC-MS/MS. Of 55 identified proteins, about 60% were integral membrane or membrane-associated proteins. We identified novel surface coat proteins, lipid-metabolizing enzymes, a new family of membrane proteins of unknown function, ion transporters, small GTP-binding proteins, and heat shock proteins. The abundance of 20 protein spots increased and that of two protein spots decreased under high salt. The major salt-regulated proteins were implicated in protein and membrane structure stabilization and within signal transduction pathways. The migration profiles of native protein complexes on blue native gels revealed oligomerization or co-migration of major surface-exposed proteins, which may indicate mechanisms of stabilization at high salinity.     

Assaying three new fungi against citrus mites in the laboratory,and a field trial

The Basidiomycotine fungi Meira geulakonigii, Meira argovae and Acaromyces ingoldii were assayed in the laboratory against five species of herbivorous mites: Phyllocoptruta oleivora (Eriophyidae), Panonychus citri, Eutetranychus orientalis, Tetranychus urticae and Tetranychus cinnabarinus (all four Tetranychidae). All fungi caused significantly high mortality rates (as compared to controls) after 14 days, some after 1 week. Phyllocoptruta oleivora was the most susceptible, showing >80% mortality even after 1 week. In a field trial, grapefruits sprayed either once a month or once a season with M. geulakonigii had significantly fewer P. oleivora and less damage than unsprayed fruit. These results suggest that M. geulakonigii may protect grapefruits against the injurious P. oleivora.     

Circulating plasma microRNAs in colorectal neoplasia: A pilot study in assessing response to therapy

IntroductionCurrent serological surveillance markers to monitor colorectal cancer (CRC) or colorectal advanced adenomas (CAA) are hampered by poor sensitivity and specificity. The aim of this study is to identify and validate a panel of plasma microRNAs which change in expression after resection of such lesions.MethodsA prospectively maintained colorectal surgery database was queried for patients in whom both pre- and post-procedural serum samples had been obtained. An initial screening analysis of CRC and CAA patients (5 each) was conducted using screening cards for 380 miRNAs. Four identified miRNAs were combined with a previously described panel of 7 miRNAs that were diagnostically predictive of CRC and CAA. Differential miRNA expression was assessed using quantitative real-time polymerase chain reaction(qRT-PCR).ResultsFifty patients were included (n = 27 CRC, n = 23 CAA). There was no difference in age, gender, or race profile of CRC patients compared to CAA patients. Six miRNA were significantly increased after CRC resection (miR-324, let7b, miR-454, miR-374a, miR-122, miR-19b, all p<0.05), while three miRNAs were significantly increased following CAA resection (miR-454, miR-374a, miR-122, all p<0.05). Three miRNA were increased in common for both (miR-454, miR-374a, miR-122).DiscussionThe expression of miRNAs associated with neoplasia (either CRC or CAA) was significantly increased following surgical resection or endoscopic removal of CRC or CAA. Future studies should focus on the evaluation of these miRNAs in CRC and CAA prognosis.     

Adenosine-derived non-phosphate antagonists for P2Y(1) purinoceptors

Novel type antagonists for P2Y(1) adenine nucleotide receptors were synthesized by coupling of adenosine 5'-OH group with oligo-aspartate chain via a carbonyl linker. All these conjugates (AdoOC(O)Asp(n), n = 1-4) inhibited the 2MeSADP-stimulated synthesis of inositol phosphates in 1321N1 human astrocytoma cells stably expressing human P2Y(1) receptors. This inhibitory effect followed the rank order AdoOC(O)Asp(2)> AdoOC(O)Asp(3)> AdoOC(O)Asp(1)> AdoOC(O)Asp(4) with antagonistic constant pA(2) = 5.4 for AdoOC(O)Asp(2). Potency of this non-phosphate inhibitor was comparable with the previously known adenosine 3',5'- and 2', 5'-bisphosphates. Chemical and biological stabilities of these novel adenosine derived antagonists of the nucleotide receptor provide perspectives of their pharmacological implication.     

SV40 immortalizes myogenic cells: DNA synthesis and mitosis in differentiating myotubes

Primary skeletal muscle myoblasts have a limited proliferative capacity in cell culture and cease to proliferate after several passages. We examined the effects of several oncogenes on the immortalization and differentiation of primary cultures of rat skeletal muscle myoblasts. Retroviruses containing a SV40 large T antigen (LT) gene very efficiently immortalize myogenic cells. The immortalized cell lines retain a very high differentiation capacity and form, in the appropriate culture conditions, a very dense network of muscle fibers. As in primary culture, cell fusion is associated with the synthesis of large amounts of muscle-specific proteins. However, unlike normal myoblasts (and previously established myogenic cell lines), nuclei in the multinucleated fibers of SV40-immortalized cells synthesize DNA and enter mitosis. Thus, withdrawal from DNA synthesis is not obligatory for cell fusion and biochemical differentiation. Using a retrovirus coding for a temperature-sensitive SV40 LT, myogenic cell lines were produced in which the SV40 LT could be inactivated by a shift from 33 degrees C to 39 degrees C. The inactivation of LT induced massive cell fusion and synthesis of muscle proteins. The nuclei in those fibers did not synthesize DNA, nor did they undergo mitosis. This approach enabled the reproducible establishment of myogenic cell lines from very small populations of myoblasts or single primary myogenic clones. Activated p53 also readily immortalized cells in primary muscle cultures, however the cells of eight out of the nine cell lines isolated had a fibroblastic morphology and could not be induced to form multinucleated fibers.     

Native homing endonucleases can target conserved genes in humans and in animal models

In recent years, both homing endonucleases (HEases) and zinc-finger nucleases (ZFNs) have been engineered and selected for the targeting of desired human loci for gene therapy. However, enzyme engineering is lengthy and expensive and the off-target effect of the manufactured endonucleases is difficult to predict. Moreover, enzymes selected to cleave a human DNA locus may not cleave the homologous locus in the genome of animal models because of sequence divergence, thus hampering attempts to assess the in vivo efficacy and safety of any engineered enzyme prior to its application in human trials. Here, we show that naturally occurring HEases can be found, that cleave desirable human targets. Some of these enzymes are also shown to cleave the homologous sequence in the genome of animal models. In addition, the distribution of off-target effects may be more predictable for native HEases. Based on our experimental observations, we present the HomeBase algorithm, database and web server that allow a high-throughput computational search and assignment of HEases for the targeting of specific loci in the human and other genomes. We validate experimentally the predicted target specificity of candidate fungal, bacterial and archaeal HEases using cell free, yeast and archaeal assays.     

A Fundamental Trade-off in Covalent Switching and Its Circumvention by Enzyme Bifunctionality in Glucose Homeostasis

Covalent modification provides a mechanism for modulating molecular state and regulating physiology. A cycle of competing enzymes that add and remove a single modification can act as a molecular switch between “on” and “off” and has been widely studied as a core motif in systems biology. Here, we exploit the recently developed “linear framework” for time scale separation to determine the general principles of such switches. These methods are not limited to Michaelis-Menten assumptions, and our conclusions hold for enzymes whose mechanisms may be arbitrarily complicated. We show that switching efficiency improves with increasing irreversibility of the enzymes and that the on/off transition occurs when the ratio of enzyme levels reaches a value that depends only on the rate constants. Fluctuations in enzyme levels, which habitually occur due to cellular heterogeneity, can cause flipping back and forth between on and off, leading to incoherent mosaic behavior in tissues, that worsens as switching becomes sharper. This trade-off can be circumvented if enzyme levels are correlated. In particular, if the competing catalytic domains are on the same protein but do not influence each other, the resulting bifunctional enzyme can switch sharply while remaining coherent. In the mammalian liver, the switch between glycolysis and gluconeogenesis is regulated by the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2). We suggest that bifunctionality of PFK-2/FBPase-2 complements the metabolic zonation of the liver by ensuring coherent switching in response to insulin and glucagon.