Impacts of Nutrient Reduction on Coastal Communities

Eutrophication due to high anthropogenic nutrient loading has greatly impacted ecological processes in marine coastal waters and, therefore, much effort has been put into reducing nitrogen and phosphorus discharges into European and North-American waters. Nutrient enrichment usually resulted in increase of biomass and production of phytoplankton and microphytobenthos, often coinciding with shifts in species composition within the primary producer community. Consequences of increasing eutrophication for higher trophic levels are still being disputed, and even less is known about the consequences of nutrient reduction on coastal food webs. Here, we present 30-year concurrent field observations on phytoplankton, macrozoobenthos and estuarine birds in the Dutch Wadden Sea, which has been subject to decades of nutrient enrichment and subsequent nutrient reduction. We demonstrate that long-term variations in limiting nutrients (phosphate and silicon) were weakly correlated with biomass and more strongly with community structures of phytoplankton, macrozoobenthos and estuarine birds. Although we cannot conclusively determine if, and if so to what extent, nutrient enrichment and subsequent nutrient reduction actually contributed to the concurrent trends in these communities, it appears likely that part of the variance in the studied coastal communities is related to changes in nutrient loads. Our results imply that nutrient reduction measures should not ignore the potential consequences for policies aimed at bird conservation and exploitation of marine living resources. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Conquest of the deep,old and cold: an exceptional limpet radiation in Lake Baikal

Lake Baikal is the deepest, oldest and most speciose ancient lake in the world. The lake is characterized by high levels of molluscan species richness and endemicity, including the limpet family Acroloxidae with 25 endemic species. Members of this group generally inhabit the littoral zone, but have been recently found in the abyssal zone at hydrothermal vents and oil-seeps. Here, we use mitochondrial and nuclear data to provide a first molecular phylogeny of the Lake Baikal limpet radiation, and to date the beginning of intra-lacustrine diversification. Divergence time estimates suggest a considerably younger age for the species flock compared with lake age estimates, and the beginning of extensive diversification is possibly related to rapid deepening and cooling during rifting. Phylogenetic relationships and divergence time estimates do not clearly indicate when exactly the abyssal was colonized but suggest a timeframe coincident with the formation of the abyssal in the northern basin (Middle to Late Pleistocene).     

Remarkable heterogeneity in myosin heavy-chain composition of the human young masseter compared with young biceps brachii

Adult human jaw muscles differ from limb and trunk muscles in enzyme-histochemical fibre type composition. Recently, we showed that the human masseter and biceps differ in fibre type pattern already at childhood. The present study explored the myosin heavy-chain (MyHC) expression in the young masseter and biceps muscles by means of gel electrophoresis (GE) and immuno-histochemical (IHC) techniques. Plasticity in MyHC expression during life was evaluated by comparing the results with the previously reported data for adult muscles. In young masseter, GE identified MyHC-I, MyHC-IIa MyHC-IIx and small proportions of MyHC-fetal and MyHC-α cardiac. Western blots confirmed the presence of MyHC-I, MyHC-IIa and MyHC-IIx. IHC revealed in the masseter six isomyosins, MyHC-I, MyHC-IIa, MyHC-IIx, MyHC-fetal, MyHC α-cardiac and a previously not reported isoform, termed MyHC-IIx'. The majority of the masseter fibres co-expressed two to four isoforms. In the young biceps, both GE and IHC identified MyHC-I, MyHC-IIa and MyHC-IIx. MyHC-I predominated in both muscles. Young masseter showed more slow and less-fast and fetal MyHC than the adult and elderly masseter. These results provide evidence that the young masseter muscle is unique in MyHC composition, expressing MyHC-α cardiac and MyHC-fetal isoforms as well as hitherto unrecognized potential spliced isoforms of MyHC-fetal and MyHC-IIx. Differences in masseter MyHC expression between young adult and elderly suggest a shift from childhood to adulthood towards more fast contractile properties. Differences between masseter and biceps are proposed to reflect diverse evolutionary and developmental origins and confirm that the masseter and biceps present separate allotypes of muscle.     

The impact of confounders on the test performance of natriuretic peptides for cardiac dysfunction in subjects aged 80 and older

The hypothesis that natriuretic peptides could be used to identify ‘pancardiac’ damage has been proposed. However, multiple factors are known to influence circulating levels of natriuretic peptides, especially in the very old. Therefore, the impact of confounders on the association between natriuretic peptide levels and cardiac dysfunction was further explored in subjects aged 80 and older. A diagnostic cross-sectional study embedded within the BELFRAIL study (n = 567) was performed. Baseline BNP and NT-proBNP levels were measured and echocardiograms were performed at the subject's home. Cardiac dysfunction was defined as systolic dysfunction, valvular heart disease or isolated severe diastolic dysfunction. Several functional and structural echocardiographic parameters were independently related to circulating levels of natriuretic peptides. Cystatin C, BMI, β blockers, diabetes, heart frequency, usCRP, age and sex were identified as confounders. The prevalence of cardiac dysfunction was 17.1% in the subjects without and 30.8% in the subjects with chronic atrial fibrillation (CAF) or pacemaker (PM). Only in subjects with CAF or PM the C statistic for cardiac dysfunction improved after correcting for confounders. The post-test probability for a negative test (PTP−) ranged from 3.7% to 12.2% and the PTP+ ranged from 21.9% to 62.2% in different strata of confounders. According to these data adjusting for identified confounders does not improve the diagnostic accuracy of the natriuretic peptides for cardiac dysfunction, except in subjects with CAF or PM. Stratifying for individual confounders showed that different cut-off values could be used to optimize the diagnostic characteristics of natriuretic peptides.     

Alternatively folded proteins with unexpected beneficial functions

HAMLET (human α-lactalbumin made lethal to tumour cells) and its related partially unfolded protein-fatty acid complexes are novel biomolecular nanoparticles that possess relatively selective cytotoxic activities towards tumour cells. One of the key characteristics is the requirement for the protein to be partially unfolded, hence endowing native proteins with additional functions in the alternatively folded states. Beginning with the history of its discovery and development, the cellular targets that appear to be strongly correlated with tumour cell death are introduced in the present article.     

Multilevel grouped regression for analyzing self-reported health in relation to environmental factors: the model and its application

A method for modeling the relationship of polychotomous health ratings with predictors such as area characteristics, the distance to a source of environmental contamination, or exposure to environmental pollutants is presented. The model combines elements of grouped regression and multilevel analysis. The statistical model describes the entire response distribution as a function of the predictors so that any measure that summarizes this distribution can be calculated from the model. With the model, polychotomous health ratings can be used, and there is no need for a priori dichotomizing such variables which would lead to loss of information. It is described how, according to the model, various measures describing the response distribution are related to the exposure, and the confidence and tolerance intervals for these relationships are presented. Specific attention is given to the incorporation of random factors in the model. The application that here serves as an example, concerns annoyance from transportation noise. Exposure-response relationships obtained with the described method of modeling are presented for aircraft, road traffic, and railway noise.     

Structure and function of human α-lactalbumin made lethal to tumor cells (HAMLET)-type complexes

Human α-lactalbumin made lethal to tumor cells (HAMLET) and equine lysozyme with oleic acid (ELOA) are complexes consisting of protein and fatty acid that exhibit cytotoxic activities, drastically differing from the activity of their respective proteinaceous compounds. Since the discovery of HAMLET in the 1990s, a wealth of information has been accumulated, illuminating the structural, functional and therapeutic properties of protein complexes with oleic acid, which is summarized in this review. In vitro, both HAMLET and ELOA are produced by using ion-exchange columns preconditioned with oleic acid. However, the complex of human α-lactalbumin with oleic acid with the antitumor activity of HAMLET was found to be naturally present in the acidic fraction of human milk, where it was discovered by serendipity. Structural studies have shown that α-lactalbumin in HAMLET and lysozyme in ELOA are partially unfolded, 'molten-globule'-like, thereby rendering the complexes dynamic and in conformational exchange. HAMLET exists in the monomeric form, whereas ELOA mostly exists as oligomers and the fatty acid stoichiometry varies, with HAMLET holding an average of approximately five oleic acid molecules, whereas ELOA contains a considerably larger number (11- 48). Potent tumoricidal activity is found in both HAMLET and ELOA, and HAMLET has also shown strong potential as an antitumor drug in different in vivo animal models and clinical studies. The gain of new, beneficial function upon partial protein unfolding and fatty acid binding is a remarkable phenomenon, and may reflect a significant generic route of functional diversification of proteins via varying their conformational states and associated ligands.     

Single nucleotide polymorphism (SNP) allele frequency estimation in DNA pools using Pyrosequencing

Identifying the genetic variation underlying complex disease requires analysis of many single nucleotide polymorphisms (SNPs) in a large number of samples. Several high-throughput SNP genotyping techniques are available; however, their cost promotes the use of association screening with pooled DNA. This protocol describes the estimation of SNP allele frequencies in pools of DNA using the quantitative sequencing method Pyrosequencing (PSQ). PSQ is a relatively recently described high-throughput method for genotyping, allele frequency estimation and DNA methylation analysis based on the detection of real-time pyrophosphate release during synthesis of the complementary strand to a PCR product. The protocol involves the following steps: (i) quantity and quality assessment of individual DNA samples; (ii) DNA pooling, which may be undertaken at the pre- or post-PCR stage; (iii) PCR amplification of PSQ template containing the variable sequence region of interest; and (iv) PSQ to determine the frequency of alleles at a particular SNP site. Once the quantity and quality of individual DNA samples has been assessed, the protocol usually requires a few days for setting up pre-PCR pools, depending on sample number. After PCR amplification, preparation and analysis of PCR amplicon by PSQ takes 1 h per plate.     

An integrative view on vacuolar pH homeostasis in Arabidopsis thaliana: Combining mathematical modeling and experimentation

The acidification of plant vacuoles is of great importance for various physiological processes, as a multitude of secondary active transporters utilize the proton gradient established across the vacuolar membrane. Vacuolar-type H⁺-translocating ATPases and a pyrophosphatase are thought to enable vacuoles to accumulate protons against their electrochemical potential. However, recent studies pointed to the ATPase located at the trans-Golgi network/early endosome (TGN/EE) to contribute to vacuolar acidification in a manner not understood as of now. Here, we combined experimental data and computational modeling to test different hypotheses for vacuolar acidification mechanisms. For this, we analyzed different models with respect to their ability to describe existing experimental data. To better differentiate between alternative acidification mechanisms, new experimental data have been generated. By fitting the models to the experimental data, we were able to prioritize the hypothesis in which vesicular trafficking of Ca²⁺/H⁺-antiporters from the TGN/EE to the vacuolar membrane and the activity of ATP-dependent Ca²⁺-pumps at the tonoplast might explain the residual acidification observed in Arabidopsis mutants defective in vacuolar proton pump activity. The presented modeling approach provides an integrative perspective on vacuolar pH regulation in Arabidopsis and holds potential to guide further experimental work.