Overwintering and migration behaviour of post-spawned Atlantic salmon Salmo salar in a large hydropower-regulated river and reservoir

Tracking 47 post-spawned adult Atlantic salmon Salmo salar L. in a hydropower-regulated river through autumn, winter and spring revealed that winter survival was 56% and 75% in two study years, respectively, with higher mortality of males than females (50% vs. 33% and 100% vs. 13%, respectively). Some kelts (n = 7) displayed nondirected movements that were interpreted as a reconditioning period for an average of 9–17 days prior to directed downstream movements indicating the initiation of migration. Survival after the initiation of migration in spring was 83% and 94% to the hydropower dam in the first and second study years, and decreased to 60 and 63%, respectively, after dam passage. There were no further losses in the downriver reach in the second year, with the first year having a cumulative survival estimate of 53% to the river mouth. Kelts approached the dam when the spillway gates were available as a passage option most of the time (64%–75%), but some kelts arrived at the dam or had not yet passed when spillways were closed (n = 6) and the only remaining passage option was restricted to the turbines. However, all but one kelt that must have passed via turbine were successful in reaching the river mouth. Migratory delay presumably due to searching behaviour caused by low water flow was estimated at approximately 6 days as migration rates were significantly slower in the reservoir (median ± s.e. 8.5 ± 2.5 km day⁻¹) than up- (29.7 ± 5.0 km day⁻¹) or downriver (22.1 ± 3.1 km day⁻¹). The proportion of time (median 30%) that kelts spent swimming upstream (searching behaviour) in the reservoir was a significant variable for migration success.     

The N2A region of titin has a unique structural configuration

The N2A segment of titin is a main signaling hub in the sarcomeric I-band that recruits various signaling factors and processing enzymes. It has also been proposed to play a role in force production through its Ca²⁺-regulated association with actin. However, the molecular basis by which N2A performs these functions selectively within the repetitive and extensive titin chain remains poorly understood. Here, we analyze the structure of N2A components and their association with F-actin. Specifically, we characterized the structure of its Ig domains by elucidating the atomic structure of the I81-I83 tandem using x-ray crystallography and computing a homology model for I80. Structural data revealed these domains to present heterogeneous and divergent Ig folds, where I81 and I83 have unique loop structures. Notably, the I81-I83 tandem has a distinct rotational chain arrangement that confers it a unique multi-domain topography. However, we could not identify specific Ca²⁺-binding sites in these Ig domains, nor evidence of the association of titin N2A components with F-actin in transfected C2C12 myoblasts or C2C12-derived myotubes. In addition, F-actin cosedimentation assays failed to reveal binding to N2A. We conclude that N2A has a unique architecture that predictably supports its selective recruitment of binding partners in signaling, but that its mechanical role through interaction with F-actin awaits validation.     

The pharmacokinetics and drug-drug interactions of ivermectin in Aedes aegypti mosquitoes

Mosquitoes are vectors of major diseases such as dengue fever and malaria. Mass drug administration of endectocides to humans and livestock is a promising complementary approach to current insecticide-based vector control measures. The aim of this study was to establish an insect model for pharmacokinetic and drug-drug interaction studies to develop sustainable endectocides for vector control. Female Aedes aegypti mosquitoes were fed with human blood containing either ivermectin alone or ivermectin in combination with ketoconazole, rifampicin, ritonavir, or piperonyl butoxide. Drug concentrations were quantified by LC-MS/MS at selected time points post-feeding. Primary pharmacokinetic parameters and extent of drug-drug interactions were calculated by pharmacometric modelling. Lastly, the drug effect of the treatments was examined. The mosquitoes could be dosed with a high precision (%CV: ≤13.4%) over a range of 0.01–1 μg/ml ivermectin without showing saturation (R²: 0.99). The kinetics of ivermectin were characterised by an initial lag phase of 18.5 h (CI_90%: 17.0–19.8 h) followed by a slow zero-order elimination rate of 5.5 pg/h (CI_90%: 5.1–5.9 pg/h). By contrast, ketoconazole, ritonavir, and piperonyl butoxide were immediately excreted following first order elimination, whereas rifampicin accumulated over days in the mosquitoes. Ritonavir increased the lag phase of ivermectin by 11.4 h (CI_90%: 8.7–14.2 h) resulting in an increased exposure (+29%) and an enhanced mosquitocidal effect. In summary, this study shows that the pharmacokinetics of drugs can be investigated and modulated in an Ae. aegypti animal model. This may help in the development of novel vector-control interventions and further our understanding of toxicology in arthropods.     

Neuronal Differentiation of LUHMES Cells Induces Substantial Changes of the Proteome

Neuronal cell lines are important model systems to study mechanisms of neurodegenerative diseases. One example is the Lund Human Mesencephalic (LUHMES) cell line, which can differentiate into dopaminergic‐like neurons and is frequently used to study mechanisms of Parkinson's disease and neurotoxicity. Neuronal differentiation of LUHMES cells is commonly verified with selected neuronal markers, but little is known about the proteome‐wide protein abundance changes during differentiation. Using mass spectrometry and label‐free quantification (LFQ), the proteome of differentiated and undifferentiated LUHMES cells and of primary murine midbrain neurons are compared. Neuronal differentiation induced substantial changes of the LUHMES cell proteome, with proliferation‐related proteins being strongly down‐regulated and neuronal and dopaminergic proteins, such as L1CAM and α‐synuclein (SNCA) being up to 1,000‐fold up‐regulated. Several of these proteins, including MAPT and SYN1, may be useful as new markers for experimentally validating neuronal differentiation of LUHMES cells. Primary midbrain neurons are slightly more closely related to differentiated than to undifferentiated LUHMES cells, in particular with respect to the abundance of proteins related to neurodegeneration. In summary, the analysis demonstrates that differentiated LUHMES cells are a suitable model for studies on neurodegeneration and provides a resource of the proteome‐wide changes during neuronal differentiation. (ProteomeXchange identifier PXD020044).     

PKD controls mitotic Golgi complex fragmentation through a Raf–MEK1 pathway

Before entering mitosis, the stacks of the Golgi cisternae are separated from each other, and inhibiting this process delays entry of mammalian cells into mitosis. Protein kinase D (PKD) is known to be involved in Golgi-to–cell surface transport by controlling the biogenesis of specific transport carriers. Here we show that depletion of PKD1 and PKD2 proteins from HeLa cells by small interfering RNA leads to the accumulation of cells in the G2 phase of the cell cycle and prevents cells from entering mitosis. We further provide evidence that inhibition of PKD blocks mitotic Raf-1 and mitogen-activated protein kinase kinase (MEK) activation, and, as a consequence, mitotic Golgi fragmentation, which could be rescued by expression of active MEK1. Finally, Golgi fluorescence recovery after photobleaching analyses demonstrate that PKD is crucial for the cleavage of the noncompact zones of Golgi membranes in G2 phase. Our findings suggest that PKD controls interstack Golgi connections in a Raf-1/MEK1–dependent manner, a process required for entry of the cells into mitosis.     

Assessing the Environmental Impact of Water Consumption by Energy Crops Grown in Spain

The environmental impact of the water consumption of four typical crop rotations grown in Spain, including energy crops, was analyzed and compared against Spanish agricultural and natural reference situations. The life cycle assessment (LCA) methodology was used for the assessment of the potential environmental impact of blue water (withdrawal from water bodies) and green water (uptake of soil moisture) consumption. The latter has so far been disregarded in LCA. To account for green water, two approaches have been applied: the first accounts for the difference in green water demand of the crops and a reference situation. The second is a green water scarcity index, which measures the fraction of the soil‐water plant consumption to the available green water. Our results show that, if the aim is to minimize the environmental impacts of water consumption, the energy crop rotations assessed in this study were most suitable in basins in the northeast of Spain. In contrast, the energy crops grown in basins in the southeast of Spain were associated with the greatest environmental impacts. Further research into the integration of quantitative green water assessment in LCA is crucial in studies of systems with a high dependence on green water resources.     

Differences in mRNA Expression,Protein Content,and Enzyme Activity of Superoxide Dismutases in Type II Pneumocytes of Acute and Chronic Lung Injury

The lung is protected against oxidative stress by a variety of antioxidants and type II pneumocytes seem to play an important role in antioxidant defense. Previous studies have shown that inhalation of NO₂ results in acute and chronic lung injury. How the expression and enzyme activity of antioxidant enzymes are influenced in type II cells of different inflammatory stages has yet not been studied. To elucidate this question, we exposed rats to 10 ppm NO₂ for 3 or 20 days to induce acute or chronic lung injury. From these and air-breathing rats, type II pneumocytes were isolated. The mRNA expression and protein content of CuZnSOD and MnSOD as well as total SOD-specific enzyme activity were determined. For the acute lung injury (3 d NO₂), the expression of CuZnSOD mRNA was significantly increased, while MnSOD expression was significantly reduced after 3 days of NO 2 exposure. For the chronic lung injury (20 d NO₂), CuZnSOD expression was still enhanced, while MnSOD expression was comparable to control. In parallel to CuZnSOD mRNA expression, the protein amount was significantly increased in acute and chronic lung injury however MnSOD protein content exhibited no intergroup differences. Total SOD enzyme activity showed a significant decrease after 3 days of NO₂ exposure and was similar to control after 20 days. We conclude that during acute and chronic lung injury in type II pneumocytes expression and protein synthesis of CuZnSOD and MnSOD are regulated differently.     

Chemical genetic screen in fission yeast reveals roles for vacuolar acidification,mitochondrial fission,and cellular GMP levels in lifespan extension

The discovery that genetic mutations in several cellular pathways can increase lifespan has lent support to the notion that pharmacological inhibition of aging pathways can be used to extend lifespan and to slow the onset of age‐related diseases. However, so far, only few compounds with such activities have been described. Here, we have conducted a chemical genetic screen for compounds that cause the extension of chronological lifespan of Schizosaccharomyces pombe. We have characterized eight natural products with such activities, which has allowed us to uncover so far unknown anti‐aging pathways in S. pombe. The ionophores monensin and nigericin extended lifespan by affecting vacuolar acidification, and this effect depended on the presence of the vacuolar ATPase (V‐ATPase) subunits Vma1 and Vma3. Furthermore, prostaglandin J₂ displayed anti‐aging properties due to the inhibition of mitochondrial fission, and its effect on longevity required the mitochondrial fission protein Dnm1 as well as the G‐protein‐coupled glucose receptor Git3. Also, two compounds that inhibit guanosine monophosphate (GMP) synthesis, mycophenolic acid (MPA) and acivicin, caused lifespan extension, indicating that an imbalance in guanine nucleotide levels impinges upon longevity. We furthermore have identified diindolylmethane (DIM), tschimganine, and the compound mixture mangosteen as inhibiting aging. Taken together, these results reveal unanticipated anti‐aging activities for several phytochemicals and open up opportunities for the development of novel anti‐aging therapies.     

Proteomic Differences Between Hepatocellular Carcinoma and Nontumorous Liver Tissue Investigated by a Combined Gel-based and Label-free Quantitative Proteomics Study

Proteomics-based clinical studies have been shown to be promising strategies for the discovery of novel biomarkers of a particular disease. Here, we present a study of hepatocellular carcinoma (HCC) that combines complementary two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography (LC-MS)-based approaches of quantitative proteomics. In our proteomic experiments, we analyzed a set of 14 samples (7 × HCC versus 7 × nontumorous liver tissue) with both techniques. Thereby we identified 573 proteins that were differentially expressed between the experimental groups. Among these, only 51 differentially expressed proteins were identified irrespective of the applied approach. Using Western blotting and immunohistochemical analysis the regulation patterns of six selected proteins from the study overlap (inorganic pyrophosphatase 1 (PPA1), tumor necrosis factor type 1 receptor-associated protein 1 (TRAP1), betaine-homocysteine S-methyltransferase 1 (BHMT)) were successfully verified within the same sample set. In addition, the up-regulations of selected proteins from the complements of both approaches (major vault protein (MVP), gelsolin (GSN), chloride intracellular channel protein 1 (CLIC1)) were also reproducible. Within a second independent verification set (n = 33) the altered protein expression levels of major vault protein and betaine-homocysteine S-methyltransferase were further confirmed by Western blots quantitatively analyzed via densitometry. For the other candidates slight but nonsignificant trends were detectable in this independent cohort. Based on these results we assume that major vault protein and betaine-homocysteine S-methyltransferase have the potential to act as diagnostic HCC biomarker candidates that are worth to be followed in further validation studies.Hepatocellular carcinoma (HCC)¹ currently is the fifth most common malignancy worldwide with an annual incidence up to 500 per 100,000 individuals depending on the geographic region investigated. Whereas 80% of new cases occur in developing countries, the incidence increases in industrialized nations including Western Europe, Japan, and the United States (1). To manage patients with HCC, tumor markers are very important tools for diagnosis, indicators of disease progression, outcome prediction, and evaluation of treatment efficacy. Several tumor markers have been reported for HCC, including α-fetoprotein (AFP) (2), Lens culinaris agglutinin-reactive fraction of AFP (AFP-L3) (3), and des-γ-carboxyl prothrombin (DCP) (4). However, none of these tumor markers show 100% sensitivity or specificity, which calls for new and better biomarkers.To identify novel biomarkers of HCC, many clinical studies using “omics”-based methods have been reported over the past decade (5–6). In particular, the proteomics-based approach has turned out to be a promising one, offering several quantification techniques to reveal differences in protein expression that are caused by a particular disease. In most studies, the well-established 2D-DIGE technique has been applied for protein quantification followed by identification via mass spectrometry (7–15). Even if the quantification is very accurate and sensitive in this gel-based approach, the relatively high amount of protein sample necessary for protein identification is the major disadvantage of this technique. Several mass-spectrometry-based quantitative studies using labeling-techniques like SILAC (stable isotope labeling by amino acids in cell culture) or iTRAQ (isobaric tags for relative and absolute quantification) have also been carried out for biomarker discovery of HCC (16–18). Here, the concomitant protein quantification and identification in a mass spectrometer allows high-throughput analyses. However, such experiments imply additional labeling reactions (in case of iTRAQ) or are limited to tissue culture systems (in case of SILAC). In the latter case, one can overcome the limitation by using the isotope-labeled proteins obtained from tissue culture as an internal standard added to a corresponding tissue sample. This approach is known as CDIT (culture-derived isotope tags) and was applied in a HCC study, very recently (19). Label-free proteomics approaches based on quantification by ion-intensities or spectral counting offer another possibility for biomarker discovery. These approaches are relatively cheap compared with the labeling approaches, because they do not require any labeling reagents and furthermore they allow for high-throughput and sensitive analyses in a mass spectrometer. A quantitative study of HCC using spectral counting has been reported (20), whereas to our knowledge an ion-intensity-based study has not been performed yet. Apart from these quantification strategies, protein alterations in HCC have been studied by MALDI imaging, as well. Here, the authors could show that based on its proteomic signature, hepatocellular carcinoma can be discriminated with high accuracy from liver metastasis samples or other cancer types (21) as well as liver cirrhosis (22). Based on these results, it could be assumed that MALDI imaging might be a promising alternative to standard histological methods in the future.Here, we report a quantitative proteomic study that combines two different techniques, namely the well-established 2D-DIGE approach and a label-free ion-intensity-based quantification via mass spectrometry and liquid chromatography. To our knowledge this is the first time such a combined study was performed with regard to hepatocellular carcinoma. By comparing the results of both studies, we aim to identify high-confident biomarker candidates of HCC, as gel- and LC-MS-based techniques are complementary. To verify the differential protein expressions detected in our proteomic studies we performed additional immunological verifications for selected proteins within two different sample sets (Fig. 1).Open in a separate windowFig. 1.Schematic representation of the applied workflow.