The genus Polyarthra in Lake Peipsi

In Lake Peipsi (Estonia) Polyarthra species often dominate the rotifer community, and are represented by 6 species: Polyarthra dolichoptera Idelson, P. longiremis Carlin, P. luminosa Kutikova, P. major Burckhardt, P. remata Skorikov, P. vulgaris Carlin. The highest diversity occurs in August–September, with P. remata and P. luminosa most abundant. Morphometric data including measurements of body, fins and various types of eggs are given. The seasonal development of the different species and their life cycles are considered.     

The role of endocytosis on the uptake kinetics of luciferin-conjugated cell-penetrating peptides

Cell-penetrating peptides (CPPs) are short cationic/amphipathic peptides that can be used to deliver a variety of cargos into cells. However, it is still debated which routes CPPs employ to gain access to intracellular compartments. To assess this, most previously conducted studies have relied on information which is gained by using fluorescently labeled CPPs. More relevant information whether the internalized conjugates are biologically available has been gathered using end-point assays with biological readouts. Uptake kinetic studies have shed even more light on the matter because the arbitrary choice of end-point might have profound effect how the results could be interpreted. To elucidate uptake mechanisms of CPPs, here we have used a bioluminescence based assay to measure cytosolic delivery kinetics of luciferin-CPP conjugates in the presence of endocytosis inhibitors. The results suggest that these conjugates are delivered into cytosol mainly via macropinocytosis; clathrin-mediated endocytosis and caveolae/lipid raft dependent endocytosis are involved in a smaller extent. Furthermore, we demonstrate how the involved endocytic routes and internalization kinetic profiles can depend on conjugate concentration in case of certain peptides, but not in case of others. The employed internalization route, however, likely dictates the intracellular fate and subsequent trafficking of internalized ligands, therefore emphasizing the importance of our novel findings for delivery vector development.     

Dilation of human atria: Increased diffusion restrictions for ADP,overexpression of hexokinase 2 and its coupling to oxidative phosphorylation in cardiomyocytes

Cardiac energy metabolism with emphasis on mitochondria was addressed in atrial tissue from patients with overload-induced atrial dilation. Structural remodeling of dilated (D) atria manifested as intracellular accumulation of fibrillar aggregates, lipofuscin, signs of myolysis and autophagy. Despite impaired complex I dependent respiration and increased diffusion restriction for ADP, no changes regarding adenylate and creatine kinase occurred. We observed 7-fold overexpression of HK2 gene in D atria with concomitant 2-fold greater activation of mitochondrial oxygen consumption by glucose, which might represent an adaption to increased energy requirements and impaired mitochondrial function by effectively joining glycolysis and oxidative phosphorylation.     

Transfection of Infectious RNA and DNA/RNA Layered Vectors of Semliki Forest Virus by the Cell-Penetrating Peptide Based Reagent PepFect6

Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to therapeutics. Recombinant viral vectors are usually constructed using methods of reverse genetics to obtain the genetic material of the viral vector. The physicochemical properties of DNA and RNA make them unable to access cells by themselves, and they require assistance to achieve intracellular delivery. Non-viral delivery vectors can be used for this purpose if they enable efficient intracellular delivery without interfering with the viral life cycle. In this report, we utilize Semliki Forest virus (genus alphavirus) based RNA and DNA vectors to study the transfection efficiency of the non-viral cell-penetrating peptide-based delivery vector PepFect6 in comparison with that of the cationic liposome-based Lipofectamine 2000, and assess their impact on viral replication. The optimal conditions for transfection were determined for both reagents. These results demonstrate, for the first time, the ability of PepFect6 to transport large (13-19 kbp) constructs across the cell membrane. Curiously, DNA molecules delivered using the PepFect6 reagent were found to be transported to the cell nucleus approximately 1.5 hours later than DNA molecules delivered using the Lipofectamine 2000 reagent. Finally, although both PepFect6 and Lipofectamine 2000 reagents can be used for alphavirus research, PepFect6 is preferred because it does not induce changes in the normal cellular phenotype and it does not affect the normal replication-infection cycle of viruses in previously transfected cells.     

Proteome analysis of chemically induced mouse liver tumors with different genotype

Mouse liver tumors frequently harbor mutations in Ha-ras, B-raf, or Ctnnb1 (encoding beta-catenin). We conducted a proteome analysis with protein extracts from normal mouse liver and from liver tumors which were induced by a single injection of N-nitrosodiethylamine (DEN) as initiator followed by multiple injections of two different polychlorinated biphenyls (PCBs) as tumor promoters, or corn oil as a control. Liver tumors were stratified into two classes: they were either mutated in Ctnnb1 and positive for the marker glutamine synthetase (GS(+)), or they lacked Ctnnb1 mutations and were therefore GS-negative (GS(-)). Proteome analysis by 2-DE and MS revealed 98 significantly deregulated proteins, 44 in GS(+) and 54 in GS(-) tumors. Twelve of these proteins showed expression changes in both tumor types, but only seven of them were deregulated in the same direction. Several of the identified enzymes could be assigned to fundamental metabolic or other cellular pathways with characteristically different alterations in GS(+) and GS(-) tumors such as ammonia and amino acid turnover, cellular energy supply, and calcium homeostasis. Our data suggest that GS(+) and GS(-) tumor cells show a completely different biology and use divergent evolutionary strategies to gain a selective advantage over normal hepatocytes.     

Potentilla L. sect.Rivales Wolf and related taxa in the Baltic states

Morphological variation ofPotentilla norvegica L.,P. heidenreichii Zimmeter andP. supina L. usually treated within the sectionRivales Wolf,P. recta L. (sect.Rectae Wolf),P. canescens Bess.,P. argentea L. s.l.,P. collina Wibel (sect.Argenteae Wolf) andP. goldbachii Rupr. (sect.Chrysanthae Wolf) was studied using multivariate statistical methods. According to k-means clustering,P. canescens stands closer toP. heidenreichii of the sect.Rivales than toP. argentea. P. collina, the other representative of sect.Argenteae, is not connected withP. canescens at all. Therefore,P. canescens should belong to sect.Rivales and not to sect.Argenteae. InPotentilla argentea s.l.,P. impolita Wahlenb.,P. argentea L. var.argentea, var.decumbens (Jord.)Lehm., var.demissa (Jord.) Lehm., var.grandiceps (Zimmeter) Rouy etE.G. Camus and var.tenerrima (Velen.) Wolf were identified.P. impolita specimens did not cluster out into a separate cluster as didP. collina, P. canescens andP. heidenreichii, but formed mixed clusters with different varieties ofP. argentea s.str. Therefore,P. impolita is not worthy of the rank of species and evidently not even that of subspecies, and should be treated as a variety—P. argentea var.incanescens (Opiz) Focke.     

Effects of prolonged drought on the anatomy of sun and shade needles in young Norway spruce trees

Predicted increases in the frequency and duration of drought are expected to negatively affect tree vitality, but we know little about how water shortage will influence needle anatomy and thereby the trees’ photosynthetic and hydraulic capacity. In this study, we evaluated anatomical changes in sun and shade needles of 20‐year‐old Norway spruce trees exposed to artificial drought stress. Canopy position was found to be important for needle structure, as sun needles had significantly higher values than shade needles for all anatomical traits (i.e., cross‐sectional needle area, number of tracheids in needle, needle hydraulic conductivity, and tracheid lumen area), except proportion of xylem area per cross‐sectional needle area. In sun needles, drought reduced all trait values by 10–40%, whereas in shade needles, only tracheid maximum diameter was reduced by drought. Due to the relatively weaker response of shade needles than sun needles in drought‐stressed trees, the difference between the two needle types was reduced by 25% in the drought‐stressed trees compared to the control trees. The observed changes in needle anatomy provide new understanding of how Norway spruce adapts to drought stress and may improve predictions of how forests will respond to global climate change.     

Whole genome amplification on DNA from filter paper blood spot samples: an evaluation of selected systems

As the number of single-nucleotide polymorphism (SNP) screening and other mutation scanning studies have increased explosively, following the development of high-throughput instrumentation, it becomes even more important to have sufficient template DNA. The source of DNA is often limited, especially in epidemiological studies, which require many samples as well as enough DNA to perform numerous SNP screenings or mutation scannings. Therefore, the aim is to solve the problem of stock DNA limitation. This need has been an important reason for the development of whole genome amplification (WGA) methods. Several systems are based on Phi29 polymerase multiple displacement amplification (MDA) or on DNA fragmentation (OmniPlex). Using TaqMan SNP genotyping assays, we have tested four WGA systems -- AmpliQ Genomic Amplifier Kit, GenomiPhi, Repli-g, and GenomePlex -- on DNA extracted from Guthrie cards to evaluate the amplification bias, concordance- and call rates, cost efficiency, and flexibility. All systems successfully amplified picograms of DNA from Guthrie cards to micrograms of product without loss of heterozygosity and with minimal allelic bias. A modified AmpliQ set up was chosen for further evaluation. In all, 2,000 SNP genotyping results from amplified and nonamplified samples were compared and the concordance rates between the samples were 99.7%. The call rate using the TaqMan system was 99.8%. DNA extracted from Guthrie cards and amplified with one of the four evaluated WGA systems is applicable in epidemiological genetic screenings. System choice should be based on requirements for system flexibility, product yield, and use in subsequent analysis.