Design and performance of combined infrared canopy and belowground warming in the B4WarmED (Boreal Forest Warming at an Ecotone in Danger) experiment

Conducting manipulative climate change experiments in complex vegetation is challenging, given considerable temporal and spatial heterogeneity. One specific challenge involves warming of both plants and soils to depth. We describe the design and performance of an open‐air warming experiment called Boreal Forest Warming at an Ecotone in Danger (B4WarmED) that addresses the potential for projected climate warming to alter tree function, species composition, and ecosystem processes at the boreal‐temperate ecotone. The experiment includes two forested sites in northern Minnesota, USA, with plots in both open (recently clear‐cut) and closed canopy habitats, where seedlings of 11 tree species were planted into native ground vegetation. Treatments include three target levels of plant canopy and soil warming (ambient, +1.7 °C, +3.4 °C). Warming was achieved by independent feedback control of voltage input to aboveground infrared heaters and belowground buried resistance heating cables in each of 72‐7.0 m² plots. The treatments emulated patterns of observed diurnal, seasonal, and annual temperatures but with superimposed warming. For the 2009 to 2011 field seasons, we achieved temperature elevations near our targets with growing season overall mean differences (?T_below) of +1.84 °C and +3.66 °C at 10 cm soil depth and (?T^above) of +1.82 °C and +3.45 °C for the plant canopies. We also achieved measured soil warming to at least 1 m depth. Aboveground treatment stability and control were better during nighttime than daytime and in closed vs. open canopy sites in part due to calmer conditions. Heating efficacy in open canopy areas was reduced with increasing canopy complexity and size. Results of this study suggest the warming approach is scalable: it should work well in small‐statured vegetation such as grasslands, desert, agricultural crops, and tree saplings (<5 m tall).     

C-terminal amino acids are essential for human heat shock protein 70 dimerization

The human inducible heat shock protein 70 (hHsp70), which is involved in several major pathologies, including neurodegenerative disorders and cancer, is a key molecular chaperone and contributes to the proper protein folding and maintenance of a large number of protein structures. Despite its role in disease, the current structural knowledge of hHsp70 is almost exclusively based on its Escherichia coli homolog, DnaK, even though these two proteins only share ~50 % amino acid identity. For the first time, we describe a complete heterologous production and purification strategy that allowed us to obtain a large amount of soluble, full-length, and non-tagged hHsp70. The protein displayed both an ATPase and a refolding activity when combined to the human Hsp40. Multi-angle light scattering and bio-layer interferometry analyses demonstrated the ability of hHsp70 to homodimerize. The role of the C-terminal part of hHsp70 was identified and confirmed by a study of a truncated version of hHsp70 that could neither dimerize nor present refolding activity.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-014-0526-3) contains supplementary material, which is available to authorized users.     

First efficient CRISPR‐Cas9‐mediated genome editing in Leishmania parasites

Protozoan pathogens that cause leishmaniasis in humans are relatively refractory to genetic manipulation. In this work, we implemented the CRISPR‐Cas9 system in Leishmania parasites and demonstrated its efficient use for genome editing. The Cas9 endonuclease was expressed under the control of the Dihydrofolate Reductase‐Thymidylate Synthase (DHFR‐TS) promoter and the single guide RNA was produced under the control of the U6snRNA promoter and terminator. As a proof of concept, we chose to knockout a tandemly repeated gene family, the paraflagellar rod‐2 locus. We were able to obtain null mutants in a single round of transfection. In addition, we confirmed the absence of off‐target editions by whole genome sequencing of two independent clones. Our work demonstrates that CRISPR‐Cas9‐mediated gene knockout represents a major improvement in comparison with existing methods. Beyond gene knockout, this genome editing tool opens avenues for a multitude of functional studies to speed up research on leishmaniasis.     

Study of prolactin permeation through the pericardium and its bioavailability

The prolactin (PRL) permeation through the pericardium depending on the species of origin (porcine, bovine and ovine) was studied, and the parameters of its bioavailability were calculated. An in vitro model using pericardium as a natural membrane and Frantz cell method was applied. Significant differences in permeation were observed depending on the species of origin. Within 5 h, 17.5% of bovine PRL, 27.2% of porcine PRL and 90.3% of ovine PRL permeated the pericardium. The amount of permeated ovine PRL was 3.3-fold higher than porcine PRL and 5.2-fold higher than bovine PRL. The maximum concentration of permeated PRL was reached in the thirtieth minute of the experiment and was the highest for ovine PRL (C(max) = 677.21 μg/cm2) and the lowest for bovine PRL (C(max) = 259.97 μg/cm2). Bioavailability of PRL through the pericardium is 3.3-fold greater for ovine PRL in comparison to porcine or bovine PRL. The relative extent of bioavailability for bovine and ovine prolactin versus the porcine PRL standard was 85.6% and 229.3%, respectively.     

Efficient mammalian germline transgenesis by cis-enhanced <Emphasis Type="Italic">Sleeping Beauty</Emphasis> transposition

Heightened interest in relevant models for human disease increases the need for improved methods for germline transgenesis. We describe a significant improvement in the creation of transgenic laboratory mice and rats by chemical modification of Sleeping Beauty transposons. Germline transgenesis in mice and rats was significantly enhanced by in vitro cytosine-phosphodiester-guanine methylation of transposons prior to injection. Heritability of transgene alleles was also greater from founder mice generated with methylated versus non-methylated transposon. The artificial methylation was reprogrammed in the early embryo, leading to founders that express the transgenes. We also noted differences in transgene insertion number and structure (single-insert versus concatemer) based on the influence of methylation and plasmid conformation (linear versus supercoiled), with supercoiled substrate resulting in efficient transpositional transgenesis (TnT) with near elimination of concatemer insertion. Combined, these substrate modifications resulted in increases in both the frequency of transgenic founders and the number of transgenes per founder, significantly elevating the number of potential transgenic lines. Given its simplicity, versatility and high efficiency, TnT with enhanced Sleeping Beauty components represents a compelling non-viral approach to modifying the mammalian germline.     

Surgical porcine myocardial infarction model through permanent coronary occlusion

Using domestic pigs as an animal model, we here validated a reproducible and standardized myocardial infarction (MI) surgical model, to achieve the largest possible infarct extent with the lowest morbidity and mortality. To this end, we included several anesthetic and perisurgical precautions to minimize surgical complications. Mortality and morbidity rates were compared among groups of pigs that underwent permanent occlusion at different locations of either the left circumflex or left anterior descending artery. In addition, to compare the resulting MI between groups, data were collected by using cardiac biomarkers (including troponin I), electrocardiography, and echocardiography. These data were correlated to the final mean infarct size calculated by microscopic studies. Proximal occlusions lead to high mortality rates, whereas distal occlusions induced rather small MI areas. The optimal occlusion site in terms of morbidity, mortality, and lesion extent was the midpoint of the left anterior descending artery. In this group, only one pig died, and group cardiac data showed a rise in biomarker levels, marked left ventricular dysfunction on electrocardiography and echocardiography, and well-defined transmural MI in both ventricles. Infarct size quantitated through histologic studies revealed an average 15% ventricular lesion. Because interanimal variability in results from this group was negligible, we consider that the induced myocardial injury of this model is reliable.     

Applications of the ETS-NOCV method in descriptions of chemical reactions

The present study characterizes changes in the electronic structure of reactants during chemical reactions based on the combined charge and energy decomposition scheme, ETS-NOCV (extended transition state–natural orbitals for chemical valence). Decomposition of the activation barrier, ΔE ^#, into stabilizing (orbital interaction, ΔE _orb, and electrostatic, ΔE _elstat) and destabilizing (Pauli repulsion, ΔE _Pauli, and geometry distortion energy, ΔE _dist) factors is discussed in detail for the following reactions: (I) hydrogen cyanide to hydrogen isocyanide, HCN → CNH isomerization; (II) Diels-Alder cycloaddition of ethene to 1,3-butadiene; and two catalytic processes, i.e., (III) insertion of ethylene into the metal-alkyl bond using half-titanocene with phenyl-phenoxy ligand catalyst; and (IV) B–H bond activation catalyzed by an Ir-containing catalyst. Various reference states for fragments were applied in ETS-NOCV analysis. We found that NOCV-based deformation densities (Δρ _i) and the corresponding energies ΔE _orb(i) obtained from the ETS-NOCV scheme provide a very useful picture, both qualitatively and quantitatively, of electronic density reorganization along the considered reaction pathways. Decomposition of the barrier ΔE^# into stabilizing and destabilizing contributions allowed us to conclude that the main factor responsible for the existence of positive values of ΔE ^# for all processes (I, II, III and IV) is Pauli interaction, which is the origin of steric repulsion. In addition, in the case of reactions II, III and IV, a significant degree of structural deformation of the reactants, as measured by the geometry distortion energy, plays an important role. Depending on the reaction type, stabilization of the transition state (relatively to the reactants) originating either from the orbital interaction term or from electrostatic attraction can be of vital importance. Finally, use of the ETS-NOCV method to describe catalytic reactions allows extraction of information on the role of catalysts in determination of ΔE ^#.     

Impedance Analysis of Complex Formation Equilibria in Phosphatidylcholine Bilayers Containing Decanoic Acid or Decylamine

Bilayer lipid membranes composed of phosphatidylcholine and decanoic acid or phosphatidylcholine and decylamine were investigated using electrochemical impedance spectroscopy. Interaction between membrane components causes significant deviations from the additivity rule. Area, capacitance, and stability constant values for the complexes were calculated based on the model assuming 1:1 stoichiometry, and the model was validated by comparison of these values to experimental results. We established that phosphatidylcholine and decylamine form highly stable 1:1 complexes. In the case of decanoic acid-modified phosphatidylcholine membranes, complexes with stoichiometries other than 1:1 should be taken into consideration.     

Influence of cinnamon and catnip on the stereotypical pacing of oncilla cats (Leopardus tigrinus) in captivity

Nonhuman animals in captivity can experience environmental privation that results in their exhibiting abnormal behaviors. Environmental enrichment techniques can help improve their welfare. This study investigated the behavior of 8 zoo-housed oncilla cats (Leopardus tigrinus) in response to 2 odors (catnip and cinnamon) introduced individually into the animals' enclosures for 3 consecutive days. Proportion of scans spent engaging in stereotypical pacing were compared before, during, and after treatments. The addition of cinnamon reduced the proportion of pacing during and after enrichment (Wilcoxon: Z = 3.16, p < .001; Z = 3.16, p < .001, respectively), indicating a prolonged effect of the enrichment on the animals' behavior. Catnip appears to have elicited no significant difference in the stereotypic pacing before, during, or after the enrichment (Friedman: X(2) = 2.69; p = .260). The results highlight the potential use of cinnamon as a method of environmental enrichment for small captive-housed cats.