The mechanisms of refilling of xylem conduits and bleeding of tall birch during spring

Seasonal variations in osmolality and components of xylem sap in tall birch trees were determined using several techniques. Xylem sap was extracted from branch and trunk sections of 58 trees using the very rapid gas bubble-based jet-discharge method. The 5-cm long wood pieces were taken at short intervals over the entire tree height. The data show that large biphasic osmolality gradients temporarily exist within the conducting xylem conduits during leaf emergence (up to 272 mosmol x kg(-1) at the apex). These gradients (arising mainly from glucose and fructose) were clearly held within the xylem conduit as demonstrated by (1)H NMR imaging of intact twigs. Refilling experiments with benzene, sucrose infusion, electron and light microscopy, as well as (1)H NMR chemical shift microimaging provided evidence that the xylem of birch represents a compartment confined by solute-reflecting barriers (radial: lipid linings/lipid bodies; axial: presumably air-filled spaces). These features allow transformation of osmolality gradients into osmotic pressure gradients. Refilling of the xylem occurs by a dual mechanism: from the base (by root pressure) and from the top (by hydrostatic pressure generated by xylem-bound osmotic pressure). The generation of osmotic pressure gradients was accompanied by bleeding. Bleeding could be observed at a height of up to 21 m. Bleeding rates measured at a given height decreased exponentially with time. Evidence is presented that the driving force for bleeding is the weight of the static water columns above the bleeding point. The pressure exerted by the water columns and the bleeding volume depend on the water-filling status of (communicating) vessels.     

Comparison of classifier fusion methods for predicting response to anti HIV-1 therapy

Background

Analysis of the viral genome for drug resistance mutations is state-of-the-art for guiding treatment selection for human immunodeficiency virus type 1 (HIV-1)-infected patients. These mutations alter the structure of viral target proteins and reduce or in the worst case completely inhibit the effect of antiretroviral compounds while maintaining the ability for effective replication. Modern anti-HIV-1 regimens comprise multiple drugs in order to prevent or at least delay the development of resistance mutations. However, commonly used HIV-1 genotype interpretation systems provide only classifications for single drugs. The EuResist initiative has collected data from about 18,500 patients to train three classifiers for predicting response to combination antiretroviral therapy, given the viral genotype and further information. In this work we compare different classifier fusion methods for combining the individual classifiers.

Principal Findings

The individual classifiers yielded similar performance, and all the combination approaches considered performed equally well. The gain in performance due to combining methods did not reach statistical significance compared to the single best individual classifier on the complete training set. However, on smaller training set sizes (200 to 1,600 instances compared to 2,700) the combination significantly outperformed the individual classifiers (p<0.01; paired one-sided Wilcoxon test). Together with a consistent reduction of the standard deviation compared to the individual prediction engines this shows a more robust behavior of the combined system. Moreover, using the combined system we were able to identify a class of therapy courses that led to a consistent underestimation (about 0.05 AUC) of the system performance. Discovery of these therapy courses is a further hint for the robustness of the combined system.

Conclusion

The combined EuResist prediction engine is freely available at http://engine.euresist.org.     

Inducing Cross-Clade Neutralizing Antibodies against HIV-1 by Immunofocusing

Background

Although vaccines are important in preventing viral infections by inducing neutralizing antibodies (nAbs), HIV-1 has proven to be a difficult target and escapes humoral immunity through various mechanisms. We sought to test whether HIV-1 Env mimics may serve as immunogens.

Methodology/Principal Findings

Using random peptide phage display libraries, we identified the epitopes recognized by polyclonal antibodies of a rhesus monkey that had developed high-titer, broadly reactive nAbs after infection with a simian-human immunodeficiency virus (SHIV) encoding env of a recently transmitted HIV-1 clade C (HIV-C). Phage peptide inserts were analyzed for conformational and linear homology using computational analysis; some peptides mimicked various domains of the original HIV-C Env, such as conformational V3 loop epitopes and the conserved linear region of the gp120 C-terminus. Next, we devised a novel prime/boost strategy to test the immunogenicity of such phage-displayed peptides and primed mice only once with HIV-C gp160 DNA followed by boosting with mixtures of recombinant phages.

Conclusions/Significance

This strategy, which was designed to focus the immune system on a few Env epitopes (immunofocusing), not only induced HIV-C gp160 binding antibodies and cross-clade nAbs, but also linked a conserved HIV Env region for the first time to the induction of nAbs: the C-terminus of gp120. The identification of conserved antigen mimics may lead to novel immunogens capable of inducing broadly reactive nAbs.     

Density-dependent reproduction in the European rabbit: a consequence of individual response and age-dependent reproductive performance

Density dependence of reproduction has generally been proposed to be caused by habitat heterogeneity and by the individual response of reproductive output. However, a further mechanism might generate density dependence of average reproductive rates. High density situations might be associated with a high proportion of first-season breeders which often show a principally lower reproductive performance. We tested for the existence of the latter mechanism as well as for density-dependent individual changes of reproductive effort in a population of European rabbits living in a homogeneous grassland habitat. The study was conducted over a period of eleven years. Overall, a strong relationship between mean reproductive rates and the breeding density of females was apparent. All necessary conditions for the presence of a density-dependent effect caused by age-dependent reproduction were fulfilled: Fluctuations of breeding density were paralleled by variations in the proportion of one-year-old females. These one-year-old, first-season breeders showed a consistently lower reproductive performance than older females, which might be caused by their lower body mass and their lower social rank. However, we also found strong evidence for density-dependent response of individual reproductive effort: Individual changes in fecundity over successive years were explained by changes in the breeding density of females. The results suggest that density dependence of reproduction in European rabbits is due to an interaction of age-dependent reproductive performance together with short-term fluctuations in breeding density, and a density-dependent, individual based response of reproductive rates. We further conclude that the lower reproductive performance of first-season breeders in age-structured animal populations may contribute substantially to interannual, and under particular circumstances to density-dependent variations of mean reproductive rates.     

At‐line Determination of Glucose,Ammonia, and Acetate in High Cell Density Cultivations of Escherichia coli

A sophisticated measurement system for at‐line determination of the main C‐source glucose, the by‐product acetate, and the N‐source ammonium for high cell density cultivations (HCDC) of Escherichia coli K12 TG1 is presented. One flow diffusion technique (FDA) system is used for glucose measurement in the range of 0.5 up to 40 gL^–1 in the cultivation broth. Another FDA system detects the amount of the undesired by‐product acetate. The ammonium concentration in the range of 0.2 to 2.5 gL^–1 is determined on‐line by a flow injection analysis (FIA) system. For verification purposes, an HPLC system which is also connected to the bioreactor for at‐line measurements is utilized. Several HCDC with cell densities of more than 100 gL^–1 have been carried out. The courses of growth‐determining substrates have been detected at‐line. All used systems have shown an excellent compliance with off‐line measurements.     

Double quantum filtering homonuclear MAS NMR correlation spectra: a tool for membrane protein studies

13C homonuclear correlation spectra based on proton driven spin diffusion (PDSD) are becoming increasingly important for obtaining distance constraints from multiply labeled biomolecules by MAS NMR. One particular challenging situation arises when such constraints are to be obtained from spectra with a large natural abundance signal background which causes detrimental diagonal peak intensities. They obscure cross peaks, and furthermore impede the calculation of a buildup rates matrix which may be used to derive distance constraints, as carried out in "NMR crystallography". Here, we combine double quantum (DQ) filtering with 13C-13C dipolar assisted rotational resonance (DARR) experiments to yield correlation spectra free of natural abundance contributions. Two experimental schemes, using DQ filtering prior to evolution (DOPE), and after mixing (DOAM), have been evaluated. Diagonal peak intensities along the spectrum diagonal are removed completely, and crosspeaks close to the diagonal are easily identifiable. For DOAM spectra with negligible mixing times, it is possible to carry out 'assignment walks' which simplify peak identification substantially. The method is demonstrated on 13C-cys labeled proteorhodopsin, a 27 kDa membrane protein. The magnetization transfer characteristics were studied using buildup curves obtained on uniformly 13C labelled crystalline tripeptide MLF. Our data show that DQ filtered DARR experiments pave the way for obtaining through space constraints for structural studies on ligands, bound to membrane receptors, or on small fragments within large proteins.     

Discovery of triazolinone non-nucleoside inhibitors of HIV reverse transcriptase

Novel non-nucleoside inhibitors of HIV-RT that contain pyridazinone isosteres were prepared, and a series of triazolinones were found to be potent inhibitors of HIV replication. These compounds were active against several NNRTI-resistant virus strains. Pharmacokinetic studies indicated that inhibitor 7e has good bioavailability in rats. Several fragments of inhibitor 7c were prepared, and the binding of these compounds to HIV-RT was analyzed by surface plasmon resonance spectroscopy.     

Regulatory interactions between IRG resistance GTPases in the cellular response to Toxoplasma gondii

Members of the immunity-related GTPase (IRG) family are interferon-inducible resistance factors against a broad spectrum of intracellular pathogens including Toxoplasma gondii. The molecular mechanisms governing the function and regulation of the IRG resistance system are largely unknown. We find that IRG proteins function in a system of direct, nucleotide-dependent regulatory interactions between family members. After interferon induction but before infection, the three members of the GMS subfamily of IRG proteins, Irgm1, Irgm2 and Irgm3, which possess an atypical nucleotide-binding site, regulate the intracellular positioning of the conventional GKS subfamily members, Irga6 and Irgb6. Following infection, the normal accumulation of Irga6 protein at the parasitophorous vacuole membrane (PVM) is nucleotide dependent and also depends on the presence of all three GMS proteins. We present evidence that an essential role of the GMS proteins in this response is control of the nucleotide-bound state of the GKS proteins, preventing their GTP-dependent activation before infection. Accumulation of IRG proteins at the PVM has previously been shown to be associated with a block in pathogen replication: our results relate for the first time the enzymatic properties of IRG proteins to their role in pathogen resistance.     

Stomatal oscillations at small apertures: indications for a fundamental insufficiency of stomatal feedback-control inherent in the stomatal turgor mechanism.

Continuous measurements of stomatal aperture simultaneously with gas exchange during periods of stomatal oscillations are reported for the first time. Measurements were performed in the field on attached leaves of undisturbed Sambucus nigra L. plants which were subjected to step-wise increases of PPFD. Oscillations only occurred when stomatal apertures were small under high water vapour mole fraction difference between leaf and atmosphere (DeltaW). They consisted of periodically repeated opening movements transiently leading to very small apertures. Measurements of the area of the stomatal complex in parallel to the determination of aperture were used to record volume changes of guard cells even if stomata were closed. Stomatal opening upon a light stimulus required an antecedent guard cell swelling before a slit occurred. After opening of the slit the guard cells again began to shrink which, with some delay, led to complete closure. Opening and closing were rhythmically repeated. The time-lag until initial opening was different for each individual stoma. This led to counteracting movements of closely adjacent stomata. The tendency to oscillate at small apertures is interpreted as being a failure of smoothly damped feedback regulation at the point of stomatal opening: Volume changes are ineffective for transpiration if stomata are still closed; however, at the point of initial opening transpiration rate rises steeply. This discontinuity together with the rather long time constants inherent in the stomatal turgor mechanism makes oscillatory overshooting responses likely if at high DeltaW the 'nominal value' of gas exchange demands a small aperture.