Staphylococcus aureus-Fibronectin Interactions with and without Fibronectin-Binding Proteins and Their Role in Adhesion and Desorption

Adhesion and residence-time-dependent desorption of two Staphylococcus aureus strains with and without fibronectin (Fn) binding proteins (FnBPs) on Fn-coated glass were compared under flow conditions. To obtain a better understanding of the role of Fn-FnBP binding, the adsorption enthalpies of Fn with staphylococcal cell surfaces were determined using isothermal titration calorimetry (ITC). Interaction forces between staphylococci and Fn coatings were measured using atomic force microscopy (AFM). The strain with FnBPs adhered faster and initially stronger to an Fn coating than the strain without FnBPs, and its Fn adsorption enthalpies were higher. The initial desorption was high for both strains but decreased substantially within 2 s. These time scales of staphylococcal bond ageing were confirmed by AFM adhesion force measurement. After exposure of either Fn coating or staphylococcal cell surfaces to bovine serum albumin (BSA), the adhesion of both strains to Fn coatings was reduced, suggesting that BSA suppresses not only nonspecific but also specific Fn-FnBP interactions. Adhesion forces and adsorption enthalpies were only slightly affected by BSA adsorption. This implies that under the mild contact conditions of convective diffusion in a flow chamber, adsorbed BSA prevents specific interactions but does allow forced Fn-FnBP binding during AFM or stirring in ITC. The bond strength energies calculated from retraction force-distance curves from AFM were orders of magnitude higher than those calculated from desorption data, confirming that a penetrating Fn-coated AFM tip probes multiple adhesins in the outermost cell surface that remain hidden during mild landing of an organism on an Fn-coated substratum, like that during convective diffusional flow.     

A magneto-optic route toward the in vivo diagnosis of malaria: preliminary results and preclinical trial data

We report the development of magneto-optic technology for the rapid quantitative diagnosis of malaria that may also be realizable in a noninvasive format. Hemozoin, the waste product of malarial parasitic action on hemoglobin, is produced in a form that under the action of an applied magnetic field gives rise to an induced optical dichroism characteristic of the hemozoin concentration. Here we show that precise measurement of this induced dichroism may be used to determine the level of malarial infection because this correlates, albeit in a complex manner throughout the infection cycle, with the concentration of hemozoin in the blood and tissues of infected patients. Under conservative assumptions for the production of hemozoin as a function of parasitemia, initial results indicate that the technique can match or exceed other current diagnostic techniques. The validity of the approach is confirmed by a small preliminary clinical trial on 13 patients, and measurements on live parasitized cells obtained from in vitro culture verify the possibility of producing in vivo diagnostic instrumentation.     

Different arabinogalactan proteins are present in carrot (Daucus carota) cell culture medium and in seeds

Arabinogalactan proteins (AGPs) were isolated by Yariv phenylglycoside precipitation from the medium of carrot ( Daucus carota L.) cell cultures and from carrot seeds. The isolates showed a different composition of AGPs. The medium AGPs contained an arabinose poor AGP fraction that had relatively high levels of glucuronic acid and rhamnose. In contrast the seed AGPs only contained arabinose and galactose-rich AGP fractions that had low levels of glucuronic acid. Linkage analysis on all fractions showed that most of the arabinose residues were terminally linked and that almost all galactose was present in the 1,3-, 1,6- and 1,3,6- form. The strongly branched type II arabinogalactans are characteristic of the carbohydrate part of AGPs. AGP characteristic amino acid residues as Hyp, Pro, Glx, Ser, Gly, Asx, Ala, Leu and Thr were detected in three different fractions.     

Female great tits Parus major do not increase their daily energy expenditure when incubating enlarged clutches

Several studies have shown that enlargement of clutches during incubation reduces the long-term survival of parents. In line with these findings, studies on the energetic costs of nocturnal incubation show an increase in energy expenditure with clutch enlargement. Studies on daily energy expenditure during incubation (DEE_inc), however, do not consistently show such a negative effect of clutch enlargement. To determine whether differential survival results from a direct increase in energy costs or rather from costs associated with compensatory behaviour, we studied the DEE_inc (kJ day⁻¹), change in body mass and nest attendance behaviour of free-living female great tits Parus major that incubated either control or experimentally enlarged clutches. DEE_inc did not differ between the two treatment groups, but was negatively related to mean ambient temperature over the 24-h measuring period, and to the fraction of daytime females spent on the nest. Controlling for these two factors, females incubating enlarged clutches did not spend more energy per 24 h period than females incubating control clutches. Clutch enlargement also did not affect body mass of incubating females, or their nest attendance behaviour. Yet, in the enlarged group body mass change and nest attentiveness were negatively correlated, suggesting that females responded differently to the experimental treatment and thereby preventing us from finding an effect of clutch enlargement.     

Cryo electron tomography of vitrified fibroblasts: microtubule plus ends in situ

Mouse embryonic fibroblasts (MEFs) are cells that have highly suitable biophysical properties for cellular cryo electron tomography. MEFs can be grown directly on carbon supported by EM grids. They stretch out and grow thinner than 500 nm over major parts of the cell, attaining a minimal thickness of 50 nm at their cortex. This facilitates direct cryo-fixation by plunge-freezing and high resolution cryo electron tomography. Both by direct cryo electron microscopy projection imaging and cryo electron tomography of vitrified MEFs we visualized a variety of cellular structures like ribosomes, vesicles, mitochondria, rough endoplasmatic reticulum, actin filaments, intermediate filaments and microtubules.MEFs are primary cells that closely resemble native tissue and are highly motile. Therefore, they are attractive for studying cytoskeletal elements. Here we report on structural investigations of microtubule plus ends. We were able to visualize single frayed protofilaments at the microtubule plus end in vitrified fibroblasts using cryo electron tomography. Furthermore, it appeared that MEFs contain densities inside their microtubules, although 2.5–3.5 times less than in neuronal cells [Garvalov, B.K., Zuber, B., Bouchet-Marquis, C., Kudryashev, M., Gruska, M., Beck, M., Leis, A., Frischknecht, F., Bradke, F., Baumeister, W., Dubochet, J., and Cyrklaff, M. 2006. Luminal particles within cellular microtubules. J. Cell Biol. 174, 759–765]. Projection imaging of cellular microtubule plus ends showed that 40% was frayed, which is two times more than expected when compared to microtubule growth and shrinkage rates in MEFs. This suggests that frayed ends might be stabilized in the cell cortex.     

Titin isoform switching is a major cardiac adaptive response in hibernating grizzly bears

The hibernation phenomenon captures biological as well as clinical interests to understand how organs adapt. Here we studied how hibernating grizzly bears (Ursus arctos horribilis) tolerate extremely low heart rates without developing cardiac chamber dilation. We evaluated cardiac filling function in unanesthetized grizzly bears by echocardiography during the active and hibernating period. Because both collagen and titin are involved in altering diastolic function, we investigated both in the myocardium of active and hibernating grizzly bears. Heart rates were reduced from 84 beats/min in active bears to 19 beats/min in hibernating bears. Diastolic volume, stroke volume, and left ventricular ejection fraction were not different. However, left ventricular muscle mass was significantly lower (300 +/- 12 compared with 402 +/- 14 g; P = 0.003) in the hibernating bears, and as a result the diastolic volume-to-left ventricular muscle mass ratio was significantly greater. Early ventricular filling deceleration times (106.4 +/- 14 compared with 143.2 +/- 20 ms; P = 0.002) were shorter during hibernation, suggesting increased ventricular stiffness. Restrictive pulmonary venous flow patterns supported this conclusion. Collagen type I and III comparisons did not reveal differences between the two groups of bears. In contrast, the expression of titin was altered by a significant upregulation of the stiffer N2B isoform at the expense of the more compliant N2BA isoform. The mean ratio of N2BA to N2B titin was 0.73 +/- 0.07 in the active bears and decreased to 0.42 +/- 0.03 (P = 0.006) in the hibernating bears. The upregulation of stiff N2B cardiac titin is a likely explanation for the increased ventricular stiffness that was revealed by echocardiography, and we propose that it plays a role in preventing chamber dilation in hibernating grizzly bears. Thus our work identified changes in the alternative splicing of cardiac titin as a major adaptive response in hibernating grizzly bears.     

Energy expenditure during flight in relation to body mass: effects of natural increases in mass and artificial load in Rose Coloured Starlings

Rose Coloured Starlings (Sturnus roseus) flew repeatedly for several hours in a wind tunnel while undergoing spontaneous variation in body mass. The treatments were as follows: flying unrestrained (U), with a control harness of 1.2% of their body mass (C), or with a harness of 7.4% of their body mass, which was either applied immediately before the flight (LS) or at least 9 days in advance (LL). Energy expenditure during flight (ef in W) was measured with the Doubly Labelled Water method. Flight costs in L(S) and LL were not significantly different and therefore were pooled (L). The harness itself did not affect ef, i.e. U and C flights were not different. ef was allometrically related with body mass m (in g). The slopes were not significantly different between the treatments, but ef was increased by 5.4% in L compared to C flights (log10(ef) = 0.050 + 0.47 x log10(m) for C, and log10(ef) = 0.073 + 0.47 x log10(m) for L). The difference in ef between C, LS and LL was best explained by taking the transported mass m transp (in g) instead of m into account (log10(ef) = -0.08 + 0.54 x log10(m transp)). Flight costs increased to a lesser extent than expected from interspecific allometric comparison or aerodynamic theory, regardless of whether the increase in mass occurred naturally or artificially. We did not observe an effect of treatment on breast muscle size and wingbeat frequency. We propose that the relatively low costs at a high mass are rather a consequence of immediate adjustments in physiology and/or flight behaviour than of long-term adaptations.     

Influence of adhesion to activated carbon particles on the viability of waterborne pathogenic bacteria under flow

In rural areas around the world, people often rely on water filtration plants using activated carbon particles for safe water supply. Depending on the carbon surface, adhering microorganisms die or grow to form a biofilm. Assays to assess the efficacy of activated carbons in bacterial removal do not allow direct observation of bacterial adhesion and the determination of viability. Here we propose to use a parallel plate flow chamber with carbon particles attached to the bottom plate to study bacterial adhesion to individual carbon particles and determine the viability of adhering bacteria. Observation and enumeration is done after live/dead staining in a confocal laser scanning microscope. Escherichiae coli adhered in higher numbers than Raoultella terrigena, except to a coconut-based carbon, which showed low bacterial adhesion compared to other wood-based carbon types. After adhesion, 83-96% of the bacteria adhering to an acidic carbon were dead, while on a basic carbon 54-56% were dead. A positively charged, basic carbon yielded 76-78% bacteria dead, while on a negatively charged coconut-based carbon only 32-37% were killed upon adhesion. The possibility to determine both adhesion as well as the viability of adhering bacteria upon adhesion to carbon particles is most relevant, because if bacteria adhere but remain viable, this still puts the water treatment system at risk, as live bacteria can grow and form a biofilm that can then be shedded to cause contamination.     

Mutation in the Fas pathway impairs CD8+ T cell memory

Fas death pathway is important for lymphocyte homeostasis, but the role of Fas pathway in T cell memory development is not clear. We show that whereas the expansion and contraction of CD8+ T cell response against Listeria monocytogenes were similar for wild-type (WT) and Fas ligand (FasL) mutant mice, the majority of memory CD8+ T cells in FasL mutant mice displayed an effector memory phenotype in the long-term in comparison with the mainly central memory phenotype displayed by memory CD8+ T cells in WT mice. Memory CD8+ T cells in FasL mutant mice expressed reduced levels of IFN-gamma and displayed poor homeostatic and Ag-induced proliferation. Impairment in CD8+ T cell memory in FasL mutant hosts was not due to defective programming or the expression of mutant FasL on CD8+ T cells, but was caused by perturbed cytokine environment in FasL mutant mice. Although adoptively transferred WT memory CD8+ T cells mediated protection against L. monocytogenes in either the WT or FasL mutant hosts, FasL mutant memory CD8+ T cells failed to mediate protection even in WT hosts. Thus, in individuals with mutation in Fas pathway, impairment in the function of the memory CD8+ T cells may increase their susceptibility to recurrent/latent infections.