Microscope-Based Imaging Platform for Large-Scale Analysis of Oral Biofilms

A microscopic method for noninvasively monitoring oral biofilms at the macroscale was developed to describe the spatial distribution of biofilms of different bacterial composition on bovine enamel surfaces (BES). For this purpose, oral biofilm was grown in situ on BES that were fixed at approximal sites of individual upper jaw acrylic devices worn by a volunteer for 3 or 5 days. Eubacteria, Streptococcus spp., and Fusobacterium nucleatum were stained using specific fluorescence in situ hybridization (FISH) probes. The resulting fluorescence signals were subsequently tested by confocal laser scanning microscopy (CLSM) and monitored by an automated wide-field microscope-based imaging platform (Scan∧R). Automated image processing and data analysis were conducted by microscope-associated software and followed by statistical evaluation of the results. The full segmentation of biofilm images revealed a random distribution of bacteria across the entire area of the enamel surfaces examined. Significant differences in the composition of the microflora were recorded across individual as well as between different enamel surfaces varying from sparsely colonized (47.26%) after 3 days to almost full surface coverage (84.45%) after 5 days. The enamel plates that were positioned at the back or in the middle of the oral cavity were found to be more suitable for the examination of biofilms up to 3 days old. In conclusion, automated microscopy combined with the use of FISH can enable the efficient visualization and meaningful quantification of bacterial composition over the entire sample surface. Due to the possibility of automation, Scan∧R overcomes the technical limitations of conventional CLSM.     

The halophilic alkalithermophile Natranaerobius thermophilus adapts to multiple environmental extremes using a large repertoire of Na+(K+)/H+ antiporters

Natranaerobius thermophilus is an unusual extremophile because it is halophilic, alkaliphilic and thermophilic, growing optimally at 3.5 M Na⁺, pH^55°C 9.5 and 53°C. Mechanisms enabling this tripartite lifestyle are essential for understanding how microorganisms grow under inhospitable conditions, but remain unknown, particularly in extremophiles growing under multiple extremes. We report on the response of N. thermophilus to external pH at high salt and elevated temperature and identify mechanisms responsible for this adaptation. N. thermophilus exhibited cytoplasm acidification, maintaining an unanticipated transmembrane pH gradient of 1 unit over the entire extracellular pH range for growth. N. thermophilus uses two distinct mechanisms for cytoplasm acidification. At extracellular pH values at and below the optimum, N. thermophilus utilizes at least eight electrogenic Na⁺(K⁺)/H⁺ antiporters for cytoplasm acidification. Characterization of these antiporters in antiporter-deficient Escherichia coli KNabc showed overlapping pH profiles (pH 7.8–10.0) and Na⁺ concentrations for activity ( K _0.5 values 1.0–4.4 mM), properties that correlate with intracellular conditions of N. thermophilus . As the extracellular pH increases beyond the optimum, electrogenic antiport activity ceases, and cytoplasm acidification is achieved by energy-independent physiochemical effects (cytoplasmic buffering) potentially mediated by an acidic proteome. The combination of these strategies allows N. thermophilus to grow over a range of extracellular pH and Na⁺ concentrations and protect biomolecules under multiple extreme conditions.     

Reproduction and Hybridisation in the Genus Rosa,Section Caninae (Ser.) Rehd.

Crossing experiments were carried out in order to investigate the importance of apogamy in relation to autogamy, geitonogamy and xenogamy in the reproductive biology of pentaploid members of the genus Rosa, section Caninae (Ser.) Rehd. About 10,000 seeds from 900 crossing experiments were examined, 4500 of them for both filling of the grain and viability of the embryo. The results were compared with experimental reports and literature dating from 1753 to 1995. Apomictic reproduction was shown to be possible in all species of the section tested, but viable seed yields were only about 5% of those produced by xenogamy. This contradicts the hypothesis of Fagerlind (1940,1944,1945,1950) and Custafsson and Hakansson (1942), but supports the results of Matsson (1912) and Schwertschlager (1915). The number of viable seeds increased in the order apogamy, autogamy, geitonogamy to xenogamy. Reciprocal crossings showed significant differences in the number of seeds produced in the hip. This contradicts the hypothesis on the genomic constitution of the pentaploid Caninae with unbalanced heterogamy, as advocated by Hurst (1927) and Custafsson and Hakansson (1942).     

Changes in heart rate variability of athletes during a training camp.

Heart rate variability (HRV) reflects regulatory processes of the cardiovascular system and reveals fractal characteristics. In this paper we investigated standard HRV parameters and scaling characteristics in ten athletes before, during, and after a 2-week training camp to assess the effects of short-term overtraining on cardiovascular control. High-resolution ECGs were recorded over 30 min under resting conditions 1 week before the training camp, after 1 week of training in the camp, and after 3-4 days of recovery. Standard HRV analysis was performed according to Task Force recommendations. Scaling characteristics were assessed, applying detrended fluctuation analysis (DFA). Standard HRV analysis showed significant changes in meanNN and rmssd during the training camp. DFA revealed three distinct regions of scale-invariance and significant alterations during the training camp. In conclusion, HRV might be used to monitor the training state in athletes.     

Isolation and characterization of a xanthophyll-rich fraction from the thylakoid membrane of Dunaliella salina(green algae).

Long-term acclimation to irradiance stress (HL) of the green alga Dunaliella salina Teod. (UTEX 1644) entails substantial accumulation of zeaxanthin along with a lowering in the relative amount of other pigments, including chlorophylls and several carotenoids. This phenomenon was investigated with wild type and the zea1 mutant of D. salina, grown under conditions of low irradiance (LL), or upon acclimation to irradiance stress (HL). In the wild type, the zeaxanthin to chlorophyll (Zea/Chl)(mol : mol) ratio was as low as 0.009 : 1 under LL and as high as 0.8 : 1 under HL conditions. In the zea1 mutant, which constitutively accumulates zeaxanthin and lacks antheraxanthin, violaxanthin and neoxanthin, the Zea/Chl ratio was 0.15 : 1 in LL and 0.57 : 1 in HL. The divergent Zea/Chl ratios were reflected in the coloration of the cells, which were green under LL and yellow under HL. In LL-grown cells, all carotenoids occurred in structural association with the Chl-protein complexes. This was clearly not the case in the HL-acclimated cells. A beta-carotene-rich fraction occurred as loosely bound to the thylakoid membrane and was readily isolated by flotation following mechanical disruption of D. salina. A zeaxanthin-rich fraction was specifically isolated, upon mild surfactant treatment and differential centrifugation, from the thylakoid membrane of either HL wild type or HL-zea1 mutant. Such differential extraction of beta-carotene and Zea, and their separation from the Chl-proteins, could not be obtained from the LL-grown wild type, although small amounts of Zea could still be differentially extracted from the LL-grown zea1 strain. It is concluded that, in LL-grown D. salina, xanthophylls (including most of Zea in the zea1 strain) are structurally associated with and stabilized by the Chl-proteins in the thylakoid membrane. Under HL-growth conditions, however, zeaxanthin appears to be embedded in the lipid bilayer, or in a domain of the chloroplast thylakoids that can easily be separated from the Chl-proteins upon mild surfactant treatment. In conclusion, this work provides biochemical evidence for the domain localization of accumulated zeaxanthin under irradiance-stress conditions in green algae, and establishes protocols for the differential extraction of this high-value pigment from the green alga D. salina.     

DNA break repair: refined rules of an already complicated game.

Of the many types of DNA-damage repair, this review concentrates on the aspects of DNA single- and double-strand break repair. Originally considered to represent separate routes based on distinct enzymatic machineries, it has recently been shown that these pathways converge and are interlinked at a number of points. Poly(ADP-ribose) polymerase-1 (PARP-1) is a central player in this complicated game. We present new data and our view on the mechanisms by which PARP-1 is guided to its respective interaction partners to coordinate or participate in repair or apoptosis.     

Plant responses to climatic extremes: within‐species variation equals among‐species variation

Within‐species and among‐species differences in growth responses to a changing climate have been well documented, yet the relative magnitude of within‐species vs. among‐species variation has remained largely unexplored. This missing comparison impedes our ability to make general predictions of biodiversity change and to project future species distributions using models. We present a direct comparison of among‐ versus within‐species variation in response to three of the main stresses anticipated with climate change: drought, warming, and frost. Two earlier experiments had experimentally induced (i) summer drought and (ii) spring frost for four common European grass species and their ecotypes from across Europe. To supplement existing data, a third experiment was carried out, to compare variation among species from different functional groups to within‐species variation. Here, we simulated (iii) winter warming plus frost for four grasses, two nonleguminous, and two leguminous forbs, in addition to eleven European ecotypes of the widespread grass Arrhenatherum elatius. For each experiment, we measured: (i) C/N ratio and biomass, (ii) chlorophyll content and biomass, and (iii) plant greenness, root ¹⁵N uptake, and live and dead tissue mass. Using coefficients of variation (CVs) for each experiment and response parameter, a total of 156 within‐ vs. among‐species comparisons were conducted, comparing within‐species variation in each of four species with among‐species variation for each seed origin (five countries). Of the six significant differences, within‐species CVs were higher than among‐species CVs in four cases. Partitioning of variance within each treatment in two of the three experiments showed that within‐species variability (ecotypes) could explain an additional 9% of response variation after accounting for the among‐species variation. Our observation that within‐species variation was generally as high as among‐species variation emphasizes the importance of including both within‐ and among‐species variability in ecological theory (e.g., the insurance hypothesis) and for practical applications (e.g., biodiversity conservation).