Glucose excretion by the symbiotic Chlorella of Spongilla fluviatilis

Chlorella sorokiniana strain 211-40c, a symbiotic Chlorella isolated from a freshwater sponge, excreted between 3% and 5% of assimilated ¹⁴CO₂ as glucose in the light, with a pH optimum around 5. This percentage increased when the illuminance was lowered (to 15% at 20 lx). Release of [¹⁴C]glucose continued in the dark and could be inhibited by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Net efflux of glucose occurred even at a concentration ratio of extracellular/intracellular glucose of 4. This, together with the sensitivity to FCCP, is taken as evidence for active transport. Exogenous [¹⁴C]glucose was taken up by the cells under conditions of net glucose efflux, showing uptake and excretion to take place simultaneously.Abbreviations FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - p.c. packed cells     

Inherited fungal symbionts enhance establishment of an invasive annual grass across successional habitats

Plants infected with vertically transmitted fungal endophytes carry their microbial symbionts with them during dispersal into new areas. Yet, whether seed-borne endophytes enhance the host plant’s ability to overcome colonisation barriers and to regenerate within invaded sites remains poorly understood. We examined how symbiosis with asexual endophytic fungi (Neotyphodium) affected establishment and seed loss to predators in the invasive annual grass Lolium multiflorum (Italian ryegrass) across contrasting successional plots. Italian ryegrass seeds with high and low endophyte incidence were sown into three communities: a 1-year-old fallow field, a 15-year-old grassland, and a 24-year-old forest, which conformed to an old-field chronosequence in the eastern Inland Pampa, Argentina. We found that endophyte infection consistently increased host population recruitment and reproductive output. Endophyte presence also enhanced aerial biomass production of ryegrass in a low recruitment year but not in a high recruitment year, suggesting that symbiotic effects on growth performance are density dependent. Endophyte presence reduced seed removal by rodents, although differential predation may not account for the increased success of infected grass populations. Overall, there was no statistical evidence for an endophyte-by-site interaction, indicating that the fungal endosymbiont benefitted host establishment regardless of large differences in biotic and abiotic environment among communities. Our results imply that hereditary endophytes may increase the chances for host grass species to pass various ecological filters associated with invasion resistance across a broad range of successional habitats.     

High throughput MLVA-16 typing for <Emphasis Type="Italic">Brucella</Emphasis> based on the microfluidics technology

Background  

Brucellosis, a zoonosis caused by the genus Brucella, has been eradicated in Northern Europe, Australia, the USA and Canada, but remains endemic in most areas of the world. The strain and biovar typing of Brucella field samples isolated in outbreaks is useful for tracing back source of infection and may be crucial for discriminating naturally occurring outbreaks versus bioterrorist events, being Brucella a potential biological warfare agent. In the last years MLVA-16 has been described for Brucella spp. genotyping. The MLVA band profiles may be resolved by different techniques i.e. the manual agarose gels, the capillary electrophoresis sequencing systems or the microfluidic Lab-on-Chip electrophoresis. In this paper we described a high throughput system of MLVA-16 typing for Brucella spp. by using of the microfluidics technology.     

Investigation of bacteriophage MS2 viral dynamics using model discrimination analysis and the implications for phage therapy

Lytic phages infect their bacterial hosts, use the host machinery to replicate, and finally lyse and kill their hosts, releasing progeny phages. Various mathematical models have been developed that describe these phage-host viral dynamics. The aim of this study was to determine which of these models best describes the viral dynamics of lytic RNA phage MS2 and its host Escherichia coli C-3000. Experimental data consisted of uninfected and infected bacterial cell densities, free phage density, and substrate concentration. Parameters of various models were either determined directly through other experimental techniques or estimated using regression analysis of the experimental data. The models were evaluated using a Bayesian-based model discrimination technique. Through model discrimination it was shown that phage-resistant cells inhibited the growth of phage population. It was also shown that the uninfected bacterial population was a quasispecies consisting of phage-sensitive and phage-resistant bacterial cells. When there was a phage attack the phage-sensitive cells died out and the phage-resistant cells were selected for and became the dominant strain of the bacterial population.     

Fluorescent, internally quenched, peptides for exploring the pH-dependent substrate specificity of cathepsin B.

Cathepsin B is a cysteine protease that in tumor tissues is localized in both acidic lysosomes and extracellular spaces. It can catalyze the cleavage of peptide bonds by two mechanisms: endoproteolytic attack with a pH optimum around 7.4, and attack from the C-terminus with a pH optimum at 4.5-5.5. In this work, seven fluorescent, internally quenched, decapeptides have been synthesized using the prototypical cathepsin B selective substrate Z-Phe-Arg-AMC as a lead, and used to identify the structural factors determining the susceptibility of peptides to hydrolysis at acidic and neutral pH values. Each peptide differs from the others in one amino acid (residue 6) and contains a highly fluorescent Nma group linked to the alpha-amino function of the N-terminal Orn residue and a Dnp group linked to the side chain of the Lys(8) residue acting as a quencher. Proteolytic cleavage was monitored by measuring the increase of fluorescence at 440 nm upon excitation at 340 nm, and the cleavage sites were determined by HPLC followed by ESI-MS analysis. Peptides containing Ala or Phe at position 6 are good substrates for the enzyme at both pH 5.0 and 7.4. By contrast, those containing Glu, Asp, Lys or Val are not cleaved at all by cathepsin B at pH 7.4, and are poorly hydrolyzed at pH 5.0. These findings provide new information for the rational design of cathepsin B-activated peptide-containing anticancer drugs.     

Sample size and sampling error in geometric morphometric studies of size and shape

Geometric morphometric studies are increasingly becoming common in systematics and palaeontology. The samples in such studies are often small, due to the paucity of material available for analysis. However, very few studies have tried to assess the impact of sampling error on analytical results. Here, this issue is addressed empirically using repeated randomized selection experiments to build progressively smaller samples from an original dataset of ∼400 vervet monkey (Cercopithecus aethiops) skulls. Size and shape parameters (including mean size and shape, size and shape variances, angles of allometric trajectories) that are commonly used in geometric morphometric studies, are estimated first in the original sample and then in the random subsamples. Estimates are then compared to give an indication of what is the minimum desirable sample size for each parameter. Mean size, standard deviation of size and variance of shape are found to be fairly accurate even in relatively small samples. In contrast, mean shapes and angles between static allometric trajectories are strongly affected by sampling error. If confirmed in other groups, our findings may have substantial implications for studies of morphological variation in present and fossil species. By performing rarefaction analyses like those presented in our study, morphometricians can be easily provided with important clues on how a simple but crucial factor like sample size can alter results of their studies.     

Perturbation of cytochrome P450, generation of oxidative stress and induction of DNA damage in Cyprinus carpio exposed in situ to potable surface water

Epidemiological evidence suggests a link between consumption of chlorinated drinking water and various cancers. Chlorination of water rich in organic chemicals produces carcinogenic organochlorine by-products (OBPs) such as trihalomethanes and haloacetic acids. Since the discovery of the first OBP in the 1970s, there have been several investigations designed to determine the biological effects of single chemicals or small artificial OBP combinations. However, there is still insufficient information regarding the general biological response to these compounds, and further studies are still needed to evaluate their potential genotoxic effects. In the current study, we evaluated the effect of three drinking water disinfectants on the activity of cytochrome P450 (CYP)-linked metabolizing enzymes and on the generation of oxidative stress in the livers of male and female Cyprinus carpio fish (carp). The fish were exposed in situ for up 20 days to surface water obtained from the Trasmene lake in Italy. The water was treated with 1-2 mg/L of either sodium hypochlorite (NaClO) or chlorine dioxide (ClO2) as traditional disinfectants or with a relatively new disinfectant product, peracetic acid (PAA). Micronucleus (MN) frequencies in circulating erythrocytes from the fish were also analysed as a biomarker of genotoxic effect. In the CYP-linked enzyme assays, a significant induction (up to a 57-fold increase in the deethylation of ethoxyresorufin with PAA treatment) and a notable inactivation (up to almost a 90% loss in hydroxylation of p-nitrophenol with all disinfectants, and of testosterone 2beta-hydroxylation with NaClO) was observed in subcellular liver preparations from exposed fish. Using the electron paramagnetic resonance (EPR) spectroscopy radical-probe technique, we also observed that CYP-modulation was associated with the production of reactive oxygen species (ROS). In addition, we found a significant increase in MN frequency in circulating erythrocytes after 10 days of exposure of fish to water treated with ClO2, while a non-significant six-fold increase in MN frequency was observed with NaClO, but not with PAA. Our data suggest that the use of ClO2 and NaClO to disinfect drinking water could generate harmful OBP mixtures that are able to perturb CYP-mediated reactions, generate oxidative stress and induce genetic damage. These data may provide a mechanistic explanation for epidemiological studies linking consumption of chlorinated drinking water to increased risk of urinary, gastrointestinal and bladder cancers.