Probiotic Bacteria May Become Dormant during Storage

The determination of bacterial viability in probiotic products is of economic, technological, and clinical significance. We compared four methods to enumerate three Bifidobacterium strains in fermented oat products during storage. A subpopulation of nonculturable cells retained a functional cell membrane typical of viable cells, indicating that probiotic bacteria become dormant during storage.     

Comparing mortality in renal patients on hemodialysis versus peritoneal dialysis using a marginal structural model

When comparing the causal effect of peritoneal dialysis (PD) and hemodialysis (HD) treatment on lowering mortality in renal patients, using observational data, it is necessary to adjust for different forms of confounding and informative censoring. Both the type of dialysis treatment that is started with and mortality are affected by baseline covariates. Longitudinal and baseline variables can affect both the probability of switching from one type of dialysis to the other, and mortality. Longitudinal and baseline variables can also affect the probability of receiving a kidney transplant, possibly causing informative censoring. Adjusting for longitudinal variables by including them as covariates in a regression model potentially causes bias, for instance by losing a possible indirect effect of dialysis on mortality via these longitudinal variables. Instead, we fitted a marginal structural model (MSM) to estimate the causal effect of dialysis type, adjusted for confounding and informative censoring. We used the MSM to compare the hazard of death as well as cumulative survival between the potential treatment trajectories "always PD" and "always HD" over time, conditional on age and diabetes mellitus status. We used inverse probability weighting (IPW) to fit the MSM.     

Rhythmic conidiation in the blue-light fungus Trichoderma pleuroticola

Trichoderma species conidiate in response to blue light, however, unlike in the blue-light model fungus Neurospora crassa, conidiation in Trichoderma spp. has been considered to be non-circadian. In this study we uncovered evidence for circadian conidiation in Trichoderma pleuroticola and identified orthologues of the key N. crassa clock components, wc-1 (blr-1) and frq.     

Egg and larval load assessment and its influence on oviposition behaviour of the leaf beetle Galerucella nymphaeae

The oviposition behaviour of the water-lily beetle Galerucella nymphaeae was examined. This species is a specialist herbivore on the floating leaves of nymphaeids Nymphaeaceae and especially on the yellow water-lily, Nuphar lutea. Females lay their eggs in clutches on the leaves, and after hatching, the larvae feed on the leaves. The quality of the leaves decreases quickly after the larvae hatch, and eventually the leaves will sink below the water surface, whereupon the eggs, 1st-instar larvae and pupae are killed by drowning. The influence of conspecific eggs, larvae and feeding tracks on the oviposition preferences of the beetles was tested. Females were allowed to choose between fresh leaves and leaves with conspecific eggs and larvae as well as between leaves with larvae and leaves with feeding tracks but no larvae. An attempt was also made to determine whether eggs and larvae affect the oviposition rate of females when they are not given the opportunity to oviposit on untouched leaves. The results indicate that females tended to avoid leaves with conspecific larvae or to exhibit a decreased oviposition rate on such leaves. Females also avoided conspecific eggs, although the oviposition rate was not influenced by the presence of conspecific eggs. When females were allowed to choose between leaves with larvae and leaves with feeding tracks, possible discrimination against leaves with larvae just fails to reach the 5% level.     

Development of a sensory neuronal cell model for the estimation of mild eye irritation

In an attempt to improve the in vitro test strategy for the estimation of eye irritation, a neuronal cell model has been developed, with cells expressing vanilloid receptor type 1 (VR1) nociceptors. The currently accepted method for measuring eye irritancy is the ethically and scientifically criticised Draize rabbit eye test, despite the fact that alternative in vitro methods are available which have proved to be reliable and reproducible for predicting severe ocular toxicity. However, no alternative tests for measuring neuronal stimulation have yet been developed, and the prediction of eye irritation in the mild range is therefore insufficient. VR1 is a nociceptor localised in C-fibre neurons innervating the cornea and the surrounding tissue, and it responds to potentially damaging stimuli by releasing Ca2+ into the cytoplasm. As a sensory endpoint, [Ca2+]i was measured in VR1 transfected cells, as well as in control cells. Short-term cell cytotoxicity studies (cell membrane rupture and morphological divergence) were used to determine the non-corrosive concentrations of the test chemicals. Preliminary results indicated that hygiene products used daily may induce eye irritation via VR1 nociceptors. The lowest toxic concentration (0.025%) of liquid hand soap, as determined by morphologic divergences of cells, generated an 80% increase in [Ca2+]i over the basal [Ca2+]i in VR1 transfected cells, whereas the non-specific [Ca2+]i increased by 33%. Furthermore, all the endpoints studied indicated that shampoo for children was less active than shampoo for adults. If this method is successfully validated with standardised reference chemicals, the model could complete the test battery of in vitro alternatives, resulting in the saving of thousands of laboratory animals.     

Adenovirus-mediated gene transfer of Lp-PLA2 reduces LDL degradation and foam cell formation in vitro

Oxidation of LDL generates biologically active platelet-activating factor (PAF)-like phospholipid derivatives, which have potent proinflammatory activity. These products are inactivated by lipoprotein-associated phospholipase A2 (Lp-PLA2), an enzyme capable of hydrolyzing PAF-like phospholipids. In this study, we generated an adenovirus (Ad) encoding human Lp-PLA2 and injected 10(8), 10(9), and 10(10) plaque-forming unit doses of Adlp-PLA2 and control AdlacZ intra-arterially into rabbits to achieve overexpression of Lp-PLA2 in liver and in vivo production of Lp-PLA2-enriched LDL. As a result, LDL particles with 3-fold increased Lp-PLA2 activity were produced with the highest virus dose. Increased Lp-PLA2 activity in LDL particles decreased the degradation rate in RAW 264 macrophages after standard in vitro oxidation to 60-80% compared with LDL isolated from LacZ-transduced control rabbits. The decrease was proportional to the virus dose and Lp-PLA2 activity. Lipid accumulation and foam cell formation in RAW 264 macrophages were also decreased when incubated with oxidized LDL containing the highest Lp-PLA2 activity. Inhibition of the Lp-PLA2 activity in the LDL particles led to an increase in lipid accumulation and foam cell formation. It is concluded that increased Lp-PLA2 activity in LDL attenuates foam cell formation and decreases LDL oxidation and subsequent degradation in macrophages.     

Influence of humic substances on bacterial and viral dynamics in freshwaters

Bacterial and viral abundances were measured in 24 lakes with dissolved organic carbon (DOC) concentrations ranging from 3 to 19 mg of C liter(-1). In addition, a laboratory experiment was performed to test the effects of different sources of carbon (i.e., glucose and fulvic acids) and nutrients on the dynamics of viruses and bacteria. In the lake survey, no correlation was found between virus abundance and DOC concentration, yet there was a significant positive correlation between bacterial abundance and DOC concentration. A negative correlation was found between the virus-to-bacteria ratio and DOC level. These results are in agreement with our findings in the laboratory, where virus counts were significantly lower in treatments with fulvic acid additions than in a control (mean, 67.4% +/- 6.5% of the control). Virus counts did not differ significantly among the control and treatments with glucose, indicating that it was the type of organic carbon and not quantity which had an impact on viruses. Results from this study suggest that the way viruses control bacterial assemblages in humic lakes is different from the mechanism in clear water systems.     

Invasion success of Fibrillanosema crangonycis, n.sp., n.g.: a novel vertically transmitted microsporidian parasite from the invasive amphipod host Crangonyx pseudogracilis

Parasitism is known to be an important factor in determining the success of biological invasions. Here we examine Crangonyx pseudogracilis, a North American amphipod invasive in the United Kingdom and describe a novel microsporidium, Fibrillanosema crangonycis n.sp., n.g. The primary site of infection is the female gonad and the parasite is transovarially transmitted to the eggs. PCR screening reveals a female bias in the distribution of parasites (96.6% of females, N=29; 22.2% of males, N=27), which is indicative of host sex ratio distortion. The morphological and molecular characterisations of this new microsporidium place it outside all currently established genera. On the basis of these differences, we erect the new genus Fibrillanosema n.g. While F. crangonycis is morphologically identical to uncharacterised microsporidia from populations of North American amphipods, it is distinct from microsporidia found in European populations of amphipods. These data support the hypothesis that vertically transmitted parasites may be selectively retained during invasion events. Furthermore where vertical transmission is combined with host sex ratio distortion these parasites may directly enhance host invasion success through increased rates of population growth.     

Influence of Humic Substances on Bacterial and Viral Dynamics in Freshwaters

Bacterial and viral abundances were measured in 24 lakes with dissolved organic carbon (DOC) concentrations ranging from 3 to 19 mg of C liter⁻¹. In addition, a laboratory experiment was performed to test the effects of different sources of carbon (i.e., glucose and fulvic acids) and nutrients on the dynamics of viruses and bacteria. In the lake survey, no correlation was found between virus abundance and DOC concentration, yet there was a significant positive correlation between bacterial abundance and DOC concentration. A negative correlation was found between the virus-to-bacteria ratio and DOC level. These results are in agreement with our findings in the laboratory, where virus counts were significantly lower in treatments with fulvic acid additions than in a control (mean, 67.4% ± 6.5% of the control). Virus counts did not differ significantly among the control and treatments with glucose, indicating that it was the type of organic carbon and not quantity which had an impact on viruses. Results from this study suggest that the way viruses control bacterial assemblages in humic lakes is different from the mechanism in clear water systems.     

The contributions of protein disulfide isomerase and its homologues to oxidative protein folding in the yeast endoplasmic reticulum

In vitro, protein disulfide isomerase (Pdi1p) introduces disulfides into proteins (oxidase activity) and provides quality control by catalyzing the rearrangement of incorrect disulfides (isomerase activity). Protein disulfide isomerase (PDI) is an essential protein in Saccharomyces cerevisiae, but the contributions of the catalytic activities of PDI to oxidative protein folding in the endoplasmic reticulum (ER) are unclear. Using variants of Pdi1p with impaired oxidase or isomerase activity, we show that isomerase-deficient mutants of PDI support wild-type growth even in a strain in which all of the PDI homologues of the yeast ER have been deleted. Although the oxidase activity of PDI is sufficient for wild-type growth, pulse-chase experiments monitoring the maturation of carboxypeptidase Y reveal that oxidative folding is greatly compromised in mutants that are defective in isomerase activity. Pdi1p and one or more of its ER homologues (Mpd1p, Mpd2p, Eug1p, Eps1p) are required for efficient carboxypeptidase Y maturation. Consistent with its function as a disulfide isomerase in vivo, the active sites of Pdi1p are partially reduced (32 +/- 8%) in vivo. These results suggest that PDI and its ER homologues contribute both oxidase and isomerase activities to the yeast ER. The isomerase activity of PDI can be compromised without affecting growth and viability, implying that yeast proteins that are essential under laboratory conditions may not require efficient disulfide isomerization.