Mechanistic overview of reactive species-induced degradation of the endothelial glycocalyx during hepatic ischemia/reperfusion injury

Endothelial cells are covered by a delicate meshwork of glycoproteins known as the glycocalyx. Under normophysiological conditions the glycocalyx plays an active role in maintaining vascular homeostasis by deterring primary and secondary hemostasis and leukocyte adhesion and by regulating vascular permeability and tone. During (micro)vascular oxidative and nitrosative stress, which prevails in numerous metabolic (diabetes), vascular (atherosclerosis, hypertension), and surgical (ischemia/reperfusion injury, trauma) disease states, the glycocalyx is oxidatively and nitrosatively modified and degraded, which culminates in an exacerbation of the underlying pathology. Consequently, glycocalyx degradation due to oxidative/nitrosative stress has far-reaching clinical implications. In this review the molecular mechanisms of reactive oxygen and nitrogen species-induced destruction of the endothelial glycocalyx are addressed in the context of hepatic ischemia/reperfusion injury as a model disease state. Specifically, the review focuses on (i) the mechanisms of glycocalyx degradation during hepatic ischemia/reperfusion, (ii) the molecular and cellular players involved in the degradation process, and (iii) its implications for hepatic pathophysiology. These topics are projected against a background of liver anatomy, glycocalyx function and structure, and the biology/biochemistry and the sources/targets of reactive oxygen and nitrogen species. The majority of the glycocalyx-related mechanisms elucidated for hepatic ischemia/reperfusion are extrapolatable to the other aforementioned disease states.     

Bad coat, ripped genes: cryptic selection on coat colour varies with ontogeny in Soay sheep

A fundamental goal for evolutionary biologists is to connect the processes of natural selection and genetic drift with the maintenance of genetic variation in the wild. There are now scores of examples in which mapping phenotypes to genotypes has identified the molecular basis of traits in natural populations, but documenting the fitness consequences of these genes has proven more elusive. A long-term study of several thousand unmanaged sheep on the remote Scottish island of Hirta in the St. Kilda archipelago provides a unique opportunity to make these connections between genotype, phenotype and fitness. Since 1985, there has been a steady decline in the frequency of sheep with a rare uniform ('self'-) pigmentation pattern as opposed to the more common wild-type pattern of dark upper body and pale belly. In this issue of Molecular Ecology, Gratten et al. (2012) link variation at the agouti signalling protein (ASIP) gene with 25 years of pedigree information and individual fitness data to investigate the contributions of natural selection and genetic drift to this decline. Consistent with the decrease in frequency of self-type sheep, Gratten et al. demonstrate that recessive genetic variants at ASIP are associated with reduced lifetime fitness. But surprisingly, these variants show the opposite trend to the self-type phenotype by increasing throughout the study period. This paradoxical result occurs because heterozygotes that harbour the majority of self-type alleles have increased in frequency. This pattern would not have been detectable if only phenotype had been monitored because the pigmentation pattern of heterozygotes is indistinguishable from that of wild-type homozygotes. The study provides an excellent example of the importance of measuring selection at both the phenotypic and genetic level, and demonstrates how long-term pedigrees can be used to link alleles to phenotypes and ultimately to fitness.     

The mammalian chitinase-like lectin, YKL-40, binds specifically to type I collagen and modulates the rate of type I collagen fibril formation

YKL-40 is expressed in arthritic cartilage and produced in large amounts by cultured chondrocytes, but its exact role is unclear, and the identities of its physiological ligands remain unknown. Purification of YKL-40 from resorbing bovine nasal cartilage and chondrocyte monolayers demonstrated the existence of three isoforms, a major and minor form from resorbing cartilage and a third species from chondrocytes. Affinity chromatography experiments with purified YKL-40 demonstrated specific binding of all three forms to collagen types I, II, and III, thus identifying collagens as potential YKL-40 ligands. Binding to immobilized type I collagen was inhibited by soluble native ligand, but not heat-denatured ligand, confirming a specific interaction. Binding of the chondrocyte-derived species to type I collagen was also demonstrated by surface plasmon resonance analysis, and the dissociation rate constant was calculated (3.42 x 10(-3) to 4.50 x 10(-3) s(-1)). The chondrocyte-derived species was found to prevent collagenolytic cleavage of type I collagen and to stimulate the rate of type I collagen fibril formation in a concentration-dependent manner. By contrast, the cartilage major form had an inhibitory effect on type I collagen fibrillogenesis. Digestion with N-glycosidase F, endoglycosidase H and lectin blotting did not reveal any difference in the carbohydrate component of these two YKL-40 species, indicating that this does not account for the opposing effects on fibril formation rate.     

Cutting edge: the phosphoinositide 3-kinase p110 delta is critical for the function of CD4+CD25+Foxp3+ regulatory T cells

CD4+CD25+Foxp3+ regulatory T cells (Tregs) contribute to the maintenance of peripheral tolerance by inhibiting the expansion and function of conventional T cells. Treg development and homeostasis are regulated by the Ag receptor, costimulatory receptors such as CD28 and CTLA-4, and cytokines such as IL-2, IL-10, and TGF-beta. Here we show that the proportions of Tregs in the spleen and lymph nodes of mice with inactive p110delta PI3K (p110deltaD910A/D910A) are reduced despite enhanced Treg selection in the thymus. p110deltaD910A/D910A CD4+CD25+Foxp3+ Tregs showed attenuated suppressor function in vitro and failed to secrete IL-10. In adoptive transfer experiments, p110deltaD910A/D910A T cells failed to protect against experimental colitis. The identification of p110delta as an intracellular signaling protein that regulates the activity of CD4+CD25+Foxp3+ Tregs may facilitate the further elucidation of the molecular mechanisms responsible for Treg-mediated suppression.     

The anaerobic degradability of thermoplastic starch: polyvinyl alcohol blends: potential biodegradable food packaging materials

A systematic study on the anaerobic degradability of a series of starch:polyvinyl alcohol (TPS:PVOH) blends was performed to determine their fate upon disposal in either anaerobic digesters or bioreactor landfills. The aims of the study were to measure the rate and extent of solubilisation of the plastics. The extent of substrate solubilisation on a COD basis reached 60% for a 90:10 (w/w) blend of TPS:PVOH, 40% for 75:25, 30% for 50:50 and 15% for PVOH only. The rate of substrate solubilisation was most rapid for the 90:10 blend (0.041 h(-1)) and decreased with the amount of starch in the blend in the following order 0.034 h(-1)(75:25); 0.023 h(-1)(50:50). The total solids that remained after 900 h were 10 wt.% (90:10); 23 wt.% (75:25); 55 wt.% (50:50); 90 wt.% (0:100). Starch containing substrates produced a higher concentration of volatile fatty acids (VFAs) and biogas, compared to the 0:100 substrate. The major outcome was that PVOH inhibited the degradation of the starch from the blend.     

Immunological Interactions between 2 Common Pathogens,Th1-Inducing Protozoan Toxoplasma gondii and the Th2-Inducing Helminth Fasciola hepatica

Background

The nature of the immune response to infection is dependent on the type of infecting organism. Intracellular organisms such as Toxoplasma gondii stimulate a Th1-driven response associated with production of IL-12, IFN-γ, nitric oxide and IgG2a antibodies and classical activation of macrophages. In contrast, extracellular helminths such as Fasciola hepatica induce Th2 responses characterised by the production of IL-4, IL-5, IL-10 and IgG1 antibodies and alternative activation of macrophages. As co-infections with these types of parasites commonly exist in the field it is relevant to examine how the various facets of the immune responses induced by each may influence or counter-regulate that of the other.

Principal Findings

Regardless, of whether F. hepatica infection preceded or succeeded T. gondii infection, there was little impact on the production of the Th1 cytokines IL-12, IFN-γ or on the development of classically-activated macrophages induced by T. gondii. By contrast, the production of helminth-specific Th2 cytokines, such as IL-4 and IL-5, was suppressed by infection with T. gondii. Additionally, the recruitment and alternative activation of macrophages by F. hepatica was blocked or reversed by subsequent infection with T. gondii. The clinical symptoms of toxoplasmosis and the survival rate of infected mice were not significantly altered by the helminth.

Conclusions

Despite previous studies showing that F. hepatica suppressed the classical activation of macrophages and the Th1-driven responses of mice to bystander microbial infection, as well as reduced their ability to reject these, here we found that the potent immune responses to T. gondii were capable of suppressing the responses to helminth infection. Clearly, the outcome of particular infections in polyparasitoses depends on the means and potency by which each pathogen controls the immune response.     

Investigation of critical inter‐related factors affecting the efficacy of pulsed light for inactivating clinically relevant bacterial pathogens

Aims: To investigate critical electrical and biological factors governing the efficacy of pulsed light (PL) for the in vitro inactivation of bacteria isolated from the clinical environment. Development of this alternative PL decontamination approach is timely, as the incidence of health care–related infections remains unacceptably high. Methods and Results: Predetermined cell numbers of clinically relevant Gram‐positive and Gram‐negative bacteria were inoculated separately on agar plates and were flashed with ≤60 pulses of broad‐spectrum light under varying operating conditions, and their inactivation measured. Significant differences in inactivation largely occurred depending on the level of the applied lamp discharge energy (range 3·2–20 J per pulse), the amount of pulsing applied (range 0–60 pulses) and the distance between light source and treatment surface (range 8–20 cm) used. Greater decontamination levels were achieved using a combination of higher lamp discharge energies, increased number of pulses and shorter distances between treatment surface and the xenon light source. Levels of microbial sensitivity also varied depending on the population type, size and age of cultures treated. Production of pigment pyocynanin and alginate slime in mucoid strains of Pseudomonas aeruginosa afforded some protection against lethal action of PL; however, this was evident only by using a combination of reduced amount of pulsing at the lower lamp discharge energies tested. A clear pattern was observed where Gram‐positive bacterial pathogens were more resistant to cidal effects of PL compared to Gram negatives. While negligible photoreactivation of PL‐treated bacterial strains occurred after full pulsing regimes at the different lamp discharge energies tested, some repair was evident when using a combination of reduced pulsing at the lower lamp discharge energies. Strains harbouring genes for multiple resistances to antibiotics were not significantly more resistant to PL treatments. Slight temperature rises (≤4·2°C) were measured on agar surfaces after extended pulsing at higher lamp discharge energies. Presence of organic matter on treatment surface did not significantly affect PL decontamination efficacy, nor did growth of PL‐treated bacteria on selective agar diminish survival compared to similarly treated bacteria inoculated and enumerated on nonselective agar plates. Conclusions: Critical inter‐related factors affecting the effective and repeatable in vitro decontamination performance of PL were identified during this study that will aid further development of this athermal process technology for applications in health care and in industry. Very rapid reductions (c. 7 log₁₀ CFU cm^?2 within ≤10 pulses) occurred using discharge energy of 20 J for all tested clinically relevant bacteria under study when treated at 8 cm distance from xenon light source. While no resistant flora is expected to develop for treatment of microbial pathogens on two‐dimensional surfaces, careful consideration of scale up factors such as design and operational usage of this PL technique will be required to assure operator safety. Significance and Impact of the Study: Findings and conclusions derived from this study will enable further development and optimization of this decontamination technique in health care and in food preparation settings, and will advance the field of nonthermal processing technologies.     

Reuse of voucher specimens provides insights into the genomic associations and taxonomic value of wing colour and genitalic differences in a pest group (Lepidoptera: Tortricidae: Choristoneura)

Subtle morphological differences can be essential to diagnosing closely related species, and an understanding of the genetic basis of these characters can contribute to understanding their divergences. We used voucher specimens from previous genetic analyses of population structure to subsequently analyse genome-wide associations linking morphology to genetic variation in spruce budworms, a group of economically important and morphologically similar forest pests. In particular, we assessed the taxonomic value and genetic architecture of two morphological traits (wing pattern and genitalic spicule abundance) that have been reported to differ among spruce budworm species. Our results suggest that phallic spicule number has greater taxonomic utility than wing pattern for distinguishing Choristoneura fumiferana (Clemens) from Choristoneura occidentalis occidentalis Freeman and Choristoneura occidentalis biennis Freeman. However, there was considerable overlap among taxa for all phenotypic characters analysed. In a genome-wide association study, wing pattern variation was significantly associated with four single nucleotide polymorphism (SNP) loci, including two adjacent SNPs. One SNP was flanked by sequence resembling RNA-directed DNA polymerase from mobile element jockey-like. This locus is a promising candidate for the study of wing pattern development in spruce budworms, as jockey-like transposable elements and polymerases have documented roles in wing spot production in other Lepidoptera. Our study links classical taxonomic characters and genomic data to provide insights into the potential genetic architecture of species differences. It also demonstrates previously untapped morphological and taxonomic value in voucher specimens from earlier molecular genetic analyses.