Conformational studies of plasminogen activator inhibitor type 1 by fluorescence spectroscopy. Analysis of the reactive centre of inhibitory and substrate forms, and of their respective reactive-centre cleaved forms.

The inhibitors that belong to the serpin family are suicide inhibitors that control the major proteolytic cascades in eucaryotes. Recent data suggest that serpin inhibition involves reactive centre cleavage followed by loop insertion, whereby the covalently linked protease is translocated away from the initial docking site. However under certain circumstances, serpins can also be cleaved like a substrate by target proteases. In this report we have studied the conformation of the reactive centre of plasminogen activator inhibitor type 1 (PAI-1) mutants with inhibitory and substrate properties. The polarized steady-state and time-resolved fluorescence anisotropies were determined for BODIPY(R) probes attached to the P1' and P3 positions of the substrate and active forms of PAI-1. The fluorescence data suggest an extended orientational freedom of the probe in the reactive centre of the substrate form as compared to the active form, revealing that the conformation of the reactive centres differ. The intramolecular distance between the P1' and P3 residues in reactive centre cleaved inhibitory and substrate mutants of PAI-1, were determined by using the donor-donor energy migration (DDEM) method. The distances found were 57+/-4 A and 63+/-3 A, respectively, which is comparable to the distance obtained between the same residues when PAI-1 is in complex with urokinase-type plasminogen activator (uPA). Following reactive centre cleavage, our data suggest that the core of the inhibitory and substrate forms possesses an inherited ability of fully inserting the reactive centre loop into beta-sheet A. In the inhibitory forms of PAI-1 forming serpin-protease complexes, this ability leads to a translocation of the cognate protease from one pole of the inhibitor to the opposite one.     

The gut microbiota of the pine weevil is similar across Europe and resembles that of other conifer‐feeding beetles

The pine weevil (Hylobius abietis, Coleoptera: Curculionidae) is an important pest of conifer seedlings in Europe. Despite its economic importance, little is known about the composition of its gut microbial community and the role it plays in mediating the weevil's ability to utilize conifers as a food source. Here, we characterized the gut bacterial communities of different populations of H. abietis across Europe and compared them to those of other beetles that occupy similar ecological niches. We demonstrate that the microbial community of H. abietis is similar at higher taxonomic levels (family and genus) across locations in Europe, with Wolbachia as the dominant microbe, followed by Enterobacteria and Firmicutes. Despite this similarity, we observed consistent differences between countries and locations, but not sexes. Our meta‐analysis demonstrates that the gut bacterial community of the pine weevil is very similar to that of bark beetles that also exploit conifers as a food source. The Enterobacteriaceae symbionts of both host taxa are especially closely related phylogenetically. Conversely, the microbiota of H. abietis is distinct from that of closely related weevils feeding on nonconifer food sources, suggesting that the microbial community of the pine weevil is determined by the environment and may be relevant to host ecology. Furthermore, several H. abietis‐associated members of the Enterobacteriaceae family are known to contain genes involved in terpenoid degradation. As such, we hypothesize that the gut microbial community is important for the utilization of conifer seedlings as a food source, either through the detoxification of plant secondary metabolites or through the supplementation of essential nutrients.     

Ontogenetic and spatial variability in trophic biomarkers of juvenile saffron cod (<Emphasis Type="Italic">Eleginus gracilis</Emphasis>) from the Beaufort,Chukchi and Bering Seas

Climate models indicate the Arctic will undergo dramatic environmental change with forecasted increases in temperature and river runoff. Saffron cod (Eleginus gracilis) is abundant in nearshore waters and appears in the diet of many Arctic sea birds and marine mammals; however, little is known about its early ecology and consequently how they might be affected by environmental changes. We aimed to characterize the mechanisms of spatial and ontogenetic variation in trophic biomarkers (lipid classes, fatty acids and bulk C and N stable isotopes) of saffron cod from the Western Arctic, Chukchi and Bering Seas. Size-standardized analyses showed a significant difference in lipid condition metrics and trophic biomarkers as a function of survey location. Both ontogeny and sampling location played an important role in determining lipid stores with elevated levels in both small offshore juveniles (<55 mm) and larger inshore juveniles (>75 mm). Higher lipid storage in Arctic juveniles was associated with elevated levels of diatom fatty acid markers, but not with nearshore carbon input. Increased lipids were found in age-1 juveniles from Prudhoe Bay in the Western Beaufort that were feeding at a lower trophic level than similarly sized age-0 juveniles from surface trawls in the Bering Sea. The use of otolith annuli revealed two discrete patterns of growth that help explain the trade-offs between energy storage and rapid growth that diverge between the Arctic and Bering Sea. Laboratory temperature-growth experiments confirmed that saffron cod have a eurythermal growth response and are able to store excess lipids at temperatures as high as 20 °C.     

High mitochondrial haplotype diversity of Coleps sp. (Ciliophora: Prostomatida)

To date the awareness of the temporal population structure in eukaryotic microbes is very limited. This is exemplified in the scarce knowledge about the intraspecific genetic variation in ciliates. To elucidate the genetic variation of Coleps (Ciliophora: Prostomatida), we employed the analysis of the mitochondrial apocytochrome b gene of the Coleps community in a young lake in Germany. The analysis of 111 isolates, sampled from April 2005 to September 2006, revealed a high genetic variation for the two dominant Coleps species (11 mitochondrial haplotypes in Coleps spetai , nine in Coleps hirtus hirtus ). The study represents one of the largest datasets of intraspecific diversity in a microbial eukaryote and demonstrates for the first time the suitability of a mitochondrial gene for the detection of genetic variation within populations of eukaryotic microbes. However, the results of our study warrant caution in the application of such an approach, as we amplified some non-orthologous cob -like sequences, whose uncritical acceptance would have led to the erroneous discovery of cryptic species.     

The histone variant macro-H2A preferentially forms "hybrid nucleosomes"

The histone domain of macro-H2A, which constitutes the N-terminal one third of this histone variant, is only 64% identical to major H2A. We have shown previously that the main structural differences in a nucleosome in which both H2A moieties have been replaced by macro-H2A reside in the only point of contact between the two histone dimers, the L1-L1 interface of macro-H2A. Here we show that the L1 loop of macro-H2A is responsible for the increased salt-dependent stability of the histone octamer, with implications for the nucleosome assembly pathway. It is unknown whether only one or both of the H2A-H2B dimers within a nucleosome are replaced with H2A variant containing nucleosomes in vivo. We demonstrate that macro-H2A preferentially forms hybrid nucleosomes containing one chain each of major H2A and macro-HA in vitro. The 2.9-A crystal structure of such a hybrid nucleosome shows significant structural differences in the L1-L1 interface when comparing with homotypic major H2A- and macro-H2A-containing nucleosomes. Both homotypic and hybrid macro-nucleosome core particles (NCPs) are resistant to chaperone-assisted H2A-H2B dimer exchange. Together, our findings suggest that the histone domain of macro-H2A modifies the dynamic properties of the nucleosome. We propose that the possibility of forming hybrid macro-NCP adds yet another level of complexity to variant nucleosome structure and function.     

Imaging immune surveillance of individual natural killer cells confined in microwell arrays

New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level.     

The fetal programming effect of prenatal smoking on Igf1r and Igf1 methylation is organ- and sex-specific

The impact of prenatal smoke exposure (PSE) on DNA methylation has been demonstrated in blood samples from children of smoking mothers, but evidence for sex-dependent smoke-induced effects is limited. As the identified differentially methylated genes can be associated with developmental processes, and insulin-like growth factors (IGFs) play a critical role in prenatal tissue growth, we hypothesized that PSE induces fetal programming of Igf1r and Igf1. Using a mouse model of smoking during pregnancy, we show that PSE alters promoter methylation of Igf1r and Igf1 and deregulates their gene expression in lung and liver of fetal (E17.5) and neonatal (D3) mouse offspring. By further comparing female versus male, lung versus liver, or fetal versus neonatal time point, our results demonstrate that CpG site-specific aberrant methylation patterns sex-dependently vary per organ and time point. Moreover, PSE reduces gene expression of Igf1r and Igf1, dependent on organ, sex, and offspring's age. Our results indicate that PSE may be a source of organ-specific rather than general systemic fetal programming. This is exemplified here by gene promoter methylation and mRNA levels of Igf1r and Igf1, together with a sex- and organ-specific naturally established correlation of both parameters that is affected by prenatal smoke exposure. Moreover, the comparison of fetuses with neonates suggests a CpG site-dependent reversibility/persistence of PSE-induced differential methylation patterns.     

The NLRP3 inflammasome is differentially activated by pneumolysin variants and contributes to host defense in pneumococcal pneumonia

Streptococcus pneumoniae is a leading cause of pneumonia, meningitis, and sepsis. Pneumococci can be divided into >90 serotypes that show differences in the pathogenicity and invasiveness. We tested the hypotheses that the innate immune inflammasome pathway is involved in fighting pneumococcal pneumonia and that some invasive pneumococcal types are not recognized by this pathway. We show that human and murine mononuclear cells responded to S. pneumoniae expressing hemolytic pneumolysin by producing IL-1β. This IL-1β production depended on the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. Some serotype 1, serotype 8, and serotype 7F bacteria, which have previously been associated with increased invasiveness and with production of toxins with reduced hemolytic activity, or bacterial mutants lacking pneumolysin did not stimulate notable IL-1β production. We further found that NLRP3 was beneficial for mice during pneumonia caused by pneumococci expressing hemolytic pneumolysin and was involved in cytokine production and maintenance of the pulmonary microvascular barrier. Overall, the inflammasome pathway is protective in pneumonia caused by pneumococci expressing hemolytic toxin but is not activated by clinically important pneumococcal sequence types causing invasive disease. The study indicates that a virulence factor polymorphism may substantially affect the recognition of bacteria by the innate immune system.     

The Tumor Suppressor PML Specifically Accumulates at RPA/Rad51-Containing DNA Damage Repair Foci but Is Nonessential for DNA Damage-Induced Fibroblast Senescence

The PML tumor suppressor has been functionally implicated in DNA damage response and cellular senescence. Direct evidence for such a role based on PML knockdown or knockout approaches is still lacking. We have therefore analyzed the irradiation-induced DNA damage response and cellular senescence in human and mouse fibroblasts lacking PML. Our data show that PML nuclear bodies (NBs) nonrandomly associate with persistent DNA damage foci in unperturbed human skin and in high-dose-irradiated cell culture systems. PML bodies do not associate with transient γH2AX foci after low-dose gamma irradiation. Superresolution microscopy reveals that all PML bodies within a nucleus are engaged at Rad51- and RPA-containing repair foci during ongoing DNA repair. The lack of PML (i) does not majorly affect the DNA damage response, (ii) does not alter the efficiency of senescence induction after DNA damage, and (iii) does not affect the proliferative potential of primary mouse embryonic fibroblasts during serial passaging. Thus, while PML NBs specifically accumulate at Rad51/RPA-containing lesions and senescence-derived persistent DNA damage foci, they are not essential for DNA damage-induced and replicative senescence of human and murine fibroblasts.