Microbial activity of suspended biomass from a nitritation–anammox SBR in dependence of operational condition and size fraction

Single-stage nitritation–anammox combines the growth of aerobic ammonium-oxidizing bacteria (AOB) and anaerobic ammonium oxidizing bacteria (AnAOB) in one reactor. The necessary compromise of their milieu conditions often leads to the growth of nitrite-oxidizing bacteria (NOB). For this study, a sequencing batch reactor (SBR) for nitritation–anammox was operated for 180 days with sewage sludge reject water (removal capacity, 0.4 kg?N?m^?3?day^?1). The growth of NOB was favored by enhanced oxygen supply rather than extended aerobic phases. Suspended-type biomass from this SBR was taken regularly and sieved into three size fractions (all of them <1,000 μm). Batch experiments as well as fluorescence in situ hybridization were performed to study the distribution and activity of AnAOB, AOB, and NOB within those size fractions. Both the measured conversion rates and detected abundances decreased with increasing size fraction. The highest anammox conversion rates (15 g NH₄ ⁺–N per kilogram VSS per hour) and the highest abundances of Brocadia fulgida were found in the medium size fraction (100–315 μm). The batch experiments proved to be accurate tools for the monitoring of multiple processes in the reactor. The results were representative for reactor performance during the 6 months of reactor operation.     

Notch-signalling is required for head regeneration and tentacle patterning in Hydra

Local self-activation and long ranging inhibition provide a mechanism for setting up organising regions as signalling centres for the development of structures in the surrounding tissue. The adult hydra hypostome functions as head organiser. After hydra head removal it is newly formed and complete heads can be regenerated. The molecular components of this organising region involve Wnt-signalling and β-catenin. However, it is not known how correct patterning of hypostome and tentacles are achieved in the hydra head and whether other signals in addition to HyWnt3 are needed for re-establishing the new organiser after head removal. Here we show that Notch-signalling is required for re-establishing the organiser during regeneration and that this is due to its role in restricting tentacle activation. Blocking Notch-signalling leads to the formation of irregular head structures characterised by excess tentacle tissue and aberrant expression of genes that mark the tentacle boundaries. This indicates a role for Notch-signalling in defining the tentacle pattern in the hydra head. Moreover, lateral inhibition by HvNotch and its target HyHes are required for head regeneration and without this the formation of the β-catenin/Wnt dependent head organiser is impaired. Work on prebilaterian model organisms has shown that the Wnt-pathway is important for setting up signalling centres for axial patterning in early multicellular animals. Our data suggest that the integration of Wnt-signalling with Notch-Delta activity was also involved in the evolution of defined body plans in animals.     

Bioavailability and potential carcinogenicity of polycyclic aromatic hydrocarbons from wood combustion particulate matter in vitro

Due to increasing energy demand and limited fossil fuels, renewable energy sources have gained in importance. Particulate matter (PM) in general, but also PM from the combustion of wood is known to exert adverse health effects in human. These are often related to specific toxic compounds adsorbed to the PM surface, such as polycyclic aromatic hydrocarbons (PAH), of which some are known human carcinogens. This study focused on the bioavailability of PAHs and on the tumor initiation potential of wood combustion PM, using the PAH CALUX® reporter gene assay and the BALB/c 3T3 cell transformation assay, respectively. For this, both cell assays were exposed to PM and their respective organic extracts from varying degrees of combustion. The PAH CALUX® experiments demonstrated a concentration–response relationship matching the PAHs detected in the samples. Contrary to expectations, PM samples from complete (CC) and incomplete combustion (IC) provided for a stronger and weaker response, respectively, suggesting that PAH were more readily bioavailable in PM from CC. These findings were corroborated via PAH spiking experiments indicating that IC PM contains organic components that strongly adsorb PAH thereby reducing their bioavailability. The results obtained with organic extracts in the cell transformation assay presented the highest potential for carcinogenicity in samples with high PAH contents, albeit PM from CC also demonstrated a carcinogenic potential. In conclusion, the in vitro assays employed emphasize that CC produces PM with low PAH content however with a general higher bioavailability and thus with a nearly similar carcinogenic potential than IC PM.     

Reflux induces DNA strand breaks and expression changes of MMP1+9+14 in a human miniorgan culture model

Gastroesophageal reflux disease has been implicated in the pathogenesis of adenocarcinoma of the oesophagus. The same applies to laryngopharyngeal reflux (LPR) and squamous cell cancer of the head and neck, but so far, this link has not been proven. The impact of low pH and bile acids has not been studied extensively in cells other than oesophageal cancer cell lines and tissue. The aims of this study were to investigate the pathogenic potential of reflux and its single components on the mucosa of the upper respiratory tract. We measured DNA stability in human miniorgan cultures (MOCs) and primary epithelial cell cultures (EpCs) in response to reflux by the alkaline comet assay. As matrix metalloproteinases (MMPs) are involved in extracellular matrix remodelling processes and may contribute to cancer progression, we studied the expression of MMP1, -9, and -14 in MOCs, EpC, UM-SCC-22B, and FADU_DD. DNA strand breaks (DNA-SBs) increased significantly at low pH and after incubation with human or artificial gastric juice. Single incubation with glycochenodeoxycholic acid also showed a significant increase in DNA-SBs. In epithelial cell cultures, human gastric juice increased the number of DNA-SBs at pH 4.5 and 5.5. Artificial gastric juice significantly up regulated the gene expression of MMP9. Western blot analysis confirmed the results of gene expression analysis, but the up regulation of MMP1, -9, and -14 was donor-specific. Reflux has the ability to promote genomic instability and may contribute to micro environmental changes suitable for the initiation of malignancy. Further functional gene analysis may elucidate the role of laryngopharyngeal reflux in the development of head neck squamous cell carcinoma (HNSCC).     

STEPWISE EVOLUTION OF RESISTANCE TO TOXIC CARDENOLIDES VIA GENETIC SUBSTITUTIONS IN THE NA+/K+‐ATPASE OF MILKWEED BUTTERFLIES (LEPIDOPTERA: DANAINI)

Despite the monarch butterfly (Danaus plexippus) being famous for its adaptations to the defensive traits of its milkweed host plants, little is known about the macroevolution of these traits. Unlike most other animal species, monarchs are largely insensitive to cardenolides, because their target site, the sodium pump (Na⁺/K⁺‐ATPase), has evolved amino acid substitutions that reduce cardenolide binding (so‐called target site insensitivity, TSI). Because many, but not all, species of milkweed butterflies (Danaini) are associated with cardenolide‐containing host plants, we analyzed 16 species, representing all phylogenetic lineages of milkweed butterflies, for the occurrence of TSI by sequence analyses of the Na⁺/K⁺‐ATPase gene and by enzymatic assays with extracted Na⁺/K⁺‐ATPase. Here we report that sensitivity to cardenolides was reduced in a stepwise manner during the macroevolution of milkweed butterflies. Strikingly, not all Danaini typically consuming cardenolides showed TSI, but rather TSI was more strongly associated with sequestration of toxic cardenolides. Thus, the interplay between bottom‐up selection by plant compounds and top‐down selection by natural enemies can explain the evolutionary sequence of adaptations to these toxins.     

Structure-based design and synthesis of potent benzothiazole inhibitors of interleukin-2 inducible T cell kinase (ITK)

Inhibition of the non-receptor tyrosine kinase ITK, a component of the T-cell receptor signalling cascade, may represent a novel treatment for allergic asthma. Here we report the structure-based optimization of a series of benzothiazole amides that demonstrate sub-nanomolar inhibitory potency against ITK with good cellular activity and kinase selectivity. We also elucidate the binding mode of these inhibitors by solving the X-ray crystal structures of several inhibitor-ITK complexes.     

MHC‐disassortative mate choice and inbreeding avoidance in a solitary primate

Sexual selection theory suggests that choice for partners carrying dissimilar genes at the major histocompatibility complex (MHC) may play a role in maintaining genetic variation in animal populations by limiting inbreeding or improving the immunity of future offspring. However, it is often difficult to establish whether the observed MHC dissimilarity among mates drives mate choice or represents a by‐product of inbreeding avoidance based on MHC‐independent cues. Here, we used 454‐sequencing and a 10‐year study of wild grey mouse lemurs (Microcebus murinus), small, solitary primates from western Madagascar, to compare the relative importance on the mate choice of two MHC class II genes, DRB and DQB, that are equally variable but display contrasting patterns of selection at the molecular level, with DRB under stronger diversifying selection. We further assessed the effect of the genetic relatedness and of the spatial distance among candidate mates on the detection of MHC‐dependent mate choice. Our results reveal inbreeding avoidance, along with disassortative mate choice at DRB, but not at DQB. DRB‐disassortative mate choice remains detectable after excluding all related dyads (characterized by a relatedness coefficient r > 0), but varies slightly with the spatial distance among candidate mates. These findings suggest that the observed deviations from random mate choice at MHC are driven by functionally important MHC genes (like DRB) rather than passively resulting from inbreeding avoidance and further emphasize the need for taking into account the spatial and genetic structure of the population in correlative tests of MHC‐dependent mate choice.     

Collagen XVI in health and disease

Collagen XVI, by structural analogy a member of the FACIT- (fibril-associated collagens with interrupted triple helices) family of collagens, is described as a minor collagen component of connective tissues. Collagen XVI is expressed in various cells and tissues without known occurrence of splice variants or isoforms. For skin and cartilage tissues its suprastructure is known. Presumably, there it acts as an adaptor protein connecting and organizing large fibrillar networks and thus modulates integrity and stability of the extracellular matrix (ECM).Collagen XVI is produced by myofibroblasts in the normal intestine and its synthesis is increased in the inflamed bowel wall where myofibroblasts develop increased numbers of focal adhesion contacts on collagen XVI. Consequently, recruitment of α1 integrin into the focal adhesions at the tip of the cells is induced followed by increased cell spreading on collagen XVI. This presumably adds to the maintenance of myofibroblasts in the inflamed intestinal regions and thus promotes fibrotic responses of the tissue. Notably, α1/α2 integrins interact with collagen XVI through an α1/α2β1 integrin binding site located in the COL 1–3 domains.Collagen XVI may act as a substrate for adhesion and invasion of connective tissue tumor cells. In glioblastoma it induces tumor invasiveness by modification of the β1-integrin activation pattern. Thus, altering the cell–matrix interaction through collagen XVI might be a molecular mechanism to further augment the invasive phenotype of glioma cells. In this line, in oral squamous cell carcinoma collagen XVI expression is induced which results in an upregulation of Kindlin-1 followed by an increased interaction with beta1-integrin. Consequently, collagen XVI induces a proliferative tumor phenotype by promoting an early S-phase entry.In summary, collagen XVI plays a decisive role in the interaction of connective tissue cells with their ECM, which is impaired in pathological situations. Alteration of tissue location and expression level of collagen XVI appears to promote tumorigenesis and to perpetuate inflammatory reactions.     

Inferring the ecology of willow warblers during their winter moult by sequential stable isotope analyses of remiges

We present a comparison of feather stable isotope (δ¹³C, δ¹⁵N) patterns representing the habitat and diet conditions for two subspecies of willow warblers Phylloscopus trochilus that breed in parapatry, but winter in different regions of sub‐Saharan Africa. Previous analyses have shown that on average winter moulted innermost primaries (P1) show subspecific differences in δ¹⁵N values, although individuals show substantial variation for both δ¹³C and δ¹⁵N within the subspecies. We examined whether corresponding variation in the timing of the winter moult, as reflected by consistent intra‐wing correlations for individual's δ¹³C and δ¹⁵N values, could explain some of the previously observed isotopic variation. Further, differential subspecific adaptations to winter precipitation patterns across Africa might result in a variable degree of site fidelity or itinerancy during moult. We found no consistent trend in isotopic values from innermost to outermost primaries, thus inter‐individual variation in the timing of moult does not explain the subspecific isotopic variation for P1. Patterns in wing feather δ¹³C and δ¹⁵N values indicated that 41% of the individuals from both subspecies shifted their diet or habitats during winter moult. Importantly, despite well‐documented itinerancy in willow warblers during the winter, 59% of the individuals had feather isotope values consistent with stable use of habitats or diets during winter moult. Repeatability analyses suggest that individuals of both subspecies initiate moult in similar habitats from year‐to‐year while feeding on isotopically similar diets.