Feeding the Probiotic Enterococcus faecium Strain NCIMB 10415 to Piglets Specifically Reduces the Number of Escherichia coli Pathotypes That Adhere to the Gut Mucosa

Feed supplementation with the probiotic Enterococcus faecium for piglets has been found to reduce pathogenic gut microorganisms. Since Escherichia coli is among the most important pathogens in pig production, we performed comprehensive analyses to gain further insight into the influence of E. faecium NCIMB 10415 on porcine intestinal E. coli. A total of 1,436 E. coli strains were isolated from three intestinal habitats (mucosa, digesta, and feces) of probiotic-supplemented and nonsupplemented (control) piglets. E. coli bacteria were characterized via pulsed-field gel electrophoresis (PFGE) for clonal analysis. The high diversity of E. coli was reflected by 168 clones. Multilocus sequence typing (MLST) was used to determine the phylogenetic backgrounds, revealing 79 sequence types (STs). Pathotypes of E. coli were further defined using multiplex PCR for virulence-associated genes. While these analyses discerned only a few significant differences in the E. coli population between the feeding groups, analyses distinguishing clones that were uniquely isolated in either the probiotic group only, the control group only, or both groups (shared group) revealed clear effects at the habitat level. Interestingly, extraintestinal pathogenic E. coli (ExPEC)-typical clones adhering to the mucosa were significantly reduced in the probiotic group. Our data show a minor influence of E. faecium on the overall population of E. coli in healthy piglets. In contrast, this probiotic has a profound effect on mucosa-adherent E. coli. This finding further substantiates a specific effect of E. faecium strain NCIMB 10415 in piglets against pathogenic E. coli in the intestine. In addition, these data question the relevance of data based on sampling fecal E. coli only.     

A developmental stage of hyphal cells shows riboflavin overproduction instead of sporulation in Ashbya gossypii

The hemiascomycete Ashbya gossypii develops a mycelium. Nutritional stress leads to its differentiation into sporangia. These generate spores. In parallel, the yellow pigment riboflavin is produced. Intracellularly accumulated riboflavin, made visible as a bright green fluorescence, was observed in only 60 % of the hyphal cells. For the remaining 40 %, it was unclear whether these cells simply export riboflavin or its biosynthesis remains down-regulated in contrast to the accumulating cells. The approach followed in this work was to convert the hyphae into protoplasts by enzymatic degradation of the cell wall. Afterwards, the protoplasts were sorted by fluorescence-activated cell sorting on the basis of riboflavin accumulation. When a reporter strain expressing lacZ under the control of the most important riboflavin biosynthesis promoter, RIB3, was used, green protoplasts were found to have more than tenfold greater reporter activity than hyaline protoplasts. This was true on the basis of total protein as well as on the basis of hexokinase specific activity, a marker for constitutive expression. These results allow the conclusion that hyphal cells of A. gossypii differ in phenotype regarding riboflavin overproduction and accumulation.     

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.