Structural and Functional Insight into the Carbohydrate Receptor Binding of F4 Fimbriae-producing Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) strains are important causes of intestinal disease in humans and lead to severe production losses in animal farming. A range of fimbrial adhesins in ETEC strains determines host and tissue tropism. ETEC strains expressing F4 fimbriae are associated with neonatal and post-weaning diarrhea in piglets. Three naturally occurring variants of F4 fimbriae (F4_ab, F4_ac, and F4_ad) exist that differ in the primary sequence of their major adhesive subunit FaeG, and each features a related yet distinct receptor binding profile. Here the x-ray structure of FaeG_ad bound to lactose provides the first structural insight into the receptor specificity and mode of binding by the poly-adhesive F4 fimbriae. A small D′-D″-α1-α2 subdomain grafted on the immunoglobulin-like core of FaeG hosts the carbohydrate binding site. Two short amino acid stretches Phe¹⁵⁰–Glu¹⁵² and Val¹⁶⁶–Glu¹⁷⁰ of FaeG_ad bind the terminal galactose in the lactosyl unit and provide affinity and specificity to the interaction. A hemagglutination-based assay with E. coli expressing mutant F4_ad fimbriae confirmed the elucidated co-complex structure. Interestingly, the crucial D′-α1 loop that borders the FaeG_ad binding site adopts a different conformation in the two other FaeG variants and hints at a heterogeneous binding pocket among the FaeG serotypes.     

Schizosaccharomyces pombe encodes a mutated AP endonuclease 1

Mutagenic and cytotoxic apurinic/apyrimidinic (AP) sites are among the most frequent lesions in DNA. Repair of AP sites is initiated by AP endonucleases and most organisms possess two or more of these enzymes. Saccharomyces cerevisiae has AP endonuclease 1 (Apn1) as the major enzymatic activity with AP endonuclease 2 (Apn2) being an important backup. Schizosaccharomyces pombe also encodes two potential AP endonucleases, and Apn2 has been found to be the main repair activity, while Apn1 has no, or only a limited role in AP site repair. Here we have identified a new 5' exon (exon 1) in the apn1 gene and show that the inactivity of S. pombe Apn1 is due to a nonsense mutation in the fifth codon of this new exon. Reversion of this mutation restored the AP endonuclease activity of S. pombe Apn1. Interestingly, the apn1 nonsense mutation was only found in laboratory strains derived from L972 h(-) and not in unrelated isolates of S. pombe. Since all S. pombe laboratory strains originate from L972 h(-), it appears that all experiments involving S. pombe have been conducted in an apn1(-) mutant strain with a corresponding DNA repair deficiency. These observations have implications both for future research in S. pombe and for the interpretation of previously conducted epistatis analysis.     

Intracellular localisation and innate immune responses following Francisella noatunensis infection of Atlantic cod (Gadus morhua) macrophages

The facultative intracellular bacterium Francisella noatunensis causes francisellosis in Atlantic cod (Gadus morhua), but little is known about its survival strategies or how these bacteria evade the host immune response. In this study we show intracellular localisation of F. noatunensis in cod macrophages using indirect immunofluorescence techniques and green fluorescent labelled bacteria. Transmission electron microscopy revealed that F. noatunensis was enclosed by a phagosomal membrane during the initial phase of infection. Bacteria were at a later stage of the infection found in large electron-lucent zones, apparently surrounded by a partially intact or disintegrated membrane. Immune electron microscopy demonstrated the release of bacterial derived vesicles from intracellular F. noatunensis, an event suspected of promoting phagosomal membrane degradation and allowing escape of the bacteria to cytoplasm. Studies of macrophages infected with F. noatunensis demonstrated a weak activation of the inflammatory response genes as measured by increased expression of the Interleukin (IL)-1β and IL-8. In comparison, a stronger induction of gene expression was found for the anti-inflammatory IL-10 indicating that the bacterium exhibits a role in down-regulating the inflammatory response. Expression of the p40 subunit of IL-12/IL-17 genes was highly induced during infection suggesting that F. noatunensis promotes T cell polarisation. The host macrophage responses studied here showed low ability to distinguish between live and inactivated bacteria, although other types of responses could be of importance for such discriminations. The immunoreactivity of F. noatunensis lipopolysaccharide (LPS) was very modest, in contrast to the strong capacity of Escherichia coli LPS to induce inflammatory responsive genes. These results suggest that F. noatunensis virulence mechanisms cover many strategies for intracellular survival in cod macrophages.     

Effects of high dietary fibre diets formulated from by-products from vegetable and agricultural industries on plasma metabolites in gestating sows

The aim of the present study was to examine the biochemical influence of feeding high dietary fibre (DF) diets formulated from by-products from the vegetable and agricultural industries to sows during early to mid-gestation. The effect of feeding frequency (once vs. twice daily) on diurnal plasma metabolites patterns was also examined. The study included a total of 48 gestating sows from four blocks (12 gestating sows in each block). The sows were fed four different diets containing varying levels of starch (304-519 g/kg dry matter (DM)) and DF (171-404 g/kg DM) but with equal amounts of net energy. The low-DF diet (control) was based on barley and wheat, and the three high-DF diets formulated by replacing barley and wheat by pectin residue, sugar beet pulp and potato pulp, respectively. The experimental design comprised two periods of 4 weeks each. Half the sows were fed once daily at 08:00 h in the first period and twice daily at 08:00 and 15:00 h during the second period, and vice versa for the other half of the sows. Plasma samples from vena jugularis were collected by venipuncture at 07:00, 09:00, 12:00 and 19:00 h. Feeding high-DF increased plasma short-chain fatty acids (p = 0.02) and non-esterified fatty acids (p < 0.001). However, there was no clear effect of DF on glucose and insulin responses. A negative correlation between amount of DF in the diets and plasma creatine (R2 = 1.00; diet effect: p = 0.02) suggested that plasma creatine concentrations was an indicator for the level of glucose-glycogen interchange. Furthermore, an explorative approach using nuclear magnetic resonance spectroscopy-based metabonomics identified betaine (p < 0.001), dimethyl sulfone (DMSO2; p < 0.001) and scyllo-inositol (p < 0.001) as biomarkers for the different by-products; pectin residue was related to high plasma levels of DMSO2, sugar beet pulp to plasma betaine, DMSO2 and scyllo-inositol, and potato pulp to plasma DMSO2 and scyllo-inositol. In conclusion, replacing starch by DF affected surprisingly few metabolites in peripheral plasma. No negative effects were found in feeding pectin residue, sugar beet pulp or potato pulp for gestating sows as judged from the minor metabolic changes.     

Micro-CT based experimental liver imaging using a nanoparticulate contrast agent: a longitudinal study in mice

Background

Micro-CT imaging of liver disease in mice relies on high soft tissue contrast to detect small lesions like liver metastases. Purpose of this study was to characterize the localization and time course of contrast enhancement of a nanoparticular alkaline earth metal-based contrast agent (VISCOVER ExiTron nano) developed for small animal liver CT imaging.

Methodology

ExiTron nano 6000 and ExiTron nano 12000, formulated for liver/spleen imaging and angiography, respectively, were intravenously injected in C57BL/6J-mice. The distribution and time course of contrast enhancement were analysed by repeated micro-CT up to 6 months. Finally, mice developing liver metastases after intrasplenic injection of colon carcinoma cells underwent longitudinal micro-CT imaging after a single injection of ExiTron nano.

Principal Findings

After a single injection of ExiTron nano the contrast of liver and spleen peaked after 4–8 hours, lasted up to several months and was tolerated well by all mice. In addition, strong contrast enhancement of abdominal and mediastinal lymph nodes and the adrenal glands was observed. Within the first two hours after injection, particularly ExiTron nano 12000 provided pronounced contrast for imaging of vascular structures. ExiTron nano facilitated detection of liver metastases and provided sufficient contrast for longitudinal observation of tumor development over weeks.

Conclusions

The nanoparticulate contrast agents ExiTron nano 6000 and 12000 provide strong contrast of the liver, spleen, lymph nodes and adrenal glands up to weeks, hereby allowing longitudinal monitoring of pathological processes of these organs in small animals, with ExiTron nano 12000 being particularly optimized for angiography due to its very high initial vessel contrast.     

Enhanced matrix metalloproteinase activity in skeletal muscles of rats with congestive heart failure

Patients with congestive heart failure (CHF) are prone to increased skeletal muscle fatigue. Elevated circulatory concentrations of tumor necrosis factor (TNF)-alpha and monocyte chemoattractant protein-1, which may stimulate matrix metalloproteinase (MMP) activity and, thereby, contribute to skeletal muscle dysfunction, are frequently found in CHF. However, whether skeletal muscle MMP activity is altered in CHF is unknown. Hence, we have used a gelatinase assay to assess the activity of MMP and tissue inhibitors of MMP in single skeletal muscles of rats with CHF 6 wk after induction of myocardial infarction. Sham-operated (Sham) rats were used as controls. We also measured the gene expression and protein contents of MMP-2 and MMP-9 in skeletal muscles of these rats. Plasma MMP activity was nearly seven times higher (P < 0.05) in CHF than in Sham rats. Concomitantly, the MMP activity within single slow- and fast-twitch skeletal muscles of CHF rats increased two- to fourfold compared with Sham animals, whereas tissue inhibitor of MMP activity did not differ (P > 0.05). Preformed MMP-2 and MMP-9 were probably activated in CHF, because neither their gene expression nor protein levels were altered (P > 0.05). Serum concentrations of TNF-alpha and monocyte chemoattractant protein-1 remained unchanged (P > 0.05) between CHF and Sham rats during the 6-wk observation period. We conclude that development of CHF in rats enhances MMP activity, which in turn may distort the normal contractile function of skeletal muscle, thereby contributing to increased skeletal muscle fatigue.     

Helicostatins: brain-gut peptides of the moth, Helicoverpa armigera (Lepidoptera: Noctuidae)

Gene expression and immunolocalisation studies have determined that the helicostatins are brain-gut peptides in larvae of the lepidopteran, Helicoverpa armigera. Mapping of the distribution of these peptides in the nervous system and alimentary canal has provided evidence for multifunctional regulatory roles. In situ hybridisation studies have shown that the helicostatin precursor gene is expressed in neurones of the central and stomatogastric nervous systems, and endocrine cells of the midgut demonstrating that the helicostatins are true brain-gut peptides. Antisera raised against Leu-callatostatin 3 (ANRYGFGL-NH(2)), a peptide isolated from the blowfly, Calliphora vomitoria was used to map the distribution of allatostatin-like immunoreactive (Ast-ir) material in H. armigera to elucidate possible functions of the helicostatins. In situ hybridisation studies verified that the helicostatin precursor gene is expressed in neurones shown to contain Ast-ir, providing strong evidence that the Ast-ir material is helicostatins. Extensive immunoreactive axonal projections into complex regions of neuropile indicate that the helicostatins may have a neuromodulatory role in the brain and segmental ganglia of the ventral nerve cord. The presence of large amounts of immunoreactive material in axons within the corpora cardiaca (CC) and transverse nerves of the perisympathetic nervous system, two known neurohaemal organs, provides evidence for a neurohormonal role. The corpora allata (CA) were innervated only sparsely by Ast-ir axons suggesting that the CA are not a neurohaemal release site or a target. Thus, it is unlikely that the helicostatins regulate juvenile hormone (JH) biosynthesis or release. Ast-ir axons extended from the frontal ganglion through the recurrent nerve and many branches were closely associated with muscles of the foregut, stomodeal valve, and anterior midgut, implicating helicostatins in regulation of foregut motility. Ast-ir material was also present in nerves associated with muscles of the pyloric valve and rectum, and in endocrine cells of the midgut.     

Marine landscapes and population genetic structure of herring (Clupea harengus L.) in the Baltic Sea

Numerically small but statistically significant genetic differentiation has been found in many marine fish species despite very large census population sizes and absence of obvious barriers to migrating individuals. Analyses of morphological traits have previously identified local spawning groups of herring (Clupea harengus L.) in the environmentally heterogeneous Baltic Sea, whereas allozyme markers have not revealed differentiation. We analysed variation at nine microsatellite loci in 24 samples of spring-spawning herring collected at 11 spawning locations throughout the Baltic Sea. Significant temporal differentiation was observed at two locations, which we ascribe to sympatrically spawning but genetically divergent 'spawning waves'. Significant differentiation was also present on a geographical scale, though pairwise F(ST) values were generally low, not exceeding 0.027. Partial Mantel tests showed no isolation by geographical distance, but significant associations were observed between genetic differentiation and environmental parameters (salinity and surface temperature) (0.001 < P < or = 0.099), though these outcomes were driven mainly by populations in the southwestern Baltic Sea, which also exhibits the steepest environmental gradients. Application of a novel method for detecting barriers to gene flow by combining geographical coordinates and genetic differentiation allowed us to identify two zones of lowered gene flow. These zones were concordant with the separation of the Baltic Sea into major basins, with environmental gradients and with differences in migration behaviour. We suggest that similar use of landscape genetics approaches may increase the understanding of the biological significance of genetic differentiation in other marine fishes.