The Gut of the Soil Microarthropod Folsomia candida (Collembola) Is a Frequently Changeable but Selective Habitat and a Vector for Microorganisms

Interaction potentials between soil microarthropods and microorganisms were investigated with Folsomia candida (Insecta, Collembola) in microcosm laboratory experiments. Microscopic analysis revealed that the volumes of the simple, rod-shaped guts of adult specimens varied with their feeding activity, from 0.7 to 11.2 nl. A dense layer of bacterial cells, associated with the peritrophic membrane, was detected in the midgut by scanning electron microscopy. Depending on the molting stage, which occurred at intervals of approximately 4 days, numbers of heterotrophic, aerobic gut bacteria changed from 4.9 × 10² to 2.3 × 10⁶ CFU per specimen. A total of 11 different taxonomic bacterial groups and the filamentous fungus Acremonium charticola were isolated from the guts of five F. candida specimens. The most abundant isolate was related to Erwinia amylovora (96.2% DNA sequence similarity to its 16S rRNA gene). F. candida preferred to feed on Pseudomonas putida and three indigenous gut isolates rather than eight different type culture strains. When luciferase reporter gene-tagged bacterial strains were pulse fed to F. candida, gut isolates were continuously shed for 8 days to several weeks but Escherichia coli HB101 was shed for only 1 day. Ratios of ingested to released bacterial cells demonstrated that populations of nonindigenous gut bacteria like Sinorhizobium meliloti L33 and E. coli HB101 were reduced by more than 4 orders of magnitude but that the population of gut isolate Alcaligenes faecalis HR4 was reduced only 500-fold. This work demonstrates that F. candida represents a frequently changeable but selective habitat for bacteria in terrestrial environments and that microarthropods have to be considered factors that modify soil microbial communities.     

Bayesian-based selection of metabolic objective functions

MOTIVATION: A critical component of in silico analysis of underdetermined metabolic systems is the identification of the appropriate objective function. A common assumption is that the objective of the cell is to maximize growth. This objective function has been shown to be consistent in a few limited experimental cases, but may not be universally appropriate. Here a method is presented to quantitatively determine the most probable objective function. RESULTS: The genome-scale metabolism of Escherichia coli growing on succinate was used as a case-study for analysis. Five different objective functions, including maximization of growth rate, were chosen based on biological plausibility. A combination of flux balance analysis and linear programming was used to simulate cellular metabolism, which was then compared to independent experimental data using a Bayesian objective function discrimination technique. After comparing rates of oxygen uptake and acetate production, minimization of the production rate of redox potential was determined to be the most probable objective function. Given the appropriate reaction network and experimental data, the discrimination technique can be applied to any bacterium to test a variety of different possible objective functions. SUPPLEMENTARY INFORMATION: Additional files, code and a program for carrying out model discrimination are available at http://www.engr.uconn.edu/~srivasta/modisc.html.     

A threonine synthase homolog from a mammalian genome

The genomes of several vertebrates contain two genes encoding proteins highly similar to threonine synthase (TS), even though the biosynthesis of l-threonine (l-Thr) is not known to occur in these animals. We report a bioinformatic analysis of the two TS-like genes, the recombinant expression of one murine TS homolog (mTSH2) and its initial biochemical characterization. Recombinant mTSH2 contained bound pyridoxal-5'-phosphate (PLP), but did not synthesize l-Thr. The enzyme did, however, bind O-phospho-homoserine (PHS; the actual TS substrate) and degraded it to alpha-ketobutyrate, phosphate, and ammonia-a known side reaction of microbial TSs. mTSH2 also degraded O-phospho-threonine (PThr) to alpha-ketobutyrate, showing that it can act as a catabolic phospho-lyase on both gamma- and beta-phosphorylated substrates. These findings suggest an unusual evolutionary origin for mTSH2, whereby an original TS enzyme became 'recycled' into a phospho-lyase upon dismissal, in metazoa, of the l-Thr biosynthetic pathway.     

Asymmetric synthesis of the four diastereoisomers of a novel non-steroidal farnesoid X receptor (FXR) agonist: Role of the chirality on the biological activity

An asymmetric synthetic strategy was designed for the preparation of the four possible diastereoisomers of 3,6-dimethyl-1-(2-methylphenyl)-4-(4-phenoxyphenyl)-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-7-one, a non-steroidal FXR agonist, we recently discovered following a virtual screening approach. The results obtained from an AlphaScreen assay clearly demonstrated that only the isomer endowed with 4R,6S absolute configuration is responsible for the biological activity. A deep investigation of the different putative binding modes adopted by these enantiomerically pure ligands using computational modeling studies confirmed the enantioselectivity of FXR towards this class of molecules.     

Characterization of oxidative stress in blood from diabetic vs. hypercholesterolaemic patients,using a novel synthesized marker

In the present study, we extend our novel concept of designing and using exogenous markers for the characterization of oxidative stress (OS) and OS-associated diseases. The aim was to use such a synthetic compound as a tool for studying OS in blood from diabetic and hypercholesterolaemic (Hc) patients. The marker used N-linoleoyl tyrosine (LT) was constructed from tyrosine and linoleic acid (LA); both components are known to be easily oxidized upon exposure to different types of reactive oxygen/nitrogen species (ROS/RNS), and to generate specific oxidized products, depending on the type of oxidants present in vivo. Using the LT probe, we showed that the ratios of oxidized LT to total LT (Ox-LT/LT) is significantly higher in blood samples obtained from diabetic patients, than in Hc patients or healthy control subjects. LC/MS analysis revealed that blood from diabetic patients oxidizes the marker with predominant formation of Ox-LT hydroperoxide (LT-OOH) and epoxide (epoxy-LT), where the LA moiety is oxidized to hydroperoxide and to epoxide, respectively. Analysis of oxysterol levels in these samples (GC/MS) revealed that the blood of both diabetic and Hc patients contained significantly more oxysterols than blood of control subjects. Consumption of pomegranate juice by diabetic patients for 3 months suppressed their blood capacity to oxidize the LT and similarly also reduced their blood oxysterol/total cholesterol ratio by 93%. The use of an exogenous marker to characterize OS in blood samples yields important information on the extent of OS, and can provide a fingerprint for the early identification of different pathological conditions associated with OS.     

Nitrogen deposition enhances moss growth,but leads to an overall decline in habitat condition of mountain moss‐sedge heath

Ecosystems are subject to multiple, natural and anthropogenic environmental influences, including nitrogen (N) deposition, land use and climate. Assessment of the relative importance of these influences on biodiversity and ecosystem functioning is crucial for guiding policy and management decisions to mitigate global change; yet, few studies consider multiple drivers. In the UK, ongoing loss of the internationally important arctic/alpine moss‐sedge community, Racomitrium heath, has been linked to elevated N deposition, high grazing pressures and their combination; however, the relative importance of these drivers remains unclear. We used environmental gradients across the habitat's European distribution (UK, Faroes, Norway and Iceland) to investigate the relative impact of N deposition and grazing pressure, as well as climate, on the condition of the dominant moss species, Racomitrium lanuginosum. Key variables including tissue chemistry, growth and cover were measured at 36 sites, and multiple linear regressions were used to examine the relative importance of the drivers across sites. Our results clearly show that regional variation in the condition of R. lanuginosum across Europe is primarily associated with the impacts of N deposition, with climate (air temperature) and grazing pressure playing secondary roles. In contrast to previous experimental studies, we found moss growth to be stimulated by elevated N deposition; this apparent discrepancy may result from the use of artificially high N concentrations in many experiments. Despite increased growth rates, we found that moss mat depth and cover declined in response to N deposition. Our results suggest that this is due to increased decomposition of material in the moss mat, which ultimately leads to loss of moss cover and habitat degradation. This study clearly demonstrates both the key role of N deposition in degradation of Racomitrium heath and the importance of observational studies along natural gradients for testing predictions from experimental studies in the real world.     

Preventing Staphylococcus aureus sepsis through the inhibition of its agglutination in blood

Staphylococcus aureus infection is a frequent cause of sepsis in humans, a disease associated with high mortality and without specific intervention. When suspended in human or animal plasma, staphylococci are known to agglutinate, however the bacterial factors responsible for agglutination and their possible contribution to disease pathogenesis have not yet been revealed. Using a mouse model for S. aureus sepsis, we report here that staphylococcal agglutination in blood was associated with a lethal outcome of this disease. Three secreted products of staphylococci--coagulase (Coa), von Willebrand factor binding protein (vWbp) and clumping factor (ClfA)--were required for agglutination. Coa and vWbp activate prothrombin to cleave fibrinogen, whereas ClfA allowed staphylococci to associate with the resulting fibrin cables. All three virulence genes promoted the formation of thromboembolic lesions in heart tissues. S. aureus agglutination could be disrupted and the lethal outcome of sepsis could be prevented by combining dabigatran-etexilate treatment, which blocked Coa and vWbp activity, with antibodies specific for ClfA. Together these results suggest that the combined administration of direct thrombin inhibitors and ClfA-antibodies that block S. aureus agglutination with fibrin may be useful for the prevention of staphylococcal sepsis in humans.