Ruminococcus bromii is a keystone species for the degradation of resistant starch in the human colon

The release of energy from particulate substrates such as dietary fiber and resistant starch (RS) in the human colon may depend on the presence of specialist primary degraders (or ‘keystone species'') within the microbial community. We have explored the roles of four dominant amylolytic bacteria found in the human colon in the degradation and utilization of resistant starches. Eubacterium rectale and Bacteroides thetaiotaomicron showed limited ability to utilize RS2- and RS3-resistant starches by comparison with Bifidobacterium adolescentis and Ruminococcus bromii. In co-culture, however, R. bromii proved unique in stimulating RS2 and RS3 utilization by the other three bacterial species, even in a medium that does not permit growth of R. bromii itself. Having previously demonstrated low RS3 fermentation in vivo in two individuals with undetectable populations of R. bromii-related bacteria, we show here that supplementation of mixed fecal bacteria from one of these volunteers with R. bromii, but not with the other three species, greatly enhanced the extent of RS3 fermentation in vitro. This argues strongly that R. bromii has a pivotal role in fermentation of RS3 in the human large intestine, and that variation in the occurrence of this species and its close relatives may be a primary cause of variable energy recovery from this important component of the diet. This work also indicates that R. bromii possesses an exceptional ability to colonize and degrade starch particles when compared with previously studied amylolytic bacteria from the human colon.     

The gut anaerobe Faecalibacterium prausnitzii uses an extracellular electron shuttle to grow at oxic–anoxic interphases

Faecalibacterium prausnitzii is one of the most abundant bacteria in the human gut ecosystem and it is an important supplier of butyrate to the colonic epithelium. Low numbers of faecalibacteria have been associated with inflammatory bowel disease. Despite being extremely oxygen sensitive, F. prausnitzii is found adherent to the gut mucosa where oxygen diffuses from epithelial cells. This paradox is now explained on the basis of gas tube experiments, flavin-dependent reduction of 5,5′-dithiobis-2-nitrobenzoate and microbial fuel cell experiments. The results show that F. prausnitzii employs an extracellular electron shuttle of flavins and thiols to transfer electrons to oxygen. Both compounds are present in the healthy human gut. Our observations may have important implications for the treatment of patients with Crohn''s disease, for example, with flavin- or antioxidant rich diets, and they provide a novel key insight in host–microbe interactions at the gut barrier.     

Crystal structure of a cellulosomal family 3 carbohydrate esterase from Clostridium thermocellum provides insights into the mechanism of substrate recognition

The microbial degradation of the plant cell wall is of increasing industrial significance, exemplified by the interest in generating biofuels from plant cell walls. The majority of plant cell-wall polysaccharides are acetylated, and removal of the acetyl groups through the action of carbohydrate esterases greatly increases the efficiency of polysaccharide saccharification. Enzymes in carbohydrate esterase family 3 (CE3) are common in plant cell wall-degrading microorganisms but there is a paucity of structural and biochemical information on these biocatalysts. Clostridium thermocellum contains a single CE3 enzyme, CtCes3, which comprises two highly homologous (97% sequence identity) catalytic modules appended to a C-terminal type I dockerin that targets the esterase into the cellulosome, a large protein complex that catalyses plant cell wall degradation. Here, we report the crystal structure and biochemical properties of the N-terminal catalytic module (CtCes3-1) of CtCes3. The enzyme is a thermostable acetyl-specific esterase that exhibits a strong preference for acetylated xylan. CtCes3-1 displays an α/β hydrolase fold that contains a central five-stranded parallel twisted β-sheet flanked by six α-helices. In addition, the enzyme contains a canonical catalytic triad in which Ser44 is the nucleophile, His208 is the acid-base and Asp205 modulates the basic nature of the histidine. The acetate moiety is accommodated in a hydrophobic pocket and the negative charge of the tetrahedral transition state is stabilized through hydrogen bonds with the backbone N of Ser44 and Gly95 and the side-chain amide of Asn124.     

Molecular interactions in the insulin-like growth factor (IGF) axis: a surface plasmon resonance (SPR) based biosensor study

This review describes a comprehensive analysis of a surface plasmon resonance (SPR)-based biosensor study of molecular interactions in the insulin-like growth factor (IGF) molecular axis. In this study, we focus on the interaction between the polypeptide growth factors IGF-I and IGF-II with six soluble IGF binding proteins (IGFBP 1-6), which occur naturally in various biological fluids. We have describe the conditions required for the accurate determination of kinetic rate constants for these interactions and highlight the experimental and theoretical pitfalls, which may be encountered in the early stages of such a study. We focus on IGFBP-5 and describe a site-directed mutagenesis study, which examines the contribution of various residues in the protein to high affinity interaction with IGF-I and -II. We analyse the interaction of IGFBP-5 (and IGFBP-3) with heparin and other biomolecules and describe experiments, which were designed to monitor multi-protein complex formation in this molecular axis.     

Animal models of psychiatric disease

Animal models of psychiatric diseases are useful tools for screening new drugs and for investigating the mechanisms of those disorders. Despite the difficulties inherent in modelling human psychiatric phenotypes in animals, there has been recent success identifying mutations in mice that give rise to some of the characteristic features of anxiety, depression, schizophrenia, autism, obsessive-compulsive disorder and bipolar disorder. In some cases these models have the additional strength that drugs used to treat the human condition alleviate the symptoms in mice. Robust genetic evidence of the involvement of multiple susceptibility genes in psychiatric disease will enable future studies to move from single-gene models to models with multiple modified loci, with the promise of better representing the complexity of the human diseases.     

Photochemistry of dyes and fluorochromes used in biology and medicine: some physicochemical background and current applications

An overview of the basic principles of photochemistry is presented to facilitate discussion of fluorescence, quenching and quantum yields. These topics in turn provide the foundation for an account of fluorescence spectroscopy and its application to microscopy. A brief overview of light microscopy and the application of fluorescence microscopy is given. The influences of molecular features, such as aromatic character and substitution patterns, on color and fluorescence are described. The concept of color fading is considered with particular reference to its effect on microscopic preparations. A survey of representative fluorescent probes is provided, and their sensitivity, application, and limitations are described. The phototoxicity of fluorescent molecules is discussed using biomembranes and DNA as examples of targets of toxicity. Photodynamic therapy, a relatively new clinical application of phototoxicity, is described. Both anticancer and antimicrobial applications are noted, and an assessment is given of the current ideas on the ideal physicochemical properties of the sensitizing agents for such applications.     

A biological spectral weighting function for ozone depletion research with higher plants

Biological spectral weighting functions (BSWF) play a key role in assessing implications of stratospheric ozone reduction. They are used to calculate the increase in biologically effective solar UV radiation due to ozone reduction (radiation amplification factor, RAF), assess current latitudinal gradients of solar UV radiation, and compare solar UV radiation with that from lamps and filters used in experiments. As a basis for a BSWF, we developed an action spectrum for growth responses of light-grown oat ( Avena sativa L. cv. Otana) seedlings exposed to narrowband UV radiation from a large double water-prism monochromator. Five UV wavelength peaks were used (275, 297, 302, 313 and 366 nm) in the absence of any visible radiation. Growth responses were measured from 1 to 10 days after the treatments. At all these wavelengths, the UV radiation inhibited height growth, including the height at which the first leaf separated from the stem. Radiation at all wavelengths used, except the one UV-A wavelength, promoted the length of the second leaf. The resulting action spectrum closely resembles the commonly used generalized plant response function except that it indicates continued sensitivity into the UV-A region. When used as a BSWF for the ozone depletion problem, this new function for plant growth would suggest substantially less impact of ozone depletion because it results in only a modest increment of biologically effective UV for a given level of ozone depletion (a lower RAF). Yet this new BSWF also suggests that experimental treatments based on previous BSWF with less emphasis on the UV-A may have resulted in simulations of less pronounced ozone depletion than intended. The validity of this new BSWF with UV-A sensitivity, designated the UV plant growth weighting function, was verified in field experiments as described in the companion paper.     

Oligonucleotide Probes That Detect Quantitatively Significant Groups of Butyrate-Producing Bacteria in Human Feces

16S rRNA-targeted oligonucleotide probes were designed for butyrate-producing bacteria from human feces. Three new cluster-specific probes detected bacteria related to Roseburia intestinalis, Faecalibacterium prausnitzii, and Eubacterium hallii at mean populations of 2.3, 3.8, and 0.6%, respectively, in samples from 10 individuals. Additional species-level probes accounted for no more than 1%, with a mean of 7.7%, of the total human fecal microbiota identified as butyrate producers in this study. Bacteria related to E. hallii and the genera Roseburia and Faecalibacterium are therefore among the most abundant known butyrate-producing bacteria in human feces.     

CD4 T Cells Are the Only Lymphocytes Needed To Protect Mice against Rotavirus Shedding after Intranasal Immunization with a Chimeric VP6 Protein and the Adjuvant LT(R192G)

Intranasal immunization of mice with a chimeric VP6 protein and the mucosal adjuvant Escherichia coli heat labile toxin LT(R192G) induces nearly complete protection against murine rotavirus (strain EDIM [epizootic diarrhea of infant mice virus]) shedding for at least 1 year. The aim of this study was to identify the protective lymphocytes elicited by this new vaccine candidate. Immunization of mouse strains lacking one or more lymphocyte populations revealed that protection was dependent on alphabeta T cells but mice lacking gammadelta T cells and B cells remained fully protected. Furthermore, depletion of CD8 T cells in immunized B-cell-deficient mice before challenge resulted in no loss of protection, while depletion of CD4 T cells caused complete loss of protection. Therefore, alphabeta CD4 T cells appeared to be the only lymphocytes required for protection. As confirmation, purified splenic T cells from immunized mice were intraperitoneally injected into Rag-2 mice chronically infected with EDIM. Transfer of 2 x 10(6) CD8 T cells had no effect on shedding, while transfer of 2 x 10(5) CD4 T cells fully resolved shedding in 7 days. Interestingly, transfer of naive splenic CD4 T cells also resolved shedding but more time and cells were required. Together, these results establish CD4 T cells as effectors of protection against rotavirus after intranasal immunization of mice with VP6 and LT(R192G).