Selection of bacteria originating from a human intestinal microbiota in the gut of previously germ-free rats

Denaturing gradient gel electrophoresis (DGGE) was applied to separate PCR-amplified 16S rRNA genes originating from human microbiota associated (HMA) rat faeces as well as from the human faecal sample used for inoculation of the animals. Subsequently, a total of 15 dominant bands were excised from the DGGE gels, cloned and sequenced. Comparison of the obtained sequences with the Ribosomal Database revealed that species of Bacteroides/Prevotella and Faecalibacterium gave rise to the majority of the dominant bands in the human sample and in the HMA rats. In the HMA rats, two dominant bands, which were not present in the human DGGE profile, originated from species of Ruminococcus. With the exception of the Ruminococcus sequences, sequences originating from both rats and human samples were represented in all major branches of a maximum parsimony tree, indicating that the rat feed and gut environment allows colonization of the dominant taxonomic units from the human microbiota, but additionally selects for Ruminococci. Bands representing Prevotella and Faecalibacterium, which were found in identical positions of the DGGE gels originating from human and HMA rat faecal samples, originated from completely identical sequences, indicating that the same strains of these species were dominating in the human and rat samples.     

Die Übereinstimmung Zwischen Entwicklungsstadien und Phaenotypen Verschiedener Genotypen

Zusammenfassung BeiLinum usitatissimum kommen ausser einfarbigen auch bunte Samen vor.Die Samen der einzelnen Pflanzen zeigen eine bedeutende Variabilität des Buntheitsgrades.Es gibt mehrere voneinander verschiedene erbliche buntsamige Typen.Die genotypisch bedingten Unterschiede der buntsamigen Typen beziehen sich 1. auf das Verhältnis der hell- und der dunkel gefärbten Teile der Samenschale und 2. auf das Verhältnis der Anzahl der Samen mit verschiedenem Buntheitsgrade und eventual mit einfarbiger Schale. Die verschiedenen erblichen buntsamigen Typen stimmen in ihren Phaenotypen überein mit verschiedenen Stadien, welche die einfarbigen Samen während ihrer Entwicklung durchlaufen.Die Unterschiede zwischen den verschiedenen buntsamigen Typen und zwischen diesen und dem einfarbiten Typus sind quantitativ; es sind Unterschiede in der Reaktionsgeschwindigkeit mit der der Farbstoff in der Samenschale gebildet wird.Mit 2 Figuren     

Genetic polymorphisms ofQ region genes from wild-derived mice: Implications forQ region evolution

Both serological and DNA sequence analyses were performed to determine the extent of genetic polymorphism inQ region genes. A panel of Qa-2-specific monoclonal antibodies (mAbs) was tested on 35 wildderived and inbred mouse strains. Members of this reagent panel recognize multiple and distinct epitopes on the Qa-2-bearing molecule(s). Although quantitative variations in Qa-2 levels were observed, no structural polymorphisms were detected. All strains were either entirely positive or entirely negative with the complete set of reagents. Moreover, cell surface Qa-2 expression was not significantly affected by differences in age or sex of the mouse or cell cycle status. To confirm this apparent lack of genetic polymorphism, the polymerase chain reaction (PCR) technique was used to amplify a portion of the 3 end of theQ region genes,Q4 toQ9, from several independent wild-derived strains of mice. Sequence analysis of the amplified material revealed very little evidence of nucleotide divergence. All strains tested had aQ even DNA sequence identical to that ofQ6/Q8 in the B10 strain. Likewise, all tested strains had aQ odd DNA sequence identical toQ7/Q9 in the B10 strain. Two strains showed additionalQ even sequences, while all strains tested possessed additionalQ odd sequences. The observed lack of polymorphism suggests that theQ genes have evolved in a different manner fromH-2K andH-2D. Moreover, duplications of these genes appear to have arisen prior to nucleotide sequence divergence.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M30896-30902.     

Anti-MHC immunity detected prior to intentional alloimmunization

Cell fusion was performed between spleen cells from young BALB/cBy (H-2 ^d) mice which have never been immunized and SP2/0 mouse plasmacytoma cells. A monoclonal H-2-specific cytotoxic IgM antibody was obtained (By-1) which detected a new public biregional H-2 specificity, H-2.m210. The mcAb By-1 reacted strongly with H-2K^d, D^d, and H-2^s antigens, gave weak cross-reactions with H-2K^k, D^q, H-2^r, and H-2^v antigens and was negative with H-2^b, H-2^f, H-2^p, and H-2L^d antigens. A polymorphic reaction pattern was also observed on a panel of lymphocytes from B 10.W strains. The intriguing finding on this reaction pattern was the reactivity on H-2^d cells, including the syngeneic BALB/cBy and truly autologous cells. As shown by capping and immunoprecipitation experiments on H-2^d cells and by studies on H-2^d-transfected mouse L cells, the target molecules for McAb By-1 were H-2K^d and H-2D^d molecules. The BALB/cBy mouse, from whose spleen cells the McAb By-1 was obtained, survived after the fusion experiment, and serum was examined for the presence of cytotoxic H-2-specific antibodies during the rest of its life. At the time of the fusion, no autoreactive serum antibodies were found, but about 4 months later, we found in the serum of this mouse autoreactive H-2-specific cytotoxic IgM antibodies. The serum antibodies followed the same reaction pattern as that of the McAb By-1. As far as we know, this is the first report of autoreactive H-2-specific antibodies in serum of a mouse which has never been immunized and of the first natural autoreactive H-2-specific monoclonal antibody.Abbreviations McAb monoclonal antibody - MHC major histocompatibility complex - H-2 major histocompatibility complex of mice - CTLs cytotoxic T cells - FMF flow microfluorometry - FITC fluorescein isothiocyanate - LPS lipopolysaccharide W.E. coli 0111:134 - PBS phosphate-buffered saline - Iodogen 1,3,4,6,-tetrachloro-3,6-diphenylglycoluril - GAMIg goat-antimouse immunoglobulin - Staph-A Staphylococcus aureus Cowan I     

Design and synthesis of tetrahydropyridopyrimidine based Toll-Like Receptor (TLR) 7/8 dual agonists

In a continuing effort to discover novel TLR agonists, herein we report on the discovery and structure–activity relationship of novel tetrahydropyridopyrimidine TLR 7/8 agonists. Optimization of this series towards dual agonist activity and a high clearance profile resulted in the identification of compound 52a1. Evaluation in vivo revealed an interferon stimulated response (ISG) in mice with limited systemic exposure and demonstrated the potential in antiviral treatment or as a vaccine adjuvant.     

Action of different types of endoxylanases on eucalyptus xylan in situ

Most studies of the mode of action of industrially important endoxylanases have been done on alkali extracted-plant xylan. In just few cases, the native form of the polysaccharide, acetylated xylan, was used as a substrate. In this work action of xylanases belonging to three glycoside hydrolase families, GH10, GH11, and GH30 was investigated on acetylglucuronoxylan directly in hardwood cell walls. Powdered eucalyptus wood was used as xylanase substrate. Enzyme-generated fragments were characterized by TLC, MALDI ToF MS, and NMR spectroscopy. All three xylanases generated from eucalyptus wood powder acetylated xylooligosaccharides. Those released by GH10 enzyme were the shortest, and those released by GH30 xylanase were of the largest diversity. For GH30 xylanase the 4-O-methyl-D-glucuronic acid (MeGlcA) side residues function as substrate specificity determinants regardless the acetylation of the neighboring hydroxyl group. Much simpler xylooligosaccharide patterns were observed when xylanases were applied in combination with carbohydrate esterase family 6 acetylxylan esterase. In the presence of the esterase, all aldouronic acids remained 3-O-acetylated on the xylopyranosyl (Xylp) residue substituted with MeGlcA. The 3-O-acetyl group, in contrast to the acetyl groups of otherwise unsubstituted Xylp residues, does not affect the mode of action of endoxylanases, but contributes to recalcitrance of the acidic xylan fragments. The results confirm importance of acetylxylan esterases in microbial degradation of acetylated hardwood glucuronoxylan. They also point to still unresolved question of efficient enzymatic removal of the 3-O-acetyl group on MeGlcA-substituted Xylp residues negatively affecting the saccharification yields.

     

World Health Organization Estimates of the Relative Contributions of Food to the Burden of Disease Due to Selected Foodborne Hazards: A Structured Expert Elicitation

Background

The Foodborne Disease Burden Epidemiology Reference Group (FERG) was established in 2007 by the World Health Organization (WHO) to estimate the global burden of foodborne diseases (FBDs). This estimation is complicated because most of the hazards causing FBD are not transmitted solely by food; most have several potential exposure routes consisting of transmission from animals, by humans, and via environmental routes including water. This paper describes an expert elicitation study conducted by the FERG Source Attribution Task Force to estimate the relative contribution of food to the global burden of diseases commonly transmitted through the consumption of food.

Methods and Findings

We applied structured expert judgment using Cooke’s Classical Model to obtain estimates for 14 subregions for the relative contributions of different transmission pathways for eleven diarrheal diseases, seven other infectious diseases and one chemical (lead). Experts were identified through international networks followed by social network sampling. Final selection of experts was based on their experience including international working experience. Enrolled experts were scored on their ability to judge uncertainty accurately and informatively using a series of subject-matter specific ‘seed’ questions whose answers are unknown to the experts at the time they are interviewed. Trained facilitators elicited the 5^th, and 50^th and 95^th percentile responses to seed questions through telephone interviews. Cooke’s Classical Model uses responses to the seed questions to weigh and aggregate expert responses. After this interview, the experts were asked to provide 5^th, 50^th, and 95^th percentile estimates for the ‘target’ questions regarding disease transmission routes. A total of 72 experts were enrolled in the study. Ten panels were global, meaning that the experts should provide estimates for all 14 subregions, whereas the nine panels were subregional, with experts providing estimates for one or more subregions, depending on their experience in the region. The size of the 19 hazard-specific panels ranged from 6 to 15 persons with several experts serving on more than one panel. Pathogens with animal reservoirs (e.g. non-typhoidal Salmonella spp. and Toxoplasma gondii) were in general assessed by the experts to have a higher proportion of illnesses attributable to food than pathogens with mainly a human reservoir, where human-to-human transmission (e.g. Shigella spp. and Norovirus) or waterborne transmission (e.g. Salmonella Typhi and Vibrio cholerae) were judged to dominate. For many pathogens, the foodborne route was assessed relatively more important in developed subregions than in developing subregions. The main exposure routes for lead varied across subregions, with the foodborne route being assessed most important only in two subregions of the European region.

Conclusions

For the first time, we present worldwide estimates of the proportion of specific diseases attributable to food and other major transmission routes. These findings are essential for global burden of FBD estimates. While gaps exist, we believe the estimates presented here are the best current source of guidance to support decision makers when allocating resources for control and intervention, and for future research initiatives.