Diagnosing atopic dermatitis in infancy using established diagnostic criteria: a cohort study.

BACKGROUND: Diagnosing atopic dermatitis (AD) in infants is challenging. OBJECTIVES: To determine the incidence and persistence of eczema and AD in infants using the UK Working Party (UKWP) and Hanifin and Rajka (H&R) criteria. METHODS: A cohort of 1834 infants was examined clinically at 3, 6 and 12 months of age. AD was diagnosed by UKWP (3, 6 and 12 months) and H&R (12 months) criteria. Logistic regression models were used to assess the relationship between AD and eczema. RESULTS: Eczema was observed in 628 (34·2%) infants (n = 240, n = 359 and n = 329 at 3, 6 and 12 months, respectively), with AD diagnosed in 212 (33·7%) infants with any eczema and in 64/78 (82%) infants with eczema at all three visits. The odds of AD were lower with first presentation of eczema at 6 [odds ratio (OR) 0·33, 95% confidence interval (CI) 0·22-0·48] or 12 months (OR 0·49, 95% CI 0·32-0·74) than at 3 months, and higher in infants with eczema at three (OR 23·1, 95% CI 12·3-43·6) or two (OR 6·5, 95% CI 4·3-9·9) visits vs. one visit only. At 12 months, 156/329 (47·4%) fulfilled the UKWP and/or H&R criteria; 27 (8%) fulfilled the UKWP criteria only and 65 (20%) only the H&R criteria. Of the 129 infants who fulfilled the H&R criteria, 44 (34·1%) did not meet the itch criterion. CONCLUSIONS: Used in combination and at multiple timepoints, the UKWP and H&R criteria for AD may be useful in clinical research but may have limited value in most other clinical settings.

Filaggrin mutations in relation to skin barrier and atopic dermatitis in early infancy.

BACKGROUND: Loss-of-function mutations in the skin barrier gene filaggrin (FLG) increase the risk of atopic dermatitis (AD), but their role in skin barrier function, dry skin and eczema in infancy is unclear. OBJECTIVES: To determine the role of FLG mutations in impaired skin barrier function, dry skin, eczema and AD at 3 months of age and throughout infancy. METHODS: FLG mutations were analysed in 1836 infants in the Scandinavian population-based PreventADALL study. Transepidermal water loss (TEWL), dry skin, eczema and AD were assessed at 3, 6 and 12 months of age. RESULTS: FLG mutations were observed in 166 (9%) infants. At 3 months, carrying FLG mutations was not associated with impaired skin barrier function (TEWL > 11·3 g m-2  h-1 ) or dry skin, but was associated with eczema [odds ratio (OR) 2·89, 95% confidence interval (CI) 1·95-4·28; P < 0·001]. At 6 months, mutation carriers had significantly higher TEWL than nonmutation carriers [mean 9·68 (95% CI 8·69-10·68) vs. 8·24 (95% CI 7·97-8·15), P < 0·01], and at 3 and 6 months mutation carriers had an increased risk of dry skin on the trunk (OR 1·87, 95% CI 1·25-2·80; P = 0·002 and OR 2·44, 95% CI 1·51-3·95; P < 0·001) or extensor limb surfaces (OR 1·52, 95% CI 1·04-2·22; P = 0·028 and OR 1·74, 95% CI 1·17-2·57; P = 0·005). FLG mutations were associated with eczema and AD in infancy. CONCLUSIONS: FLG mutations were not associated with impaired skin barrier function or dry skin in general at 3 months of age, but increased the risk for eczema, and for dry skin on the trunk and extensor limb surfaces at 3 and 6 months.

Prokaryote species richness is positively correlated with eukaryote abundance in wastewater treatment biofilms.

Biological treatment represents a key step in nutrient removal from wastewater. Until now these process has mainly been considered prokaryotic, with the interactions between prokaryotes and eukaryotes not being properly explored. We therefore investigated the co-occurrence of eukaryotes and prokaryotes in biological nitrogen removal biofilms. We found that biofilms in the nitrifying reactor contained the highest diversity and abundance of both prokaryotes and eukaryotes, with nearly three times higher prokaryote species richness than for the denitrifying reactor. The positive associations between eukaryote abundance and prokaryote diversity could potentially be explained by mutualism - and/or predator/prey interactions. Further mechanistic insight, however, is needed to determine the main diversifying mechanisms. In summary, eukaryote and prokaryote interactions seem to play a fundamental yet underexplored role in biological wastewater treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Eukaryote and prokaryote interactions may play an important role in wastewater treatment. This study found that prokaryote species richness was nearly three times higher in the aerobe nitrification than in an anaerobe denitrification reactor, coinciding with the highest level of eukaryotes. This knowledge can be important in process control, and potentially in the development of novel approaches based on nitrate accumulating denitrifying eukaryotes.

Transition from infant- to adult-like gut microbiota.

Transition from an infant to an adult associated gut microbiota with age through establishment of strict anaerobic bacteria remains one of the key unresolved questions in gut microbial ecology. Here a comprehensive comparative analysis of stool microbiota in a large cohort of mothers and their children sampled longitudinally up until 2 years of age using sequencing analysis tool was presented that allows realistic microbial diversity estimates. In this work, evidence for the switch from children to adult associated microbial profile between 1 and 2 years of age was provided, suggestively driven by Bifidobacterium breve. An Operational Taxonomic Unit (OTU) belonging to B. breve was highly prevalent in the population throughout the first year of life, and was negatively associated with detection of a range of adult-like OTUs. Although an adult profile was not fully established by 2 years of age, it was demonstrated that with regards to the most prevalent OTUs, their prevalence in the child population by then already resembled that of the adult population. Taken together, it was proposed that late-colonizing OTUs were recruited at a later stage and were not acquired at birth with the recruitment being controlled by gatekeeping OTUs until the age of 1 year.

Bifidobacterial succession and correlation networks in a large unselected cohort of mothers and their children.

Bifidobacteria are a major microbial component of infant gut microbiota, which is believed to promote health benefits for the host and stimulate maturation of the immune system. Despite their perceived importance, very little is known about the natural development of and possible correlations between bifidobacteria in human populations. To address this knowledge gap, we analyzed stool samples from a randomly selected healthy cohort of 87 infants and their mothers with >90% of vaginal delivery and nearly 100% breast-feeding at 4 months. Fecal material was sampled during pregnancy, at 3 and 10 days, at 4 months, and at 1 and 2 years after birth. Stool samples were predicted to be rich in the species Bifidobacterium adolescentis, B. bifidum, B. dentium, B. breve, and B. longum. Due to high variation, we did not identify a clear age-related structure at the individual level. Within the population as a whole, however, there were clear age-related successions. Negative correlations between the B. longum group and B. adolescentis were detected in adults and in 1- and 2-year-old children, whereas negative correlations between B. longum and B. breve were characteristic for newborns and 4-month-old infants. The highly structured age-related development of and correlation networks between bifidobacterial species during the first 2 years of life mirrors their different or competing nutritional requirements, which in turn may be associated with specific biological functions in the development of healthy gut.

Estimation of metagenome size and structure in an experimental soil microbiota from low coverage next-generation sequence data.

AIMS: A major challenge in metagenome studies is to estimate the true size of all combined genomes. Here, we present a novel approach to estimate the size of all combined genomes for low coverage next-generation sequencing (NGS) data through empirically determined copy numbers of random DNA fragments. METHODS AND RESULTS: Size estimates were made based on analyses of two experimental soil micro-ecosystems - simulating soil with and without earthworms. Our analyses showed combined genome sizes of about log 11 nucleotides for each of the soil micro-ecosystems, as estimated from qPCR determined copy numbers of random DNA fragments. This corresponds to more than 20000 unique bacterial genomes in each sample. There seemed, however, to be a bacterial subpopulation in the earthworm soil, not being present in the nonearthworm soil. To describe the structure of the metagenomes, both total DNA and amplified 16S rRNA gene sequence libraries were generated with 454-sequencing. Bioinformatic analysis of 454 sequence libraries showed a large functional but low taxonomic overlap between the samples with and without earthworms. A neutrality test indicated that rare species have a competitive advantage over abundant species in both micro-ecosystems providing a potential explanation for the large metagenome sizes. CONCLUSIONS: We have shown that the soil metagenome is very large and that the large size is probably a consequence of top-down selection of the dominant bacterial species. SIGNIFICANCE AND IMPACT OF THE STUDY: Estimates of metagenome size from low coverage NGS data will be important for guiding future NGS set-ups.

Abrupt temporal fluctuations in the chicken fecal microbiota are explained by its gastrointestinal origin.

One of the main challenges in understanding the composition of fecal microbiota is that it can consist of microbial mixtures originating from different gastrointestinal (GI) segments. Here, we addressed this challenge for broiler chicken feces using a direct 16S rRNA gene-sequencing approach combined with multivariate statistical analyses. Broiler feces were chosen because of easy sampling and the importance for pathogen transmission to the human food chain. Feces were sampled daily for 16 days from chickens with and without a feed structure-induced stimulation of the gastric barrier function. Overall, we found four dominant microbial phylogroups in the feces. Two of the phylogroups were related to clostridia, one to lactobacilli, and one to Escherichia/Shigella. The relative composition of these phylogroups showed apparent stochastic temporal fluctuations in feces. Analyses of dissected chickens at the end of the experiment, however, showed that the two clostridial phylogroups were correlated to the microbiota in the cecum/colon and the small intestine, while the upper gut (crop and gizzard) microbiota was correlated to the lactobacillus phylogroup. In addition, chickens with a stimulated gizzard also showed less of the proximate GI dominating bacterial group in the feces, supporting the importance of the gastric barrier function. In conclusion, our results suggest that GI origin is a main determinant for the chicken fecal microbiota composition. This knowledge will be important for future understanding of factors affecting shedding of both harmful and beneficial gastrointestinal bacteria through feces.

Reduced spread of Campylobacter jejuni in broiler chickens by stimulating the bird's natural barriers.

AIM: We have tested the effect of feed structure and feeding regime to prevent the spread of the zoonotic pathogen Campylobacter jejuni in broiler chicken flocks. METHODS AND RESULTS: Birds were offered two types of feed, control diet and a diet supplemented with 15% oat/barley hulls for structure. In addition, the birds were either fed ad libitum or intermittent. One bird in each treatment group was infected with a three-strain-mix of Camp. jejuni, and the spread of Camp. jejuni within the group was investigated. Feed structure increased the gizzard weight, delayed the spread of Camp. jejuni within the group and reduced the relative amount of Camp. jejuni in the caecum compared with the control diet. CONCLUSIONS: Our results show that stimulating the bird's natural barriers is a novel and promising intervention strategy to reduce the spread of Camp. jejuni in chicken flocks. SIGNIFICANCE AND IMPACT OF THE STUDY: Preventing Camp. jejuni in broiler chicken flocks is essential to ensure food safety because this bacterium is transferred to chicken carcasses during the slaughter process and readily survive in unprocessed poultry products. We have evaluated a novel approach for stimulation of the bird's natural barriers in the upper digestive tract with promising results.

Modelling bacterial transmission in human allergen-specific IgE sensitization.

AIMS: The impact of bacterial transmission from mother to child on human allergy development is poorly understood. The aim of the present work was therefore to use a temporal collected dataset of 117 mothers and their children to model the potential effect of mother-to-child bacterial transmission on allergy (IgE) sensitization. METHODS AND RESULTS: We have recently shown a negative IgE correlation to high Escherichia coli levels until the age of 1 year, with a shift to positive correlation to high Bacteroides fragilis levels at the age of 2. In the present work, we used the previous published data to model the persistence and interaction effects of E. coli and B. fragilis with respect to IgE sensitization. Temporal modelling was made by first defining a stochastic model for sensitization state based on Markov chains and regression tree analyses. Subsequent simulations were used to determine the impact of mother-to-infant bacterial transmission. The regression tree analyses showed that E. coli colonization within 4 days was negatively correlated to sensitization, while lack of E. coli colonization at day 4 combined with B. fragilis colonization after 4 months was positively correlated. With Markov chain analyses, we found that E. coli was highly persistent in infants until the age of 4 months, while the persistence of B. fragilis increased with age. CONCLUSIONS: Simulations showed that the mother's bacterial composition correlated significantly to the child's IgE sensitization state at the age of 2 years. High E. coli and low B. fragilis levels in the mother were negatively correlated, while low E. coli and high B. fragilis were positively correlated to IgE. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results support that allergy could partly be communicable, being transferred from mother to infant through the gut microbiota.

Rapid lactic acid bacteria identification in dairy products by high-resolution melt analysis of DGGE bands.

AIM: To investigate the application of high-resolution melt (HRM) analysis for rapid species-level identification of lactic acid bacteria (LAB) communities in dairy products, as well as for bacterial community profiling and monitoring. METHODS AND RESULTS: First, comparisons of HRM profiles of known reference strains of LAB and their denaturing gradient gel electrophoresis (DGGE) bands showed very good agreement, allowing species recognition and identification from DGGE bands by HRM. Second, samples of cheese, kefir grains and kefir were characterized by PCR-DGGE, and melting profiles of DGGE bands were compared with known reference strains. Of the 13 DGGE bands, ten were identified by HRM by comparison with the reference strains and only three required sequencing for identification. Use of HRM profiling for comparison and monitoring of total LAB communities from dairy products or starter cultures was also evaluated, and good agreement was found when comparing clustering of DGGE band profiles with clustering of HRM melting profiles. CONCLUSION: Identification of DGGE bands is possible by comparison of HRM melting profiles with known reference strains. SIGNIFICANCE AND IMPACT OF THE STUDY: HRM profiling is suggested as an additional approach for identification of DGGE bands.

Strain-level characterization of nonstarter lactic acid bacteria in Norvegia cheese by high-resolution melt analysis.

The nonstarter lactic acid bacteria (NSLAB) constitute an important microbial group found during cheese ripening and they are thought to be fundamental to the quality of cheese. Rapid and accurate diagnostic tests for NSLAB are important for cheese quality control and in understanding the cheese ripening process. Here, we present a novel rapid approach for strain-level characterization through combined 16S rRNA gene and repetitive sequence-based high-resolution melt analysis (HRM). The approach was demonstrated through the characterization of 94 isolates from Norvegia, a Gouda-type cheese. The HRM profiles of the V1 and V3 variable regions of the 16S rRNA gene of the isolates were compared with the HRM profiles of 13 reference strains. The HRM profile comparison of the V1 and V3 regions of the 16S rRNA gene allowed discrimination of isolates and reference strains. Among the cheese isolates, Lactobacillus casei/paracasei (62 isolates) and Lactobacillus plantarum/Lactobacillus pentosus (27 isolates) were the dominant species, whereas Lactobacillus curvatus/Lactobacillus sakei were found occasionally (5 isolates). The HRM profiling of repetitive sequence-based PCR using the (GTG)(5) primer was developed for strain-level characterization. The clustering analysis of the HRM profiles showed high discriminatory power, similar to that of cluster analysis based on the gel method. In conclusion, the HRM approach in this study may be applied as a fast, accurate, and reproducible method for characterization of the NSLAB microflora in cheese and may be applicable to other microbial environments following selective plate culturing.

Bimodal distribution of seafloor microbiota diversity and function are associated with marine aquaculture.

The aim of the current work was to investigate the impact of marine aquaculture on seafloor biogeochemistry and diversity from pristine environments in the northern part of Norway. Our analytical approach included analyses of 182 samples from 16 aquaculture sites using 16S and 18S rRNA, shotgun analyses, visual examination of macro-organisms, in addition to chemical measurements. We observed a clear bimodal distribution of the prokaryote composition and richness, determined by analyses of 16S rRNA gene operational taxonomic units (OTUs). The high OTU richness cluster was associated with non-perturbed environments and farness from the aquaculture sites, while the low OTU richness cluster was associated with perturbed environments and proximity to the aquaculture sites. Similar patterns were also observed for eukaryotes using 18S rRNA gene analyses and visual examination, but without a bimodal distribution of OTU richness. Shotgun sequencing showed the archaeum Nitrosopumilus as dominant for the high OTU richness cluster, and the epsilon protobacterium Sulfurovum as dominant for the low OTU richness cluster. Metabolic reconstruction of Nitrosopumilus indicates nitrification as the main metabolic pathway. Sulfurovum, on the other hand, was associated with sulfur oxidation and denitrification. Changes in nitrogen and sulfur metabolism is proposed as a potential explanation for the difference between the high and low OTU richness clusters. In conclusion, these findings suggest that pollution from elevated loads of organic waste drives the microbiota towards a complete alteration of respiratory routes and species composition, in addition to a collapse in prokaryote OTU richness.

Temporal variations in early gut microbial colonization are associated with allergen-specific immunoglobulin E but not atopic eczema at 2 years of age.

BACKGROUND: Intestinal microbiota undergoes substantial development during the first 2 years of life, important for intestinal immunologic development and maturation influencing systemic immune responses. OBJECTIVE: We aimed to investigate, using a prospective study design, whether allergen-specific IgE (sIgE) and atopic eczema are associated with variations in gut microbial colonization patterns in an unselected population during the first 2 years of life. METHODS: Faeces from 94 infants were repeatedly sampled from 10 days, 4 months, 1 and 2 years postnatal and analysed for 12 different bacterial species by quantitative real-time PCR. Venous blood samples from the infants were collected at 2 years of age and were analysed for sIgE for 12 specific allergens. The temporal gut colonization patterns for 42 sIgE-positive (sIgE≥0.35 kU/L) and 52 sIgE-negative children (sIgE<0.1 kU/L) were then compared. The association between colonization pattern and phenotype as atopic eczema according to UK Working Party (UKWP) criteria were also described. RESULTS: Subjects with atopic sensitization had lower levels of Escherichia coli at 4 months and 1 year, higher levels of Bifidobacterium longum at 1 year and lower levels of Bacteroides fragilis at 2 years. For E. coli and B. longum, the differences were only transient and had disappeared by 2 years of age. For other species, there were no differences in colonization patterns, and we found no association between colonization pattern and atopic eczema. CONCLUSIONS AND CLINICAL RELEVANCE: We found temporal and transient variations in gut microbial colonization patterns associated with differences in sIgE sensitization at 2 years of age. A full understanding of the principles and mechanisms that underlie intestinal microbial colonization and diversity and host-microbiota relationships will be pivotal for the development of therapeutic approaches that manipulate the intestinal microbiota to maintain human health. [ REGISTRATION NUMBER: ISRCTN28090297].

Mother-to-child transmission of and multiple-strain colonization by Bacteroides fragilis in a cohort of mothers and their children.

Bacteroides fragilis represents an early infant colonizer with important host interactions. Our knowledge about the diversity, transmission, and persistence of this bacterium, however, is limited. Here, we addressed these questions using a combination of multilocus sequence typing (MLST) and variable-number tandem repeat (VNTR) sequence analyses. We used both culture-dependent and -independent typing. We genotyped B. fragilis in fecal samples from a cohort of 93 mothers and their children, with samples taken from the mothers and from the children at the ages 1 to 10 days, 4 months, 1 year, and 2 years. By MLST we found two main B. fragilis groups, which we denoted clades A and B. Direct typing of stool samples using the icd gene revealed seven sequence types, five within clade A and two within clade B. A single clade A sequence type, however, represented 79% of all the sequences. This sequence type was further subtyped using VNTR. VNTR subtyping revealed 16 different VNTR types. Based on the distribution patterns of these, we show mother-to-child transmission and multiple-strain colonization. We argue that negative host selection promotes the coexistence of multiple strains. The significance of our findings is that we have started unraveling the transmission and persistence patterns of one of the most important human gut colonizers.

Prevention of intestinal Campylobacter jejuni colonization in broilers by combinations of in-feed organic acids.

AIM: We have tested the effect of various combinations of formic acid and sorbate on Campylobacter jejuni colonization in broiler chickens to reduce the colonization of this zoonotic pathogen in broiler chicken flocks. METHODS AND RESULTS: Chickens were offered feed supplemented with different concentrations and combinations of formic acid and/or potassium sorbate. We found little or no effect on the Camp. jejuni colonization levels in chickens that were given feed supplemented with formic acid alone. A combination of 1.5% formic acid and 0.1% sorbate reduced the colonization of Camp. jejuni significantly, while a concentration of 2.0% formic acid in combination with 0.1% sorbate prevented Camp. jejuni colonization in chickens. This inhibition was replicated in two independent trials with a combination of three different Camp. jejuni strains. CONCLUSIONS: Our results show a novel and promising intervention strategy to reduce the incidence of Camp. jejuni in poultry products and to obtain safer food. SIGNIFICANCE AND IMPACT OF THE STUDY: To ensure food safety, a reduction of the carcass contamination with Camp. jejuni through reduced colonization of this pathogen in broiler chicken flocks is important. A range of organic acids as additives in feed and drinking water have already been evaluated for this purpose. However, no studies have yet shown a complete inhibition of Camp. jejuni colonization in broiler chickens.

Ratios between Alpha-, Beta- and Gamma-proteobacteria in tap water determined by the ProteoQuant assay.

AIMS: Proteobacteria are widespread on earth. Recently, it has been discovered that a diverse repertoire of proteobacteria are also dominant in tap water. It is therefore important to use high-throughput monitoring tools for tap water. Here, the high-throughput assay ProteoQuant was developed to quantify the main proteobacterial phyla in tap water. METHODS AND RESULTS: The principle of ProteoQuant is proteobacterial-selective 23S rRNA gene PCR amplification, with multiple competitive TaqMan probes for quantifying the phyla Alpha-, Beta- and Gamma-proteobacteria. The ProteoQuant assay was evaluated, analysing both designed proteobacterial mixes and rRNA gene clone libraries from tap water. These evaluations showed a good coverage and accuracy of the ProteoQuant assay. CONCLUSIONS: Large-scale tap water screening using ProteoQuant revealed a dominance of Beta-proteobacteria and a potential interaction between Alpha- and Beta-proteobacteria. Gamma-proteobacteria, on the other hand, seemed independent of the two other phyla. SIGNIFICANCE AND IMPACT OF THE STUDY: The ProteoQuant assay will potentially be important for future understanding of the ecological forces shaping the tap water microbiota.

Association of gut microbiota with metabolism in juvenile Atlantic salmon.

The gut microbiome plays a key role in animal health and metabolism through the intricate functional interconnection between the feed, gut microbes, and the host. Unfortunately, in aquaculture, the links between gut microbes and fish genetics and production phenotypes are not well understood.In this study, we investigate the associations between gut microbial communities, fish feed conversion, and fish genetics in the domestic Atlantic salmon. Microbial community composition was determined for 230 juvenile fish from 23 full-sib families and was then regressed on growth, carbon and nitrogen metabolism, and feed efficiency. We only found weak associations between host genetics and microbial composition. However, we did identify significant (p < 0.05) associations between the abundance of three microbial operational taxonomical units (OTUs) and fish metabolism phenotypes. Two OTUs were associated with both carbon metabolism in adipose tissue and feed efficiency, while a third OTU was associated with weight gain.In conclusion, this study demonstrates an intriguing association between host lipid metabolism and the gut microbiota composition in Atlantic salmon. Video Abstract.

Evaluation of DNA Extraction Methods for Processing Fingerprint Powder-Coated Forensic Evidence.

In unison, fingerprinting and DNA analysis have played a pivotal role in forensic investigations. Fingerprint powders that are available on the market can come in a range of colors and with specific properties. This study evaluated the efficiency of DNA extraction from samples coated with 3 brands of fingerprint powders: Lightning, Sirchie, and SupraNano, covering a range of colors and properties. A total of 23 fingerprint powders were tested using the Chelex, Promega DNA IQ™, and Applied Biosystems™ PrepFiler™ DNA extraction protocols. The DNA IQ™ and PrepFiler™ methods extracted higher yields of DNA in comparison to Chelex, which also accounted for better quality of PowerPlex x00AE; 21 DNA profiles recovered. There were no signs of degradation or inhibition in the quantification data, indicating that samples returning low DNA yield was due to interference during DNA extraction and not PCR inhibition. DNA profiles were recovered from the majority of fingerprint powders with only a single powder, Sirchie Magnetic Silver, failing to produce a profile using any of the methods tested. A link was observed between the DNA extraction chemistry, fingerprint powder property, that is, nonmagnetic, magnetic and aqueous, and the brand of fingerprint powder. Overall, the DNA IQ™ method was favorable for nonmagnetic fingerprint powders, while magnetic fingerprint powders produced more DNA profiles when extracted with the PrepFiler™ chemistry. This study highlights the importance of screening DNA extraction chemistries for the type of fingerprint powder used, as there is not a single DNA extraction method that suits all fingerprint powder brands and properties.

Novel 16S rRNA gene analyses reveal new in vitro effects of insoluble barley fibres on the human faecal microbiota.

AIMS: The aim of this work was to analyse the growth of human faecal microbiota on barley dietary fibres (DF). It is generally accepted that insoluble DF are health promoting, but the information is scarce about how these fibres affect the gastrointestinal (GI) microbiota. A major reason for the limited knowledge is that there are currently no proper tools to analyse the complete GI microbiota. METHODS AND RESULTS: Here we present a novel 16S rRNA gene analytical approach that enables the analyses of the complete microbiota, including the part that has not yet been characterized. The basic principle of the method is use of 16S rRNA gene signature sequences to determine both the phylogenetic relatedness and the distribution of bacteria in the samples analysed. Using this approach, we analysed the microbiota after in vitro fermentation of different barley fractions with human faeces. Our main finding was that groups of actinobacteria were selectively enriched by growth on the insoluble DF fractions. CONCLUSIONS: Our novel analytical approaches revealed new enrichment patterns in the taxa that respond to insoluble DF. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results may have major implications for future understanding of insoluble DF health effects.

Reduced expression of the inhibitory synapse scaffolding protein gephyrin in Alzheimer's disease.

Excitotoxicity may contribute to neuronal and synaptic loss in Alzheimer's disease (AD). Aberrant levels of gephyrin, a post-synaptic receptor-stabilizing protein, could affect the inhibitory modulation of excitatory impulses. We assayed gephyrin protein in two brain areas susceptible to neuronal loss in AD, and in a spared area, in autopsy tissue from normal subjects (n=15) and AD patients (n=5). Quantification was by in-gel immunodetection against known concentrations of a recombinant truncated gephyrin standard. Gephyrin abundance was significantly reduced (P<0.01) in AD. Area-wise analysis showed that gephyrin levels were reduced in both spared and susceptible regions, indicating a global phenomenon. When samples were categorized on an index of pathological severity, gephyrin levels decreased with increasing severity until a moderate index was reached, and then increased, suggesting that higher gephyrin levels might compensate for excitotoxic damage in late stages of the disease. AD males showed a more pronounced reduction in gephyrin levels than AD females cf same-sex controls. A major splice variant of gephyrin was detected in all cases and in all three brain areas. This is the first study of gephyrin expression in AD.