Uropygial gland microbiota differ between free-living and captive songbirds.

Symbiotic microbes can affect host behavior and fitness. Gut microbiota have received the most study, with less attention to other important microbial communities like those of scent-producing glands such as mammalian anal glands and the avian uropygial gland. However, mounting evidence suggests that microbes inhabiting scent-producing glands play an important role in animal behavior by contributing to variation in chemical signals. Free-living and captive conditions typically differ in social environment, food diversity and availability, disease exposure, and other factors-all of which can translate into differences in gut microbiota. However, whether extrinsic factors such as captivity alter microbial communities in scent glands remains an open question. We compared the uropygial gland microbiota of free-living and captive song sparrows (Melospiza melodia) and tested for an effect of dietary manipulations on the gland microbiota of captive birds. As predicted, the uropygial gland microbiota was significantly different between free-living and captive birds. Surprisingly, microbial diversity was higher in captive than free-living birds, and we found no effect of dietary treatments on captive bird microbiota. Identifying the specific factors responsible for microbial differences among groups and determining whether changes in symbiotic microbiota alter behavior and fitness are important next steps in this field.

Preen gland microbiota covary with major histocompatibility complex genotype in a songbird.

Pathogen-mediated selection at the major histocompatibility complex (MHC) is thought to promote MHC-based mate choice in vertebrates. Mounting evidence implicates odour in conveying MHC genotype, but the underlying mechanisms remain uncertain. MHC effects on odour may be mediated by odour-producing symbiotic microbes whose community structure is shaped by MHC genotype. In birds, preen oil is a primary source of body odour and similarity at MHC predicts similarity in preen oil composition. Hypothesizing that this relationship is mediated by symbiotic microbes, we characterized MHC genotype, preen gland microbial communities and preen oil chemistry of song sparrows (Melospiza melodia). Consistent with the microbial mediation hypothesis, pairwise similarity at MHC predicted similarity in preen gland microbiota. Counter to this hypothesis, overall microbial similarity did not predict chemical similarity of preen oil. However, permutation testing identified a maximally predictive set of microbial taxa that best reflect MHC genotype, and another set of taxa that best predict preen oil chemical composition. The relative strengths of relationships between MHC and microbes, microbes and preen oil, and MHC and preen oil suggest that MHC may affect host odour both directly and indirectly. Thus, birds may assess MHC genotypes based on both host-associated and microbially mediated odours.

The acute effects of inulin and resistant starch on postprandial serum short-chain fatty acids and second-meal glycemic response in lean and overweight humans.

BACKGROUND/OBJECTIVES: Colonic fermentation of dietary fiber to short-chain fatty acids (SCFA) may protect against obesity and diabetes, but excess production of colonic SCFA has been implicated in the promotion of obesity. We aimed to compare the effects of two fermentable fibers on postprandial SCFA and second-meal glycemic response in healthy overweight or obese (OWO) vs lean (LN) participants. SUBJECTS/METHODS: Using a randomized crossover design, 13 OWO and 12 LN overnight fasted participants were studied for 6 h on three separate days after consuming 300 ml water containing 75 g glucose (GLU) as control or with 24 g inulin (IN) or 28 g resistant starch (RS). A standard lunch was served 4 h after the test drink. RESULTS: Within the entire group, compared with control, IN significantly increased serum SCFA (P<0.001) but had no effect on free-fatty acids (FFA) or second-meal glucose and insulin responses. In contrast, RS had no significant effect on SCFA but reduced FFA rebound (P<0.001) and second-meal glucose (P=0.002) and insulin responses (P=0.024). OWO had similar postprandial serum SCFA and glucose concentrations but significantly greater insulin and FFA than LN. However, the effects of IN and RS on SCFA, glucose, insulin and FFA responses were similar in LN and OWO. CONCLUSIONS: RS has favorable second-meal effects, likely related to changes in FFA rather than SCFA concentrations. However, a longer study may be needed to demonstrate an effect of RS on SCFA. We found no evidence that acute increases in SCFA after IN reduce glycemic responses in humans, and we were unable to detect a significant difference in SCFA responses between OWO vs LN subjects.

Chemical composition of preen wax reflects major histocompatibility complex similarity in songbirds.

In jawed vertebrates, genes of the major histocompatibility complex (MHC) play a key role in immunity by encoding cell-surface proteins that recognize and bind non-self antigens. High variability at MHC suggests that these loci may also function in social signalling such as mate choice and kin recognition. This requires that MHC genotype covaries with some perceptible phenotypic trait. In mammals and fish, MHC is signalled chemically through volatile and non-volatile peptide odour cues, facilitating MHC-dependent mate choice and other behaviours. In birds, despite evidence for MHC-dependent mating, candidate mechanisms for MHC signalling remain largely unexplored. However, feather preen wax has recently been implicated as a potential source of odour cues. We examined whether the chemical composition of preen wax correlates with MHC class IIß genotypes of wild song sparrows (Melospiza melodia). Pairwise chemical distance reflected amino acid distance at MHC for male-female dyads, although not for same-sex dyads. Chemical diversity did not reflect MHC diversity. We used gas chromatography-mass spectrometry (GC-MS) to characterize preen wax compounds, and identified four wax esters that best reflect MHC similarity. Provided songbirds can detect variation in preen wax composition, this cue may allow individuals to assess MHC compatibility of potential mates.

Post-acquisition filtering of salt cluster artefacts for LC-MS based human metabolomic studies.

Liquid chromatography-high resolution mass spectrometry (LC-MS) has emerged as one of the most widely used platforms for untargeted metabolomics due to its unparalleled sensitivity and metabolite coverage. Despite its prevalence of use, the proportion of true metabolites identified in a given experiment compared to background contaminants and ionization-generated artefacts remains poorly understood. Salt clusters are well documented artefacts of electrospray ionization MS, recognized by their characteristically high mass defects (for this work simply generalized as the decimal numbers after the nominal mass). Exploiting this property, we developed a method to identify and remove salt clusters from LC-MS-based human metabolomics data using mass defect filtering. By comparing the complete set of endogenous metabolites in the human metabolome database to actual plasma, urine and stool samples, we demonstrate that up to 28.5 % of detected features are likely salt clusters. These clusters occur irrespective of ionization mode, column type, sweep gas and sample type, but can be easily removed post-acquisition using a set of R functions presented here. Our mass defect filter removes unwanted noise from LC-MS metabolomics datasets, while retaining true metabolites, and requires only a list of m/z and retention time values. Reducing the number of features prior to statistical analyses will result in more accurate multivariate modeling and differential feature selection, as well as decreased reporting of unknowns that often constitute the largest proportion of human metabolomics data.

Evidence for greater production of colonic short-chain fatty acids in overweight than lean humans.

BACKGROUND: Short-chain fatty acids (SCFA) are produced by colonic microbiota from dietary carbohydrates and proteins that reach the colon. It has been suggested that SCFA may promote obesity via increased colonic energy availability. Recent studies suggest obese humans have higher faecal SCFA than lean, but it is unclear whether this difference is due to increased SCFA production or reduced absorption. OBJECTIVES: To compare rectal SCFA absorption, dietary intake and faecal microbial profile in lean (LN) versus overweight and obese (OWO) individuals. DESIGN: Eleven LN and eleven OWO individuals completed a 3-day diet record, provided a fresh faecal sample and had SCFA absorption measured using the rectal dialysis bag method. The procedures were repeated after 2 weeks. RESULTS: Age-adjusted faecal SCFA concentration was significantly higher in OWO than LN individuals (81.3±7.4 vs 64.1±10.4 mmol kg(-1), P=0.023). SCFA absorption (24.4±0.8% vs 24.7±1.2%, respectively, P=0.787) and dietary intakes were similar between the groups, except for a higher fat intake in OWO individuals. However, fat intake did not correlate with SCFAs or bacterial abundance. OWO individuals had higher relative Firmicutes abundance (83.1±4.1 vs 69.5±5.8%, respectively, P=0.008) and a higher Firmicutes:Bacteriodetes ratio (P=0.023) than LN individuals. There was a positive correlation between Firmicutes and faecal SCFA within the whole group (r=0.507, P=0.044), with a stronger correlation after adjusting for available carbohydrate (r=0.615, P=0.005). CONCLUSIONS: The higher faecal SCFA in OWO individuals is not because of differences in SCFA absorption or diet. Our results are consistent with the hypothesis that OWO individuals produce more colonic SCFA than LN individuals because of differences in colonic microbiota. However, further studies are needed to prove this.

Microbial ecosystems therapeutics: a new paradigm in medicine?

Increasing evidence indicates that the complex microbial ecosystem of the human intestine plays a critical role in protecting the host against disease. This review discusses gut dysbiosis (here defined as a state of imbalance in the gut microbial ecosystem, including overgrowth of some organisms and loss of others) as the foundation for several diseases, and the applicability of refined microbial ecosystem replacement therapies as a future treatment modality. Consistent with the concept of a 'core' microbiome encompassing key functions required for normal intestinal homeostasis, 'Microbial Ecosystem Therapeutics' (MET) would entail replacing a dysfunctional, damaged ecosystem with a fully developed and healthy ecosystem of 'native' intestinal bacteria. Its application in treating Clostridium difficile infection is discussed and possible applications to other diseases such as ulcerative colitis, obesity, necrotising enterocolitis, and regressive-type autism are reviewed. Unlike conventional probiotic therapies that are generally limited to a single strain or at most a few strains of bacteria 'Microbial Ecosystem Therapeutics' would utilise whole bacterial communities derived directly from the human gastrointestinal tract. By taking into account the intrinsic needs of the entire microbial ecosystem, MET would emphasise the rational design of healthy, resilient and robust microbial communities that could be used to maintain or restore human health. More than simply a new probiotic treatment, this emerging paradigm in medicine may lead to novel strategies in treating and managing a wide variety of human diseases.

Mutual information without the influence of phylogeny or entropy dramatically improves residue contact prediction.

MOTIVATION: Compensating alterations during the evolution of protein families give rise to coevolving positions that contain important structural and functional information. However, a high background composed of random noise and phylogenetic components interferes with the identification of coevolving positions. RESULTS: We have developed a rapid, simple and general method based on information theory that accurately estimates the level of background mutual information for each pair of positions in a given protein family. Removal of this background results in a metric, MIp, that correctly identifies substantially more coevolving positions in protein families than any existing method. A significant fraction of these positions coevolve strongly with one or only a few positions. The vast majority of such position pairs are in contact in representative structures. The identification of strongly coevolving position pairs can be used to impose significant structural limitations and should be an important additional constraint for ab initio protein folding. AVAILABILITY: Alignments and program files can be found in the Supplementary Information.

Using information theory to search for co-evolving residues in proteins.

MOTIVATION: Some functionally important protein residues are easily detected since they correspond to conserved columns in a multiple sequence alignment (MSA). However important residues may also mutate, with compensatory mutations occurring elsewhere in the protein, which serve to preserve or restore functionality. It is difficult to distinguish these co-evolving sites from other non-conserved sites. RESULTS: We used Mutual Information (MI) to identify co-evolving positions. Using in silico evolved MSAs, we examined the effects of the number of sequences, the size of amino acid alphabet and the mutation rate on two sources of background MI: finite sample size effects and phylogenetic influence. We then assessed the performance of various normalizations of MI in enhancing detection of co-evolving positions and found that normalization by the pair entropy was optimal. Real protein alignments were analyzed and co-evolving isolated pairs were often found to be in contact with each other. AVAILABILITY: All data and program files can be found at http://www.biochem.uwo.ca/cgi-bin/CDD/index.cgi

Expression of single chain antibodies (ScFvs) for c-myc oncoprotein in recombinant Escherichia coli membranes by using the ice-nucleation protein of Pseudomonas syringae.

The ice nucleation protein (INP) is a glycosyl phosphatidylinositol anchored outer membrane protein found in certain Gram-negative bacteria. In this study, the INP from Pseudomonas syringae was applied as a fusion partner with the single chain antibody fragment (ScFv) against the human oncoprotein c-myc. Two new plasmids pNinaZ-myc and pNinaZScFv-BsaA1 were constructed and cloned into Escherichia coli JM109. The expression of the fusion protein was successfully demonstrated in the cloned cells. The fusion proteins had no effect on the viability of the host cells. Ice nucleation activity measurements and flow cytometry studies were followed to investigate the membrane expression of the fusion protein.

Distinct P-element excision products in somatic and germline cells of Drosophila melanogaster.

The footprints remaining following somatic P-element excision from the Drosophila white locus were recovered and characterized. Two different types of footprints were observed. Over 75% of the footprints were short, composed of 4 or 7 nucleotides of the P-element inverted terminal repeat, and were similar to those found in a previously described plasmid excision assay. The remaining footprints were composed of 14-18 nucleotides of both inverted terminal repeats. These large footprints were indistinguishable from those recovered following germline P-element excision. Enhanced expression of the Drosophila homologue of the Ku70 protein did not affect the structure of the somatic footprints. Therefore, this protein is not a limiting factor for double-strand break repair by nonhomologous end-joining in Drosophila somatic cells.

In vivo gap repair in Drosophila: a one-way street with many destinations.

While it has long been possible to study the process of recombination in yeast and other single-celled organisms, it has been difficult to distinguish between pathways of meiotic and mitotic recombination in multicellular eukaryotes. The experimental system described here bridges the historically separated fields of Genetic Recombination and DNA Repair in Drosophila. It is now feasible to study the repair of unique double-strand breaks induced in the Drosophila genome by the excision of a P-transposable element or by cleavage at an introduced endonuclease recognition sequence. This repair can be studied in both somatic cells and mitotically dividing germ cells. The repair of these breaks occurs mainly by copying sequence from a template located anywhere in the karyoplasm, and occurs in both male and female flies. This system, which was the first of its kind in metazoan organisms, is now being used for gene targeting in Drosophila. This review summarizes results that provide new insights into the process of gap repair in Drosophila and outline some recent experiments that demonstrate the power of the gene targeting technique.

Homology requirements for targeting heterologous sequences during P-induced gap repair in Drosophila melanogaster.

The effect of homology on gene targeting was studied in the context of P-element-induced double-strand breaks at the white locus of Drosophila melanogaster. Double-strand breaks were made by excision of P-w(hd), a P-element insertion in the white gene. A nested set of repair templates was generated that contained the 8 kilobase (kb) yellow gene embedded within varying amounts of white gene sequence. Repair with unlimited homology was also analyzed. Files were scored phenotypically for conversion of the yellow gene to the white locus. Targeting of the yellow gene was abolished when all of the 3' homology was removed. Increases in template homology up to 51 base pairs (bp) did not significantly promote targeting. Maximum conversion was observed with a construct containing 493 bp of homology, without a significant increase in frequency when homology extended to the tips of the chromosome. These results demonstrate that the homology requirements for targeting a large heterologous insertion are quite different than those for a point mutation. Furthermore, heterologous insertions strongly affect the homology requirements for the conversion of distal point mutations. Several aberrant conversion tracts, which arose from templates that contained reduced homology, also were examined and characterized.

Efficient gap repair in Drosophila melanogaster requires a maximum of 31 nucleotides of homologous sequence at the searching ends.

Double-strand breaks (DSB) were generated in the Drosophila melanogaster white gene by excision of the P-w(hd) element. An ectopic P-element vector carrying a modified white gene was used as a template for DSB repair. All template-dependent repair events were examined, and four different classes of events were recovered. The two most common products observed were gene conversions external to the P-w(hd) element and gene conversions (targeted transpositions) internal to the P-w(hd) element. These two events were equally frequent. Similar numbers for both orientations of internal conversion events were recovered. The results suggest that P-element excision occurs by a staggered cut that leaves behind at least 33 nucleotides of single-stranded sequence. Our results further demonstrate that an efficient homology search is conducted by the broken end with less than 31 nucleotides.

Gene targeting of a plasmid-borne sequence to a double-strand DNA break in Drosophila melanogaster.

We report an efficient and specific gene targeting method for transforming the germ line of Drosophila melanogaster. The targeting occurs during the repair of a double-strand DNA break that is induced at the white locus by the excision of a P transposable element. The break is repaired when homologous sequence is copied from a plasmid injected into the Drosophila embryo. The procedure efficiently integrates DNA into the targeted locus of the Drosophila genome. Heterologous sequence of up to 13 kbp in length can be inserted, permitting the intergration of entire genes into a common genomic site for further study.

A role for the KP leucine zipper in regulating P element transposition in Drosophila melanogaster.

The KP element can repress P element mobility in Drosophila melanogaster. Three mutant KP elements were made that had either two amino acid substitutions or a single amino acid deletion in the putative leucine zipper domain found in the KP polypeptide. Each KP element was expressed from the actin 5C proximal promoter. The wild-type control construct strongly repressed P element mobility, measured by the GD sterility and sn(w) mutability assays, in a position-independent manner. The single amino acid deletion mutant failed to repress P mobility by the double amino acid substitution mutants was position dependent. The results show that the leucine zipper of the KP polypeptide is important for P element regulation. This supports the multimer-poisoning model of P element repression, because leucine zipper motifs are involved in protein-protein interactions.

Efficient copying of nonhomologous sequences from ectopic sites via P-element-induced gap repair.

P-element-induced gap repair was used to copy nonhomologous DNA into the Drosophila white locus. We found that nearly 8,000 bp of nonhomologous sequence could be copied from an ectopic template at essentially the same rate as a single-base substitution at the same location. An in vitro-constructed deletion was also copied into white at high frequencies. This procedure can be applied to the study of gene expression in Drosophila melanogaster, especially for genes too large to be manipulated in other ways. We also observed several types of more complex events in which the copied template sequences were rearranged such that the breakpoints occurred at direct duplications. Most of these can be explained by a model of double strand break repair in which each terminus of the break invades a template independently and serves as a primer for DNA synthesis from it, yielding two overlapping single-stranded sequences. These single strands then pair, and synthesis is completed by each using the other as a template. This synthesis-dependent strand annealing (SDSA) model as a possible general mechanism in complex organisms is discussed.