Optimizing a PCR protocol for cpn60-based microbiome profiling of samples variously contaminated with host genomic DNA.

The current recommended protocol for chaperonin-60 (cpn60) universal target based microbiome profiling includes universal PCR of microbiome samples across an annealing temperature gradient to maximize the diversity of sequences amplified. However, the value of including this gradient approach has not been formally evaluated since the optimization of a modified universal PCR primer cocktail for cpn60 PCR. PCR conditions that maximize representation of the microbiome while minimizing PCR-associated distortion of the community structure, especially in samples containing large amounts of host genomic DNA are critical. The goal of this study was to measure the effects of PCR annealing temperature and the ratio of host to bacterial DNA on the outcome of microbiota analysis, using pig microbiota as a model environment.Six samples were chosen with an anticipated range of ratios of pig to bacterial genomic DNA, and universal cpn60 PCR amplification with an annealing temperature gradient was used to create libraries for pyrosequencing, resulting in 426,477 sequences from the six samples. The sequences obtained were classified as target (cpn60) or non-target based on the percent identity of their closest match to the cpnDB reference database, and target sequences were further processed to create microbiome profiles for each sample at each annealing temperature. Annealing temperature affected the amount of PCR product generated, with more product generated at higher temperatures. Samples containing proportionally more host genomic DNA yielded more non-target reads, especially at lower annealing temperatures. However, microbiome composition for each sample across the annealing temperature gradient remained consistent at both the phylum and operational taxonomic unit levels. Although some microbial sequences were detected at only one annealing temperature, these sequences accounted for a minority of the total microbiome.These results indicate that PCR annealing temperature does have an affect on cpn60 based microbiome profiles, but that most of the differences are due to differences in detection of low abundance sequences. Higher annealing temperatures resulted in larger amounts of PCR product and lower amounts of non-target sequence amplification, especially in samples containing proportionally large amounts of host DNA. Taken together these results provide important information to guide decisions about experimental design for cpn60 based microbiome studies

A New Model of Chemical Bonding in Ionic Melts

We developed a new physical model to predict macroscopic properties of inorganic molten systems using a realistic description of inter-atomic interactions. Unlike the conventional approach, which tends to overestimate viscosity by several times, our systems consist of a set of ions with an admixture of neutral atoms. The neutral atom subsystem is a consequence of the covalent/ionic state reduction, occurring in the liquid phase. Comparison of the calculated macroscopic properties (shear viscosity and self-diffusion constants) with the experiment demonstrates good performance of our model. The presented approach is inspired by a significant degree of covalent interaction between the alkali and chlorine atoms, predicted by the coupled cluster theory

Characterization of the fecal microbiota of pigs before and after inoculation with "Brachyspira hampsonii".

Brachyspira hampsonii causes disease indistinguishable from swine dysentery, and the structure of the intestinal microbiome likely plays a role in determining susceptibility of individual pigs to infection and development of clinical disease. The objectives of the current study were to determine if the pre-inoculation fecal microbiota differed between inoculated pigs that did (INOC MH) or did not (INOC non-MH) develop mucohaemorrhagic diarrhea following challenge with B. hampsonii , and to quantify changes in the structure of the microbiome following development of clinical disease. Fecal microbiota profiles were generated based on amplification and sequencing of the cpn60 universal target sequence from 89 samples from 18 pigs collected at -8, -5, -3 and 0 days post-inoculation, and at termination. No significant differences in richness, diversity or taxonomic composition distinguished the pre-inoculation microbiomes of INOC MH and INOC non-MH pigs. However, the development of bloody diarrhea in inoculated pigs was associated with perturbation of the microbiota relative to INOC non-MH or sham-inoculated control pigs. Specifically, the fecal microbiota of INOC MH pigs was less dense (fewer total 16S rRNA copies per gram of feces), and had a lower Bacteroidetes:Firmicutes ratio. Further investigation of the potential long-term effects of Brachyspira disease on intestinal health and performance is warranted

mPUMA: a computational approach to microbiota analysis by de novo assembly of operational taxonomic units based on protein-coding barcode sequences.

BACKGROUND: Formation of operational taxonomic units (OTU) is a common approach to data aggregation in microbial ecology studies based on amplification and sequencing of individual gene targets. The de novo assembly of OTU sequences has been recently demonstrated as an alternative to widely used clustering methods, providing robust information from experimental data alone, without any reliance on an external reference database. RESULTS: Here we introduce mPUMA (microbial Profiling Using Metagenomic Assembly, http://mpuma.sourceforge.net), a software package for identification and analysis of protein-coding barcode sequence data. It was developed originally for Cpn60 universal target sequences (also known as GroEL or Hsp60). Using an unattended process that is independent of external reference sequences, mPUMA forms OTUs by DNA sequence assembly and is capable of tracking OTU abundance. mPUMA processes microbial profiles both in terms of the direct DNA sequence as well as in the translated amino acid sequence for protein coding barcodes. By forming OTUs and calculating abundance through an assembly approach, mPUMA is capable of generating inputs for several popular microbiota analysis tools. Using SFF data from sequencing of a synthetic community of Cpn60 sequences derived from the human vaginal microbiome, we demonstrate that mPUMA can faithfully reconstruct all expected OTU sequences and produce compositional profiles consistent with actual community structure. CONCLUSIONS: mPUMA enables analysis of microbial communities while empowering the discovery of novel organisms through OTU assembly

Characterization of the upper respiratory tract microbiomes of patients with pandemic H1N1 influenza.

The upper respiratory tract microbiome has an important role in respiratory health. Influenza A is a common viral infection that challenges that health, and a well-recognized sequela is bacterial pneumonia. Given this connection, we sought to characterize the upper respiratory tract microbiota of individuals suffering from the pandemic H1N1 influenza A outbreak of 2009 and determine if microbiome profiles could be correlated with patient characteristics. We determined the microbial profiles of 65 samples from H1N1 patients by cpn60 universal target amplification and sequencing. Profiles were examined at the phylum and nearest neighbor species levels using the characteristics of patient gender, age, originating health authority, sample type and designation (STAT/non-STAT). At the phylum level, Actinobacteria-, Firmicutes- and Proteobacteria-dominated microbiomes were observed, with none of the patient characteristics showing significant profile composition differences. At the nearest neighbor species level, the upper respiratory tract microbiomes were composed of 13-20 species and showed a trend towards increasing diversity with patient age. Interestingly, at an individual level, most patients had one to three organisms dominant in their microbiota. A limited number of discrete microbiome profiles were observed, shared among influenza patients regardless of patient status variables. To assess the validity of analyses derived from sequence read abundance, several bacterial species were quantified by quantitative PCR and compared to the abundance of cpn60 sequence read counts obtained in the study. A strong positive correlation between read abundance and absolute bacterial quantification was observed. This study represents the first examination of the upper respiratory tract microbiome using a target other than the 16S rRNA gene and to our knowledge, the first thorough examination of this microbiome during a viral infection

Evolution of higher torque in Campylobacter-type bacterial flagellar motors

Understanding the evolution of molecular machines underpins our understanding of the development of life on earth. A well-studied case are bacterial flagellar motors that spin helical propellers for bacterial motility. Diverse motors produce different torques, but how this diversity evolved remains unknown. To gain insights into evolution of the high-torque ε-proteobacterial motor exemplified by the Campylobacter jejuni motor, we inferred ancestral states by combining phylogenetics, electron cryotomography, and motility assays to characterize motors from Wolinella succinogenes, Arcobacter butzleri and Bdellovibrio bacteriovorus. Observation of ~12 stator complexes in many proteobacteria, yet ~17 in ε-proteobacteria suggest a “quantum leap” evolutionary event. Campylobacter-type motors have high stator occupancy in wider rings of additional stator complexes that are scaffolded by large proteinaceous periplasmic rings. We propose a model for motor evolution wherein independent inner- and outer-membrane structures fused to form a scaffold for additional stator complexes. Significantly, inner- and outer-membrane associated structures have evolved independently multiple times, suggesting that evolution of such structures is facile and poised the ε-proteobacteria to fuse them to form the high-torque Campylobacter-type motor

Tamoxifen induces cellular stress in the nervous system by inhibiting cholesterol synthesis

Background: Tamoxifen (TAM) is an important cancer therapeutic and an experimental tool for effecting genetic recombination using the inducible Cre-Lox technique. Despite its widespread use in the clinic and laboratory, we know little about its effects on the nervous system. This is of significant concern because TAM, via unknown mechanisms, induces cognitive impairment in humans. A hallmark of cellular stress is induction of Activating Transcription Factor 3 (Atf3), and so to determine whether TAM induces cellular stress in the adult nervous system, we generated a knock-in mouse in which Atf3 promoter activity drives transcription of TAM-dependent Cre recombinase (Cre-ERT2); when crossed with tdtomato reporter mice, Atf3 induction results in robust and permanent genetic labeling of cells in which it is up-regulated even transiently. Results: We found that granular neurons of the olfactory bulb and dentate gyrus, vascular cells and ependymal cells throughout the brain, and peripheral sensory neurons expressed tdtomato in response to TAM treatment. We also show that TAM induced Atf3 up-regulation through inhibition of cholesterol epoxide hydrolase (ChEH): reporter expression was mitigated by delivery in vitamin E-rich wheat germ oil (vitamin E depletes ChEH substrates), and was partially mimicked by a ChEH-specific inhibitor. Conclusions: This work demonstrates that TAM stresses cells of the adult central and peripheral nervous systems and highlights concerns about clinical and experimental use of TAM. We propose TAM administration in vitamin E-rich vehicles such as wheat germ oil as a simple remedy

Biochemical evidence for an alternate pathway in N-linked glycoprotein biosynthesis

Asparagine-linked glycosylation is a complex protein modification conserved among all three domains of life. Herein we report the in vitro analysis of N-linked glycosylation from the methanogenic archaeon Methanococcus voltae. Using a suite of synthetic and semisynthetic substrates, we show that AglK initiates N-linked glycosylation in M. voltae through the formation of α-linked dolichyl monophosphate N-acetylglucosamine, which contrasts with the polyprenyl diphosphate intermediates that feature in both eukaryotes and bacteria. Notably, AglK has high sequence homology to dolichyl phosphate β-glucosyltransferases, including Alg5 in eukaryotes, suggesting a common evolutionary origin. The combined action of the first two enzymes, AglK and AglC, afforded an α-linked dolichyl monophosphate glycan that serves as a competent substrate for the archaeal oligosaccharyl transferase AglB. These studies provide what is to our knowledge the first biochemical evidence revealing that, despite the apparent similarity of the overall pathways, there are actually two general strategies to achieve N-linked glycoproteins across the domains of life.National Institutes of Health (U.S.) (Grant GM039334