Swabs to genomes: A comprehensive workflow

© 2015 Dunitz et al. The sequencing, assembly, and basic analysis of microbial genomes, once a painstaking and expensive undertaking, has become much easier for research labs with access to standard molecular biology and computational tools. However, there are a confusing variety of options available for DNA library preparation and sequencing, and inexperience with bioinformatics can pose a significant barrier to entry for many who may be interested in microbial genomics. The objective of the present study was to design, test, troubleshoot, and publish a simple, comprehensive workflow from the collection of an environmental sample (a swab) to a published microbial genome; empowering even a lab or classroom with limited resources and bioinformatics experience to performit

PhyloSift: Phylogenetic analysis of genomes and metagenomes

Like all organisms on the planet, environmental microbes are subject to the forces of molecular evolution. Metagenomic sequencing provides a means to access the DNA sequence of uncultured microbes. By combining DNA sequencing of microbial communities with evolutionary modeling and phylogenetic analysis we might obtain new insights into microbiology and also provide a basis for practical tools such as forensic pathogen detection. In this work we present an approach to leverage phylogenetic analysis of metagenomic sequence data to conduct several types of analysis. First, we present a method to conduct phylogeny-driven Bayesian hypothesis tests for the presence of an organism in a sample. Second, we present a means to compare community structure across a collection of many samples and develop direct associations between the abundance of certain organisms and sample metadata. Third, we apply new tools to analyze the phylogenetic diversity of microbial communities and again demonstrate how this can be associated to sample metadata. These analyses are implemented in an open source software pipeline called PhyloSift. As a pipeline, PhyloSift incorporates several other programs including LAST, HMMER, and pplacer to automate phylogenetic analysis of protein coding and RNA sequences in metagenomic datasets generated by modern sequencing platforms (e.g., Illumina, 454). © 2014 Darling et al

Genomes from bacteria associated with the canine oral cavity: A test case for automated genome-based taxonomic assignment

© 2019 Coil et al. Taxonomy for bacterial isolates is commonly assigned via sequence analysis. However, the most common sequence-based approaches (e.g. 16S rRNA gene-based phylogeny or whole genome comparisons) are still labor intensive and subjective to varying degrees. Here we present a set of 33 bacterial genomes, isolated from the canine oral cavity. Taxonomy of these isolates was first assigned by PCR amplification of the 16S rRNA gene, Sanger sequencing, and taxonomy assignment using BLAST. After genome sequencing, taxonomy was revisited through a manual process using a combination of average nucleotide identity (ANI), concatenated marker gene phylogenies, and 16S rRNA gene phylogenies. This taxonomy was then compared to the automated taxonomic assignment given by the recently proposed Genome Taxonomy Database (GTDB). We found the results of all three methods to be similar (25 out of the 33 had matching genera), but the GTDB approach required fewer subjective decisions, and required far less labor. The primary differences in the non-identical taxonomic assignments involved cases where GTDB has proposed taxonomic revisions

Draft genome sequences of 26 Porphyromonas strains isolated from the canine oral microbiome

� 2015 Coil et al. We present the draft genome sequences for 26 strains of Porphyromonas (P. canoris, P. gulae, P. cangingavalis, P. macacae, and 7 unidentified) and an unidentified member of the Porphyromonadaceae family. All of these strains were isolated from the canine oral cavity, from dogs with and without early periodontal disease

<i>Teredinibacter waterburyi</i> sp. nov., a marine, cellulolytic endosymbiotic bacterium isolated from the gills of the wood-boring mollusc <i>Bankia setacea</i> (Bivalvia: Teredinidae) and emended description of the genus <i>Teredinibacter</i>

A cellulolytic, aerobic, gammaproteobacterium, designated strain Bs02T, was isolated from the gills of a marine wood-boring mollusc, Bankia setacea (Bivalvia: Teredinidae). The cells are Gram-stain-negative, slightly curved motile rods (2-5×0.4-0.6 µm) that bear a single polar flagellum and are capable of heterotrophic growth in a simple mineral medium supplemented with cellulose as a sole source of carbon and energy. Cellulose, carboxymethylcellulose, xylan, cellobiose and a variety of sugars also support growth. Strain Bs02T requires combined nitrogen for growth. Temperature, pH and salinity optima (range) for growth were 20 °C (range, 10-30 °C), 8.0 (pH 6.5-8.5) and 0.5 M NaCl (range, 0.0-0.8 M), respectively when grown on 0.5 % (w/v) galactose. Strain Bs02T does not require magnesium and calcium ion concentrations reflecting the proportions found in seawater. The genome size is approximately 4.03 Mbp and the DNA G+C content of the genome is 47.8 mol%. Phylogenetic analyses based on 16S rRNA gene sequences, and on conserved protein-coding sequences, show that strain Bs02T forms a well-supported clade with Teredinibacter turnerae. Average nucleotide identity and percentage of conserved proteins differentiate strain Bs02T from Teredinibacter turnerae at threshold values exceeding those proposed to distinguish bacterial species but not genera. These results indicate that strain Bs02T represents a novel species in the previously monotypic genus Teredinibacter for which the name Teredinibacter waterburyi sp. nov. is proposed. The strain has been deposited under accession numbers ATCC TSD-120T and KCTC 62963T

Modeling the Effect of Propofol and Remifentanil Combinations for Sedation-Analgesia in Endoscopic Procedures Using an Adaptive Neuro Fuzzy Inference System (ANFIS)

BACKGROUND: The increasing demand for anesthetic procedures in the gastrointestinal endoscopy area has not been followed by a similar increase in the methods to provide and control sedation and analgesia for these patients. In this study, we evaluated different combinations of propofol and remifentanil, administered through a target-controlled infusion system, to estimate the optimal concentrations as well as the best way to control the sedative effects induced by the combinations of drugs in patients undergoing ultrasonographic endoscopy. METHODS: One hundred twenty patients undergoing ultrasonographic endoscopy were randomized to receive, by means of a target-controlled infusion system, a fixed effect-site concentration of either propofol or remifentanil of 8 different possible concentrations, allowing adjustment of the concentrations of the other drug. Predicted effect-site propofol (C(e)pro) and remifentanil (C(e)remi) concentrations, parameters derived from auditory evoked potential, autoregressive auditory evoked potential index (AAI/2) and electroencephalogram (bispectral index [BIS] and index of consciousness [IoC]) signals, as well as categorical scores of sedation (Ramsay Sedation Scale [RSS] score) in the presence or absence of nociceptive stimulation, were collected, recorded, and analyzed using an Adaptive Neuro Fuzzy Inference System. The models described for the relationship between C(e)pro and C(e)remi versus AAI/2, BIS, and IoC were diagnosed for inaccuracy using median absolute performance error (MDAPE) and median root mean squared error (MDRMSE), and for bias using median performance error (MDPE). The models were validated in a prospective group of 68 new patients receiving different combinations of propofol and remifentanil. The predictive ability (P(k)) of AAI/2, BIS, and IoC with respect to the sedation level, RSS score, was also explored. RESULTS: Data from 110 patients were analyzed in the training group. The resulting estimated models had an MDAPE of 32.87, 12.89, and 8.77; an MDRMSE of 17.01, 12.81, and 9.40; and an MDPE of -1.86, 3.97, and 2.21 for AAI/2, BIS, and IoC, respectively, in the absence of stimulation and similar values under stimulation. P(k) values were 0.82, 0.81, and 0.85 for AAI/2, BIS, and IoC, respectively. The model predicted the prospective validation data with an MDAPE of 34.81, 14.78, and 10.25; an MDRMSE of 16.81, 15.91, and 11.81; an MDPE of -8.37, 5.65, and -1.43; and P(k) values of 0.81, 0.8, and 0.8 for AAI/2, BIS, and IoC, respectively. CONCLUSION: A model relating C(e)pro and C(e)remi to AAI/2, BIS, and IoC has been developed and prospectively validated. Based on these models, the (C(e)pro, C(e)remi) concentration pairs that provide an RSS score of 4 range from (1.8 μg·mL(-1), 1.5 ng·mL(-1)) to (2.7 μg·mL(-1), 0 ng·mL(-1)). These concentrations are associated with AAI/2 values of 25 to 30, BIS of 71 to 75, and IoC of 72 to 76. The presence of noxious stimulation increases the requirements of C(e)pro and C(e)remi to achieve the same degree of sedative effects

Nitrogen fixation in a landrace of maize is supported by a mucilage-associated diazotrophic microbiota

© 2018 Van Deynze et al. http://creativecommons.org/licenses/by/4.0/. Plants are associated with a complex microbiota that contributes to nutrient acquisition, plant growth, and plant defense. Nitrogen-fixing microbial associations are efficient and well characterized in legumes but are limited in cereals, including maize. We studied an indigenous landrace of maize grown in nitrogen-depleted soils in the Sierra Mixe region of Oaxaca, Mexico. This landrace is characterized by the extensive development of aerial roots that secrete a carbohydrate-rich mucilage. Analysis of the mucilage microbiota indicated that it was enriched in taxa for which many known species are diazotrophic, was enriched for homologs of genes encoding nitrogenase subunits, and harbored active nitrogenase activity as assessed by acetylene reduction and 15 N 2 incorporation assays. Field experiments in Sierra Mixe using 15 N natural abundance or 15 N-enrichment assessments over 5 years indicated that atmospheric nitrogen fixation contributed 29%–82% of the nitrogen nutrition of Sierra Mixe maize

A Sodium Leak Current Regulates Pacemaker Activity of Adult Central Pattern Generator Neurons in Lymnaea Stagnalis

The resting membrane potential of the pacemaker neurons is one of the essential mechanisms underlying rhythm generation. In this study, we described the biophysical properties of an uncharacterized channel (U-type channel) and investigated the role of the channel in the rhythmic activity of a respiratory pacemaker neuron and the respiratory behaviour in adult freshwater snail Lymnaea stagnalis. Our results show that the channel conducts an inward leak current carried by Na+ (ILeak-Na). The ILeak-Na contributed to the resting membrane potential and was required for maintaining rhythmic action potential bursting activity of the identified pacemaker RPeD1 neurons. Partial knockdown of the U-type channel suppressed the aerial respiratory behaviour of the adult snail in vivo. These findings identified the Na+ leak conductance via the U-type channel, likely a NALCN-like channel, as one of the fundamental mechanisms regulating rhythm activity of pacemaker neurons and respiratory behaviour in adult animals

A Cholinergic-Regulated Circuit Coordinates the Maintenance and Bi-Stable States of a Sensory-Motor Behavior during Caenorhabditis elegans Male Copulation

Penetration of a male copulatory organ into a suitable mate is a conserved and necessary behavioral step for most terrestrial matings; however, the detailed molecular and cellular mechanisms for this distinct social interaction have not been elucidated in any animal. During mating, the Caenorhabditis elegans male cloaca is maintained over the hermaphrodite's vulva as he attempts to insert his copulatory spicules. Rhythmic spicule thrusts cease when insertion is sensed. Circuit components consisting of sensory/motor neurons and sex muscles for these steps have been previously identified, but it was unclear how their outputs are integrated to generate a coordinated behavior pattern. Here, we show that cholinergic signaling between the cloacal sensory/motor neurons and the posterior sex muscles sustains genital contact between the sexes. Simultaneously, via gap junctions, signaling from these muscles is transmitted to the spicule muscles, thus coupling repeated spicule thrusts with vulval contact. To transit from rhythmic to sustained muscle contraction during penetration, the SPC sensory-motor neurons integrate the signal of spicule's position in the vulva with inputs from the hook and cloacal sensilla. The UNC-103 K+ channel maintains a high excitability threshold in the circuit, so that sustained spicule muscle contraction is not stimulated by fewer inputs. We demonstrate that coordination of sensory inputs and motor outputs used to initiate, maintain, self-monitor, and complete an innate behavior is accomplished via the coupling of a few circuit components