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University campuses have proven to play a significant role in the production of cities due to their impact as knowledge and innovation hubs. Therefore, this paper examines the impact caused by building new campuses in suburban areas to see to what extent a campus can affect the transformation process of its respective city. Kyushu University’s Ito campus area was selected to be analysed using space syntax, spacematrix and Mixed-use index to see the impact caused by the campus since 1993 till 2017. Results have shown that after Ito campus establishment, urbanization started to be expanded throughout the area after it was limited to accessible parts around major routes and stations. Therefore, the location of the campus is considered as a vital decision that needs to be taken with the engagement of the city, with stakeholders, and business owners to harness a successful campus-city relationship.

AimTo understand the spatiotemporal dynamics of bacterial succession during gingivitis, and to identify taxa with a critical role in gum health with prognostic value.Materials and methodsLongitudinal microbiome data were collected from 15 individuals after completely discontinuing all forms of oral hygiene, and subsequently reintroducing it for three and two weeks, respectively. Sequences from the 16S rRNA V4-V5 gene region from sub- and supra-gingival plaque, saliva, and tongue sites were annotated and mapped to a reference tree of Human Oral Microbiome Database sequences.ResultsSuspending oral hygiene induced gingivitis, which was resolved after its resumption to baseline. Most significant shifts in bacterial abundance were observed in dental plaque, but not in saliva and tongue sites. During gingivitis-induction, baseline microbiota dominated by Streptococcus, was superseded by increased Prevotella, Fusobacterium, Leptotrichia, and Porphyromonas genera. Converse to its decline during disease-induction, gum health restoration was accompanied by a significant increase in streptococci.ConclusionWe present the most comprehensive, spatiotemporal map of bacterial succession during gingivitis onset and resolution. We have identified taxa with potential as probiotic candidates for gum disease (i.e., perio-probiotics), and suggest tooth-associated plaque and not saliva or tongue plaque should be used in future prognostic tests.

Wastewater stabilization ponds (WSPs) are commonly used to treat municipal wastewater in the Canadian Arctic. Bacterial community structure and functionality remain mostly uncharacterized for arctic WSPs, yet are presumed important for treatment outcomes during the 3-month summer treatment season with open water in the WSPs. The objective of this study was to investigate treatment performance and related temporal and spatial changes in the structure and putative function of bacterial communities during treatment of municipal wastewater in the WSPs of Pond Inlet and Clyde River, Nunavut over two consecutive summer treatment seasons. Influent raw wastewater contained a high organic load and large bacterial communities (~9 log 16S rRNA copies/mL) belonging mainly to Proteobacteria. Although designed to be facultative ponds, both WSPs remained anaerobic with neutral pH values (7.5–7.8) throughout the summer treatment season. Water quality data showed that nutrients [measured as carbonaceous biological oxygen demand (CBOD5)], total suspended solids, and total ammonia nitrogen were progressively reduced during treatment in the ponds as the summer progressed. The pond bacterial population size and species richness depended on the pond temperature (2–18°C), with 8.5 log 16S rRNA copies/mL and the largest alpha diversities (Shannon-Wiener index of 4-4.5) observed mid-season (late July). While the phylogenetic beta diversity in raw wastewater from the two locations remained similar, pond bacterial communities underwent significant (p < 0.05) changes to dominance of Comamonadaceae, Geobacteracea, and Porphyromonadaceae. Multivariate distance based redundancy analysis and predicted gene functionalities in the microbiota agreed with water quality results that microbial removal of nutrients (e.g., CBOD5) peaked in the middle of the summer coinciding with the treatment period with the highest pond temperatures. Information from this study will be useful for further development of models to predict biological treatment outcomes, which could be used to size and assess the feasibility of WSPs in extreme climates. Higher pond temperatures resulted in optimal biological processes and nutrient removal in the middle of the summer. While it is challenging to control environmental factors in a passive wastewater treatment system there are some design considerations that could be used to optimize temperature regimes, such as the depth of the pond.

Microbiota associated with mosquito vector populations impact several traits of mosquitoes, including survival, reproduction, control, and immunity against pathogens. The influence of seasonal variations and mosquito species on mosquito gut microbiota is poorly understood. We sought to determine whether the mosquito microbiota associated with immature stages of two congeners (Culex coronator and Culex nigripalpus) differ temporally and between the two species. Using high throughput 16S rRNA gene sequence analysis, we characterized bacterial and archaeal communities found in the immature stages of the two Culex mosquito species sampled over three seasons to compare the diversity of bacteria between the two species. Beta diversity analyses of the larval microbiota sequences revealed that the two Culex species differed significantly, both temporally within each species and between the two species. Bacteria in Cx. coronator larvae were dominated by Alphaproteobacteria, mainly associated with Roseoccocus and unidentified species of Rhizobiales, and two unidentified species of Cyanobacteria. In contrast, Cx. nigripalpus was dominated by Thorsellia anophelis (Gammaproteobacteria), Clostridium, an unidentified species of Ruminococcacae (Clostridiales), and additional unidentified species associated with Erysipelotrichaceae (Erysipelotrichales), Bacteroidales, and Mollicutes. Results of our study revealed both seasonal and interspecies differences in bacterial community composition associated with the immature stages of Cx. coronator and Cx. nigripalpus vector populations in Florida. These results have important implications for our understanding of the underlying factors of variations in disease transmission among seasons, susceptibility to various pesticides, and other biotic factors, including the role of the microbiota on the spread of invasive species. In addition, our results suggest close associations of certain bacteria species with each of the two Culex species that will be further targeted for their potential in the development of microbial-based control strategies.

To investigate the effect of the application of rice husk (RH) and rice husk charcoal (RHC) on soil properties and rice production, pot experiment comprising of five treatments was conducted. Soil was mixed at the rate of 0 (control), 2% and 4% (w/w) with RH and RHC, respectively with randomized complete block design (RCBD). RHC incorporation had a potential to reduce the acidity of the soil, whereas, RH incorporation had almost no effect on the pH of the soil. RH and RHC amendment both increased the saturated hydraulic conductivity, saturated water content, plant available water and field capacity but decreased the bulk density of soil. Crop growth components at harvest revealed that the highest plant height was recorded in RH4%. However, for the panicle length, panicle weight and number of tillers, the highest value was found in RHC2%, 14.2 cm, 4.0 g and 28.8 cm, respectively. Furthermore, number of panicle, 1000-grains weight and grain yield were also found highest in RHC2%, 22.4 g and 4.41 t/ha, respectively. However, for the number of grain per panicle and percentage of filled grain, the highest value was found in RH4%, 79.0 and 88.5, respectively. The grain yield increased by 38%, 28%, 18% and 22% and the biological yield increased by 27%, 18%, 14%, and 16% for RHC2%, RHC4%, RH2%, and RH4%, respectively, compared to that of the control; however, the significant difference was found only for RHC2% for both. The harvest index increased under all application rates of RH and RHC compared to that of control.

Although mosquito microbiota are known to influence reproduction, nutrition, disease transmission, and pesticide resistance, the relationship between host-associated microbial community composition and geographical location is poorly understood. To begin addressing this knowledge gap, we characterized microbiota associated with adult females of Culex nigripalpus mosquito vectors of Saint Louis Encephalitis and West Nile viruses sampled from three locations in Florida (Vero Beach, Palmetto Inland, and Palmetto Coast). High-throughput sequencing of PCR-amplified 16S rRNA genes demonstrated significant differences among microbial communities of mosquitoes sampled from the three locations. Mosquitoes from Vero Beach (east coast Florida) were dominated by uncultivated Asaia sp. (Alphaproteobacteria), whereas microbiota associated with mosquitoes collected from two mosquito populations at Palmetto (west coast Florida) sites were dominated by uncultured Spironema culicis (Spirochaetes), Salinisphaera hydrothermalis (Gammaproteobacteria), Spiroplasma (Mollicutes), uncultured Enterobacteriaceae, Candidatus Megaira (Alphaproteobacteria; Rickettsiae), and Zymobacter (Gammaproteobacteria). The variation in taxonomic profiles of Cx. nigripalpus gut microbial communities, especially with respect to dominating taxa, is a potentially critical factor in understanding disease transmission and mosquito susceptibility to insecticides among different mosquito populations.

Microbial marker-gene sequence data can be used to generate comprehensive taxonomic profiles of the microorganisms present in a given community and for other community diversity analyses. The process of going from raw gene sequences to taxonomic profiles or diversity measures involves a series of data transformations performed by numerous computational tools. This includes tools for sequence quality checking, denoising, taxonomic classification, alignment, and phylogenetic tree building. In this chapter, we demonstrate how the Quantitative Insights Into Microbial Ecology version 2 (QIIME2) software suite can simplify 16S rRNA marker-gene analysis. We walk through an example data set extracted from the guts of bumblebees in order to show how QIIME2 can transform raw sequences into taxonomic bar plots, phylogenetic trees, principal co-ordinates analyses, and other visualizations of microbial diversity.

Aerobic granules offer enhanced biological nutrient removal and are compact and dense structures resulting in efficient settling properties. Granule instability, however, is still a challenge as understanding of the drivers of instability is poorly understood. In this study, transient instability of aerobic granules, associated with filamentous outgrowth, was observed in laboratory-scale sequencing batch reactors (SBRs). The transient phase was followed by the formation of stable granules. Loosely bound, dispersed, and pinpoint seed flocs gradually turned into granular flocs within 60 days of SBR operation. In stage 1, the granular flocs were compact in structure and typically 0.2 mm in diameter, with excellent settling properties. Filaments appeared and dominated by stage 2, resulting in poor settleability. By stage 3, the SBRs were selected for larger granules and better settling structures, which included filaments that became enmeshed within the granule, eventually forming structures 2-5 mm in diameter. Corresponding changes in sludge volume index were observed that reflected changes in settleability. The protein-to-polysaccharide ratio in the extracted extracellular polymeric substance (EPS) from stage 1 and stage 3 granules was higher (2.8 and 5.7, respectively), as compared to stage 2 filamentous bulking (1.5). Confocal laser scanning microscopic (CLSM) imaging of the biomass samples, coupled with molecule-specific fluorescent staining, confirmed that protein was predominant in stage 1 and stage 3 granules. During stage 2 bulking, there was a decrease in live cells; dead cells predominated. Denaturing gradient gel electrophoresis (DGGE) fingerprint results indicated a shift in bacterial community composition during granulation, which was confirmed by 16S rRNA gene sequencing. In particular, Janthinobacterium (known denitrifier and producer of antimicrobial pigment) and Auxenochlorella protothecoides (mixotrophic green algae) were predominant during stage 2 bulking. The chitinolytic activity of Chitinophaga is likely antagonistic towards Auxenochlorella and may have contributed to stage 3 stable granule formation. Rhodanobacter, known to support complete denitrification, were predominant in stage 1 and stage 3 granules. The relative abundance of Rhodanobacter coincided with high protein concentrations in EPS, suggesting a role in microbial aggregation and granule formation.

Summary Bacillus thuringiensis subsp. israelensis (Bti) is the most widely used biopesticide against mosquitoes and blackflies, with a history of high specificity and efficacy. High doses of Bti have been suggested for extended vector control in some environments; however, the effects of Bti application on the native microfauna in the environment are poorly understood. Two Bti (VectoBac G) treatments (high = 48·1 kg ha−1; low = 0·6 kg ha−1), in addition to an untreated control, were assigned to replicate 300 L microcosms. Culex abundance, phytoplankton biomass, sestonic particulates and nutrients were reduced significantly in the high Bti treatment. These changes affected other physicochemical variables in the water column during the 44-day field study. Bacterial communities present in the water column were assessed by Illumina sequencing of 16S rRNA genes. The most abundant aquatic bacteria in microcosms subject to a low dose of Bti and untreated control, Cyanobacteria, Cytophagales, Cyclobacteriaceae (phylum Bacteroidetes) and Sphingomonas (class Alphaproteobacteria), were suppressed in microcosms subject to a high dose of Bti. Bacteria in the high Bti treatment were dominated by Mucilaginibacter, Sediminibacterium (phylum Sphingobacteria) and Polaromonas (class Betaproteobacteria) and were more diverse than in the other treatments. Synthesis and applications. The biotic and abiotic changes resulting from a biopesticide application that significantly reduced mosquito abundance by more than 50% persisted longer than the period during which larval mosquito numbers were reduced. This warranted further investigation into the ecosystem-level effects of Bti application rates used routinely for mosquito control. Application rates greater than the label rate for a Bti biopesticide can reduce mosquito abundance for an extended period of time and therefore lessen the operational cost of repeated application, but they could exceed manufacturer's recommendations and might violate country-specific regulations governing biopesticide applications in natural habitats. Results of our study suggest that the widespread adoption of Bti application above the recommended label rate should be discouraged in habitats where algal abundance and its effect on primary production, microbial communities and nutrient cycling could affect the functioning of aquatic food webs.

Taxonomic markers such as the 16S ribosomal RNA gene are widely used in microbial community analysis. A common first step in marker-gene analysis is grouping genes into clusters to reduce data sets to a more manageable size and potentially mitigate the effects of sequencing error. Instead of clustering based on sequence identity, marker-gene data sets collected over time can be clustered based on temporal correlation to reveal ecologically meaningful associations. We present Ananke, a free and open-source algorithm and software package that complements existing sequence-identity-based clustering approaches by clustering marker-gene data based on time-series profiles and provides interactive visualization of clusters, including highlighting of internal OTU inconsistencies. Ananke is able to cluster distinct temporal patterns from simulations of multiple ecological patterns, such as periodic seasonal dynamics and organism appearances/disappearances. We apply our algorithm to two longitudinal marker gene data sets: faecal communities from the human gut of an individual sampled over one year, and communities from a freshwater lake sampled over eleven years. Within the gut, the segregation of the bacterial community around a food-poisoning event was immediately clear. In the freshwater lake, we found that high sequence identity between marker genes does not guarantee similar temporal dynamics, and Ananke time-series clusters revealed patterns obscured by clustering based on sequence identity or taxonomy. Ananke is free and open-source software available at https://github.com/beiko-lab/ananke.

Oral microbes form a complex and dynamic biofilm community, which is subjected to daily host and environmental challenges. Dysbiosis of the oral biofilm is correlated with local and distal infections and postulating a baseline for the healthy core oral microbiota provides an opportunity to examine such shifts during the onset and recurrence of disease. Here we quantified the daily, weekly, and monthly variability of the oral microbiome by sequencing the largest oral microbiota time-series to date, covering multiple oral sites in ten healthy individuals. Temporal dynamics of salivary, dental, and tongue consortia were examined by high-throughput 16S rRNA gene sequencing over 90 days, with four individuals sampled additionally 1 year later. Distinct communities were observed between dental, tongue, and salivary samples, with high levels of similarity observed between the tongue and salivary communities. Twenty-six core OTUs that classified within Streptococcus, Fusobacterium, Haemophilus, Neisseria, Prevotella, and Rothia genera were present in ≥95% samples and accounted for ~65% of the total sequence data. Phylogenetic diversity varied from person to person, but remained relatively stable within individuals over time compared to inter-individual variation. In contrast, the composition of rare microorganisms was highly variable over time, within most individuals. Using machine learning, an individual's oral microbial assemblage could be correctly assigned to them with 88–97% accuracy, depending on the sample site; 83% of samples taken a year after initial sampling could be confidently traced back to the source subject., Peering into the mouth: Bacterial turnover in plaque and saliva A study of bacteria in the mouth reveals insights into their diversity, stability, and variability among people and over time. By tracking daily, weekly, and monthly fluctuations of plaque and salivary bacteria in ten healthy volunteers, Dilani Senadheera at the Faculty of Dentistry, University of Toronto and co-researchers in Canada reveal significant differences in the “microbiome” present in dental, tongue and saliva samples over time. They found considerable variation in these communities between individuals, sufficient to identify a person with “bacterial fingerprints” using plaque or saliva even after 1 year. The researchers reveal a “core community” that spans different persons, oral sites, and time, suggesting some level of stability. This study is useful to understand the diversity and community drifts in different oral sites over time, which is important when plaque and saliva are used for bacterial analysis in diagnostic, risk-prediction, and forensic applications.

Massively parallel sequencing of 16S rRNA genes enables the comparison of terrestrial, aquatic, and host-associated microbial communities with sufficient sequencing depth for robust assessments of both alpha and beta diversity. Establishing standardized protocols for the analysis of microbial communities is dependent on increasing the reproducibility of PCR-based molecular surveys by minimizing sources of methodological bias. In this study, we tested the effects of template concentration, pooling of PCR amplicons, and sample preparation/interlane sequencing on the reproducibility associated with paired-end Illumina sequencing of bacterial 16S rRNA genes. Using DNA extracts from soil and fecal samples as templates, we sequenced pooled amplicons and individual reactions for both high (5- to 10-ng) and low (0.1-ng) template concentrations. In addition, all experimental manipulations were repeated on two separate days and sequenced on two different Illumina MiSeq lanes. Although within-sample sequence profiles were highly consistent, template concentration had a significant impact on sample profile variability for most samples. Pooling of multiple PCR amplicons, sample preparation, and interlane variability did not influence sample sequence data significantly. This systematic analysis underlines the importance of optimizing template concentration in order to minimize variability in microbial-community surveys and indicates that the practice of pooling multiple PCR amplicons prior to sequencing contributes proportionally less to reducing bias in 16S rRNA gene surveys with next-generation sequencing.

The purpose of this case series is to describe the treatment and outcomes of a series of patients presenting with frozen shoulder syndrome who received a novel chiropractic approach (OTZ Tension Adjustment).The files of 50 consecutive patients who presented to a private chiropractic practice with frozen shoulder syndrome were reviewed retrospectively. Two primary outcomes were extracted from the files for initial examination and at final evaluation: (1) the 11-point numeric pain rating scale and (2) the percentage change in shoulder abduction. Each patient received a series of chiropractic manipulative procedures that focused on the cervical and thoracic spine.Of the case files reviewed, 20 were male and 30 were female; and all were between the ages of 40 and 70 years. The median number of days under care was 28 days (range, 11 to 51 days). The median change in Numeric Pain Rating Scale score was - 7 (range, 0 to - 10). Of the 50 cases, 16 resolved completely (100% improvement), 25 showed 75% to 90% improvement, 8 showed 50% to 75% improvement, and 1 showed 0% to 50% improvement.Most patients with frozen shoulder syndrome in this case series appeared to improve with the chiropractic treatment.

Objective: Pulse oximetry is used regularly to assess oxygen saturation levels. The objective of this commentary is to discuss a rationale for using pulse oximetry in chiropractic practice. Discussion: Pulse oximetry may offer doctors of chiropractic a way to monitor patients' oxygen saturation levels. Quantification of saturation values with heart rate may give clinical aid to the managementofchiropracticpatients.Markedlyreducedsaturationlevelsmaynecessitatemedical referral, whereas mildly reduced levels could lead to changes in chiropractic management. Conclusions: Pulse oximetry has the potential to be an integral part of chiropractic practice. © 2012 National University of Health Sciences.

The ability of Panulirus ornatus phyllosoma to capture, feed and process diets of different texture and particle size were studied during developmental stages I, III and V. Diets were captured using the spines and dactyl on the second and third pereiopods and brought to the oral cavity assisted by the second and third maxillapeds. Optimal capture was obtained when diets were a gelatinous-muscular consistency, and hard particles were caught when embedded in a muscular carrier. A range of food textures, from gelatinous to hard, were able to be masticated in the oral cavity by stage V phyllosoma. Masticated particles were filtered by phyllosoma through spines and hairs in the proventriculum, during stages I–II, and a filter press in stages III–V. Maximum particle size passing into the midgut gland for digestion was 7, 3 and 0.5 µm in stage I, III and V phyllosoma, respectively. B cells were prominent in the proximal ends of the caece of stage V phyllosoma and were visible in live animals under light microscopy.

Background The native microflora associated with mosquitoes have important roles in mosquito development and vector competence. Sequencing of bacterial V3 region from 16S rRNA genes across the developmental stages of Culex mosquitoes (early and late larval instars, pupae and adults) was used to test the hypothesis that bacteria found in the larval stage of Culex are transstadially transmitted to the adult stage, and to compare the microbiomes of field-collected versus laboratory-reared mosquitoes. Results Beta diversity analysis revealed that bacterial community structure differed among three life stages (larvae, pupae and adults) of Culex tarsalis. Although only ~2 % of the total number of bacterial OTUs were found in all stages, sequences from these OTUs accounted for nearly 82 % of the total bacterial sequences recovered from all stages. Thorsellia (Gammaproteobacteria) was the most abundant bacterial taxon found across all developmental stages of field-collected Culex mosquitoes, but was rare in mosquitoes from laboratory-reared colonies. The proportion of Thorsellia sequences in the microbiomes of mosquito life stages varied ontogenetically with the greatest proportions recovered from the pupae of C. tarsalis and the lowest from newly emerged adults. The microbiome of field-collected late instar larvae was not influenced significantly by differences in the microbiota of the habitat due to habitat age or biopesticide treatments. The microbiome diversity was the greatest in the early instar larvae and the lowest in laboratory-reared mosquitoes. Conclusions Bacterial communities in early instar C. tarsalis larvae were significantly more diverse when compared to late instar larvae, pupae and newly emerged adults. Some of the bacterial OTUs found in the early instar larvae were also found across developmental stages. Thorsellia dominated the bacterial communities in field-collected immature stages but occurred at much lower relative abundance in adults. Differences in microbiota observed in larval habitats did not influence bacterial community profiles of late instar larvae or adults. However, bacterial communities in laboratory-reared C. tarsalis larvae differed significantly from the field. Determining the role of Thorsellia in mosquitoes and its distribution across different species of mosquitoes warrants further investigation. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0475-8) contains supplementary material, which is available to authorized users.

Soil microbial diversity represents the largest global reservoir of novel microorganisms and enzymes. In this study, we coupled functional metagenomics and DNA stable-isotope probing (DNA-SIP) using multiple plant-derived carbon substrates and diverse soils to characterize active soil bacterial communities and their glycoside hydrolase genes, which have value for industrial applications. We incubated samples from three disparate Canadian soils (tundra, temperate rainforest, and agricultural) with five native carbon (12C) or stable-isotope-labeled (13C) carbohydrates (glucose, cellobiose, xylose, arabinose, and cellulose). Indicator species analysis revealed high specificity and fidelity for many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Actinomycetales (Salinibacterium), Rhizobiales (Devosia), Rhodospirillales (Telmatospirillum), and Caulobacterales (Phenylobacterium and Asticcacaulis) were bacterial indicator species for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (Phenylobacterium) were associated with metabolism of cellulose, and Alphaproteobacteria were associated with the metabolism of arabinose; members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycoside hydrolase gene representation within the pooled heavy DNA. By screening 2,876 cloned fragments derived from the 13C-labeled DNA isolated from soils incubated with cellulose, we demonstrate the power of combining DNA-SIP, multiple-displacement amplification (MDA), and functional metagenomics by efficiently isolating multiple clones with activity on carboxymethyl cellulose and fluorogenic proxy substrates for carbohydrate-active enzymes., IMPORTANCE The ability to identify genes based on function, instead of sequence homology, allows the discovery of genes that would not be identified through sequence alone. This is arguably the most powerful application of metagenomics for the recovery of novel genes and a natural partner of the stable-isotope-probing approach for targeting active-yet-uncultured microorganisms. We expanded on previous efforts to combine stable-isotope probing and metagenomics, enriching microorganisms from multiple soils that were active in degrading plant-derived carbohydrates, followed by construction of a cellulose-based metagenomic library and recovery of glycoside hydrolases through functional metagenomics. The major advance of our study was the discovery of active-yet-uncultivated soil microorganisms and enrichment of their glycoside hydrolases. We recovered positive cosmid clones in a higher frequency than would be expected with direct metagenomic analysis of soil DNA. This study has generated an invaluable metagenomic resource that future research will exploit for genetic and enzymatic potential.

Anaerobic ammonia-oxidizing (anammox) bacteria are able to oxidize ammonia and reduce nitrite to produce N2 gas. After being discovered in a wastewater treatment plant (WWTP), anammox bacteria were subsequently characterized in natural environments, including marine, estuary, freshwater, and terrestrial habitats. Anammox bacteria play an important role in removing fixed N from both engineered and natural ecosystems, but broad scale anammox bacterial distributions, based on available data, have not yet been summarized. The objectives of this study were to explore global distributions and diversity of anammox bacteria and to identify factors that influence their biogeography. Over 6,000 anammox 16S rRNA gene sequences from the public database were analyzed in this current study. Data ordinations indicated that salinity was an important factor governing anammox bacterial distributions, with distinct populations inhabiting natural and engineered ecosystems. Gene phylogenies and rarefaction analysis demonstrated that freshwater environments and the marine water column harbored the highest and the lowest diversity of anammox bacteria, respectively. A co-occurrence network analysis indicated that Ca. Scalindua strongly correlated with other Ca. Scalindua taxa, whereas Ca. Brocadia co-occurred with taxa from both known and unknown anammox genera. Our survey provides a better understanding of ecological factors affecting anammox bacterial distributions and provides a comprehensive baseline for understanding the relationships among anammox communities in global environments.

High-throughput sequencing methods have been instrumental in the growing field of metagenomics, with technological improvements enabling greater throughput at decreased costs. Nonetheless, the economy of high-throughput sequencing cannot be fully leveraged in the subdiscipline of functional metagenomics. In this area of research, environmental DNA is typically cloned to generate large-insert libraries from which individual clones are isolated, based on specific activities of interest. Sequence data are required for complete characterization of such clones, but the sequencing of a large set of clones requires individual barcode-based sample preparation; this can become costly, as the cost of clone barcoding scales linearly with the number of clones processed, and thus sequencing a large number of metagenomic clones often remains cost-prohibitive. We investigated a hybrid Sanger/Illumina pooled sequencing strategy that omits barcoding altogether, and we evaluated this strategy by comparing the pooled sequencing results to reference sequence data obtained from traditional barcode-based sequencing of the same set of clones. Using identity and coverage metrics in our evaluation, we show that pooled sequencing can generate high-quality sequence data, without producing problematic chimeras. Though caveats of a pooled strategy exist and further optimization of the method is required to improve recovery of complete clone sequences and to avoid circumstances that generate unrecoverable clone sequences, our results demonstrate that pooled sequencing represents an effective and low-cost alternative for sequencing large sets of metagenomic clones.

Microbes are important for mosquito nutrition, growth, reproduction and control. In this study, we examined bacterial communities associated with larval mosquitoes and their habitats. Specifically, we characterized bacterial communities associated with late larval instars of the western encephalitis mosquito ( Culex tarsalis ), the submerged portions of two emergent macrophytes (California bulrush, Schoenoplectus californicus and alkali bulrush, Schoenoplectus maritimus ), and the associated water columns to investigate potential differential use of resources by mosquitoes in different wetland habitats. Using next-generation sequence data from 16S rRNA gene hypervariable regions, the alpha diversity of mosquito gut microbial communities did not differ between pond mesocosms containing distinct monotypic plants. Proteobacteria, dominated by the genus Thorsellia (Enterobacteriaceae), was the most abundant phylum recovered from C . tarsalis larvae. Approximately 49% of bacterial OTUs found in larval mosquitoes were identical to OTUs recovered from the water column and submerged portions of the two bulrushes. Plant and water samples were similar to one another, both being dominated by Actinobacteria, Bacteroidetes, Cyanobacteria , Proteobacteria and Verrucomicrobia phyla. Overall, the bacterial communities within C . tarsalis larvae were conserved and did not change across sampling dates and between two distinct plant habitats. Although Thorsellia spp. dominated mosquito gut communities, overlap of mosquito gut, plant and water-column OTUs likely reveal the effects of larval feeding. Future research will investigate the role of the key indicator groups of bacteria across the different developmental stages of this mosquito species.

Thank you for the opportunity to respond to this Letter to the Editor. We appreciate that there is interest in our technical article.1 It appears to us that 3 points are being raised about our article. It is being suggested that (1) our use of the Roth and Holtmann reference does not support our statement that “pulse oximetry is indicated because hypoxia is commonly found in the elderly,” (2) that our use of the article by Sommermeyer et al does not support our “position that pulse oximetry is indicated in hypoxia-related disorders,” and (3) that our use of the Braun chapter does not “support (our alleged) contention that pain is an indicator for pulse oximetry.” We feel that all 3 of these points do not accurately describe what we stated in our article. We offer an explanation and response to these points. Firstly, we use the article by Roth and Holtmann2 to support our statement that hypoxia may be found in elderly patients (see “1” in Fig 1); and this article clearly states that hypoxia is commonly found in elderly patients. We do not use this article to argue that pulse oximetry should be used in elderly patients (see “3” in Fig 1). Fig 1 Use of the article by Roth and Holtmann.2 Secondly, we use the article by Sommermeyer et al3 to support our statement that hypoxia is also commonly found in persons with “coronary vascular disease, coronary artery disease, disorders of balance, hypertension, chronic migraines, and anxiety and mood disorders.…” The article by Sommermeyer et al asserts those concepts labeled as “A” in Fig 2. It is an inherent property of pulse oximetry that, in any condition associated with oxygen desaturation, pulse oximetry is indicated (see “B” in Fig 2). Fig 2 Use of the article by Sommermeyer et al.3 Finally, we use the Braun chapter4 to support our position that pain can cause inadequate mechanical ventilation. Based upon this proposition, inadequate mechanical ventilation can possibly cause desaturation. Therefore, pain may be an indicator for pulse oximetry. We are not asserting that pain itself can cause desaturation. After reviewing the comments and our own references, it is apparent to us that, in these instances, this is a simple misinterpretation of our arguments. Hopefully, our explanations serve to clarify our positions for readers of our article.

General flexibility is a key component of health, well-being, and general physical conditioning. Reduced flexibility has both physical and mental/emotional etiologies and can lead to musculoskeletal injuries and athletic underperformance. Few studies have tested the effectiveness of a mind-body therapy on general flexibility. The aim of this study was to investigate if Neuro Emotional Technique® (NET), a mind-body technique shown to be effective in reducing stress, can also improve general flexibility. The sit-and-reach test (SR) score was used as a measure of general flexibility. Forty-five healthy participants were recruited from the general population and assessed for their initial SR score before being randomly allocated to receive (a) two 20-minute sessions of NET (experimental group); (b) two 20-minute sessions of stretching instruction (active control group); or (c) no intervention or instruction (passive control group). After intervention, the participants were reassessed in a similar manner by the same blind assessor. The participants also answered questions about demographics, usual water and caffeine consumption, and activity level, and they completed an anxiety/mood psychometric preintervention and postintervention. The mean (SD) change in the SR score was +3.1 cm (2.5) in the NET group, +1.2 cm (2.3) in the active control group and +1.0 cm (2.6) in the passive control group. Although all the 3 groups showed some improvement, the improvement in the NET group was statistically significant when compared with that of either the passive controls (p = 0.015) or the active controls (p = 0.021). This study suggests that NET could provide an effective treatment in improving general flexibility. A larger study is required to confirm these findings and also to assess longer term effectiveness of this therapy on general flexibility.

s from the WFC’s 11th Biennial Congress 159 OTZ Tension Adjustments for Frozen Shoulder Syndrome: A retrospective case series of 50 patients Francis Murphy *, Michael Hall, Anne Jensen *Corresponding author. E-mail address: fxmurphy@otzhealthed.com Introduction: Frozen Shoulder Syndrome (FSS) is a condition characterized by a restricted and painful arc of the shoulder that can persist for months to years, and has a high co-morbidity rate with diabetes mellitus, Parkinson disease, hypothyroidism, and stroke. It is speculated that with FSS there is a breakdown of the entire dynamic shoulder complex, but particularly of the scapulothoracic joint mediated by neuropathology of cranial nerve 11 causing malfunction of the trapezius muscle. The purpose of this paper is to describe the outcomes of patients presenting to a private chiropractic practice specializing in the treatment of FSS using the OTZ Tension Adjustment. Methods: Case files of 50 consecutive new patients presenting with FFS between May 2007 and March 2008 were reviewed retrospectively. Two primary outcomes were extracted each at 2 points, upon initial examination and at the final evaluation: (1) the 11-point Numeric Pain Rating Scale (NPRS), which has been shown to be a valid and reliable measure in patients with shoulder pain, and (2) the percentage change in shoulder abduction, with 100% improvement meaning the patient achieved the norm of 1808 of painfree active abduction. Each patient received a series of specific chiropractic adjustments focusing on the cervical and thoracic spine, especially the atlanto-occipital joints, cervicothoracic junction and the ribs. The specific technique used was the OTZ Tension Adjustment (OTZ Health Education Systems, Dallas, TX). Results: Of the 50 case files reviewed, 40% were male (n = 20) and 60% were female (n = 30), and all were between ages 40 and 70 years. The median number of days in the treatment program was 28 days, with a range of 11—51 days. The median initial NPRS score was 9 out of 10 with a range of 7—10. The median final NPRS score was 2 with a range of 0—10. The median change in NPRS score was 7 with a range of 0 to 10. Of the 50 cases, 16 resolved completely, regaining 1808 of painfree active shoulder abduction. Another 25 showed 75—90% improvement in active abduction, 8 showed 50—75% improvement, and 1 showed 0—50% improvement. Discussion: The results of this study are encouraging in that many of these patients’ complaints were resolved or greatly improved within 1 month of presentation, whereas in general it is thought that FSS symptoms can persist for 2 years or more. The limitations of this study are those for any case series, such as, the management of these patients occurred with a private chiropractic practice and, therefore, was an uncontrolled environment Caution is urged when drawing definitive conclusions from these results or when generalizing to other patients. This study would have been strengthened by the reporting of the length of time the patients had the FSS symptoms before presentation. Conclusion: This retrospective analysis of the outcome of treatment using the OTZ Tension Adjustment for FSS was reported with encouraging results. Future research is warranted and should consist of experimental clinical trials testing the effectiveness of the OTZ Tension Adjustment in a controlled setting. doi: 10.1016/j.clch.2011.09.022 Effects of manipulative therapy on the expressions of NADPH-diaphorase and nitric oxide synthase in the spinal motor neurons after right knee joint immobilization in the guinea pig HanSuk Jung *, JooHyun Ham, SoonJeong Jeong, JooHan Kang, MinSun Lee *Corresponding author. E-mail address: hsjung@hanseo.ac.kr Introduction: Chiropractic manipulative therapy can be helpful in alleviating some of the pain in knee joint problems; however, studies on the effects of chiropractic manipulative therapy in knee joint problem are rare. Recent interest in nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and nitric oxide synthase (NOS) has focused on its expression in motor neurons in the central nervous system (CNS) after peripheral nerve injury. The induction and up-regulation of NADPH-d and NOS have been suggested to be related to neuronal degeneration. The aim of the present study was to investigate effects of manipulative therapy in neurochemical changes of NADPH-d and NOS of the spinal motor neurons after right knee joint

We examined the specifications and design of the inspiratory pressure regulating valve of 8 continuous flow, pressure-limited infant ventilators. Two pressure regulating designs are currently available; one placing the primary pressure regulating valve on the inspiratory limb, the other placing it on the expiratory limb. Seven ventilators incorporate the latter design to limit inspiratory pressure and must have a safety pressure-relief valve located on the inspiratory limb to vent pressure in case of circuit occlusion. These pressure-relief valves are generally set by the manufacturer far in excess of pressures normally used for infant ventilation. Alarm systems are often absent or inadequate to warn of high pressure conditions during circuit obstruction. A case report detailing the fatal complication of prolonged excessive airway pressure during circuit occlusion is presented. Improvements in the pressure-relief valve designs currently available are possible, and may be necessary to provide adequate protection from barotrauma. The majority of infant ventilators currently available expose the patient to unnecessary excessive airway pressures in the case of expiratory limb occlusion, and the lack of alarm systems may leave the operator unaware of malfunction.

Five years (1978 to 1982) of respiratory care data were reviewed to determine the changes in patient charges, hospital costs, and frequency of neonatal blood gas analysis created by the introduction of transcutaneous oxygen monitoring. During the 4 years of transcutaneous oxygen monitoring (1979 to 1982), an estimated $196,000 reduction in patient charges was accomplished. When reduced patient charges were balanced with the increased cost to the hospital for equipment, supplies, and personnel time, a net reduction of more than $100,000 for health care delivery was achieved. Transcutaneous oxygen monitoring is an example of technologic achievement in which society receives both economic and medical benefits.

Background: Although high-throughput sequencing of small subunit rRNA genes has revolutionized our understanding of microbial ecosystems, these technologies generate data at depths that benefit from automated analysis. Here we present AXIOME (Automation, eXtension, and Integration Of Microbial Ecology), a highly flexible and extensible management tool for popular microbial ecology analysis packages that promotes reproducibility and customization in microbial research. Findings: AXIOME streamlines and manages analysis of small subunit (SSU) rRNA marker data in QIIME and mothur. AXIOME also implements features including the PAired-eND Assembler for Illumina sequences (PANDAseq), non-negative matrix factorization (NMF), multi-response permutation procedures (MRPP), exploring and recovering phylogenetic novelty (SSUnique) and indicator species analysis. AXIOME has a companion graphical user interface (GUI) and is designed to be easily extended to facilitate customized research workflows. Conclusions: AXIOME is an actively developed, open source project written in Vala and available from GitHub (http://neufeld.github.com/axiome) and as a Debian package. Axiometic, a GUI companion tool is also freely available (http://neufeld.github.com/axiometic). Given that data analysis has become an important bottleneck for microbial ecology studies, the development of user-friendly computational tools remains a high priority. AXIOME represents an important step in this direction by automating multi-step bioinformatic analyses and enabling the customization of procedures to suit the diverse research needs of the microbial ecology community.

IMPACT OF TRANSCUTANEOUS OXYGEN MONITORING (TCOM) ON QUALITY AND COST OF NEONATAL INTENSIVE CARE

Background Periodontitis is an infectious and inflammatory disease of polymicrobial etiology that can lead to the destruction of bones and tissues that support the teeth. The management of chronic periodontitis (CP) relies heavily on elimination or at least control of known pathogenic consortia associated with the disease. Until now, microbial plaque obtained from the subgingival (SubG) sites has been the primary focus for bacterial community analysis using deep sequencing. In addition to the use of SubG plaque, here, we investigated whether plaque obtained from supragingival (SupG) and tongue dorsum sites can serve as alternatives for monitoring CP-associated bacterial biomarkers. Results Using SubG, SupG, and tongue plaque DNA from 11 healthy and 13 diseased subjects, we sequenced V3 regions (approximately 200 bases) of the 16S rRNA gene using Illumina sequencing. After quality filtering, approximately 4.1 million sequences were collapsed into operational taxonomic units (OTUs; sequence identity cutoff of >97%) that were classified to a total of 19 phyla spanning 114 genera. Bacterial community diversity and overall composition was not affected by health or disease, and multiresponse permutation procedure (MRPP) on Bray-Curtis distance measures only supported weakly distinct bacterial communities in SubG and tongue plaque depending on health or disease status (P