ThermoPhyl : a software tool for selecting phylogenetically optimized conventional and quantitative-PCR taxon-targeted assays for use with complex samples

The ability to specifically and sensitively target genotypes of interest is critical for the success of many PCR-based analyses of environmental or clinical samples that contain multiple templates.Next-generation sequence data clearly show that such samples can harbour hundreds to thousands of operational taxonomic units; a richness which precludes the manual evaluation of candidate assay specificity and sensitivity using multiple sequence alignments. To solve this problem we have developed and validated a free software tool which automates the identification of PCR assays targeting specific genotypes in complex samples. ThermoPhyl uses user-defined target and non-target sequence databases to assess the phylogenetic sensitivity and specificity of thermodynamically optimised candidate assays derived from primer design software packages. ThermoPhyl takes its name from its central premise of testing Thermodynamically optimal assays for Phylogenetic specificity and sensitivity and can be used for two primer (traditional PCR) or two primers with an internal probe (e.g. TaqMan® qPCR) applications and potentially for oligonucleotide probes.Here we describe the use of ThermoPhyl for traditional PCR and qPCR assays. PCR assays selected using ThermoPhyl were validated using 454 pyrosequencing of a traditional specific PCR assay and with a set of four genotype-specific qPCR assays applied to estuarine sediment samples

The Effects Of Multimodal Feedback And Age On A Mouse Pointing Task

As the beneficial aspects of computers become more apparent to the elderly population and the baby boom generation moves into later adulthood there is opportunity to increase performance for older computer users. Performance decrements that occur naturally to the motor skills of older adults have shown to have a negative effect on interactions with indirect-manipulation devices, such as computer mice (Murata & Iwase, 2005). Although, a mouse will always have the traits of an indirect-manipulation interaction, the inclusion of additional sensory feedback likely increases the saliency of the task to the real world resulting in increases in performance (Biocca et al., 2002). There is strong evidence for a bimodal advantage that is present in people of all ages; additionally there is also very strong evidence that older adults are a group that uses extra sensory information to increase their everyday interactions with the environment (Cienkowski & Carney, 2002; Thompson & Malloy, 2004). This study examined the effects of having multimodal feedback (i.e., visual cues, auditory cues, and tactile cues) present during a target acquisition mouse task for young, middle-aged, and older experienced computer users. This research examined the performance and subjective attitudes when performing a mouse based pointing task when different combinations of the modalities were present. The inclusion of audio or tactile cues during the task had the largest positive effect on performance, resulting in significantly quicker task completion for all of the computer users. The presence of audio or tactile cues increased performance for all of the age groups; however the performance of the older adults tended to be positively influenced more than the other age groups due the inclusion of these modalities. Additionally, the presence of visual cues did not have as strong of an effect on overall performance in comparison to the other modalities. Although the presence of audio and tactile feedback both increased performance there was evidence of a speed accuracy trade-off. Both the audio and tactile conditions resulted in a significantly higher number of misses in comparison to having no additional cues or visual cues present. So, while the presence of audio and tactile feedback improved the speed at which the task could be completed this occurred due to a sacrifice in accuracy. Additionally, this study shows strong evidence that audio and tactile cues are undesirable to computer users. The findings of this research are important to consider prior to adding extra sensory modalities to any type of user interface. The idea that additional feedback is always better may not always hold true if the feedback is found to be distracting, annoying, or negatively affects accuracy, as was found in this study with audio and tactile cues

A canonical FtsZ protein in Verrucomicrobium spinosum, a member of the Bacterial phylum Verrucomicrobia that also includes tubulin-producing Prosthecobacter species

BACKGROUND: The origin and evolution of the homologous GTP-binding cytoskeletal proteins FtsZ typical of Bacteria and tubulin characteristic of eukaryotes is a major question in molecular evolutionary biology. Both FtsZ and tubulin are central to key cell biology processes – bacterial septation and cell division in the case of FtsZ and in the case of tubulins the function of microtubules necessary for mitosis and other key cytoskeleton-dependent processes in eukaryotes. The origin of tubulin in particular is of significance to models for eukaryote origins. Most members of domain Bacteria possess FtsZ, but bacteria in genus Prosthecobacter of the phylum Verrucomicrobia form a key exception, possessing tubulin homologs BtubA and BtubB. It is therefore of interest to know whether other members of phylum Verrucomicrobia possess FtsZ or tubulin as their FtsZ-tubulin gene family representative. RESULTS: Verrucomicrobium spinosum, a member of Phylum Verrucomicrobia of domain Bacteria, has been found to possess a gene for a protein homologous to the cytoskeletal protein FtsZ. The deduced amino acid sequence has sequence signatures and predicted secondary structure characteristic for FtsZ rather than tubulin, but phylogenetic trees and sequence analysis indicate that it is divergent from all other known FtsZ sequences in members of domain Bacteria. The FtsZ gene of V. spinosum is located within a dcw gene cluster exhibiting gene order conservation known to contribute to the divisome in other Bacteria and comparable to these clusters in other Bacteria, suggesting a similar functional role. CONCLUSION: Verrucomicrobium spinosum has been found to possess a gene for a protein homologous to the cytoskeletal protein FtsZ. The results suggest the functional as well as structural homology of the V. spinosum FtsZ to the FtsZs of other Bacteria implying its involvement in cell septum formation during division. Thus, both bacteria-like FtsZ and eukaryote-like tubulin cytoskeletal homologs occur in different species of the phylum Verrucomicrobia of domain Bacteria, a result with potential major implications for understanding evolution of tubulin-like cytoskeletal proteins and the origin of eukaryote tubulins

Evaluating the Presence of In-Vehicle Devices on Driver Performance: Methodological Issues

A central concern of Intelligent Transportation Systems (ITS) is the effect of in-vehicle devices (e.g. cell phones, navigation systems, radios) on driver performance and safety. As diverse and innovative technologies are designed and implemented for in-vehicle use, questions regarding the presence and use of these devices assume progressively greater importance. Concern for the safety of advanced driver training and require us to develop and validate reliable and effective procedures for assessing such effects. This work examines a number of candidate procedures, in particular the evaluation of cognitive workload as a strategy by which such goals might be achieved

Metabolic flexibility as a major predictor of spatial distribution in microbial communities

A better understand the ecology of microbes and their role in the global ecosystem could be achieved if traditional ecological theories can be applied to microbes. In ecology organisms are defined as specialists or generalists according to the breadth of their niche. Spatial distribution is often used as a proxy measure of niche breadth; generalists have broad niches and a wide spatial distribution and specialists a narrow niche and spatial distribution. Previous studies suggest that microbial distribution patterns are contrary to this idea; a microbial generalist genus (Desulfobulbus) has a limited spatial distribution while a specialist genus (Methanosaeta) has a cosmopolitan distribution. Therefore, we hypothesise that this counter-intuitive distribution within generalist and specialist microbial genera is a common microbial characteristic. Using molecular fingerprinting the distribution of four microbial genera, two generalists, Desulfobulbus and the methanogenic archaea Methanosarcina, and two specialists, Methanosaeta and the sulfate-reducing bacteria Desulfobacter were analysed in sediment samples from along a UK estuary. Detected genotypes of both generalist genera showed a distinct spatial distribution, significantly correlated with geographic distance between sites. Genotypes of both specialist genera showed no significant differential spatial distribution. These data support the hypothesis that the spatial distribution of specialist and generalist microbes does not match that seen with specialist and generalist large organisms. It may be that generalist microbes, while having a wider potential niche, are constrained, possibly by intrageneric competition, to exploit only a small part of that potential niche while specialists, with far fewer constraints to their niche, are more capable of filling their potential niche more effectively, perhaps by avoiding intrageneric competition. We suggest that these counter-intuitive distribution patterns may be a common feature of microbes in general and represent a distinct microbial principle in ecology, which is a real challenge if we are to develop a truly inclusive ecology

Diversity and distribution of Frankia strains symbiotic with Ceanothus in California

Frankia strains symbiotic with Ceanothus present an interesting opportunity to study the patterns and causes of Frankia diversity and distribution within a particular host infectivity group. We intensively sampled Frankia from nodules on Ceanothus plants along an elevational gradient in the southern Sierra Nevada of California, and we also collected nodules from a wider host taxonomic and geographic range throughout California. The two sampling scales comprised 36 samples from eight species of Ceanothus representing six of the seven major biogeographic regions in and around California. The primary objective of this study was to use a quantitative model to test the relative importance of geographic separation, host specificity, and environment in influencing the identity of Ceanothus Frankia symbionts as determined by ribosomal DNA sequence data. At both sampling scales, Frankia strains symbiotic with Ceanothus exhibited a high degree of genetic similarity. Frankia strains symbiotic with Chamaebatia (Rosaceae) were within the same clade as several Ceanothus symbionts. Results from a classification and regression tree model used to quantitatively explain Frankia phylogenetic groupings demonstrated that the only significant variable in distinguishing between phylogenetic groups at the more local sampling scale was host species. At the regional scale, Frankia phylogenetic groupings were explained by host species and the biogeographic province of sample collection. We did not find any significant correspondence between Frankia and Ceanothus phylogenies indicative of coevolution, but we concluded that the identity of Frankia strains inhabiting Ceanothus nodules may involve interactions between host species specificity and geographic isolation

The effects of fire on soil nitrogen associated with patches of the actinorhizal shrub Ceanothus cordulatus

Nitrogen is a limiting resource in many temperate forests and nitrogen-fixing plants are usually limited to the early stages of post-disturbance succession. In fire-dependent Sierra Nevada forests, however, Ceanothus cordulatus is relatively abundant even in old-growth forest conditions which are at least partly maintained by fire.We conducted a field experiment to determine if soil beneath Ceanothus patches represent ‘resource islands’ of available N which persist after fire. Nine plots containing discrete patches of Ceanothus, Arctostaphylos patula (manzanita; chosen as a non N-fixing reference species), and bare forest floor were subjected to either a low-intensity (n = 3) or highintensity (n = 3) bum treatment, or remained unburned as controls (n = 3). Soil temperatures during the bum were monitored by a network of thermocouples placed at the surface of the mineral soil and at ca. 10 cm depths. Soil samples were collected from the organic horizon, 0-10 cm and 15-25 cm depths within each patch type immediately before burning and 2 days, and 6, and 11 months after. Soil moisture, total C and N, and ammonium and nitrate concentrations were determined in the laboratory. Before the burn, Ceanothus patches were significantly enriched in total and inorganic N in the organic horizon relative to the other patch types. A sharp increase in inorganic N was observed in all patch types and depths immediately following burning, but by 6 months after the burn, Ceanothus patches were significantly enriched relative to the surrounding patch types and remained so at months. Resprouting Ceanothus patches will continue to be an important source of a limiting nutrient in this fire-prone ecosystem.Keywords: Frankia, Sierra Nevada, Fire, Ceanothus, Nitroge

Facilitative and competitive effecs of a N-fixing shrub on white fir saplings

In Sierra Nevada forests, shrubs are considered strong soil moisture competitors with regenerating trees, reducing seedling establishment, and slowing growth. Recent studies, however, suggest that in some circumstances shrubs can facilitate tree establishment and growth by modifying harsh microclimate conditions; increasing acquisition of water, carbon, and/or nutrients via shared mycorrhizal connections; or enhancing soil fertility, particularly under nitrogen-fixing shrubs such as Ceanothus spp. We examined the establishment dates and growth rates and patterns of white fir saplings growing in greenleaf manzanita, whitethorn ceanothus, and bare patches to examine whether establishment was correlated with past wet years, whether saplings growing in ceanothus had nitrogen-enriched foliage or faster growth rates than in the other two patches, and whether saplings in shrub patches experienced competition for light. Sapling establishment was not correlated with high precipitation or heavy snowpack years, suggesting shade-tolerant saplings do not need wet years to become established. Soils under ceanothus were nitrogen enriched, but white fir sapling foliage did not have higher nitrogen concentrations and saplings did not grow faster in ceanothus than in the other two patches. Because growth rates of saplings were comparable in all patch types examined despite significantly different edaphic and abiotic conditions, we inferred that the various competitive and facilitative interactions affecting tree growth are in net balance across the patch types examined. However, competition for light is important—a significant percentage of growth release events occurred after saplings emerged above their host shrubs. Where shrubs are present, shade-tolerant species (i.e., white fir) are favored over drought-tolerant (pine) species. Our results may help interpret changes in understory conditions that are contributing to mixed conifer’s compositional shift toward more shade-tolerant species after a century of fire-suppression.Keywords: shade tolerance, soil fertility, forest regeneration, fire suppression, nitrogen fixation, mixed conifer, plant competitio