Towards the identification of essential genes using targeted genome sequencing and comparative analysis

BACKGROUND: The identification of genes essential for survival is of theoretical importance in the understanding of the minimal requirements for cellular life, and of practical importance in the identification of potential drug targets in novel pathogens. With the great time and expense required for experimental studies aimed at constructing a catalog of essential genes in a given organism, a computational approach which could identify essential genes with high accuracy would be of great value. RESULTS: We gathered numerous features which could be generated automatically from genome sequence data and assessed their relationship to essentiality, and subsequently utilized machine learning to construct an integrated classifier of essential genes in both S. cerevisiae and E. coli. When looking at single features, phyletic retention, a measure of the number of organisms an ortholog is present in, was the most predictive of essentiality. Furthermore, during construction of our phyletic retention feature we for the first time explored the evolutionary relationship among the set of organisms in which the presence of a gene is most predictive of essentiality. We found that in both E. coli and S. cerevisiae the optimal sets always contain host-associated organisms with small genomes which are closely related to the reference. Using five optimally selected organisms, we were able to improve predictive accuracy as compared to using all available sequenced organisms. We hypothesize the predictive power of these genomes is a consequence of the process of reductive evolution, by which many parasites and symbionts evolved their gene content. In addition, essentiality is measured in rich media, a condition which resembles the environments of these organisms in their hosts where many nutrients are provided. Finally, we demonstrate that integration of our most highly predictive features using a probabilistic classifier resulted in accuracies surpassing any individual feature. CONCLUSION: Using features obtainable directly from sequence data, we were able to construct a classifier which can predict essential genes with high accuracy. Furthermore, our analysis of the set of genomes in which the presence of a gene is most predictive of essentiality may suggest ways in which targeted sequencing can be used in the identification of essential genes. In summary, the methods presented here can aid in the reduction of time and money invested in essential gene identification by targeting those genes for experimentation which are predicted as being essential with a high probability

Health effects in fish of long-term exposure to effluents from wastewater treatment works

The effects of simple mixtures of chemicals, with similar mechanisms of action, can be predicted using the concentration addition model (CA). The ability of this model to predict the estrogenic effects of more complex mixtures such as effluent discharges, however, has yet to be established. Effluents from 43 U.K. wastewater treatment works were analyzed for the presence of the principal estrogenic chemical contaminants, estradiol, estrone, ethinylestradiol, and nonylphenol. The measured concentrations were used to predict the estrogenic activity of each effluent, employing the model of CA, based on the relative potencies of the individual chemicals in an in vitro recombinant yeast estrogen screen (rYES) and a short-term (14-day) in vivo rainbow trout vitellogenin induction assay. Based on the measured concentrations of the four chemicals in the effluents and their relative potencies in each assay, the calculated in vitro and in vivo responses compared well and ranged between 3.5 and 87 ng/L of estradiol equivalents (E2 EQ) for the different effluents. In the rYES, however, the measured E2 EQ concentrations in the effluents ranged between 0.65 and 43 ng E2 EQ/L, and they varied against those predicted by the CA model. Deviations in the estimation of the estrogenic potency of the effluents by the CA model, compared with the measured responses in the rYES, are likely to have resulted from inaccuracies associated with the measurement of the chemicals in the extracts derived from the complex effluents. Such deviations could also result as a consequence of interactions between chemicals present in the extracts that disrupted the activation of the estrogen response elements in the rYES. E2 EQ concentrations derived from the vitellogenic response in fathead minnows exposed to a series of effluent dilutions were highly comparable with the E2 EQ concentrations derived from assessments of the estrogenic potency of these dilutions in the rYES. Together these data support the use of bioassays for determining the estrogenic potency of WwTW effluents, and they highlight the associated problems for modeling approaches that are reliant on measured concentrations of estrogenic chemicals

In-Situ Biofilm Formation in Hyper Alkaline Environments

Lime manufacture in the UK has resulted in the generation of a number of alkaline sites (>pH 11.0) with complex indigenous microbial populations. Within the present study, retrievable cotton samples were used to investigate the fate of cellulose, the primary carbon source, within three sites aged from ≈25 to 140 years. Following 3 months incubation in situ, biofilms had formed on all cotton samples in these extreme pH conditions; with matrices comprised of carbohydrates, proteins, lipids and eDNA. Biofilms from the older sites contained greater amounts of eDNA, a structural component that aids the production of a denser biofilm. The age of the sites correlated with a shift from polysaccharides composed of β 1,4 and β 1,3 linked sugars to those composed of pyranosyl sugars within the older sites. These changes were reflected in the active biofilm communities which shifted from being Clostridiales dominated in the youngest site to Proteobacteria dominated in the older sites. The study demonstrates that the microbial communities resident in anthropogenic alkaline sites are able to form biofilms at pH values > pH 11.0 and that these biofilms evolve toward Proteobacteria dominated communities employing eDNA and pyranosyl sugar based polysaccharides to build the biofilm matrix

Perfect Fluid Quantum Anisotropic Universe: Merits and Challenges

The present paper deals with quantization of perfect fluid anisotropic cosmological models. Bianchi type V and IX models are discussed following Schutz's method of expressing fluid velocities in terms of six potentials. The wave functions are found for several examples of equations of state. In one case a complete wave packet could be formed analytically. The initial singularity of a zero proper volume can be avoided in this case, but it is plagued by the usual problem of non-unitarity of anisotropic quantum cosmological models. It is seen that a particular operator ordering alleviates this problem.Comment: 13 pages, 4 figures; Accepted for publication in Gen Relativ Gravi

Ozone’s Impact on Public Health: Contributions from Indoor Exposures to Ozone and Products of Ozone-Initiated Chemistry

OBJECTIVE: The associations between ozone concentrations measured outdoors and both morbidity and mortality may be partially due to indoor exposures to ozone and ozone-initiated oxidation products. In this article I examine the contributions of such indoor exposures to overall ozone-related health effects by extensive review of the literature as well as further analyses of published data. FINDINGS: Daily inhalation intakes of indoor ozone (micrograms per day) are estimated to be between 25 and 60% of total daily ozone intake. This is especially noteworthy in light of recent work indicating little, if any, threshold for ozone’s impact on mortality. Additionally, the present study estimates that average daily indoor intakes of ozone oxidation products are roughly one-third to twice the indoor inhalation intake of ozone alone. Some of these oxidation products are known or suspected to adversely affect human health (e.g., formaldehyde, acrolein, hydroperoxides, fine and ultrafine particles). Indirect evidence supports connections between morbidity/mortality and exposures to indoor ozone and its oxidation products. For example, cities with stronger associations between outdoor ozone and mortality tend to have residences that are older and less likely to have central air conditioning, which implies greater transport of ozone from outdoors to indoors. CONCLUSIONS: Indoor exposures to ozone and its oxidation products can be reduced by filtering ozone from ventilation air and limiting the indoor use of products and materials whose emissions react with ozone. Such steps might be especially valuable in schools, hospitals, and childcare centers in regions that routinely experience elevated outdoor ozone concentrations

Remission of severe restless legs syndrome and periodic limb movements in sleep after bilateral excision of multiple foot neuromas: a case report

<p>Abstract</p> <p>Introduction</p> <p>Restless legs syndrome is a sensorimotor neurological disorder characterized by an urge to move the legs in response to uncomfortable leg sensations. While asleep, 70 to 90 percent of patients with restless legs syndrome have periodic limb movements in sleep. Frequent periodic limb movements in sleep and related brain arousals as documented by polysomnography are associated with poorer quality of sleep and daytime fatigue. Restless legs syndrome in middle age is sometimes associated with neuropathic foot dysesthesias. The causes of restless legs syndrome and periodic limb movements in sleep are unknown, but the sensorimotor symptoms are hypothesized to originate in the central nervous system. We have previously determined that bilateral forefoot digital nerve impingement masses (neuromas) may be a cause of both neuropathic foot dysesthesias and the leg restlessness of restless legs syndrome. To the best of our knowledge, this case is the first report of bilateral foot neuromas as a cause of periodic limb movements in sleep.</p> <p>Case presentation</p> <p>A 42-year-old Caucasian woman with severe restless legs syndrome and periodic limb movements in sleep and bilateral neuropathic foot dysesthesias was diagnosed as having neuromas in the second, third, and fourth metatarsal head interspaces of both feet. The third interspace neuromas represented regrowth (or 'stump') neuromas that had developed since bilateral third interspace neuroma excision five years earlier. Because intensive conservative treatments including repeated neuroma injections and various restless legs syndrome medications had failed, radical surgery was recommended. All six neuromas were excised. Leg restlessness, foot dysesthesias and subjective sleep quality improved immediately. Assessment after 18 days showed an 84 to 100 percent reduction of visual analog scale scores for specific dysesthesias and marked reductions of pre-operative scores of the Pittsburgh sleep quality index, fatigue severity scale, and the international restless legs syndrome rating scale (36 to 4). Polysomnography six weeks post-operatively showed improved sleep efficiency, a marked increase in rapid eye movement sleep, and marked reductions in hourly rates of both periodic limb movements in sleep with arousal (135.3 to 3.3) and spontaneous arousals (17.3 to 0).</p> <p>Conclusion</p> <p>The immediate and near complete remission of symptoms, the histopathology of the excised tissues, and the marked improvement in polysomnographic parameters documented six weeks after surgery together indicate that this patient's severe restless legs syndrome and periodic limb movements in sleep was of peripheral nerve (foot neuroma) origin. Further study of foot neuromas as a source of periodic limb movements in sleep and as a cause of sleep dysfunction in patients with or without concomitant restless legs syndrome, is warranted.</p

Establishment of clonal myogenic cell lines from severely affected dystrophic muscles - CDK4 maintains the myogenic population

<p>Abstract</p> <p>Background</p> <p>A hallmark of muscular dystrophies is the replacement of muscle by connective tissue. Muscle biopsies from patients severely affected with facioscapulohumeral muscular dystrophy (FSHD) may contain few myogenic cells. Because the chromosomal contraction at 4q35 linked to FSHD is thought to cause a defect within myogenic cells, it is important to study this particular cell type, rather than the fibroblasts and adipocytes of the endomysial fibrosis, to understand the mechanism leading to myopathy.</p> <p>Results</p> <p>We present a protocol to establish clonal myogenic cell lines from even severely dystrophic muscle that has been replaced mostly by fat, using overexpression of CDK4 and the catalytic component of telomerase (human telomerase reverse transcriptase; hTERT), and a subsequent cloning step. hTERT is necessary to compensate for telomere loss during <it>in vitro </it>cultivation, while CDK4 prevents a telomere-independent growth arrest affecting CD56+ myogenic cells, but not their CD56- counterpart, <it>in vitro</it>.</p> <p>Conclusions</p> <p>These immortal cell lines are valuable tools to reproducibly study the effect of the FSHD mutation within myoblasts isolated from muscles that have been severely affected by the disease, without the confounding influence of variable amounts of contaminating connective-tissue cells.</p

Dynamic Analysis of Vascular Morphogenesis Using Transgenic Quail Embryos

Background: One of the least understood and most central questions confronting biologists is how initially simple clusters or sheet-like cell collectives can assemble into highly complex three-dimensional functional tissues and organs. Due to the limits of oxygen diffusion, blood vessels are an essential and ubiquitous presence in all amniote tissues and organs. Vasculogenesis, the de novo self-assembly of endothelial cell (EC) precursors into endothelial tubes, is the first step in blood vessel formation [1]. Static imaging and in vitro models are wholly inadequate to capture many aspects of vascular pattern formation in vivo, because vasculogenesis involves dynamic changes of the endothelial cells and of the forming blood vessels, in an embryo that is changing size and shape. Methodology/Principal Findings: We have generated Tie1 transgenic quail lines Tg(tie1:H2B-eYFP) that express H2B-eYFP in all of their endothelial cells which permit investigations into early embryonic vascular morphogenesis with unprecedented clarity and insight. By combining the power of molecular genetics with the elegance of dynamic imaging, we follow the precise patterning of endothelial cells in space and time. We show that during vasculogenesis within the vascular plexus, ECs move independently to form the rudiments of blood vessels, all while collectively moving with gastrulating tissues that flow toward the embryo midline. The aortae are a composite of somatic derived ECs forming its dorsal regions and the splanchnic derived ECs forming its ventral region. The ECs in the dorsal regions of the forming aortae exhibit variable mediolateral motions as they move rostrally; those in more ventral regions show significant lateral-to-medial movement as they course rostrally. Conclusions/Significance: The present results offer a powerful approach to the major challenge of studying the relative role(s) of the mechanical, molecular, and cellular mechanisms of vascular development. In past studies, the advantages of the molecular genetic tools available in mouse were counterbalanced by the limited experimental accessibility needed for imaging and perturbation studies. Avian embryos provide the needed accessibility, but few genetic resources. The creation of transgenic quail with labeled endothelia builds upon the important roles that avian embryos have played in previous studies of vascular development

Linking a dermal permeation and an inhalation model to a simple pharmacokinetic model to study airborne exposure to di(n-butyl) phthalate

Six males clad only in shorts were exposed to high levels of airborne di(n-butyl) phthalate (DnBP) and diethyl phthalate (DEP) in chamber experiments conducted in 2014. In two 6 h sessions, the subjects were exposed only dermally while breathing clean air from a hood, and both dermally and via inhalation when exposed without a hood. Full urine samples were taken before, during, and for 48 h after leaving the chamber and measured for key DnBP and DEP metabolites. The data clearly demonstrated high levels of DnBP and DEP metabolite excretions while in the chamber and during the first 24 h once leaving the chamber under both conditions. The data for DnBP were used in a modeling exercise linking dose models for inhalation and transdermal permeation with a simple pharmacokinetic model that predicted timing and mass of metabolite excretions. These models were developed and calibrated independent of these experiments. Tests included modeling of the “hood-on” (transdermal penetration only), “hood-off” (both inhalation and transdermal) scenarios, and a derived “inhalation-only” scenario. Results showed that the linked model tended to duplicate the pattern of excretion with regard to timing of peaks, decline of concentrations over time, and the ratio of DnBP metabolites. However, the transdermal model tended to overpredict penetration of DnBP such that predictions of metabolite excretions were between 1.1 and 4.5 times higher than the cumulative excretion of DnBP metabolites over the 54 h of the simulation. A similar overprediction was not seen for the “inhalation-only” simulations. Possible explanations and model refinements for these overpredictions are discussed. In a demonstration of the linked model designed to characterize general population exposures to typical airborne indoor concentrations of DnBP in the United States, it was estimated that up to one-quarter of total exposures could be due to inhalation and dermal uptake