Quantifying and resolving multiple vector transformants in S. cerevisiae plasmid libraries

<p>Abstract</p> <p>Background</p> <p>In addition to providing the molecular machinery for transcription and translation, recombinant microbial expression hosts maintain the critical genotype-phenotype link that is essential for high throughput screening and recovery of proteins encoded by plasmid libraries. It is known that <it>Escherichia coli </it>cells can be simultaneously transformed with multiple unique plasmids and thusly complicate recombinant library screening experiments. As a result of their potential to yield misleading results, bacterial multiple vector transformants have been thoroughly characterized in previous model studies. In contrast to bacterial systems, there is little quantitative information available regarding multiple vector transformants in yeast. <it>Saccharomyces cerevisiae </it>is the most widely used eukaryotic platform for cell surface display, combinatorial protein engineering, and other recombinant library screens. In order to characterize the extent and nature of multiple vector transformants in this important host, plasmid-born gene libraries constructed by yeast homologous recombination were analyzed by DNA sequencing.</p> <p>Results</p> <p>It was found that up to 90% of clones in yeast homologous recombination libraries may be multiple vector transformants, that on average these clones bear four or more unique mutant genes, and that these multiple vector cells persist as a significant proportion of library populations for greater than 24 hours during liquid outgrowth. Both vector concentration and vector to insert ratio influenced the library proportion of multiple vector transformants, but their population frequency was independent of transformation efficiency. Interestingly, the average number of plasmids born by multiple vector transformants did not vary with their library population proportion.</p> <p>Conclusion</p> <p>These results highlight the potential for multiple vector transformants to dominate yeast libraries constructed by homologous recombination. The previously unrecognized prevalence and persistence of multiply transformed yeast cells have important implications for yeast library screens. The quantitative information described herein should increase awareness of this issue, and the rapid sequencing approach developed for these studies should be widely useful for identifying multiple vector transformants and avoiding complications associated with cells that have acquired more than one unique plasmid.</p

Evaluation of Unsaturated Flow in Fissured Sediments in the Chihuahuan Desert, Texas

Localized flow in fissured sediments in arid settings has important implications for waste disposal in these regions. Fissures are surface features or gullies that are underlain by partially open or sediment-filled fractures. The objectives of this study were to compare unsaturated flow beneath different fissures, investigate the vertical and lateral extent of increased flow associated with fissured sediments, and examine different techniques for evaluating flow in fissured zones. Boreholes were drilled directly beneath four fissures and at distances of 10 and 50 m from the fissures. Sediment samples were analyzed for hydraulic parameters such as water content and water potential and environmental tracers such as Cl, 36Cl, 3H, 2H, and 18O. A trench was dug beneath one fissure for detailed sampling. Electromagnetic induction was used to measure apparent electrical conductivity in transects perpendicular to the fissures. Unsaturated flow is relatively high beneath fissures, as evidenced by higher water potentials and lower chloride concentrations there than in surrounding sediments. The lateral extent of high water flux was restricted to the zone directly beneath one fissure but extended to profiles 10 m from two other fissures. The profiles 50 m distant from all fissures had low water fluxes, as indicated by low water potentials and high maximum chloride concentrations. The vertical extent of high water fluxes was restricted to the upper 10 to 20 m, as shown by water potential and chloride fronts within the upper 10 m zone beneath one fissure and by chloride fronts in the upper 20 m zone beneath and 10 m distant from another fissure. Additional evidence for localized water flux was provided by high tritium levels, less-enriched 2H and 18O, and higher plant water potentials in fissured sediments than in nonfissured sediments. Apparent electrical conductivity was higher in two of the four fissures. Multiple independent lines of evidence indicate that subsurface water fluxes are higher at shallow depths beneath fissures; however, the various techniques differ in their effectiveness in delineating higher water fluxes beneath fissures. Multiple profiles drilled in one fissure indicate that there is large variability in flow along this fissure that is attributed to topographic variations and degree of ponding.Bureau of Economic Geolog

Asymmetries in the Value of Existence

According to asymmetric comparativism, it is worse for a person to exist with a miserable life than not to exist, but it is not better for a person to exist with a happy life than not to exist. My aim in this paper is to explain how asymmetric comparativism could possibly be true. My account of asymmetric comparativism begins with a different asymmetry, regarding the (dis)value of early death. I offer an account of this early death asymmetry, appealing to the idea of conditional goods, and generalize it to explain how asymmetric comparativism could possibly be true. I also address the objection that asymmetric comparativism has unacceptably antinatalist implications

Bioengineered Lysozyme Reduces Bacterial Burden and Inflammation in a Murine Model of Mucoid Pseudomonas aeruginosa Lung Infection

The spread of drug-resistant bacterial pathogens is a growing global concern and has prompted an effort to explore potential adjuvant and alternative therapies derived from nature\u27s repertoire of bactericidal proteins and peptides. In humans, the airway surface liquid layer is a rich source of antibiotics, and lysozyme represents one of the most abundant and effective antimicrobial components of airway secretions. Human lysozyme is active against both Gram-positive and Gram-negative bacteria, a

Chemically reacting hypersonic flows over 3D cavities : flowfield structure characterisation

In this paper, a computational investigation of hypersonic rarefied gas flows in the transitional flow regime over 3D cavities is carried out by using the direct simulation Monte Carlo method. Such cavities give rise to geometric discontinuities that are often present at the surface of reentry vehicles. This work is focused on the flowfield structure characterisation under a rarefied environment and in the presence of chemical reactions. The cavities are investigated with different length-to-depth ratios, and the different flow structures are studied. In particular, for length-to-depth ratios of 1 and 2, a single recirculation is observed inside the cavities and the main flow is not able to enter the cavity due to the recirculation structure and high particle density. In the case of length-to-depth ratio 3, the flow is able to partially enter the cavity resulting in an elongated recirculation and the beginning of a secondary recirculation core is noticed. For the case of values 4 and 5, the main flow is able to penetrate deeper into the cavities and two recirculation zones are observed; however, for the length-to-depth ratio 5 the flow impinges directly on the bottom surface, which is a behaviour that is only observed in the continuum regime with a cavity length-to-depth ratio greater than 14

Characteristics of HIV-infected adolescents enrolled in a disclosure intervention trial in western Kenya

Knowledge of one's own HIV status is essential for long-term disease management, but there are few data on how disclosure of HIV status to infected children and adolescents in sub-Saharan Africa is associated with clinical and psychosocial health outcomes. We conducted a detailed baseline assessment of the disclosure status, medication adherence, HIV stigma, depression, emotional and behavioral difficulties, and quality of life among a cohort of Kenyan children enrolled in an intervention study to promote disclosure of HIV status. Among 285 caregiver-child dyads enrolled in the study, children's mean age was 12.3 years. Caregivers were more likely to report that the child knew his/her diagnosis (41%) compared to self-reported disclosure by children (31%). Caregivers of disclosed children reported significantly more positive views about disclosure compared to caregivers of non-disclosed children, who expressed fears of disclosure related to the child being too young to understand (75%), potential psychological trauma for the child (64%), and stigma and discrimination if the child told others (56%). Overall, the vast majority of children scored within normal ranges on screenings for behavioral and emotional difficulties, depression, and quality of life, and did not differ by whether or not the child knew his/her HIV status. A number of factors were associated with a child's knowledge of his/her HIV diagnosis in multivariate regression, including older age (OR 1.8, 95% CI 1.5-2.1), better WHO disease stage (OR 2.5, 95% CI 1.4-4.4), and fewer reported caregiver-level adherence barriers (OR 1.9, 95% CI 1.1-3.4). While a minority of children in this cohort knew their HIV status and caregivers reported significant barriers to disclosure including fears about negative emotional impacts, we found that disclosure was not associated with worse psychosocial outcomes

Comprehensive evaluation of caregiver-reported antiretroviral therapy adherence for HIV-infected children

For HIV-infected children, adherence to antiretroviral therapy (ART) is often assessed by caregiver report but there are few data on their validity. We conducted prospective evaluations with 191 children ages 0-14 years and their caregivers over 6 months in western Kenya to identify questionnaire items that best predicted adherence to ART. Medication Event Monitoring Systems(®) (MEMS, MWV/AARDEX Ltd., Switzerland) electronic dose monitors were used as external criterion for adherence. We employed a novel variable selection tool using the LASSO technique with logistic regression to identify items best correlated with dichotomized MEMS adherence (≥90 or <90 % doses taken). Nine of 48 adherence items were identified as the best predictors of adherence, including missed or late doses in the past 7 days, problems giving the child medicines, and caregiver-level factors like not being present at medication taking. These items could be included in adherence assessment tools for pediatric patients

Open source Direct Simulation Monte Carlo (DSMC) chemistry modelling for hypersonic flows

An open source implementation of chemistry modelling for the direct simulation Monte Carlo (DSMC) method is presented. Following the recent work of Bird [1] an approach known as the quantum kinetic (Q-K) method has been adopted to describe chemical reactions in a 5-species air model using DSMC procedures based on microscopic gas information. The Q-K technique has been implemented within the framework of the dsmcFoam code, a derivative of the open source CFD code OpenFOAM. Results for vibrational relaxation, dissociation and exchange reaction rates for an adiabatic bath demonstrate the success of the Q-K model when compared with analytical solutions for both inert and reacting conditions. A comparison is also made between the Q-K and total collision energy (TCE) chemistry approaches for a hypersonic flow benchmark case

Lifespan extension and the doctrine of double effect

Recent developments in biogerontology—the study of the biology of ageing—suggest that it may eventually be possible to intervene in the human ageing process. This, in turn, offers the prospect of significantly postponing the onset of age-related diseases. The biogerontological project, however, has met with strong resistance, especially by deontologists. They consider the act of intervening in the ageing process impermissible on the grounds that it would (most probably) bring about an extended maximum lifespan—a state of affairs that they deem intrinsically bad. In a bid to convince their deontological opponents of the permissibility of this act, proponents of biogerontology invoke an argument which is grounded in the doctrine of double effect. Surprisingly, their argument, which we refer to as the ‘double effect argument’, has gone unnoticed. This article exposes and critically evaluates this ‘double effect argument’. To this end, we first review a series of excerpts from the ethical debate on biogerontology in order to substantiate the presence of double effect reasoning. Next, we attempt to determine the role that the ‘double effect argument’ is meant to fulfil within this debate. Finally, we assess whether the act of intervening in ageing actually can be justified using double effect reasoning

Aerothermodynamic comparison of two- and three-dimensional rarefied hypersonic cavity flows

The thermal protection system is a key element in atmospheric re-entry missions of aerospace vehicles. Usually, in the thermal load calculations, the analysis assumes that the vehicle has a smooth surface. However, discontinuities or imperfections are often present on the aerospace vehicle surfaces due to fabrication tolerances, sensor installations, spaces between the thermal protection plates, and differential expansion or ablation rates between non-similar materials. In the present work, rarefied hypersonic flows over two- and three-dimensional cavities at an altitude of 80km in the Earth’s atmosphere are studied numerically. To model flows in the transitional regime, where the validity of the Navier Stokes equations is questionable, the direct simulation Monte Carlo method has been used. The primary goal is to assess the sensitivity of heat transfer, pressure, and skin-friction coefficients for a family of two- and three-dimensional cavities defined by different length-to-depth ratios. The analysis shows that an assumption of two-dimensionality plays a key role in the over prediction of the aerodynamic properties. Previous work using a continuum approach shows that two recirculation regions and flow attachment occurs when the length-to-depth ratio is equal to 14; however, the same phenomena are observed in the transitional regime when the cavity length-to-depth ratio is equal to 4. A study of the influence of the cavity width has also been conducted. It is shown that increasing the cavity width results in an augmentation of the surface aerothermodynamic quantitie