Next-generation sequencing of HIV-1 RNA genomes: determination of error rates and minimizing artificial recombination.

Next-generation sequencing (NGS) is a valuable tool for the detection and quantification of HIV-1 variants in vivo. However, these technologies require detailed characterization and control of artificially induced errors to be applicable for accurate haplotype reconstruction. To investigate the occurrence of substitutions, insertions, and deletions at the individual steps of RT-PCR and NGS, 454 pyrosequencing was performed on amplified and non-amplified HIV-1 genomes. Artificial recombination was explored by mixing five different HIV-1 clonal strains (5-virus-mix) and applying different RT-PCR conditions followed by 454 pyrosequencing. Error rates ranged from 0.04-0.66% and were similar in amplified and non-amplified samples. Discrepancies were observed between forward and reverse reads, indicating that most errors were introduced during the pyrosequencing step. Using the 5-virus-mix, non-optimized, standard RT-PCR conditions introduced artificial recombinants in a fraction of at least 30% of the reads that subsequently led to an underestimation of true haplotype frequencies. We minimized the fraction of recombinants down to 0.9-2.6% by optimized, artifact-reducing RT-PCR conditions. This approach enabled correct haplotype reconstruction and frequency estimations consistent with reference data obtained by single genome amplification. RT-PCR conditions are crucial for correct frequency estimation and analysis of haplotypes in heterogeneous virus populations. We developed an RT-PCR procedure to generate NGS data useful for reliable haplotype reconstruction and quantification.

EVALUATION OF INTENSITY MODULATED RADIATION THERAPY (IMRT) DELIVERY ERROR DUE TO IMRT TREATMENT PLAN COMPLEXITY AND IMPROPERLY MATCHED DOSIMETRY DATA

Intensity modulated radiation therapy (IMRT) is a technique that delivers a highly conformal dose distribution to a target volume while attempting to maximally spare the surrounding normal tissues. IMRT is a common treatment modality used for treating head and neck (H&N) cancers, and the presence of many critical structures in this region requires accurate treatment delivery. The Radiological Physics Center (RPC) acts as both a remote and on-site quality assurance agency that credentials institutions participating in clinical trials. To date, about 30% of all IMRT participants have failed the RPC’s remote audit using the IMRT H&N phantom. The purpose of this project is to evaluate possible causes of H&N IMRT delivery errors observed by the RPC, specifically IMRT treatment plan complexity and the use of improper dosimetry data from machines that were thought to be matched but in reality were not. Eight H&N IMRT plans with a range of complexity defined by total MU (1460-3466), number of segments (54-225), and modulation complexity scores (MCS) (0.181-0.609) were created in Pinnacle v.8m. These plans were delivered to the RPC’s H&N phantom on a single Varian Clinac. One of the IMRT plans (1851 MU, 88 segments, and MCS=0.469) was equivalent to the median H&N plan from 130 previous RPC H&N phantom irradiations. This average IMRT plan was also delivered on four matched Varian Clinac machines and the dose distribution calculated using a different 6MV beam model. Radiochromic film and TLD within the phantom were used to analyze the dose profiles and absolute doses, respectively. The measured and calculated were compared to evaluate the dosimetric accuracy. All deliveries met the RPC acceptance criteria of ±7% absolute dose difference and 4 mm distance-to-agreement (DTA). Additionally, gamma index analysis was performed for all deliveries using a ±7%/4mm and ±5%/3mm criteria. Increasing the treatment plan complexity by varying the MU, number of segments, or varying the MCS resulted in no clear trend toward an increase in dosimetric error determined by the absolute dose difference, DTA, or gamma index. Varying the delivery machines as well as the beam model (use of a Clinac 6EX 6MV beam model vs. Clinac 21EX 6MV model), also did not show any clear trend towards an increased dosimetric error using the same criteria indicated above.

Gibbs point process approximation: Total variation bounds using Stein's method

We obtain upper bounds for the total variation distance between the distributions of two Gibbs point processes in a very general setting. Applications are provided to various well-known processes and settings from spatial statistics and statistical physics, including the comparison of two Lennard-Jones processes, hard core approximation of an area interaction process and the approximation of lattice processes by a continuous Gibbs process. Our proof of the main results is based on Stein's method. We construct an explicit coupling between two spatial birth-death processes to obtain Stein factors, and employ the Georgii-Nguyen-Zessin equation for the total bound.

Analytical Models of Exoplanetary Atmospheres. II. Radiative Transfer via the Two-Stream Approximation

We present a comprehensive analytical study of radiative transfer using the method of moments and include the effects of non-isotropic scattering in the coherent limit. Within this unified formalism, we derive the governing equations and solutions describing two-stream radiative transfer (which approximates the passage of radiation as a pair of outgoing and incoming fluxes), flux-limited diffusion (which describes radiative transfer in the deep interior) and solutions for the temperature-pressure profiles. Generally, the problem is mathematically under-determined unless a set of closures (Eddington coefficients) is specified. We demonstrate that the hemispheric (or hemi-isotropic) closure naturally derives from the radiative transfer equation if energy conservation is obeyed, while the Eddington closure produces spurious enhancements of both reflected light and thermal emission. We concoct recipes for implementing two-stream radiative transfer in stand-alone numerical calculations and general circulation models. We use our two-stream solutions to construct toy models of the runaway greenhouse effect. We present a new solution for temperature-pressure profiles with a non-constant optical opacity and elucidate the effects of non-isotropic scattering in the optical and infrared. We derive generalized expressions for the spherical and Bond albedos and the photon deposition depth. We demonstrate that the value of the optical depth corresponding to the photosphere is not always 2/3 (Milne's solution) and depends on a combination of stellar irradiation, internal heat and the properties of scattering both in optical and infrared. Finally, we derive generalized expressions for the total, net, outgoing and incoming fluxes in the convective regime.

High Incorrect Use of the Standard Error of the Mean (SEM) in Original Articles in Three Cardiovascular Journals Evaluated for 2012.

RATIONALE In biomedical journals authors sometimes use the standard error of the mean (SEM) for data description, which has been called inappropriate or incorrect. OBJECTIVE To assess the frequency of incorrect use of SEM in articles in three selected cardiovascular journals. METHODS AND RESULTS All original journal articles published in 2012 in Cardiovascular Research, Circulation: Heart Failure and Circulation Research were assessed by two assessors for inappropriate use of SEM when providing descriptive information of empirical data. We also assessed whether the authors state in the methods section that the SEM will be used for data description. Of 441 articles included in this survey, 64% (282 articles) contained at least one instance of incorrect use of the SEM, with two journals having a prevalence above 70% and "Circulation: Heart Failure" having the lowest value (27%). In 81% of articles with incorrect use of SEM, the authors had explicitly stated that they use the SEM for data description and in 89% SEM bars were also used instead of 95% confidence intervals. Basic science studies had a 7.4-fold higher level of inappropriate SEM use (74%) than clinical studies (10%). LIMITATIONS The selection of the three cardiovascular journals was based on a subjective initial impression of observing inappropriate SEM use. The observed results are not representative for all cardiovascular journals. CONCLUSION In three selected cardiovascular journals we found a high level of inappropriate SEM use and explicit methods statements to use it for data description, especially in basic science studies. To improve on this situation, these and other journals should provide clear instructions to authors on how to report descriptive information of empirical data.

In the aftermath of medical error : Caring for patients, family, and the healthcare workers involved

Medical errors, in particular those resulting in harm, pose a serious situation for patients ("first victims") and the healthcare workers involved ("second victims") and can have long-lasting and distressing consequences. To prevent a second traumatization, appropriate and empathic interaction with all persons involved is essential besides error analysis. Patients share a nearly universal, broad preference for a complete disclosure of incidents, regardless of age, gender, or education. This includes the personal, timely and unambiguous disclosure of the adverse event, information relating to the event, its causes and consequences, and an apology and sincere expression of regret. While the majority of healthcare professionals generally support and honest and open disclosure of adverse events, they also face various barriers which impede the disclosure (e.g., fear of legal consequences). Despite its essential importance, disclosure of adverse events in practice occurs in ways that are rarely acceptable to patients and their families. The staff involved often experiences acute distress and an intense emotional response to the event, which may become chronic and increase the risk of depression, burnout and post-traumatic stress disorders. Communication with peers is vital for people to be able to cope constructively and protectively with harmful errors. Survey studies among healthcare workers show, however, that they often do not receive sufficient individual and institutional support. Healthcare organizations should prepare for medical errors and harmful events and implement a communication plan and a support system that covers the requirements and different needs of patients and the staff involved.

Functional error modeling for uncertainty quantification in hydrogeology

Approximate models (proxies) can be employed to reduce the computational costs of estimating uncertainty. The price to pay is that the approximations introduced by the proxy model can lead to a biased estimation. To avoid this problem and ensure a reliable uncertainty quantification, we propose to combine functional data analysis and machine learning to build error models that allow us to obtain an accurate prediction of the exact response without solving the exact model for all realizations. We build the relationship between proxy and exact model on a learning set of geostatistical realizations for which both exact and approximate solvers are run. Functional principal components analysis (FPCA) is used to investigate the variability in the two sets of curves and reduce the dimensionality of the problem while maximizing the retained information. Once obtained, the error model can be used to predict the exact response of any realization on the basis of the sole proxy response. This methodology is purpose-oriented as the error model is constructed directly for the quantity of interest, rather than for the state of the system. Also, the dimensionality reduction performed by FPCA allows a diagnostic of the quality of the error model to assess the informativeness of the learning set and the fidelity of the proxy to the exact model. The possibility of obtaining a prediction of the exact response for any newly generated realization suggests that the methodology can be effectively used beyond the context of uncertainty quantification, in particular for Bayesian inference and optimization.

Experimental dissolution of molybdenum-sulphides at low oxygen concentrations: A first-order approximation of late Archean atmospheric conditions

The abundance of atmospheric oxygen and its evolution through Earth's history is a highly debated topic. The earliest change of the Mo concentration and isotope composition of marine sediments are interpreted to be linked to the onset of the accumulation of free O2 in Earth's atmosphere. The O2 concentration needed to dissolve significant amounts of Mo in water is not yet quantified, however. We present laboratory experiments on pulverized and surface-cleaned molybdenite (MoS2) and a hydrothermal breccia enriched in Mo-bearing sulphides using a glove box setup. Duration of an experiment was 14 days, and first signs of oxidation and subsequent dissolution of Mo compounds start to occur above an atmospheric oxygen concentration of 72 ± 20 ppmv (i.e., 2.6 to 4.6 × 10−4 present atmospheric level (PAL)). This experimentally determined value coincides with published model calculations supporting atmospheric O2 concentrations between 1 × 10−5 to 3 × 10−4 PAL prior to the Great Oxidation Event and sets an upper limit to the molecular oxygen needed to trigger Mo accumulation and Mo isotope variations recorded in sediments. In combination with the published Mo isotope composition of the rock record, this result implies an atmospheric oxygen concentration prior to 2.76 Ga of below 72 ± 20 ppmv.

Saddlepoint approximations

The saddlepoint method provides accurate approximations for the distributions of many test statistics, estimators and for important probabilities arising in various stochastic models. The saddlepoint approximation is a large deviations technique which is substantially more accurate than limiting normal or Edgeworth approximations, especially in presence of very small sample sizes or very small probabilities. The outstanding accuracy of the saddlepoint approximation can be explained by the fact that it has bounded relative error.