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.

Anmerkungen zu den (spät)persischen Samaria-Bullen aus dem Wadi ed-Dalije. Hellenisches, Persisches und Lokaltraditionen im Grenzgebiet der Provinz Jehud

The present article deals exemplarily with the remarkable iconographic attestations connected with the Wadi ed-Daliye (WD) findings. The discussed bullae were attached to papyri which provide a clear dating of the hoard between 375-335 BCE. Considering style and convention the preserved motives are to be classified as Persian, Greek or Greco-Persian. A major goal of the following presentation is the contextualization of the very material; this is achieved by taking into account local parallels as well as relevant attestations of the dominant / “imperial” cultures of Persia and Greece. The correlation of motives with the (often more complex, more detailed or more contoured) examples stemming from the “source-cultures” follows a clear agenda: It is methodologically based on the approach that was employed by Silvia Schroer and Othmar Keel throughout the project „Die Ikonographie Palästinas/Israels und der Alte Orient (IPIAO). Eine Religionsgeschichte in Bildern” (2005, 23ff). The WD-findings demand a careful analysis since the influencing cultures behind the imagery are deeply rooted in the field of Greek mythology and iconography. Special attention has to be drawn to the bullae, as far as excavated, from a huge Punic temple archive of Carthago (Berges 1997 and 2002) as well as those from the archive of the satrap seat in Daskyleion in the Northwest of Asia Minor (Kaptan 2002), which are chronologically close (end 5th and 4th century BCE) to the WD-finds. Not each and every single motive and artifact of the WD-corpus comprising more than 120 items can be dealt with in detail throughout the following pages. We refer to the editio princeps by Leith (1990, 1997) respectively to the concerning chapter in Keel’s Corpus volume II (Keel 2010, 340-379). The article gives a brief history of research (2.), some basic remarks on the development of style (3.) and a selection of detail-studies (4.). A crosscheck with other relevant corpora of stamp-seals (5.) as well as a compressed synthesis (6.) are contributions in order to characterize and classify the unique iconographic assemblage. There are rather few references to the late Persian coins from Samaria (Meshorer/Qedar 1999), which have been impressed about contemporaneous with the WD-bullae (372-333 BCE), as there is an article by Patrick Wyssmann in this volume about that specific corpus. Through the perspective of the late Persian iconography, Samaria appears as a dazzling metropolis at the crossroads of Greek and Persian culture, which is far away from a strict and revolutionary religious orthodoxy

CYTOPLASMIC MALATE DEHYDROGENASE IS THE AROMATIC ALPHA-KETO ACID REDUCTASE IN MAN (PHENYLKETONURIA, PKU, TYROSINE)

This dissertation presents evidence to support the hypothesis that cytoplasmic malate dehydrogenase (MDH-1) is the enzyme in humans which catalyzes the reduction of aromatic alpha-keto acids in the presence of NADH, and the enzyme which has been described in the literature as aromatic alpha-keto acid reductase (KAR; E.C. 1.1.1.96) is actually a secondary activity of cytoplasmic malate dehydrogenase.^ Purified MDH and purified KAR have the same molecular weight, subunit structure, heat-inactivation profile and tissue distribution. After starch gel electrophoresis, and using p-hydroxyphenylpyruvic acid (HPPA) as substrate, KAR activity co-migrates with MDH-1 in all species studied except some marine animals. Inhibition with malate, the end-product of malate dehydrogenase, substantially reduces or totally eliminates KAR activity. Purified cytoplasmic MDH from human erythrocytes has an alpha-keto acid reductase activity with identical mobility. All electrophoretic variants of MDH-1 seen in the fresh-water bony fish Xiphophorus, the amphibians Rana and humans exhibited identical variation for KAR, and the two traits co-segregated in the small group of offspring from one Rana heterozygote studied. Both enzymes show almost no electrophoretic variation among humans from many ethnic groups, and among several inbred strains of mice both MDH-s and KAR co-migrate with no variation. MDH-1 and KAR in mouse and Chinese hamster fibroblasts show identical mobility differences between species. Antisera raised against purified chicken cytoplasmic MDH totally inhibited both MDH-1 and KAR in chickens and humans. Mitochondrial MDH from tissue homogenates has no detectable KAR activity but purified MDH-2 does.^ The previous claim that the gene for KAR is on human chromosome 12 is disputed because both MDH-1 and LDH bands appear with slightly different mobility approximately midway between the human and hamster controls in somatic cell hybrid studies, and the meaning of this artifact is discussed. ^

Impact of a 4th generation iterative reconstruction technique on image quality in low-dose computed tomography of the chest in immunocompromised patients

PURPOSE To determine the image quality of an iterative reconstruction (IR) technique in low-dose MDCT (LDCT) of the chest of immunocompromised patients in an intraindividual comparison to filtered back projection (FBP) and to evaluate the dose reduction capability. MATERIALS AND METHODS 30 chest LDCT scans were performed in immunocompromised patients (Brilliance iCT; 20-40 mAs; mean CTDIvol: 1.7 mGy). The raw data were reconstructed using FBP and the IR technique (iDose4™, Philips, Best, The Netherlands) set to seven iteration levels. 30 routine-dose MDCT (RDCT) reconstructed with FBP served as controls (mean exposure: 116 mAs; mean CDTIvol: 7.6 mGy). Three blinded radiologists scored subjective image quality and lesion conspicuity. Quantitative parameters including CT attenuation and objective image noise (OIN) were determined. RESULTS In LDCT high iDose4™ levels lead to a significant decrease in OIN (FBP vs. iDose7: subscapular muscle 139.4 vs. 40.6 HU). The high iDose4™ levels provided significant improvements in image quality and artifact and noise reduction compared to LDCT FBP images. The conspicuity of subtle lesions was limited in LDCT FBP images. It significantly improved with high iDose4™ levels (> iDose4). LDCT with iDose4™ level 6 was determined to be of equivalent image quality as RDCT with FBP. CONCLUSION iDose4™ substantially improves image quality and lesion conspicuity and reduces noise in low-dose chest CT. Compared to RDCT, high iDose4™ levels provide equivalent image quality in LDCT, hence suggesting a potential dose reduction of almost 80%.

Monoenergetic computed tomography reconstructions reduce beam hardening artifacts from dental restorations

The aim of this study was to assess the potential of monoenergetic computed tomography (CT) images to reduce beam hardening artifacts in comparison to standard CT images of dental restoration on dental post-mortem CT (PMCT). Thirty human decedents (15 male, 58 ± 22 years) with dental restorations were examined using standard single-energy CT (SECT) and dual-energy CT (DECT). DECT data were used to generate monoenergetic CT images, reflecting the X-ray attenuation at energy levels of 64, 69, 88 keV, and at an individually adjusted optimal energy level called OPTkeV. Artifact reduction and image quality of SECT and monoenergetic CT were assessed objectively and subjectively by two blinded readers. Subjectively, beam artifacts decreased visibly in 28/30 cases after monoenergetic CT reconstruction. Inter- and intra-reader agreement was good (k = 0.72, and k = 0.73 respectively). Beam hardening artifacts decreased significantly with increasing monoenergies (repeated-measures ANOVA p < 0.001). Artifact reduction was greatest on monoenergetic CT images at OPTkeV. Mean OPTkeV was 108 ± 17 keV. OPTkeV yielded the lowest difference between CT numbers of streak artifacts and reference tissues (-163 HU). Monoenergetic CT reconstructions significantly reduce beam hardening artifacts from dental restorations and improve image quality of post-mortem dental CT.

A neurophysiological fingerprint of the empty-chair dialogue

The empty chair dialogue is a validated technique used in Gestalt and emotion focused therapy to help clients overcome unresolved interpersonal grievances. It aims at influencing emotional processing in a way that emotional states characterized by advanced meaning making, thus by the integration of cognition and affect, are facilitated. Even though a variety of studies demonstrated the effectiveness of this technique as well as the usefulness of improvements in emotional processing, it remains unclear how these changes are characterized on a neuronal level. The present study aimed at tracing changes induced by the empty-chair dialogue with electrophysiological methods. Subjects reporting long-standing interpersonal grievances were recruited. After informed consent, an experienced therapist guided subjects to work on their individual interpersonal theme using the empty chair dialogue. During this one-session intervention, multichannel EEG was recorded and the session was video-taped. Afterwards, a validated observational rating instrument was used to identify time periods representing emotional states characterized by either high or low meaning making and the preprocessed, artifact-free EEG-data was labeled accordingly. Thus the comparison of neurophysiological activity during two distinct types of emotional processing becomes possible. EEG-data will be analyzed with modern methods of frequency analysis. Furthermore global field synchronization will be compared between the two types of emotional processing.

The effect of surgical titanium rods on proton therapy delivered for cervical bone tumors: experimental validation using an anthropomorphic phantom.

To investigate the effect of metal implants in proton radiotherapy, dose distributions of different, clinically relevant treatment plans have been measured in an anthropomorphic phantom and compared to treatment planning predictions. The anthropomorphic phantom, which is sliced into four segments in the cranio-caudal direction, is composed of tissue equivalent materials and contains a titanium implant in a vertebral body in the cervical region. GafChromic® films were laid between the different segments to measure the 2D delivered dose. Three different four-field plans have then been applied: a Single-Field-Uniform-Dose (SFUD) plan, both with and without artifact correction implemented, and an Intensity-Modulated-Proton-Therapy (IMPT) plan with the artifacts corrected. For corrections, the artifacts were manually outlined and the Hounsfield Units manually set to an average value for soft tissue. Results show a surprisingly good agreement between prescribed and delivered dose distributions when artifacts have been corrected, with > 97% and 98% of points fulfilling the gamma criterion of 3%/3 mm for both SFUD and the IMPT plans, respectively. In contrast, without artifact corrections, up to 18% of measured points fail the gamma criterion of 3%/3 mm for the SFUD plan. These measurements indicate that correcting manually for the reconstruction artifacts resulting from metal implants substantially improves the accuracy of the calculated dose distribution.

Body weight estimation based on postmortem CT data-validation of a multiplication factor

Postmortem computed tomography (pmCT) is increasingly applied in forensic medicine as a documentation and diagnostic tool. The present study investigated if pmCT data can be used to estimate the corpse weight. In 50 forensic cases, pmCT examinations were performed prior to autopsy and the pmCT data were used to determine the body volume using an automated segmentation tool. PmCT was performed within 48 h postmortem. The body weights assessed prior to autopsy and the body volumes assessed using the pmCT data were used to calculate individual multiplication factors. The mean postmortem multiplication factor for the study cases was 1.07 g/ml. Using this factor, the body weight may be estimated retrospectively when necessary. Severe artifact causing foreign bodies within the corpses limit the use of pmCT data for body weight estimations.

A New Approach for Automatic Removal of Movement Artifacts in Near-Infrared Spectroscopy Time Series by Means of Acceleration Data

Abstract: Near-infrared spectroscopy (NIRS) enables the non-invasive measurement of changes in hemodynamics and oxygenation in tissue. Changes in light-coupling due to movement of the subject can cause movement artifacts (MAs) in the recorded signals. Several methods have been developed so far that facilitate the detection and reduction of MAs in the data. However, due to fixed parameter values (e.g., global threshold) none of these methods are perfectly suitable for long-term (i.e., hours) recordings or were not time-effective when applied to large datasets. We aimed to overcome these limitations by automation, i.e., data adaptive thresholding specifically designed for long-term measurements, and by introducing a stable long-term signal reconstruction. Our new technique (“acceleration-based movement artifact reduction algorithm”, AMARA) is based on combining two methods: the “movement artifact reduction algorithm” (MARA, Scholkmann et al. Phys. Meas. 2010, 31, 649–662), and the “accelerometer-based motion artifact removal” (ABAMAR, Virtanen et al. J. Biomed. Opt. 2011, 16, 087005). We describe AMARA in detail and report about successful validation of the algorithm using empirical NIRS data, measured over the prefrontal cortex in adolescents during sleep. In addition, we compared the performance of AMARA to that of MARA and ABAMAR based on validation data.

Quantifying the vestibulo-ocular reflex with video-oculography: nature and frequency of artifacts

Video-oculography devices are now used to quantify the vestibulo-ocular reflex (VOR) at the bedside using the head impulse test (HIT). Little is known about the impact of disruptive phenomena (e.g. corrective saccades, nystagmus, fixation losses, eye-blink artifacts) on quantitative VOR assessment in acute vertigo. This study systematically characterized the frequency, nature, and impact of artifacts on HIT VOR measures. From a prospective study of 26 patients with acute vestibular syndrome (16 vestibular neuritis, 10 stroke), we classified findings using a structured coding manual. Of 1,358 individual HIT traces, 72% had abnormal disruptive saccades, 44% had at least one artifact, and 42% were uninterpretable. Physicians using quantitative recording devices to measure head impulse VOR responses for clinical diagnosis should be aware of the potential impact of disruptive eye movements and measurement artifacts.

Effects of Study Area Size on Geographic Characterizations of Health Events: Prostate Cancer Incidence in Southern New England, USA, 1994-1998.

ABSTRACT : BACKGROUND : We consider how representations of geographic variation in prostate cancer incidence across Southern New England, USA may be affected by selection of study area and/or properties of the statistical analysis. METHOD : A spatial scan statistic was used to monitor geographic variation among 35,167 incident prostate cancer cases diagnosed in Massachusetts, Connecticut and Rhode Island from 1994 to 1998, in relation to the 1990 populations of men 20+ years of age living in that region. Results from the combined-states analysis were compared to those from single-states. Impact of scanning procedures set to examine up to 50% or no more than10% of at-risk populations also was evaluated. RESULTS : With scanning set to 50%, 5 locations in the combined-states analysis were identified with markedly distinct incidence rates. Fewer than expected cases were estimated for nearly all Connecticut, Rhode Island and West Central Massachusetts, whereas census tracts on and around Cape Cod, and areas of Southwestern Connecticut and adjacent to greater Boston were estimated to have yielded more than expected incidence. Results of single-state analyses exhibited several discrepancies from the combined-states analysis. More conservative scanning found many more locations with varying incidence, but discrepancies between the combined- and single-state analysis were fewer. CONCLUSION : It is important to acknowledge the conditional nature of spatial analyses and carefully consider whether a true cluster of events is identified or artifact stemming from selection of study area size and/or scanning properties.

Multiple gradient echo propeller (MGREP) MRI: Technical development and potential applications

The PROPELLER (Periodically Rotated Overlapping Parallel Lines with Enhanced Reconstruction) magnetic resonance imaging (MRI) technique has inherent advantages over other fast imaging methods, including robust motion correction, reduced image distortion, and resistance to off-resonance effects. These features make PROPELLER highly desirable for T2*-sensitive imaging, high-resolution diffusion imaging, and many other applications. However, PROPELLER has been predominantly implemented as a fast spin-echo (FSE) technique, which is insensitive to T2* contrast, and requires time-inefficient signal averaging to achieve adequate signal-to-noise ratio (SNR) for many applications. These issues presently constrain the potential clinical utility of FSE-based PROPELLER. ^ In this research, our aim was to extend and enhance the potential applications of PROPELLER MRI by developing a novel multiple gradient echo PROPELLER (MGREP) technique that can overcome the aforementioned limitations. The MGREP pulse sequence was designed to acquire multiple gradient-echo images simultaneously, without any increase in total scan time or RF energy deposition relative to FSE-based PROPELLER. A new parameter was also introduced for direct user-control over gradient echo spacing, to allow variable sensitivity to T2* contrast. In parallel to pulse sequence development, an improved algorithm for motion correction was also developed and evaluated against the established method through extensive simulations. The potential advantages of MGREP over FSE-based PROPELLER were illustrated via three specific applications: (1) quantitative T2* measurement, (2) time-efficient signal averaging, and (3) high-resolution diffusion imaging. Relative to the FSE-PROPELLER method, the MGREP sequence was found to yield quantitative T2* values, increase SNR by ∼40% without any increase in acquisition time or RF energy deposition, and noticeably improve image quality in high-resolution diffusion maps. In addition, the new motion algorithm was found to improve the performance considerably in motion-artifact reduction. ^ Overall, this work demonstrated a number of enhancements and extensions to existing PROPELLER techniques. The new technical capabilities of PROPELLER imaging, developed in this thesis research, are expected to serve as the foundation for further expanding the scope of PROPELLER applications. ^

The imaging and dosimetric capabilities of a CT/MR-suitable, anatomically adaptive, shielded intracavitary brachytherapy applicator for the treatment of cervical cancer

Introduction. Investigations into the shortcomings of current intracavitary brachytherapy (ICBT) technology has lead us to design an Anatomically Adaptive Applicator (A3). The goal of this work was to design and characterize the imaging and dosimetric capabilities of this device. The A3 design incorporates a single shield that can both rotate and translate within the colpostat. We hypothesized that this feature, coupled with specific A3 component construction materials and imaging techniques, would facilitate artifact-free CT and MR image acquisition. In addition, by shaping the delivered dose distribution via the A3 movable shield, dose delivered to the rectum will be less compared to equivalent treatments utilizing current state-of-the-art ICBT applicators. ^ Method and materials. A method was developed to facilitate an artifact-free CT imaging protocol that used a "step-and-shoot" technique: pausing the scanner midway through the scan and moving the A 3 shield out of the path of the beam. The A3 CT imaging capabilities were demonstrated acquiring images of a phantom that positioned the A3 and FW applicators in a clinically-applicable geometry. Artifact-free MRI imaging was achieved by utilizing MRI-compatible ovoid components and pulse-sequences that minimize susceptibility artifacts. Artifacts were qualitatively compared, in a clinical setup. For the dosimetric study, Monte-Carlo (MC) models of the A3 and FW (shielded and unshielded) applicators were validated. These models were incorporated into a MC model of one cervical cancer patient ICBT insertion, using 192Ir (mHDR v2 source). The A3 shield's rotation and translation was adjusted for each dwell position to minimize dose to the rectum. Superposition of dose to rectum for all A3 dwell sources (4 per ovoid) was applied to obtain a comparison of equivalent FW treatments. Rectal dose-volume histograms (absolute and HDR/PDR biologically effective dose (BED)) and BED to 2 cc (BED2cc ) were determined for all applicators and compared. ^ Results. Using a "step-and-shoot" CT scanning method and MR compliant materials and optimized pulse-sequences, images of the A 3 were nearly artifact-free for both modalities. The A3 reduced BED2cc by 18.5% and 7.2% for a PDR treatment and 22.4% and 8.7% for a HDR treatment compared to treatments delivered using an uFW and sFW applicator, respectively. ^ Conclusions. The novel design of the A3 facilitated nearly artifact-free image quality for both CT and MR clinical imaging protocols. The design also facilitated a reduction in BED to the rectum compared to equivalent ICBT treatments delivered using current, state-of-the-art applicators. ^

Socioeconomic disparities in thyroid cancer incidence within the Surveillance, Epidemiology, and End Results (SEER) registry, 1980--2008

Background. In the past two decades, the incidence of thyroid cancer in the United States (US) has been increasing. There has been debate on whether the increase is real or an artifact of improved diagnostic scrutiny. Methods. We linked SEER9 database with 2000 US Census to obtain county-level SES (Socioeconomic Status) and compared thyroid cancer incidence trends between high and low SES counties. Joinpoint analysis was used to assess the thyroid cancer incidence trends. Annual Percentage Changes (APCs) were calculated to evaluate incidence trends. Results . The thyroid cancer incidence in high SES counties increased moderately (APC1=+2.5*, *P<0.05) before late 1990s and dramatically increased (APC2=+6.3*) after late 1990s, whereas incidence in low SES counties increased moderately (APC=+3.5*) during the entire time period (1980–2008). For smaller tumors (≤4cm), the APCs in high and low SES counties are similar to each other before late 1990s, but the incidence in high SES counties increased dramatically after late 1990s while that in low SES counties continued at a moderate increase. For large tumors (>4cm), the incidence trends in high SES counties are similar to those of low SES counties, which had a steady moderate increase. Conclusion. Our findings indicate that enhanced detection likely contributed to the increased thyroid cancer incidence in the past decades but cannot fully explain the increase, suggesting that a true increase also exists. Efforts should be made on identifying the cause of this observed increased incidence as well as more refined/selected screening and prevention measures.^

UNDERSTANDING NURSE CREATED COGNITIVE ARTIFACTS: PERSONALLY-CREATED-COGNITIVE-ARTIFACTS AS EXTERNAL REPRESENTATIONS OF DISTRIBUTED COGNITION

Manuscript 1: “Conceptual Analysis: Externalizing Nursing Knowledge” We use concept analysis to establish that the report tool nurses prepare, carry, reference, amend, and use as a temporary data repository are examples of cognitive artifacts. This tool, integrally woven throughout the work and practice of nurses, is important to cognition and clinical decision-making. Establishing the tool as a cognitive artifact will support new dimensions of study. Such studies can characterize how this report tool supports cognition, internal representation of knowledge and skills, and external representation of knowledge of the nurse. Manuscript 2: “Research Methods: Exploring Cognitive Work” The purpose of this paper is to describe a complex, cross-sectional, multi-method approach to study of personal cognitive artifacts in the clinical environment. The complex data arrays present in these cognitive artifacts warrant the use of multiple methods of data collection. Use of a less robust research design may result in an incomplete understanding of the meaning, value, content, and relationships between personal cognitive artifacts in the clinical environment and the cognitive work of the user. Manuscript 3: “Making the Cognitive Work of Registered Nurses Visible” Purpose: Knowledge representations and structures are created and used by registered nurses to guide patient care. Understanding is limited regarding how these knowledge representations, or cognitive artifacts, contribute to working memory, prioritization, organization, cognition, and decision-making. The purpose of this study was to identify and characterize the role a specific cognitive artifact knowledge representation and structure as it contributed to the cognitive work of the registered nurse. Methods: Data collection was completed, using qualitative research methods, by shadowing and interviewing 25 registered nurses. Data analysis employed triangulation and iterative analytic processes. Results: Nurse cognitive artifacts support recall, data evaluation, decision-making, organization, and prioritization. These cognitive artifacts demonstrated spatial, longitudinal, chronologic, visual, and personal cues to support the cognitive work of nurses. Conclusions: Nurse cognitive artifacts are an important adjunct to the cognitive work of nurses, and directly support patient care. Nurses need to be able to configure their cognitive artifact in ways that are meaningful and support their internal knowledge representations.