Characterization of Chelonus inanitus polydnavirus segments: sequences and analysis, excision site and demonstration of clustering

Polydnaviruses (genera Ichnovirus and Bracovirus) have a segmented genome of circular double-stranded DNA molecules, replicate in the ovary of parasitic wasps and are essential for successful parasitism of the host. Here we show the first detailed analysis of various segments of a bracovirus, the Chelonus inanitus virus (CiV). Four segments were sequenced and two of them, CiV12 and CiV14, were found to be closely related while CiV14.5 and CiV16.8 were unrelated. CiV12, CiV14.5 and CiV16.8 are unique while CiV14 occurs also nested in another larger segment. All four segments are predicted to contain genes and predictions could be substantiated in most cases. Comparison with databases revealed no significant similarities at either the nucleotide or amino acid level. Inverted repeats with identities between 77% and 92% and lengths between 26 bp and 100 bp were found on all segments outside of predicted genes. Hybridization experiments indicate that CiV12 and CiV14 are both flanked by other virus segments, suggesting that proviral CiV segments are clustered in the genome of the wasp. The integration/excision site of CiV14 was analysed and compared to that of CiV12. On both termini of proviral CiV12 and CiV14 as well as in the excised circular molecule and the rejoined DNA a very similar repeat of 14 bp was found. A model to illustrate where the terminal repeats might recombine to yield the circular molecule is presented. Excision of CiV12 and CiV14 is restricted to the female and sets in at a very specific time-point in pupal-adult development.

Conserved sequences and cell-specific DNase I hypersensitive sites upstream from the co-ordinately expressed alpha I- and alpha II-globin genes of Xenopus laevis

The globin gene family of Xenopus laevis comprises pairs of closely related genes that are arranged in two clusters, each pair of genes being co-ordinately and stage-specifically expressed. To get information on putative regulatory elements, we compared the DNA sequences and the chromatin conformation 5' to the co-ordinately expressed adult alpha-globin genes. Sequence analysis revealed a relatively conserved region from the cap site up to position -289, and further upstream seven distinct boxes of homology, separated by more diverged sequences or deletions/insertions. The homology boxes comprise 22 to 194 base-pairs showing 78 to 95% homology. Analysis of chromatin conformation showed that DNase I preferentially cuts the upstream region of both genes at similar positions, 5' to the T-A-T-A and the C-C-A-A-T boxes, only in chromatin of adult erythroblasts and erythrocytes, where adult globin genes are expressed, but not in chromatin of adult liver cells or larval erythrocytes, where these genes are silent. This suggests that cell- and stage-specific activation of these genes coincides with specific changes in chromatin conformation within the proximal upstream region. No difference was found in the nucleotide sequence within the DNase I hypersensitive region proximal to the adult alpha 1-globin gene in DNA from embryonic cells, in which this gene is inactive, and adult erythrocytes, expressing this gene.

A unified approach to architecture conformance checking

Architectural decisions can be interpreted as structural and behavioral constraints that must be enforced in order to guarantee overarching qualities in a system. Enforcing those constraints in a fully automated way is often challenging and not well supported by current tools. Current approaches for checking architecture conformance either lack in usability or offer poor options for adaptation. To overcome this problem we analyze the current state of practice and propose an approach based on an extensible, declarative and empirically-grounded specification language. This solution aims at reducing the overall cost of setting up and maintaining an architectural conformance monitoring environment by decoupling the conceptual representation of a user-defined rule from its technical specification prescribed by the underlying analysis tools. By using a declarative language, we are able to write tool-agnostic rules that are simple enough to be understood by untrained stakeholders and, at the same time, can be can be automatically processed by a conformance checking validator. Besides addressing the issue of cost, we also investigate opportunities for increasing the value of conformance checking results by assisting the user towards the full alignment of the implementation with respect to its architecture. In particular, we show the benefits of providing actionable results by introducing a technique which automatically selects the optimal repairing solutions by means of simulation and profit-based quantification. We perform various case studies to show how our approach can be successfully adopted to support truly diverse industrial projects. We also investigate the dynamics involved in choosing and adopting a new automated conformance checking solution within an industrial context. Our approach reduces the cost of conformance checking by avoiding the need for an explicit management of the involved validation tools. The user can define rules using a convenient high-level DSL which automatically adapts to emerging analysis requirements. Increased usability and modular customization ensure lower costs and a shorter feedback loop.

Medication double-checking procedures in clinical practice: a cross-sectional survey of oncology nurses' experiences.

BACKGROUND Double-checking is widely recommended as an essential method to prevent medication errors. However, prior research has shown that the concept of double-checking is not clearly defined, and that little is known about actual practice in oncology, for example, what kind of checking procedures are applied. OBJECTIVE To study the practice of different double-checking procedures in chemotherapy administration and to explore nurses' experiences, for example, how often they actually find errors using a certain procedure. General evaluations regarding double-checking, for example, frequency of interruptions during and caused by a check, or what is regarded as its essential feature was assessed. METHODS In a cross-sectional survey, qualified nurses working in oncology departments of 3 hospitals were asked to rate 5 different scenarios of double-checking procedures regarding dimensions such as frequency of use in practice and appropriateness to prevent medication errors; they were also asked general questions about double-checking. RESULTS Overall, 274 nurses (70% response rate) participated in the survey. The procedure of jointly double-checking (read-read back) was most commonly used (69% of respondents) and rated as very appropriate to prevent medication errors. Jointly checking medication was seen as the essential characteristic of double-checking-more frequently than 'carrying out checks independently' (54% vs 24%). Most nurses (78%) found the frequency of double-checking in their department appropriate. Being interrupted in one's own current activity for supporting a double-check was reported to occur frequently. Regression analysis revealed a strong preference towards checks that are currently implemented at the responders' workplace. CONCLUSIONS Double-checking is well regarded by oncology nurses as a procedure to help prevent errors, with jointly checking being used most frequently. Our results show that the notion of independent checking needs to be transferred more actively into clinical practice. The high frequency of reported interruptions during and caused by double-checks is of concern.

Use of a novel DNA melting profile assay for the identification of PCR-amplified genomic sequences encoding for variant-specific surface proteins from the clonal GS/M-83-H7 line of Giardia lamblia.

During infections, Giardia lamblia undergoes a continuous change of its major surface antigens, the variant-specific surface proteins (VSPs). Many studies on antigenic variation have been performed using G. lamblia clone GS/M-83-H7, which expresses surface antigen VSP H7. The present study was focused on the identification and characterization of vsp gene sequences within the genome of the clonal G. lamblia GS/M-83-H7 line. For this purpose, we applied a PCR which specifically amplified truncated sequences from the 3'-terminal region of the vsp genes. Upon cloning, most of the vsp gene amplification products were shown to be approximately identical in size and thus could not be distinguished from each other by conventional gel electrophoresis. In order to pre-estimate the sequence complexity within the large panel of vsp clones isolated, we elaborated a novel concept which facilitated our large-scale genetic screening approach: PCR products from cloned DNA molecules were generated and then subjected to a DNA melting profile assay based on the use of the LightCycler Instrument. This high-throughput assay system proved to be well suited to monitor sequence differences between the amplification products from closely related vsp genes and thus could be used for the primary, sequence-related discrimination of the corresponding clones. After testing 50 candidates, vsp clones could be divided into five groups, each characterized by an individual DNA melting profile of the corresponding amplification products. Sequence analysis of some of these 50 candidates confirmed data from the aforementioned assay in that clones were demonstrated to be identical within, but different between, the distinct groups. The nucleotide and deduced amino acid sequences of five representative vsp clones showed high similarities both among each other and also with the corresponding gene segment of the variant-specific surface antigen (VSP H7) expressed by the original GS/M-83-H7 variant type. Furthermore, three of the genomic vsp sequences turned out to be identical to vsp sequences that represented previously characterized transcription products from in vivo- or in vitro-switched GS/M-83-H7 trophozoites. In conclusion, the DNA melting profile assay seems to be a versatile tool for the PCR-based genotyping of moderately or highly diversified sequence orthologues.

A Unified Approach to Architecture Conformance Checking

Software erosion can be controlled by periodically checking for consistency between the de facto architecture and its theoretical counterpart. Studies show that this process is often not automated and that developers still rely heavily on manual reviews, despite the availability of a large number of tools. This is partially due to the high cost involved in setting up and maintaining tool-specific and incompatible test specifications that replicate otherwise documented invariants. To reduce this cost, our approach consists in unifying the functionality provided by existing tools under the umbrella of a common business-readable DSL. By using a declarative language, we are able to write tool-agnostic rules that are simple enough to be understood by non-technical stakeholders and, at the same time, can be interpreted as a rigorous specification for checking architecture conformance

On the Evaluation of a DSL for Architectural Consistency Checking

Software architecture erodes over time and needs to be constantly monitored to be kept consistent with its original intended design. Consistency is rarely monitored using automated techniques. The cost associated to such an activity is typically not considered proportional to its benefits. To improve this situation, we propose Dicto, a uniform DSL for specifying architectural invariants. This language is designed to reduce the cost of consistency checking by offering a framework in which existing validation tools can be matched to newly-defined language constructs. In this paper we discuss how such a DSL can be qualitatively and qualitatively evaluated in practice.

Genotypic Resistance Tests Sequences Reveal the Role of Marginalized Populations in HIV-1 Transmission in Switzerland.

Targeting hard-to-reach/marginalized populations is essential for preventing HIV-transmission. A unique opportunity to identify such populations in Switzerland is provided by a database of all genotypic-resistance-tests from Switzerland, including both sequences from the Swiss HIV Cohort Study (SHCS) and non-cohort sequences. A phylogenetic tree was built using 11,127 SHCS and 2,875 Swiss non-SHCS sequences. Demographics were imputed for non-SHCS patients using a phylogenetic proximity approach. Factors associated with non-cohort outbreaks were determined using logistic regression. Non-B subtype (univariable odds-ratio (OR): 1.9; 95% confidence interval (CI): 1.8-2.1), female gender (OR: 1.6; 95% CI: 1.4-1.7), black ethnicity (OR: 1.9; 95% CI: 1.7-2.1) and heterosexual transmission group (OR:1.8; 95% CI: 1.6-2.0), were all associated with underrepresentation in the SHCS. We found 344 purely non-SHCS transmission clusters, however, these outbreaks were small (median 2, maximum 7 patients) with a strong overlap with the SHCS'. 65% of non-SHCS sequences were part of clusters composed of >= 50% SHCS sequences. Our data suggests that marginalized-populations are underrepresented in the SHCS. However, the limited size of outbreaks among non-SHCS patients in-care implies that no major HIV outbreak in Switzerland was missed by the SHCS surveillance. This study demonstrates the potential of sequence data to assess and extend the scope of infectious-disease surveillance.

A Comprehensive Discussion of HMBC Pulse Sequences: 4. Establishing Two-Bond Correlations from HMBC and Related Experiments

The utility of the HMBC experiment for structure elucidation is unquestionable, but the nature of the coupling pathways leading to correlations in an HMBC experiment creates the potential for misinterpretation. This misinterpretation potential is intimately linked to the size of the long-range heteronuclear couplings involved, and may become troublesome in those cases of a particularly strong 2JCH correlation that might be mistaken for a 3JCH correlation or a 4JCH correlation of appreciable strength that could be mistaken for a weaker 3JCH correlation. To address these potential avenues of confusion, work from several laboratories has been focused on the development of what might be considered “coupling pathway edited” long-range heteronuclear correlation experiments that are derived from or related to the HMBC experiment. The first example of an effort to address the problems associated with correlation path length was seen in the heteronucleus-detected XCORFE experiment described by Reynolds and co-workers that predated the development of the HMBC experiment. Proton-detected analogs of the HMBC experiment intended to differentiate 2JCH correlations from nJCH correlations where n = 3, 4, include the 2J,3J-HMBC, HMBC-RELAY, H2BC, edited-HMBC, and HAT H2BC experiments. The principles underlying the critical components of each of these experiments are discussed and experimental verification of the results that can be obtained using model compounds are shown. This contribution concludes with a brief discussion of the 1,1-ADEQUATE experiments that provide an alternative means of identifying adjacent protonated and non-protonated carbon correlations by exploiting 1JCC correlations at natural abundance.