The 1.14 Å Crystal Structure of Yeast Cytosine Deaminase Evolution of Nucleotide Salvage Enzymes and Implications for Genetic Chemotherapy

AbstractCytosine deaminase (CD) catalyzes the deamination of cytosine and is only present in prokaryotes and fungi, where it is a member of the pyrimidine salvage pathway. The enzyme is of interest both for antimicrobial drug design and gene therapy applications against tumors. The structure of Saccharomyces cerevisiae CD has been determined in the presence and absence of a mechanism-based inhibitor, at 1.14 and 1.43 Å resolution, respectively. The enzyme forms an α/β fold similar to bacterial cytidine deaminase, but with no similarity to the α/β barrel fold used by bacterial cytosine deaminase or mammalian adenosine deaminase. The structures observed for bacterial, fungal, and mammalian nucleic acid deaminases represent an example of the parallel evolution of two unique protein folds to carry out the same reaction on a diverse array of substrates

Identifying Complexity in Infectious Diseases Inpatient Settings: An Observation Study

Background Understanding complexity in healthcare has the potential to reduce decision and treatment uncertainty. Therefore, identifying both patient and task complexity may offer better task allocation and design recommendation for next-generation health information technology system design. Objective To identify specific complexity-contributing factors in the infectious disease domain and the relationship with the complexity perceived by clinicians. Method We observed and audio recorded clinical rounds of three infectious disease teams. Thirty cases were observed for a period of four consecutive days. Transcripts were coded based on clinical complexity-contributing factors from the clinical complexity model. Ratings of complexity on day 1 for each case were collected. We then used statistical methods to identify complexity-contributing factors in relationship to perceived complexity of clinicians. Results A factor analysis (principal component extraction with varimax rotation) of specific items revealed three factors (eigenvalues \u3e 2.0) explaining 47% of total variance, namely task interaction and goals (10 items, 26%, Cronbach’s Alpha = 0.87), urgency and acuity (6 items, 11%, Cronbach’s Alpha = 0.67), and psychosocial behavior (4 items, 10%, Cronbach’s alpha = 0.55). A linear regression analysis showed no statistically significant association between complexity perceived by the physicians and objective complexity, which was measured from coded transcripts by three clinicians (Multiple R-squared = 0.13, p = 0.61). There were no physician effects on the rating of perceived complexity. Conclusion Task complexity contributes significantly to overall complexity in the infectious diseases domain. The different complexity-contributing factors found in this study can guide health information technology system designers and researchers for intuitive design. Thus, decision support tools can help reduce the specific complexity-contributing factors. Future studies aimed at understanding clinical domain-specific complexity-contributing factors can ultimately improve task allocation and design for intuitive clinical reasoning

Dissemination and Sustainability of a Hospital-Wide Hand Hygiene Program Emphasizing Positive Reinforcement

Objective. To increase and sustain hospital-wide compliance with hand hygiene through a long-term ongoing multidimensional improvement program emphasizing behavioral factors. Design. Quasi-experimental short study (August 2000-November 2001) and descriptive time series (April 2003-December 2006). Setting. A 450-bed teaching tertiary-care hospital. Interventions. An initial intervention bundle was introduced in pilot locations that addressed cognitive behavioral factors, which included access to alcohol sanitizer, education, and ongoing audit and feedback. The bundle was subsequently disseminated hospital-wide, along with a novel approach focused on behavior modification through positive reinforcement and annually changing incentives. Results. A total of 36,123 hand hygiene opportunities involving all categories of healthcare workers from 12 inpatient units were observed from October 2000 to October 2006. The rate of compliance with hand hygiene significantly improved after the intervention in 2 cohorts over the first year (from 40% to 64% of opportunities and from 34% to 49% of opportunities; P< .001, compared with the control group). Mean compliance rates ranged from 19% to 41% of 4174 opportunities (at baseline), increased to the highest levels of 73%-84% of 6,420 opportunities 2 years after hospital-wide dissemination, and remained improved at 59%-81% of 4,990 opportunities during year 6 of the program. Conclusion. This interventional cohort study used a behavioral change approach and is one of the earliest and largest institution-wide programs promoting alcohol sanitizer from the United States that has shown significant and sustained improvements in hand hygiene compliance. This creative campaign used ongoing frequent audit and feedback with novel use of immediate positive reinforcement at an acceptable cost to the institutio

Computational reprogramming of homing endonuclease specificity at multiple adjacent base pairs

Site-specific homing endonucleases are capable of inducing gene conversion via homologous recombination. Reprogramming their cleavage specificities allows the targeting of specific biological sites for gene correction or conversion. We used computational protein design to alter the cleavage specificity of I-MsoI for three contiguous base pair substitutions, resulting in an endonuclease whose activity and specificity for its new site rival that of wild-type I-MsoI for the original site. Concerted design for all simultaneous substitutions was more successful than a modular approach against individual substitutions, highlighting the importance of context-dependent redesign and optimization of protein–DNA interactions. We then used computational design based on the crystal structure of the designed complex, which revealed significant unanticipated shifts in DNA conformation, to create an endonuclease that specifically cleaves a site with four contiguous base pair substitutions. Our results demonstrate that specificity switches for multiple concerted base pair substitutions can be computationally designed, and that iteration between design and structure determination provides a route to large scale reprogramming of specificity

Nonresponse to 17-alpha hydroxyprogesterone caproate for recurrent spontaneous preterm birth prevention: clinical prediction and generation of a risk scoring system

Spontaneous preterm birth (SPTB) remains a leading cause of neonatal morbidity and mortality amongst non-anomalous neonates in the United States. SPTB tends to recur at similar gestational ages. Intramuscular 17-alpha hydroxyprogesterone caproate (17OHP-C) reduces the risk of recurrent SPTB. Unfortunately, one-third of high-risk women will have a recurrent SPTB despite 17OHP-C therapy; the reasons for this variability in response are unknown

Activity, specificity and structure of I-Bth0305I: a representative of a new homing endonuclease family

Novel family of putative homing endonuclease genes was recently discovered during analyses of metagenomic and genomic sequence data. One such protein is encoded within a group I intron that resides in the recA gene of the Bacillus thuringiensis 0305ϕ8–36 bacteriophage. Named I-Bth0305I, the endonuclease cleaves a DNA target in the uninterrupted recA gene at a position immediately adjacent to the intron insertion site. The enzyme displays a multidomain, homodimeric architecture and footprints a DNA region of ∼60 bp. Its highest specificity corresponds to a 14-bp pseudopalindromic sequence that is directly centered across the DNA cleavage site. Unlike many homing endonucleases, the specificity profile of the enzyme is evenly distributed across much of its target site, such that few single base pair substitutions cause a significant decrease in cleavage activity. A crystal structure of its C-terminal domain confirms a nuclease fold that is homologous to very short patch repair (Vsr) endonucleases. The domain architecture and DNA recognition profile displayed by I-Bth0305I, which is the prototype of a homing lineage that we term the ‘EDxHD’ family, are distinct from previously characterized homing endonucleases

Trends in Antibiotic Use and Nosocomial Pathogens in Hospitalized Veterans With Pneumonia at 128 Medical Centers, 2006-2010

This national Department of Veterans Affairs study of hospitalizations for pneumonia found a dramatic increase in broad-spectrum antibiotic use from 2006 to 2010, without an increase in nosocomial pathogens or improvement in the match between coverage and pathoge

The American Association for the Surgery of Trauma renal injury grading scale: Implications of the 2018 revisions for injury reclassification and predicting bleeding interventions.

BackgroundIn 2018, the American Association for the Surgery of Trauma (AAST) published revisions to the renal injury grading system to reflect the increased reliance on computed tomography scans and non-operative management of high-grade renal trauma (HGRT). We aimed to evaluate how these revisions will change the grading of HGRT and if it outperforms the original 1989 grading in predicting bleeding control interventions.MethodsData on HGRT were collected from 14 Level-1 trauma centers from 2014 to 2017. Patients with initial computed tomography scans were included. Two radiologists reviewed the scans to regrade the injuries according to the 1989 and 2018 AAST grading systems. Descriptive statistics were used to assess grade reclassifications. Mixed-effect multivariable logistic regression was used to measure the predictive ability of each grading system. The areas under the curves were compared.ResultsOf the 322 injuries included, 27.0% were upgraded, 3.4% were downgraded, and 69.5% remained unchanged. Of the injuries graded as III or lower using the 1989 AAST, 33.5% were upgraded to grade IV using the 2018 AAST. Of the grade V injuries, 58.8% were downgraded using the 2018 AAST. There was no statistically significant difference in the overall areas under the curves between the 2018 and 1989 AAST grading system for predicting bleeding interventions (0.72 vs. 0.68, p = 0.34).ConclusionAbout one third of the injuries previously classified as grade III will be upgraded to grade IV using the 2018 AAST, which adds to the heterogeneity of grade IV injuries. Although the 2018 AAST grading provides more anatomic details on injury patterns and includes important radiologic findings, it did not outperform the 1989 AAST grading in predicting bleeding interventions.Level of evidencePrognostic and Epidemiological Study, level III