Simultaneous randomisation of eight key active site residues in E. coli NfsA to generate superior nitroreductases for prodrug activation

There is a substantial gap between the levels of enzyme activity Nature can evolve and those that scientists can engineer in the lab. This suggests that conventional directed evolution techniques involving incremental improvements in enzyme activity may frequently fail to ascend even local fitness maxima. This is most likely due to an inability of step-wise evolutionary approaches to effectively retain mutations that are beneficial in combination with one another, but on an individual basis are neutral or even slightly deleterious (i.e., exhibit positive epistasis). To overcome this limitation, we are seeking to “jump” straight to an enzyme with peak activity by conducting simultaneous mass randomisation of eight key active site residues in Escherichia coli NfsA, a nitroreductase enzyme that has several diverse applications in biotechnology. Using degenerate codons, we generated a diverse library containing 425 million unique variants. We then applied a powerful selection system using either or both of two recently identified positive selection compounds, which has enabled us to recover a diverse range of highly active nitroreductase variants. These have been screened against a panel of prodrug substrates to identify variants that are improved with specific prodrug substrates of interest. A primary focus has been developing nitroreductases as tools for targeted cell ablation in zebrafish. The basic system involves co-expression of a nitroreductase and fluorescent reporter under the control of a cell type specific promoter in a transgenic fish. Expression of the nitroreductase selectively sensitises target cells to a prodrug which, following nitroreduction, yields a cytotoxic compound that causes precise targeted cell ablation. We have identified several nil-bystander prodrugs that are able to selectively ablate nitroreductase expressing cells with no harm to nearby cells, and have paired these with highly specialised NfsA variants to improve the efficacy and accuracy of cell ablation. We have also screened our mass-randomisation libraries to recover nitroreductases that have non-overlapping prodrug specificities, to be used in a multiplex cell ablation system. This expands upon the previous system, by using pairs of selective nitroreductases and two different prodrugs to facilitate independent ablation of multiple cell types. For example, we have identified a specialist NfsA variant that has activity for tinidazole and not for metronidazole, achieved by including metronidazole as a simultaneous counter-selection during the initial positive selection process. This elegant positive/negative selection eliminated activity with metronidazole, while still ensuring that some level of nitroreductase activity was retained overall

Discordant antibiotic therapy and length of stay in children hospitalized for urinary tract infection

BACKGROUND: Urinary tract infections (UTIs) are a common reason for pediatric hospitalizations. OBJECTIVE: To determine the effect of discordant antibiotic therapy (in vitro nonsusceptibility of the uropathogen to initial antibiotic) on clinical outcomes for children hospitalized for UTI. DESIGN/SETTING: Multicenter retrospective cohort study in children aged 3 days to 18 years, hospitalized at 5 children's hospitals with a laboratory‐confirmed UTI. Data were obtained from medical records and the Pediatric Hospital Information System (PHIS) database. PARTICIPANTS: Patients with laboratory‐confirmed UTI. MAIN EXPOSURE: Discordant antibiotic therapy. MEASUREMENTS: Length of stay and fever duration. Covariates included age, sex, insurance, race, vesicoureteral reflux, antibiotic prophylaxis, genitourinary abnormality, and chronic care conditions. RESULTS: The median age of the 216 patients was 2.46 years (interquartile range [IQR]: 0.27, 8.89) and 25% were male. The most common causative organisms were E. coli and Klebsiella species. Discordant therapy occurred in 10% of cases and most commonly in cultures positive for Klebsiella species, Enterobacter species, and mixed organisms. In adjusted analyses, discordant therapy was associated with a 1.8 day (95% confidence interval [CI]: 1.5, 2.1) longer length of stay [LOS], but not with fever duration. CONCLUSIONS: Discordant antibiotic therapy for UTI is common and associated with longer hospitalizations. Further research is needed to understand the clinical factors contributing to the increased LOS and to inform decisions for empiric antibiotic selection in children with UTIs. Journal of Hospital Medicine 2012; © 2012 Society of Hospital MedicinePeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/94298/1/1960_ftp.pd

A Genetic Signature of Spina Bifida Risk from Pathway-Informed Comprehensive Gene-Variant Analysis

Despite compelling epidemiological evidence that folic acid supplements reduce the frequency of neural tube defects (NTDs) in newborns, common variant association studies with folate metabolism genes have failed to explain the majority of NTD risk. The contribution of rare alleles as well as genetic interactions within the folate pathway have not been extensively studied in the context of NTDs. Thus, we sequenced the exons in 31 folate-related genes in a 480-member NTD case-control population to identify the full spectrum of allelic variation and determine whether rare alleles or obvious genetic interactions within this pathway affect NTD risk. We constructed a pathway model, predetermined independent of the data, which grouped genes into coherent sets reflecting the distinct metabolic compartments in the folate/one-carbon pathway (purine synthesis, pyrimidine synthesis, and homocysteine recycling to methionine). By integrating multiple variants based on these groupings, we uncovered two provocative, complex genetic risk signatures. Interestingly, these signatures differed by race/ethnicity: a Hispanic risk profile pointed to alterations in purine biosynthesis, whereas that in non-Hispanic whites implicated homocysteine metabolism. In contrast, parallel analyses that focused on individual alleles, or individual genes, as the units by which to assign risk revealed no compelling associations. These results suggest that the ability to layer pathway relationships onto clinical variant data can be uniquely informative for identifying genetic risk as well as for generating mechanistic hypotheses. Furthermore, the identification of ethnic-specific risk signatures for spina bifida resonated with epidemiological data suggesting that the underlying pathogenesis may differ between Hispanic and non-Hispanic groups

In their own words: Safety and quality perspectives from families of hospitalized children with medical complexity

BACKGROUND: Children with medical complexity (CMC) experience adverse events due to multiorgan impairment, frequent hospitalizations, subspecialty care, and dependence on multiple medications/equipment. Their families are well-versed in care and can help identify safety/quality gaps to inform improvements. Although previous studies have shown families identify important safety/quality gaps in hospitals, studies of inpatient safety/quality experience of CMC and their families are limited. To address this gap and identify otherwise unrecognized, family-prioritized areas for improving safety/quality of CMC, we conducted a secondary qualitative analysis of safety reporting surveys among families of CMC. OBJECTIVE: Explore safety reports from families of hospitalized CMC to identify areas to improve safety/quality. DESIGNS, SETTINGS AND PARTICIPANTS: We analyzed free-text responses from predischarge safety reporting surveys administered to families of CMC at a quaternary children\u27s hospital from April 2018 to November 2020. Using a qualitative descriptive approach, we categorized responses into standard clinical categories. Three team members inductively generated an initial codebook to apply iteratively to responses. Reviewers coded responses collaboratively, resolved discrepancies through consensus, and generated themes. MAIN OUTCOME AND MEASURES: Outcomes: family-reported areas of safety/quality improvement. MEASURES: pre-discharge family surveys. RESULTS: Two hundred and eight/two hundred and thirty-seven (88%) families completed surveys; 83 families offered 138 free-text safety responses about medications, feeds, cares, and other categories. Themes included unmet expectations of hospital care/environment, lack of consistency, provider-patient communication lapses, families\u27 expertise about care, and the value of transparency. CONCLUSION: To improve care of CMC and their families, hospitals can manage expectations about hospital limitations, improve consistency of care/communication, acknowledge family expertise, and recognize that family-observed quality concerns can have safety implications. Soliciting family input can help hospitals improve care in meaningful, otherwise unrecognized ways

Intracellular complexities of acquiring a new enzymatic function revealed by mass-randomisation of active-site residues

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