Development of an in vivo Himar1 transposon mutagenesis system for use in Streptococcus equi subsp. equi

Streptococcus equi subsp. equi is the causative agent of the equine disease strangles. In this study we describe the development of an in vivo Himar1 transposon system for the random mutagenesis of S. equi and, potentially, other Gram-positive bacteria. We demonstrate efficient and random transposition of a modified mini-transposon onto the chromosome by Southern blot analysis and insertion site sequencing. Non-haemolytic mutants were isolated at a frequency of 0.2%, and acapsular mutants at a frequency of 0.04%. Taken together, these data demonstrate that in vivo Himar1 mutagenesis can be used for genomic-scale mutational analysis of S. equi, and is likely to be applicable to the study of other streptococci.     

Laboratory Test Surveillance following Acute Kidney Injury

Background

Patients with hospitalized acute kidney injury (AKI) are at increased risk for accelerated loss of kidney function, morbidity, and mortality. We sought to inform efforts at improving post-AKI outcomes by describing the receipt of renal-specific laboratory test surveillance among a large high-risk cohort.

Methods

We acquired clinical data from the Electronic health record (EHR) of 5 Veterans Affairs (VA) hospitals to identify patients hospitalized with AKI from January 1^st, 2002 to December 31^st, 2009, and followed these patients for 1 year or until death, enrollment in palliative care, or improvement in renal function to estimated GFR (eGFR) ≥60 L/min/1.73 m². Using demographic data, administrative codes, and laboratory test data, we evaluated the receipt and timing of outpatient testing for serum concentrations of creatinine and any as well as quantitative proteinuria recommended for CKD risk stratification. Additionally, we reported the rate of phosphorus and parathyroid hormone (PTH) monitoring recommended for chronic kidney disease (CKD) patients.

Results

A total of 10,955 patients admitted with AKI were discharged with an eGFR<60 mL/min/1.73 m². During outpatient follow-up at 90 and 365 days, respectively, creatinine was measured on 69% and 85% of patients, quantitative proteinuria was measured on 6% and 12% of patients, PTH or phosphorus was measured on 10% and 15% of patients.

Conclusions

Measurement of creatinine was common among all patients following AKI. However, patients with AKI were infrequently monitored with assessments of quantitative proteinuria or mineral metabolism disorder, even for patients with baseline kidney disease.     

The Inner Membrane Complex Sub-compartment Proteins Critical for Replication of the Apicomplexan Parasite Toxoplasma gondii Adopt a Pleckstrin Homology Fold

Toxoplasma gondii, an apicomplexan parasite prevalent in developed nations, infects up to one-third of the human population. The success of this parasite depends on several unique structures including an inner membrane complex (IMC) that lines the interior of the plasma membrane and contains proteins important for gliding motility and replication. Of these proteins, the IMC sub-compartment proteins (ISPs) have recently been shown to play a role in asexual T. gondii daughter cell formation, yet the mechanism is unknown. Complicating mechanistic characterization of the ISPs is a lack of sequence identity with proteins of known structure or function. In support of elucidating the function of ISPs, we first determined the crystal structures of representative members TgISP1 and TgISP3 to a resolution of 2.10 and 2.32 Å, respectively. Structural analysis revealed that both ISPs adopt a pleckstrin homology fold often associated with phospholipid binding or protein-protein interactions. Substitution of basic for hydrophobic residues in the region that overlays with phospholipid binding in related pleckstrin homology domains, however, suggests that ISPs do not retain phospholipid binding activity. Consistent with this observation, biochemical assays revealed no phospholipid binding activity. Interestingly, mapping of conserved surface residues combined with crystal packing analysis indicates that TgISPs have functionally repurposed the phospholipid-binding site likely to coordinate protein partners. Recruitment of larger protein complexes may also be aided through avidity-enhanced interactions resulting from multimerization of the ISPs. Overall, we propose a model where TgISPs recruit protein partners to the IMC to ensure correct progression of daughter cell formation.     

Sensitivity of marine fish thermal habitat models to fishery data sources

Statistical models built using different data sources and methods can exhibit conflicting patterns. We used the northern stock of black sea bass (Centropristis striata) as a case study to assess the impacts of using different fisheries data sources and laboratory‐derived physiological metrics in the development of thermal habitat models for marine fishes. We constructed thermal habitat models using generalized additive models (GAMs) based on various fisheries datasets as input, including the NOAA Northeast Fisheries Science Center (NEFSC) bottom trawl surveys, various inshore fisheries‐independent trawl surveys (state waters), NEFSC fisheries‐dependent observer data, and laboratory‐based physiological metrics. We compared each model''s GAM response curve and coupled them to historical ocean conditions in the U.S. Northeast Shelf using bias‐corrected ocean temperature output from a regional ocean model. Thermal habitat models based on shelf‐wide data (NEFSC fisheries‐dependent observer data and fisheries‐independent spring and fall surveys) explained the most variation in black sea bass presence/absence data at ~15% deviance explained. Models based on a narrower range of sampled thermal habitat from inshore survey data in the Northeast Area Monitoring and Assessment Program (NEAMAP) and the geographically isolated Long Island Sound data performed poorly. All models had similar lower thermal limits around 8.5℃, but thermal optima, when present, ranged from 16.7 to 24.8℃. The GAMs could reliably predict habitat from years excluded from model training, but due to strong seasonal temperature fluctuations in the region, could not be used to predict habitat in seasons excluded from training. We conclude that survey data source can greatly impact development and interpretation of thermal habitat models for marine fishes. We suggest that model development be based on data sources that sample the widest range of ocean temperature and physical habitat throughout multiple seasons when possible, and encourage thorough consideration of how data gaps may influence model uncertainty.     

Restoring Savanna Using Fire: Impact on the Breeding Bird Community

Restoration of many terrestrial plant communities involves the reintroduction of fire. However, there have been few studies of the effects of fire on the avifauna during the restoration process. To study the effects of oak savanna restoration on avian communities, breeding birds were censused and the vegetation structure documented in seven experimental burn units (8–18 ha) that had experienced different frequencies of controlled burns during the past 31 years (0–26 burns). Data were analyzed with both direct and indirect gradient analyses using multivariate techniques. The results showed that, as savanna restoration proceeded, there was a general decline in predominantly insectivorous species, particularly those that feed in the upper canopy region (leaves and air space), and a general increase in omnivorous species, particularly those that feed on the ground and in the lower canopy. Insectivorous bark gleaners (woodpeckers) also increased during restoration and were correlated with the increase in standing dead trees resulting from the fires. Overall, savanna restoration resulted in increases in the abundance of many open country bird species, including many species that have been declining in central and eastern North America, including red‐headed woodpecker, Baltimore oriole, eastern kingbird, vesper sparrow, field sparrow, lark sparrow, brown thrasher, American goldfinch, and brown‐headed cowbird. The shifts in species and guilds were correlated with changes in burn frequency and the macro vegetation structure—tree and shrub density, leaf area index, and relative proportion of standing dead trees. The findings show that savanna restoration can increase bird diversity and provide important habitat for uncommon or declining bird species. These birds are most likely attracted to one or more of the distinctive habitat features of the restored savanna environments, including scattered mature trees, standing dead trees and snags, and presence of both shrubby and grassland vegetation. The findings also suggest that restoration ecologists and wildlife biologists will need to work together to achieve desired goals, since different types of savanna restoration efforts may produce different effects on the breeding bird community.     

Coliform Bacteria and Nitrogen Fixation in Pulp and Paper Mill Effluent Treatment Systems

The majority of pulp and paper mills now biotreat their combined effluents using activated sludge. On the assumption that their wood-based effluents have negligible fixed N, and that activated-sludge microorganisms will not fix significant N, these mills routinely spend large amounts adding ammonia or urea to their aeration tanks (bioreactors) to permit normal biomass growth. N₂ fixation in seven Eastern Canadian pulp and paper mill effluent treatment systems was analyzed using acetylene reduction assays, quantitative nitrogenase (nifH) gene probing, and bacterial isolations. In situ N₂ fixation was undetectable in all seven bioreactors but was present in six associated primary clarifiers. One primary clarifier was studied in greater detail. Approximately 50% of all culturable cells in the clarifier contained nifH, of which >90% were Klebsiella strains. All primary-clarifier coliform bacteria growing on MacConkey agar were identified as klebsiellas, and all those probed contained nifH. In contrast, analysis of 48 random coliform isolates from other mill water system locations showed that only 24 (50%) possessed the nifH gene, and only 13 (27%) showed inducible N₂-fixing activity. Thus, all the pulp and paper mill primary clarifiers tested appeared to be sites of active N₂ fixation (0.87 to 4.90 mg of N liter⁻¹ day⁻¹) and a microbial community strongly biased toward this activity. This may also explain why coliform bacteria, especially klebsiellas, are indigenous in pulp and paper mill water systems.     

Single-molecule dilution and multiple displacement amplification for molecular haplotyping

Separate haploid analysis is frequently required for heterozygous genotyping to resolve phase ambiguity or confirm allelic sequence. We demonstrate a technique of single-molecule dilution followed by multiple strand displacement amplification to haplotype polymorphic alleles. Dilution of DNA to haploid equivalency, or a single molecule, is a simple method for separating di-allelic DNA. Strand displacement amplification is a robust method for non-specific DNA expansion that employs random hexamers and phage polymerase Phi29 for double-stranded DNA displacement and primer extension, resulting in high processivity and exceptional product length. Single-molecule dilution was followed by strand displacement amplification to expand separated alleles to microgram quantities of DNA for more efficient haplotype analysis of heterozygous genes.