UK-wide major trauma center tertiary trauma survey pro forma review and aggregation and consolidation into a redesigned document.

Objectives: The trauma tertiary survey (TTS) is an essential part of the continued care for major trauma patients which is performed to ensure that all injuries have been identified and none have been overlooked during the patient's stay. Although the Advanced Trauma Life Support Course states a need for a tertiary survey, there is currently no standard for what this survey comprises. Methods: Using local consultant expert opinion and a literature search we identified a set of 32 TTS potential features that may be included within a TTS pro forma. Major trauma center (MTC) documents were requested from every MTC within the UK. 4 investigators sequentially interrogated each MTC TTS document looking for (1) presence of each feature and (2) how well the feature was represented on the document (0 to 4 Likert Scale). Any previously unidentified potential TTS features were noted and later reviewed for a second round of document analysis. Results: A total of 21 out of all 26 UK MTCs had a TTS pro forma document. A total of 68 possible features were identified. Respiratory and Abdominal assessment sections were the most frequently identified features (present in 90.4% of the TTS pro formas; n=19. Neck assessment and neurological assessment were included within 85.7% of the TTS pro formas (n=18). Further aspects identified for Round 2 analysis typically included features that were thought to be important but highly specific. For example, pregnancy test and DNACPR discussions were found in 1 MTC TTS each (4%). Conclusion: This article presents a review of the existing documents at 21 MTCs in the UK, identification of features used and proposes a gold standard TTS which can be used by any doctor to perform the tertiary survey and reduce the risk of missed injuries in trauma patients. Level of Evidence: 3.

Bayesian hierarchical spatial models: Implementing the Besag York Mollié model in stan.

This report presents a new implementation of the Besag-York-Mollié (BYM) model in Stan, a probabilistic programming platform which does full Bayesian inference using Hamiltonian Monte Carlo (HMC). We review the spatial auto-correlation models used for areal data and disease risk mapping, and describe the corresponding Stan implementations. We also present a case study using Stan to fit a BYM model for motor vehicle crashes injuring school-age pedestrians in New York City from 2005 to 2014 localized to census tracts. Stan efficiently fit our multivariable BYM model having a large number of observations (n=2095 census tracts) with small outcome counts < 10 in the majority of tracts. Our findings reinforced that neighborhood income and social fragmentation are significant correlates of school-age pedestrian injuries. We also observed that nationally-available census tract estimates of commuting methods may serve as a useful indicator of underlying pedestrian densities.

Alternative Water Transport and Storage Containers: Assessing Sustained Use of the PackH2O in Rural Haiti.

The PackH2O water backpack carrier was developed to provide safe storage and relieve stress of head-loading during water transport with traditional containers such as buckets and jerry cans. We conducted an evaluation to assess both self-reported and observed use over a 6-month period between November 2014 and May 2015. A total of 866 packs were distributed to 618 households in six communities in rural Haiti, and 431 and 441 households were surveyed at midline and end line, respectively. We performed linear regression to assess change of self-reported use over time. Although 79.3% of respondents reported continued use of the 20-L pack after 6 months, other measures of self-reported use were low, with only 16.8% reporting to have used the pack the last time they collected water and 10.3% preferring the pack over other water collection containers. In addition, only 10.2% of all people collecting water at community sources were observed using packs and 12.0% of all households surveyed had water in the pack at the time of visit. Pack use varied by community and demographics. Although women were targeted during distribution, men preferred the pack and were more commonly observed using it at the community water sources. In conclusion, the use of the PackH2O was not widely adopted in rural Haiti; however, further research is needed to assess the pack acceptance in areas where back-loading is more common and in emergency settings.

Proluciferin acetals as bioluminogenic substrates for cytochrome P450 activity and probes for CYP3A inhibition.

Cytochrome P450 (P450) assays use probe substrates to interrogate the influence of new chemical entities toward P450 enzymes. We report the synthesis and study of a family of bioluminogenic luciferin acetal substrates that are oxidized by P450 enzymes to form luciferase substrates. The luciferin acetals were screened against a panel of purified P450 enzymes. In particular, one proluciferin acetal has demonstrated sensitive and selective CYP3A4-catalyzed oxidation to a luciferin ester-K(m) and k(cat) are 2.88 µM and 5.87 pmol metabolite · min(-1) · pmol enzyme(-1), respectively. The proluciferin acetal was used as a probe substrate to measure IC(50) values of known inhibitors against recombinant CYP3A4 or human liver microsomes. IC(50) values for the known inhibitors correlate strongly with IC(50) values calculated from the traditional high-performance liquid chromatography-based probe substrate testosterone. Luciferin acetals are rapidly oxidized to unstable hemi-orthoesters by CYP3A resulting in luciferin esters and, therefore, are conducive to simple rapid CYP3A bioluminescent assays.

Affinity chromatography: general methods.

Affinity chromatography is one of the most diverse and powerful chromatographic methods for purification of a specific molecule or a group of molecules from complex mixtures. It is based on highly specific biological interactions between two molecules, such as interactions between enzyme and substrate, receptor and ligand, or antibody and antigen. These interactions, which are typically reversible, are used for purification by placing one of the interacting molecules, referred to as affinity ligand, onto a solid matrix to create a stationary phase while the target molecule is in the mobile phase. Successful affinity purification requires a certain degree of knowledge and understanding of the nature of interactions between the target molecule and the ligand to help determine the selection of an appropriate affinity ligand and purification procedure. With the growing popularity of affinity purification, many of the commonly used ligands coupled to affinity matrices are now commercially available and are ready to use. However, in some cases new affinity chromatographic material may need to be developed by coupling the ligand onto the matrix such that the ligand retains specific binding affinity for the molecule of interest. In this chapter, we discuss factors which are important to consider when selecting the ligand, proper attachment chemistry, and the matrix. In recent years, matrices with unique features which overcome some of the limitations of more traditional materials have been developed and these are also described. Affinity purification can provide significant time savings and several hundred-fold or higher purification, but the success depends on the method used. Thus, it is important to optimize the purification protocol to achieve efficient capture and maximum recovery of the target.

HaloTag7: a genetically engineered tag that enhances bacterial expression of soluble proteins and improves protein purification.

Over-expression and purification of soluble and functional proteins remain critical challenges for many aspects of biomolecular research. To address this, we have developed a novel protein tag, HaloTag7, engineered to enhance expression and solubility of recombinant proteins and to provide efficient protein purification coupled with tag removal. HaloTag7 was designed to bind rapidly and covalently with a unique synthetic linker to achieve an essentially irreversible attachment. The synthetic linker may be attached to a variety of entities such as fluorescent dyes and solid supports, permitting labeling of fusion proteins in cell lysates for expression screening, and efficient capture of fusion proteins onto a purification resin. The combination of covalent capture with rapid binding kinetics overcomes the equilibrium-based limitations associated with traditional affinity tags and enables efficient capture even at low expression levels. Following immobilization on the resin, the protein of interest is released by cleavage at an optimized TEV protease recognition site, leaving HaloTag7 bound to the resin and pure protein in solution. Evaluation of HaloTag7 for expression of 23 human proteins in Escherichia coli relative to MBP, GST and His(6)Tag revealed that 74% of the proteins were produced in soluble form when fused to HaloTag7 compared to 52%, 39% and 22%, respectively, for the other tags. Using a subset of the test panel, more proteins fused to HaloTag7 were successfully purified than with the other tags, and these proteins were of higher yield and purity.

HaloTag: a novel protein labeling technology for cell imaging and protein analysis.

We have designed a modular protein tagging system that allows different functionalities to be linked onto a single genetic fusion, either in solution, in living cells, or in chemically fixed cells. The protein tag (HaloTag) is a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands (HaloTag ligands). The synthetic ligands comprise a chloroalkane linker attached to a variety of useful molecules, such as fluorescent dyes, affinity handles, or solid surfaces. Covalent bond formation between the protein tag and the chloroalkane linker is highly specific, occurs rapidly under physiological conditions, and is essentially irreversible. We demonstrate the utility of this system for cellular imaging and protein immobilization by analyzing multiple molecular processes associated with NF-kappaB-mediated cellular physiology, including imaging of subcellular protein translocation and capture of protein--protein and protein--DNA complexes.

Detection of canine microalbuminuria using semiquantitative test strips designed for use with human urine.

BACKGROUND: Commercial testing for microalbuminuria in human urine is often performed with point-of-care semiquantitative test strips followed by quantitative testing when indicated. An ELISA that quantifies canine urine albumin concentration has been developed, but semiquantitative test strips for use in the dog are not available. OBJECTIVE: The purpose of this study was to prospectively determine the concordance of canine urine albumin concentrations measured by a commercial human test strip and by ELISA. METHODS: Urine samples were obtained from 67 dogs evaluated for a variety of clinical conditions. Dipstick urinalyses were performed on all samples; clinician discretion determined method of urine collection and performance of urine sediment examination and/or urine culture. Urine albumin concentration was determined using test strips (Clinitek Microalbumin, Bayer Corporation, Elkhart, Ind, USA), and results were compared with those obtained by ELISA. RESULTS: The Clinitek strips correctly determined albumin concentration in 42 of 67 (63%) urine samples tested. Concordance was lowest (48%) for dogs with microalbuminuria (10-300 microg/mL by ELISA). Clinitek strip sensitivity and specificity for correct identification of microalbuminuria were 48% and 75%, respectively. Concordance was lower in dogs with urinary tract infection or hematuria and in samples collected by catheterization. Sensitivity and specificity for correct identification of microalbuminuria after exclusion of dogs with urinary tract infection or hematuria were 59% and 83%, respectively. CONCLUSIONS: These results suggest that the Clinitek strips lack sufficient concordance with results obtained by ELISA to be a reliable screening for test microalbuminuria in the dog. A reliable semiquantitative point-of-care test for canine urine albumin concentrations below those detected by standard urine dipsticks is still needed.

Use of serologic tests to predict resistance to feline herpesvirus 1, feline calicivirus, and feline parvovirus infection in cats.

OBJECTIVE: To determine whether detection of virus-specific serum antibodies correlates with resistance to challenge with virulent feline herpesvirus 1 (FHV-1), feline calicivirus (FCV), and feline parvovirus (FPV) in cats and to determine percentages of client-owned cats with serum antibodies to FHV-1, FCV, and FPV. DESIGN: Prospective experimental study. ANIMALS: 72 laboratory-reared cats and 276 client-owned cats. PROCEDURES: Laboratory-reared cats were vaccinated against FHV-1, FCV, and FPV, using 1 of 3 commercial vaccines, or maintained as unvaccinated controls. Between 9 and 36 months after vaccination, cats were challenged with virulent virus. Recombinant-antigen ELISA for detection of FHV-1-, FCV-, and FPV-specific antibodies were developed, and results were compared with results of hemagglutination inhibition (FPV) and virus neutralization (FHV-1 and FCV) assays and with resistance to viral challenge. RESULTS: For vaccinated laboratory-reared cats, predictive values of positive results were 100% for the FPV and FCV ELISA and 90% for the FHV-1 ELISA. Results of the FHV-1, FCV, and FPV ELISA were positive for 195 (70.7%), 255 (92.4%), and 189 (68.5%), respectively, of the 276 client-owned cats. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that for cats that have been vaccinated, detection of FHV-1-, FCV-, and FPV-specific antibodies is predictive of whether cats are susceptible to disease, regardless of vaccine type or vaccination interval. Because most client-owned cats had detectable serum antibodies suggestive of resistance to infection, use of arbitrary booster vaccination intervals is likely to lead to unnecessary vaccination of some cats.