Impact of trauma centre capacity and volume on the mortality risk of incoming new admissions.

INTRODUCTION: Trauma centre capacity and surge volume may affect decisions on where to transport a critically injured patient and whether to bypass the closest facility. Our hypothesis was that overcrowding and high patient acuity would contribute to increase the mortality risk for incoming admissions. METHODS: For a 6-year period, we merged and cross-correlated our institutional trauma registry with a database on Trauma Resuscitation Unit (TRU) patient admissions, movement and discharges, with average capacity of 12 trauma bays. The outcomes of overall hospital and 24 hours mortality for new trauma admissions (NEW) were assessed by multivariate logistic regression. RESULTS: There were 42 003 (mean=7000/year) admissions having complete data sets, with 36 354 (87%) patients who were primary trauma admissions, age ≥18 and survival ≥15 min. In the logistic regression model for the entire cohort, NEW admission hospital mortality was only associated with NEW admission age and prehospital Glasgow Coma Scale (GCS) and Shock Index (SI) (all p<0.05). When TRU occupancy reached ≥16 patients, the factors associated with increased NEW admission hospital mortality were existing patients (TRU >1 hour) with SI ≥0.9, recent admissions (TRU ≤1 hour) with age ≥65, NEW admission age and prehospital GCS and SI (all p<0.05). CONCLUSION: The mortality of incoming patients is not impacted by routine trauma centre overcapacity. In conditions of severe overcrowding, the number of admitted patients with shock physiology and a recent surge of elderly/debilitated patients may influence the mortality risk of a new trauma admission.

How are we currently training and maintaining clinical readiness of US and UK military surgeons responsible for managing head, face and neck wounds on deployment?

INTRODUCTION: The conflicts in Iraq and Afghanistan provided military surgeons from the USA and the UK with extensive experience into the management of injuries to the head, face and neck (HFN) from high energy bullets and explosive weaponry. The challenge is now to maintain the expertise in managing such injuries for future military deployments. METHODS: The manner in which each country approaches four parameters required for a surgeon to competently treat HFN wounds in deployed military environments was compared. These comprised initial surgical training (residency/registrar training), surgical fellowships, hospital type and appointment as an attending (USA) or consultant (UK) and predeployment training. RESULTS: Neither country has residents/registrars undertaking surgical training that is military specific. The Major Trauma and Reconstructive Fellowship based in Birmingham UK and the Craniomaxillofacial Trauma fellowship at Duke University USA provide additional training directly applicable to managing HFN trauma on deployment. Placement in level 1 trauma/major trauma centres is encouraged by both countries but is not mandatory. US surgeons attend one of three single-service predeployment courses, of which HFN skills are taught on both cadavers and in a 1-week clinical placement in a level 1 trauma centre. UK surgeons attend the Military Operational Surgical Training programme, a 1-week course that includes 1 day dedicated to teaching HFN injury management on cadavers. CONCLUSIONS: Multiple specialties of surgeon seen in the civilian environment are unlikely to be present, necessitating development of extended competencies. Military-tailored fellowships are capable of generating most of these skills early in a career. Regular training courses including simulation are required to maintain such skills and should not be given only immediately prior to deployment. Strong evidence exists that military consultants and attendings should only work at level 1/major trauma centres.

Potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody.

The botulinum neurotoxins (BoNTs) cause the paralytic human disease botulism and are one of the highest-risk threat agents for bioterrorism. To generate a pharmaceutical to prevent or treat botulism, monoclonal antibodies (mAbs) were generated by phage display and evaluated for neutralization of BoNT serotype A (BoNT/A) in vivo. Although no single mAb significantly neutralized toxin, a combination of three mAbs (oligoclonal Ab) neutralized 450,000 50% lethal doses of BoNT/A, a potency 90 times greater than human hyperimmune globulin. The potency of oligoclonal Ab was primarily due to a large increase in functional Ab binding affinity. The results indicate that the potency of the polyclonal humoral immune response can be deconvoluted to a few mAbs binding nonoverlapping epitopes, providing a route to drugs for preventing and treating botulism and diseases caused by other pathogens and biologic threat agents.

Expression of single-chain Fv-Fc fusions in Pichia pastoris.

Phage display technology makes possible the direct isolation of monovalent single-chain Fv antibody fragments. For many applications, however, it is useful to restore Fc mediated antibody functions such as avidity, effector functions and a prolonged serum half-life. We have constructed vectors for the convenient, rapid expression of a single-chain antibody Fv domain (scFv) fused to the Fc portion of human IgG1 in the methylotrophic yeast Pichia pastoris. The scFv-Fc fusion protein is secreted and recovered from the culture medium as a disulfide-linked, glycosylated homodimer. The increased size of the dimer (approximately 106 kDa vs. approximately 25 kDa for a scFv) results in a prolonged serum half-life in vivo, with t(1/2) of the beta phase of clearance increasing from 3.5 h for a typical scFv to 93 h for a scFv-Fc fusion in mice. The scFv-Fc fusion is capable of mediating antibody-dependent cellular cytotoxicity against tumor target cells using human peripheral blood mononuclear cells as effectors. Finally, the Fc domain is a convenient, robust affinity handle for purification and immunochemical applications, eliminating the need for proteolytically sensitive epitope and/or affinity tags on the scFv.

Molecular modeling of substrate binding in wild-type and mutant Corynebacteria 2,5-diketo-D-gluconate reductases.

2,5-diketo-D-gluconic acid reductase (2,5-DKGR; E.C. 1.1.1.-) catalyzes the Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent stereo-specific reduction of 2, 5-diketo-D-gluconate (2,5-DKG) to 2-keto-L-gulonate (2-KLG), a precursor in the industrial production of vitamin C (L-ascorbate). Microorganisms that naturally ferment D-glucose to 2,5-DKG can be genetically modified to express the gene for 2,5-DKGR, and thus directly produce vitamin C from D-glucose. Two naturally occurring variants of DKGR (DKGR A and DKGR B) have been reported. DKGR B exhibits higher specific activity toward 2,5-DKG than DKGR A; however, DKGR A exhibits a greater selectivity for this substrate and significantly higher thermal stability. Thus, a modified form of DKGR, combining desirable properties from both enzymes, would be of substantial commercial interest. In the present study we use a molecular dynamics-based approach to understand the conformational changes in DKGR A as the active site is mutated to include two active site residue changes that occur in the B form. The results indicate that the enhanced kinetic properties of the B form are due, in part, to residue substitutions in the binding pocket. These substitutions augment interactions with the substrate or alter the alignment with respect to the putative proton donor group. Proteins 2000;39:68-75.

Crystal structure of 2,5-diketo-D-gluconic acid reductase A complexed with NADPH at 2.1-A resolution.

The three-dimensional structure of Corynebacterium 2, 5-diketo-D-gluconic acid reductase A (2,5-DKGR A; EC 1.1.1.-), in complex with cofactor NADPH, has been solved by using x-ray crystallographic data to 2.1-A resolution. This enzyme catalyzes stereospecific reduction of 2,5-diketo-D-gluconate (2,5-DKG) to 2-keto-L-gulonate. Thus the three-dimensional structure has now been solved for a prokaryotic example of the aldo-keto reductase superfamily. The details of the binding of the NADPH cofactor help to explain why 2,5-DKGR exhibits lower binding affinity for cofactor than the related human aldose reductase does. Furthermore, changes in the local loop structure near the cofactor suggest that 2,5-DKGR will not exhibit the biphasic cofactor binding characteristics observed in aldose reductase. Although the crystal structure does not include substrate, the two ordered water molecules present within the substrate-binding pocket are postulated to provide positional landmarks for the substrate 5-keto and 4-hydroxyl groups. The structural basis for several previously described active-site mutants of 2,5-DKGR A is also proposed. Recent research efforts have described a novel approach to the synthesis of L-ascorbate (vitamin C) by using a genetically engineered microorganism that is capable of synthesizing 2,5-DKG from glucose and subsequently is transformed with the gene for 2,5-DKGR. These modifications create a microorganism capable of direct production of 2-keto-L-gulonate from D-glucose, and the gulonate can subsequently be converted into vitamin C. In economic terms, vitamin C is the single most important specialty chemical manufactured in the world. Understanding the structural determinants of specificity, catalysis, and stability for 2,5-DKGR A is of substantial commercial interest.

Stereolithography: a historical review and indications for use in the management of trauma.

BACKGROUND: Stereolithography (SL) is a new adjunct for treatment planning in complex maxillofacial trauma. It uses an argon/ion laser to polymerize acrylic resin models of reformatted computerized tomographic radiographs. SL provides superior anatomical detail and translucency, and it can be sterilized and transported to the operative field, if necessary. DISCUSSION: A review of the historical development of SL technology is presented, along with three clinical cases, showing the advantages of SL models in the treatment of patients with complex maxillofacial trauma. CONCLUSION: SL technology provides superior understanding of anatomic relationships, the ability to perform presurgical simulation of the proposed procedure, preoperative adaptation of biomaterials, decreased time in the operating room and associated patient morbidity, and improved residency training and patient education. The primary disadvantages of SL are availability and cost.

The effect of the one-chain to two-chain conversion in tissue plasminogen activator: characterization of mutations at position 275.

Tissue plasminogen activator (t-PA) is homologous to other serine proteases and contains an apparent activation cleavage site at arginine 275. It has been demonstrated that this arginine-275 can be replaced with either glutamic acid (Tate, K. M., Higgins, D. L., Holmes, W. E., Winkler, M. E., Heyneker, H. L., and Vehar, G. A. Biochemistry 26, 338-343, 1987) or glycine (Peterson, L. C., Johannessen, M., Foster, D., Kumar, A., and Mulvihill, E. Biochim. Biophys. Acta 952, 245-254, 1988; Boose, J. A., Kuismanen, E., Gerard, R., Sambrook, J. and Gething, M.-J. Biochemistry 28, 635-643, 1989) so that the product of the plasminogen activation reaction, plasmin, can no longer hydrolyze the one-chain form of t-PA to the two-chain form. These "one-chain" t-PA variants had diminished activity, compared to wild-type t-PA, in the absence of a cofactor, but in the presence of the fibrin(ogen) cofactor the two variants had activity similar to wild-type t-PA. In order to compare the effects of all possible substitutions, t-PA variants with each of the other nineteen amino acids besides arginine at position 275 were produced by site-directed mutagenesis. All were recovered from cell culture supernatants completely in the one-chain form, except for R275 (wild-type) and R275K, which were partially converted to the two-chain form. These latter two species could be completely converted to the two-chain form by plasmin. In addition, these two forms showed significantly more plasminogen activating activity in the absence of a fibrin(ogen) cofactor, compared to the other 18 variants. In the presence of a cofactor, all of the t-PA mutants had plasminogen activating activity equivalent to wild-type t-PA, except for R275C. The R275C t-PA had comparatively less clot lysis and fibrin binding activity as well. Presumably the new cysteine in this variant was involved in a mixed disulfide or caused misfolding of the molecule resulting in decreased activity. The difference in the plasminogen activating activity of one- and two-chain forms of t-PA was investigated by determining the apparent Michaelis constants and the apparent turnover numbers for R275E t-PA, which remains in the one-chain form throughout the assay, and two-chain R275 t-PA. The kinetic constants were measured in both the presence and the absence of plasmin-digested fibrinogen.(ABSTRACT TRUNCATED AT 400 WORDS)

Designing substrate specificity by protein engineering of electrostatic interactions.

Protein engineering of electrostatic interactions between charged substrates and complementary charged amino acids, at two different sites in the substrate binding cleft of the protease subtilisin BPN', increases kcat/Km toward complementary charged substrates (up to 1900 times) and decreases kcat/Km toward similarly charged substrates. From kinetic analysis of 16 mutants of subtilisin and the wild type, the average free energies for enzyme-substrate ion-pair interactions at the two different sites are calculated to be -1.8 +/- 0.5 and -2.3 +/- 0.6 kcal/mol (1 cal = 4.18 J) [at 25 degrees C in 0.1 M Tris X HCl (pH 8.6)]. The combined electrostatic effects are roughly additive. These studies demonstrate the feasibility for rational design of charged ligand binding sites in proteins by tailoring of electrostatic interactions.

Probing steric and hydrophobic effects on enzyme-substrate interactions by protein engineering.

Steric and hydrophobic effects on substrate specificity were probed by protein engineering of subtilisin. Subtilisin has broad peptidase specificity and contains a large hydrophobic substrate binding cleft. A conserved glycine (Gly(166)), located at the bottom of the substrate binding left, was replaced by 12 nonionic amino acids by the cassette mutagenesis method. Mutant enzymes showed large changes in specificity toward substrates of increasing size and hydrophobicity. In general, the catalytic efficiency (k(cat)/K(m)) toward small hydrophobic substrates was increased (up to 16 times) by hydrophobic substitutions at position 166 in the binding cleft. Exceeding the optimal binding volume of the cleft ( approximately 160 A(3)), by enlarging either the substrate side chain or the side chain at position 166, evoked precipitous drops in catalytic efficiency (k(cat)/K(m)) (up to 5000 times) as a result of steric hindrance.

In vivo formation and stability of engineered disulfide bonds in subtilisin.

Computer modeling suggested that a disulfide bond could be built into Bacillus amyloliquefaciens subtilisin between positions 22 (wild-type, Thr) and 87 (Ser) or between positions 24 (Ser) and 87 (Ser). Single cysteines were introduced into this cysteine-free protease at positions 22, 24, or 87 by site-directed mutagenesis of the cloned subtilisin gene. The corresponding double-cysteine mutants were constructed, and recombinant plasmids were expressed in Bacillus subtilis. Double-cysteine mutant enzymes were secreted as efficiently as wild-type, and disulfide bonds were formed quantitatively in vivo. These disulfide bonds were introduced approximately 24 A away from the catalytic site and had no detectable effect on either the specific activities or the pH optima of the mutant enzymes. The equilibrium constants for the reduction of the mutant disulfide bonds by dithiothreitol were determined to be 82 +/- 22 and 20 +/- 5 for Cys22/Cys87 and Cys24/Cys87, respectively. Studies of autoproteolytic inactivation of wild-type subtilisin support a relationship between autolytic stability and conformational stability of the protein. The stabilities of Cys24/Cys87 and wild-type enzymes to autolysis were essentially the same; however, Cys22/Cys87 was actually less stable to autolysis. Reduction of the disulfide cross-bridge lowered the autolytic stability of both double-cysteine mutants relative to their disulfide forms. This correlates with a lowered autolytic stability for the Cys22 and Cys87 single-cysteine mutants, and the fact that an intramolecular hydrogen bond between the hydroxyl groups of Thr22 and Ser87 is likely to be disrupted in the Cys22 and Cys87 single-cysteine mutant proteins.

Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites.

A method is described for the efficient insertion of mutagenic oligodeoxynucleotide cassettes which allow saturation of a target amino acid codon with multiple mutations. Restriction sites are introduced by oligonucleotide-directed mutagenesis procedures to flank closely the target codon in the plasmid containing the gene. The restriction sites to be introduced are chosen based on their uniqueness to the plasmid, proximity to the target codon and conservation of the final amino acid coding sequence. The flanking restriction sites in the plasmid are digested with the cognate restriction enzymes, and short synthetic duplex DNA cassettes (10-25 bp) are inserted. The mutagenic cassette is designed to restore fully the wild-type coding sequence, except over the target codon, and to eliminate one or both restriction sites. Elimination of a restriction site facilitates selection of clones containing the mutagenic oligodeoxynucleotide cassette. To make the cassettes, single-stranded oligodeoxynucleotides and their complements are synthesized in separate pools containing different codons over the target. This method has been successfully applied to generate 19 amino acid substitutions at position 222 in the subtilisin protein sequence.

Synergistic antiviral and antiproliferative activities of Escherichia coli-derived human alpha, beta, and gamma interferons.

The antiviral and antiproliferative effects of highly purified Escherichia coli-derived human interferons (IFNs) were examined in human melanoma cells (Hs294T). Antiproliferative activity was monitored by measuring inhibition of cell multiplication, and antiviral activity was determined by inhibition of herpes simplex virus type 1 replication. Treatment of cells with IFN-gamma in combination with IFN-alpha A or IFN-beta 1 resulted in potentiation of both antiproliferative and antiviral activities. In contrast, combination treatments composed of IFN-alpha A and IFN beta 1 yielded inconsistent results. Some combinations reflected additive responses, whereas others were antagonistic. To examine correlations between IFN-induced biological activities and interactions of the different IFNs with cell surface receptors, in vivo [35S]methionine-labeled IFN-alpha A was prepared. Binding studies indicated the presence of 2,980 +/- 170 receptors per cell, each with an apparent Kd of (8.4 +/- 1.3) X 10(-11) M. Results from competitive binding studies suggested that Hs294T cells possess at least two types of IFN receptors: one which binds IFN-alpha A and IFN-beta 1 and another to which IFN-gamma binds.