Identification of amino acid domains of Borrelia burgdorferi P66 that are surface exposed and important for localization, oligomerization, and porin function of the protein.

P66, a bifunctional integral outer membrane protein, is necessary for Borrelia burgdorferi to establish initial infection and to disseminate in mice. The integrin binding function of P66 facilitates extravasation and dissemination, but the role of its porin function during murine infection has not been investigated. A limitation to studying P66 porin function during mammalian infection has been the lack of structural information for P66. In this study, we experimentally characterized specific domains of P66 with regard to structure and function. First, we aligned the amino acid sequences of P66 from Lyme disease-causing Borrelia and relapsing fever-causing Borrelia to identify conserved and unique domains between these disease-causing clades. Then, we examined whether specific domains of P66 are exposed on the surface of the bacteria by introducing c-Myc epitope tags into each domain of interest. The c-Myc epitope tag inserted C-terminally to E33 (highly conserved domain), to T187 (integrin binding region domain and a non-conserved domain), and to E334 (non-conserved domain) were all detected on the surface of Borrelia burgdorferi. The c-Myc epitope tag inserted C-terminally to E33 and D303 in conserved domains disrupted P66 oligomerization and porin function. In a murine model of infection, the E33 and D303 mutants exhibited decreased infectivity and dissemination. Taken together, these results suggest the importance of these conserved domains, and potentially P66 porin function, in vivo.

Non-invasive real-time monitoring of cell concentration and viability using Doppler ultrasound.

Bioprocess optimization towards higher productivity and better quality control relies on real-time process monitoring tools to measure process and culture parameters. Cell concentration and viability are among the most important parameters to be monitored during bioreactor operations that are typically determined using optical methods on an extracted sample. In this paper, we have developed an online non-invasive sensor to measure cell concentration and viability based on Doppler ultrasound. An ultrasound transducer is mounted outside the bioreactor vessel and emits a high frequency tone burst (15 MHz) through the vessel wall. Acoustic backscatter from cells in the bioreactor depends on cell concentration and viability. The backscattered signal is collected through the same transducer and analyzed using multivariate data analysis (MVDA) to characterize and predict the cell culture properties. We have developed accurate MVDA models to predict the Chinese hamster ovary (CHO) cell concentration in a broad range from 0.1 × 106 cells/mL to 100 × 106 cells/mL, and cell viability from 3% to 99%. The non-invasive monitoring is ideal for single use bioreactor and the in-situ measurements removes the burden for offline sampling and dilution steps. This method can be similarly applied to other suspension cell culture modalities.

Heterologous production of the adhesin LIC13411 from pathogenic Leptospira facilitates binding of non-pathogenic Leptospira in vitro and in vivo.

Leptospirosis is an important cause of morbidity and mortality worldwide. Disease severity ranges from asymptomatic colonization to widespread hemorrhage and multiorgan dysfunction. The causative agents, Leptospira spp., are zoonotic Gram-negative spirochetes. One important step in pathogenesis is binding of bacterial adhesins to host components. Previously our laboratory identified two L. interrogans candidate adhesins, LIC11574 and LIC13411, that bind to VE-cadherin in vitro. In the current study, we demonstrate the ability of two strains of pathogenic L. interrogans to disrupt the localization of VE-cadherin, a protein important to maintaining inter-endothelial junctions. Purified MBP-LIC11574 and MBP-LIC13411 bind human dermal microvascular endothelial cells in a pattern reminiscent of VE-cadherin, but do not disrupt VE-cadherin localization. Genes encoding the candidate adhesins from pathogenic Leptospira were cloned in an overexpression vector and introduced into non-pathogenic L. biflexa, creating gain-of-function strains producing LIC11574 or LIC13411. Protein production and localization to the outer membrane were confirmed by Triton X-114 fractionation. Although these strains do not disrupt VE-cadherin localization, production of LIC13411 increases binding of non-pathogenic Leptospira to human endothelial cells and specifically to VE-cadherin. In a short-term murine model of infection, LIC13411 production led to increased burdens of the non-pathogen in the lung, liver, kidney, and bladder. These data confirm the role of LIC13411 as an adhesin in Leptospira spp. and implicate it in dissemination to multiple organs. Importantly, anti-adhesin therapy has been shown to have many benefits over classical antibiotics. Taken together, this work provides novel insight into the pathogenesis of Leptospira spp. and identifies LIC13411 as a potential prophylactic and therapeutic target.

Hematogenous dissemination of pathogenic and non-pathogenic Leptospira in a short-term murine model of infection.

Leptospirosis is an emerging zoonosis caused by pathogenic Leptospira spp. Because rodents are natural hosts of Leptospira, rodent models of pathogenesis have been limited, but are valuable to understand infection in reservoir animals even in the absence of disease. Mouse models of infection provide advantages due to genetic tractability, so developing murine models of Leptospira infection is crucial for further understanding the biology of this organism. Previously our laboratory developed a short-term murine model of Borrelia burgdorferi hematogenous dissemination to investigate the role of adhesion proteins on bacterial survival and dissemination within a host. Here we adapt this model to Leptospira. C3H/HeJ mice are anesthetized, inoculated intravenously, and then bacteria are allowed to circulate for up to twenty-four hours. Mice are euthanized, perfused with saline, and tissues are harvested for culture and DNA purification. Bacterial burdens are determined by quantitative PCR. Reproducible burdens of bacteria were found in tissues upon inoculation with pathogens and non-pathogens, demonstrating the utility of this model to probe different Leptospira species and strains. Pathogenic L. interrogans has a significantly higher burden in blood, liver, kidney, and bladder at one-hour post-inoculation when compared to non-pathogenic L. biflexa. Colonization of the kidney is essential to the life cycle of pathogenic Leptospira in nature. Measurable burdens of non-pathogenic L. biflexa were found in numerous organs and live leptospires were recovered from blood samples for at least three hours post-inoculation, contrary to the previous belief that non-pathogenic leptospires are rapidly cleared. This short-term murine model of Leptospira hematogenous dissemination will allow for the interrogation of virulence factors potentially important for tissue colonization and evasion of host defenses, and represents a novel animal model for investigating determinants of Leptospira infection.

VlsE, the nexus for antigenic variation of the Lyme disease spirochete, also mediates early bacterial attachment to the host microvasculature under shear force.

Hematogenous dissemination is a critical step in the evolution of local infection to systemic disease. The Lyme disease (LD) spirochete, which efficiently disseminates to multiple tissues, has provided a model for this process, in particular for the key early event of pathogen adhesion to the host vasculature. This occurs under shear force mediated by interactions between bacterial adhesins and mammalian cell-surface proteins or extracellular matrix (ECM). Using real-time intravital imaging of the Lyme spirochete in living mice, we previously identified BBK32 as the first LD spirochetal adhesin demonstrated to mediate early vascular adhesion in a living mouse; however, deletion of bbk32 resulted in loss of only about half of the early interactions, suggesting the existence of at least one other adhesin (adhesin-X) that promotes early vascular interactions. VlsE, a surface lipoprotein, was identified long ago by its capacity to undergo rapid antigenic variation, is upregulated in the mammalian host and required for persistent infection in immunocompetent mice. In immunodeficient mice, VlsE shares functional overlap with OspC, a multi-functional protein that displays dermatan sulfate-binding activity and is required for joint invasion and colonization. In this research, using biochemical and genetic approaches as well as intravital imaging, we have identified VlsE as adhesin-X; it is a dermatan sulfate (DS) adhesin that efficiently promotes transient adhesion to the microvasculature under shear force via its DS binding pocket. Intravenous inoculation of mice with a low-passage infectious B. burgdorferi strain lacking both bbk32 and vlsE almost completely eliminated transient microvascular interactions. Comparative analysis of binding parameters of VlsE, BBK32 and OspC provides a possible explanation why these three DS adhesins display different functionality in terms of their ability to promote early microvascular interactions.

BBB07 contributes to, but is not essential for, Borrelia burgdorferi infection in mice.

Borrelia burgdorferi, a causative agent of Lyme disease, encodes a protein BBB07 on the genomic plasmid cp26. BBB07 was identified as a candidate integrin ligand based on the presence of an RGD tripeptide motif, which is present in a number of mammalian ligands for ß1 and ß3 integrins . Previous work demonstrated that BBB07 in recombinant form binds to ß1 integrins and induces inflammatory responses in synovial cells in culture. Several transposon mutants in bbb07 were attenuated in an in vivo screen of the transposon library in mice. We therefore tested individual transposon mutant clones in single-strain infections in mice and found that they were attenuated in terms of ID50 but did not have significantly reduced tissue burdens in mice. Based on data presented here we conclude that BBB07 is not essential for, but does contribute to, B. burgdorferi infectivity in mice.

Effects of humeral intraosseous epinephrine in a pediatric hypovolemic cardiac arrest porcine model.

BACKGROUND: Aims of the study were to determine the effects of humerus intraosseous (HIO) versus intravenous (IV) administration of epinephrine in a hypovolemic, pediatric pig model. We compared concentration maximum (Cmax), time to maximum concentration (Tmax), mean concentration (MC) over time and return of spontaneous circulation (ROSC). METHODS: Pediatric pig were randomly assigned to each group (HIO (n=7); IV (n=7); cardiopulmonary resuscitation (CPR)+defibrillation (defib) (n=7) and CPR-only group (n=5)). The pig were anesthetized; 35% of the blood volume was exsanguinated. pigs were in arrest for 2 min, and then CPR was performed for 2 min. Epinephrine 0.01 mg/kg was administered 4 min postarrest by either route. Samples were collected over 5 min. After sample collection, epinephrine was administered every 4 min or until ROSC. The Cmax and MC were analyzed using high-performance liquid chromatography. Defibrillation began at 3 min postarrest and administered every 2 min or until ROSC or endpoint at 20 min after initiation of CPR. RESULTS: Analysis indicated that the Cmax was significantly higher in the IV versus HIO group (p=0.001). Tmax was shorter in the IV group but was not significantly different (p=0.789). The MC was significantly greater in the IV versus HIO groups at 90 and 120 s (p<0.05). The IV versus HIO had a significantly higher MC (p=0.001). χ2 indicated the IV group (5 out of 7) had significantly higher rate of ROSC than the HIO group (1 out of 7) (p=0.031). One subject in the CPR+defib and no subjects in the CPR-only groups achieved ROSC. DISCUSSION: Based on the results of our study, the IV route is more effective than the HIO route.

Outcomes of the collapse of a large bubble in water at high ambient pressures.

Presented here are observations of the outcomes of the collapses of large single bubbles in H_{2}O and D_{2}O at high ambient pressures. Experiments were carried out in a high-pressure spherical resonator at ambient pressures of up to 30 MPa and acoustic pressures up to 35 MPa. Monitoring of the collapse events and their outcomes was accomplished using multiframe high-speed photography. Among the observations to be presented are the temporal and spatial evolution of light emissions produced by the collapse events, which were observed to last on the order of 30 ns and have time independent radii on the order of 30µm; the production of Rayleigh-Taylor jets which were observed to travel distances of up to 70µm at speeds in excess of 4500 m/s; the entrainment of the light emitting regions in the jets' remnants; the production of spheroidal objects around the collapse points of the bubbles, far from any surface of the resonator; and the traversal and emergence of the Rayleigh-Taylor jets through the spherical objects. These spheroidal objects appear to behave as amorphous solids and form at locations where hydrodynamics predicts pressures in excess of the known transition pressures of water into the high-pressure crystalline ices, Ice-VI and Ice-VII.

Analytical Method for Sugar Profile in Pet Food and Animal Feeds by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection.

There is a need for a standardized, accurate, rugged, and consistent method to measure for sugars in pet foods and animal feeds. Many traditional standard sugar methods exist for other matrixes, but when applied in collaborative studies there was poor agreement and sources of error identified with those standard methods. The advancement in technology over the years has given us the ability to improve on these standard methods of analysis. A method is described here that addresses these common issues and was subjected to a single-laboratory validation to assess performance on a wide variety of pet foods and animal feeds. Of key importance to the method performance is the sample preparation before extraction, type of extraction solvent, postextraction cleanup, and, finally, optimized chromatography using high-performance anion exchange chromatography with pulsed amperometric detection. The results obtained from the validation demonstrate how typical issues seen with these matrixes can influence performance of sugar analysis. The results also demonstrate that this method is fit-for-purpose and can meet the challenges of sugar analysis in pet food and animal feeds to lay the foundation for a standardized method of analysis.

Assessing the rates of error and adverse events in the ED.

BACKGROUND: The incidence of errors and adverse events in emergency medicine is poorly characterized. OBJECTIVE: The objective was to systematically determine the rates and types of errors and adverse events in an academic, tertiary care emergency department (ED). METHODS: Prospective data were collected on all patients presenting to a tertiary-care academic medical center ED with an annual census of 55,000 patients between January 2009 and November 2012. Cases of patients meeting predetermined criteria were systematically identified by an electronic medical record system. Criteria for review included patients who (1) returned to the ED within 72 hours and were admitted on their second visit, (2) were admitted from the ED to the floor and then transferred to the intensive care unit (ICU) within 24 hours, (3) expired within 24 hours of ED arrival, (4) required airway management, or (5) were referred to the QA committee as the result of complaints. Cases were randomly assigned to individual physicians not involved with the care. All cases were reviewed using a structured electronic tool that assessed the occurrence of error and adverse events. Institutional review board jurisdiction was waived by the Beth Israel Deaconess Medical Center IRB. RESULTS: During the study period, 152,214 cases were screened and 2131 cases (1.4%) met prespecified criteria for review. The incidence of error in these cases was 9.5% (95% confidence interval [CI], 8.3%-10.8%), representing an overall incidence of 0.13% among all ED patients. In cases that involved error, 50.5% occurred among patients who returned to the ED within 72 hours; 17.3% occurred among floor-to-ICU transfers; 5.4% occurred among mortality cases; 2.0% occurred among airway cases; and 24.8% occurred among cases referred as the result of complaints. The incidence of adverse events in the reviewed cohort was 8.3% (CI, 7.2%-9.6%), representing an overall incidence of 0.11% among all ED patients. In cases that involved adverse events, 48.6% occurred among patients who returned to the ED within 72 hours; 16.4% occurred among floor-to-ICU transfers; 9.0% occurred among mortality cases; 1.1% occurred among airway cases; and 24.9% occurred among cases referred as the result of complaints. CONCLUSION: Although the overall incidence of error and adverse events in EDs is low, the likelihood of such events is markedly increased among patients who return to the ED within 72 hours, among patients who require floor-to-ICU transfer within 24 hours, and among those whose cases come to attention as the result of complaints.

Combining an optical resonance biosensor with enzyme activity kinetics to understand protein adsorption and denaturation.

Understanding protein adsorption and resultant conformation changes on modified and unmodified silicon dioxide surfaces is a subject of keen interest in biosensors, microfluidic systems and for medical diagnostics. However, it has been proven difficult to investigate the kinetics of the adsorption process on these surfaces as well as understand the topic of the denaturation of proteins and its effect on enzyme activity. A highly sensitive optical whispering gallery mode (WGM) resonator was used to study a catalytic enzyme's adsorption processes on different silane modified glass substrates (plain glass control, DETA, 13 F, and SiPEG). The WGM sensor was able to obtain high resolution kinetic data of glucose oxidase (GO) adsorption with sensitivity of adsorption better than that possible with SPR. The kinetic data, in combination with a functional assay of the enzyme activity, was used to test hypotheses on adsorption mechanisms. By fitting numerical models to the WGM sensograms for protein adsorption, and by confirming numerical predictions of enzyme activity in a separate assay, we were able to identify mechanisms for GO adsorption on different alkylsilanes and infer information about the adsorption of protein on nanostructured surfaces.

Temporally and spatially resolved imaging of laser-nucleated bubble cloud sonoluminescence.

Imaging techniques have been used to capture the temporal and spatial evolution of light emissions from collapsing bubble clouds at high static pressures. Emission events lasting up to 70 ns with peak diameters nearing 1 mm have been observed. Observations of the cloud evolution before and after emission events have been made. Photomultiplier tube monitoring has been employed in conjunction with imaging to study the temporal characteristics of light emission.

Whispering gallery mode biosensor quantification of fibronectin adsorption kinetics onto alkylsilane monolayers and interpretation of resultant cellular response.

A Whispering Gallery Mode (WGM) biosensor was constructed to measure the adsorption of protein onto alkysilane self-assembled monolayers (SAMs) at solution concentrations unattainable with other techniques. The high sensitivity was provided by a WGM resonance excited in a silica microsphere that was functionalized with alkylsilane SAMs and integrated in a microfluidic flow cell under laminar flow conditions. It was found that FN adsorbed at biologically relevant surface densities, however, the adsorption kinetics and concentration dependent saturation values varied significantly from work published utilizing alkanethiol SAMs. Mathematical models were applied to the experimental results to interpret the observed kinetics of FN adsorption. Embryonic hippocampal neurons and skeletal myoblasts were cultured on the modified surfaces, a live--dead assay was used to determine the viability of the FN surfaces for cell culture, and major differences were noted in the biological response to the different SAMs. The high sensitivity and simplicity of the WGM biosensor, combined with its ability to quantify the adsorption of any dilute protein in a label-free assay, establishes the importance of this technology for the study of surface accretion and its effect on cellular function, which can affect biomaterials for both in vivo and in vitro applications.

Optical nucleation of bubble clouds in a high pressure spherical resonator.

An experimental setup for nucleating clouds of bubbles in a high-pressure spherical resonator is described. Using nanosecond laser pulses and multiple phase gratings, bubble clouds are optically nucleated in an acoustic field. Dynamics of the clouds are captured using a high-speed CCD camera. The images reveal cloud nucleation, growth, and collapse and the resulting emission of radially expanding shockwaves. These shockwaves are reflected at the interior surface of the resonator and then reconverge to the center of the resonator. As the shocks reconverge upon the center of the resonator, they renucleate and grow the bubble cloud. This process is repeated over many acoustic cycles and with each successive shock reconvergence, the bubble cloud becomes more organized and centralized so that subsequent collapses give rise to stronger, better defined shockwaves. After many acoustic cycles individual bubbles cannot be distinguished and the cloud is then referred to as a cluster. Sustainability of the process is ultimately limited by the detuning of the acoustic field inside the resonator. The nucleation parameter space is studied in terms of laser firing phase, laser energy, and acoustic power used.

Biomarker profiles as descriptors of left ventricular remodeling after acute myocardial infarction.

BACKGROUND: Biomarker expression can predict subsequent cardiovascular events. The goal of this study was to determine the pattern of expression in blood of a broad array of cytokines and growth factors taken 24-72 h after an ST elevation myocardial infarction (STEMI) involving the left anterior descending (LAD) coronary artery. METHODS: Blood was taken from 16 patients with LAD STEMI. Cytokine and growth factor expressions were quantified with the use of a Milliplex cytokine/chemokine array analysis that tested 42 analytes. Results from patients were compared with those in blood from 20 healthy volunteers. RESULTS: Most (15/16) participants had positive remodeling without reduction in left ventricular function during follow-up. Analytes were grouped based on their function into those that activate class 1 T-helper cells (Th1 activates cell-mediated immunity), those that activated a Th2 response (activates humoral immunity and attenuates cell-mediated immunity), chemokines (attract leukocytes), and growth factors (promote a healing response). Elevation of cytokines involved in the Th2 response predominated over the Th1 response demonstrating a balance favoring tolerance and limiting activation of cell-mediated immunity. The concentration of selected chemokines favoring cell-mediated immunity was not elevated. The concentration of selected growth factors was increased. CONCLUSION: The cytokine expression, 24-72 h after an LAD STEMI, suggests that positive ventricular remodeling is associated with growth factor expression and limitation of cell-mediated immunity. Subsequent studies are warranted to determine whether deviation from this pattern identifies patients at an increased risk of adverse remodeling after myocardial infarction.

Two hour bivalirudin infusion after PCI for ST elevation myocardial infarction.

The standard of care for STEMI PCI for the past decade has been aspirin, clopidogrel, heparin, and a glycoprotein IIbIIIa receptor inhibitor (GPI). A bivalirudin strategy was shown to be superior to a GPI strategy in the HORIZONS AMI trial for net adverse clinical events (combined MACE and bleeding). An increased risk of acute stent thrombosis in the bivalirudin arm may have prevented broader adoption of bivalirudin for this indication. We hypothesized that acute stent thrombosis risk could be ameliorated by a 2 h infusion of bivalirudin following STEMI PCI. We implemented a multicenter, prospective registry for all STEMI patients in Vermont treated at a single PCI center. Each patient was routinely pre-loaded with dual antiplatelet therapy and 75% received an unfractionated heparin bolus prior to PCI. The utilization of bivalirudin bolus and continued 2 h infusion after PCI was routine with GPI bailout optional. 128 consecutive STEMI patients underwent primary PCI from October 1, 2008 to September 30, 2009. 92% of primary PCI patients received bivalrudin during and after the procedure with a 9% rate of bail out GPI. There was one case of probable or definite acute stent thrombosis (0.7%), and this single case occurred despite use of bailout GPI. Despite the prolonged infusion of bivalirudin, major bleeding occurred in only 1.7% of STEMI patients. In conclusion, prolonging bivalirudin for 2 h after STEMI PCI may be a promising method to alleviate acute stent thrombosis risk without losing the bleeding complication benefit of the bivalirudin strategy.

[Description of a U.S. emergency department and its patient population during a year]. / Beskrivelse af en amerikansk "emergency department" og dens patientpopulation gennem et år.

INTRODUCTION: In the United States, Emergency Medicine (EM) is recognized as an independent medical speciality, which focuses on the initial evaluation and treatment of all patients with acute illness or injury. Patients have free access to the emergency department (ED), which serves as the main portal of entry for most acute patients coming into the hospital. The ED is staffed with EM specialists at all hours. The purpose of this study was to describe a U.S. tertiary care hospital ED and the patient population that is cared for there. MATERIAL AND METHODS: A descriptive retrospective study of the patient population at Beth Israel Deaconess Medical Center Emergency Department, from 1 January to 31 December 2003. Demographic data were collected for each patient, as well as estimated severity index score, diagnosis and ED disposition. RESULTS: 46,936 patients with a median age of 46.5 years where seen at the ED in 2003. Women represented 56%. The majority of patients had an ESI triage score = 3. All of the main categories of ICD-9 diagnostic codes where represented among patients' ED discharge diagnoses. 65% of ED patients where discharged home from the department. Approximately one third where admitted to the hospital. CONCLUSION: A broad range of patients are seen in the ED, which sets high demands for the knowledge and skills of the ED staff as well as co-operation between the ED and other specialties and hospital departments.

Method for the direct determination of available carbohydrates in low-carbohydrate products using high-performance anion exchange chromatography.

An improved method for direct determination of available carbohydrates in low-level products has been developed and validated for a low-carbohydrate soy infant formula. The method involves modification of an existing direct determination method to improve specificity, accuracy, detection levels, and run times through a more extensive enzymatic digestion to capture all available (or potentially available) carbohydrates. The digestion hydrolyzes all common sugars, starch, and starch derivatives down to their monosaccharide components, glucose, fructose, and galactose, which are then quantitated by high-performance anion-exchange chromatography with photodiode array detection. Method validation consisted of specificity testing and 10 days of analyzing various spike levels of mixed sugars, maltodextrin, and corn starch. The overall RSD was 4.0% across all sample types, which contained within-day and day-to-day components of 3.6 and 3.4%, respectively. Overall average recovery was 99.4% (n = 10). Average recovery for individual spiked samples ranged from 94.1 to 106% (n = 10). It is expected that the method could be applied to a variety of low-carbohydrate foods and beverages.

Carboxymethyl-beta-cyclodextrin mitigates toxicity of cadmium, cobalt, and copper during naphthalene biodegradation.

Hazardous waste sites are commonly contaminated with both organic and metal pollutants. Many metal pollutants have been shown to inhibit organic pollutant biodegradation. We investigated the ability of a modified, polydentate cyclodextrin (carboxymethyl-beta-cyclodextrin, CMCD) to reduce the toxicity of 33.4 microM cadmium, cobalt or copper during naphthalene degradation by a Burkholderia sp. in 120 h aerobic, batch studies. The highest investigated concentration of CMCD, 3340 microM, reduced cadmium, cobalt, and copper toxicity. With each metal, the length of the lag phase was reduced (by as much as 108 h with cobalt or copper), the cell yield was increased (by as much as a factor of 16 with cobalt), and the growth rate was increased (by as much as a factor of 31 with cobalt). The degrader was unable to use CMCD as the sole source of carbon and energy. Our data suggest that the ability of CMCD to complex metals plays an important role in its ability to mitigate metal toxicity and that CMCD has the potential to enhance biodegradation in organic and metal co-contaminated environments.

The Tuscan Emergency Medicine Initiative.

Italy lacks standardized specialty training in emergency medicine. There is no system of national or regional accreditation of the knowledge base or skill set of physicians working in regional emergency departments (ED), which results in variability of emergency medical care delivery not only between hospital EDs but also within individual EDs. To address this need, the Tuscan Minister of Health chose to develop a partnership with emergency medicine specialists from the United States to help expedite the growth of the specialty in Tuscany. The collaboration called the Tuscan Emergency Medicine Initiative consists of the regional health care service, the Tuscan university system, Harvard Medical International, and the Beth Israel Deaconess Medical Center Department of Emergency Medicine. We describe that effort and process, with an expectation of more than 625 physicians completing the program by June 2008.