miR-34a is a tumor suppressor in zebrafish and its expression levels impact metabolism, hematopoiesis and DNA damage.

Li-Fraumeni syndrome is caused by inherited TP53 tumor suppressor gene mutations. MicroRNA miR-34a is a p53 target and modifier gene. Interestingly, miR-34 triple-null mice exhibit normal p53 responses and no overt cancer development, but the lack of miR-34 promotes tumorigenesis in cancer-susceptible backgrounds. miR-34 genes are highly conserved and syntenic between zebrafish and humans. Zebrafish miR-34a and miR-34b/c have similar expression timing in development, but miR-34a is more abundant. DNA damage by camptothecin led to p53-dependent induction of miR-34 genes, while miR-34a mutants were adult-viable and had normal DNA damage-induced apoptosis. Nevertheless, miR-34a-/- compound mutants with a gain-of-function tp53R217H/ R217H or tp53-/- mutants were more cancer-prone than tp53 mutants alone, confirming the tumor-suppressive function of miR-34a. Through transcriptomic comparisons at 28 hours post-fertilization (hpf), we characterized DNA damage-induced transcription, and at 8, 28 and 72 hpf we determined potential miR-34a-regulated genes. At 72 hpf, loss of miR-34a enhanced erythrocyte levels and up-regulated myb-positive hematopoietic stem cells. Overexpression of miR-34a suppressed its reporter mRNA, but not p53 target induction, and sensitized injected embryos to camptothecin but not to γ-irradiation.

CIAO1 and MMS19 deficiency: A lethal neurodegenerative phenotype caused by cytosolic Fe-S cluster protein assembly disorders.

PURPOSE: The functionality of many cellular proteins depends on cofactors; yet, they have only been implicated in a minority of Mendelian diseases. Here, we describe the first 2 inherited disorders of the cytosolic iron-sulfur protein assembly system. METHODS: Genetic testing via genome sequencing was applied to identify the underlying disease cause in 3 patients with microcephaly, congenital brain malformations, progressive developmental and neurologic impairments, recurrent infections, and a fatal outcome. Studies in patient-derived skin fibroblasts and zebrafish models were performed to investigate the biochemical and cellular consequences. RESULTS: Metabolic analysis showed elevated uracil and thymine levels in body fluids but no pathogenic variants in DPYD, encoding dihydropyrimidine dehydrogenase. Genome sequencing identified compound heterozygosity in 2 patients for missense variants in CIAO1, encoding cytosolic iron-sulfur assembly component 1, and homozygosity for an in-frame 3-nucleotide deletion in MMS19, encoding the MMS19 homolog, cytosolic iron-sulfur assembly component, in the third patient. Profound alterations in the proteome, metabolome, and lipidome were observed in patient-derived fibroblasts. We confirmed the detrimental effect of deficiencies in CIAO1 and MMS19 in zebrafish models. CONCLUSION: A general failure of cytosolic and nuclear iron-sulfur protein maturation caused pleiotropic effects. The critical function of the cytosolic iron-sulfur protein assembly machinery for antiviral host defense may well explain the recurrent severe infections occurring in our patients.

PLPHP deficiency: clinical, genetic, biochemical, and mechanistic insights.

Biallelic pathogenic variants in PLPBP (formerly called PROSC) have recently been shown to cause a novel form of vitamin B6-dependent epilepsy, the pathophysiological basis of which is poorly understood. When left untreated, the disease can progress to status epilepticus and death in infancy. Here we present 12 previously undescribed patients and six novel pathogenic variants in PLPBP. Suspected clinical diagnoses prior to identification of PLPBP variants included mitochondrial encephalopathy (two patients), folinic acid-responsive epilepsy (one patient) and a movement disorder compatible with AADC deficiency (one patient). The encoded protein, PLPHP is believed to be crucial for B6 homeostasis. We modelled the pathogenicity of the variants and developed a clinical severity scoring system. The most severe phenotypes were associated with variants leading to loss of function of PLPBP or significantly affecting protein stability/PLP-binding. To explore the pathophysiology of this disease further, we developed the first zebrafish model of PLPHP deficiency using CRISPR/Cas9. Our model recapitulates the disease, with plpbp-/- larvae showing behavioural, biochemical, and electrophysiological signs of seizure activity by 10 days post-fertilization and early death by 16 days post-fertilization. Treatment with pyridoxine significantly improved the epileptic phenotype and extended lifespan in plpbp-/- animals. Larvae had disruptions in amino acid metabolism as well as GABA and catecholamine biosynthesis, indicating impairment of PLP-dependent enzymatic activities. Using mass spectrometry, we observed significant B6 vitamer level changes in plpbp-/- zebrafish, patient fibroblasts and PLPHP-deficient HEK293 cells. Additional studies in human cells and yeast provide the first empirical evidence that PLPHP is localized in mitochondria and may play a role in mitochondrial metabolism. These models provide new insights into disease mechanisms and can serve as a platform for drug discovery.

Applicants with Prior Training.

Emergency medicine (EM) has its challenges, downsides, advantages, and accompanying lifestyle. Additionally, graduates of EM residency programs have abundant job opportunities. Accordingly, there is an increased interest in residency training in EM, even among residents with prior training. Transitioning from another specialty to EM can be complicated yet achievable, especially if EM is the transitioning physician's passion and career goal. Therefore, in this article, we elaborate on the transition process from another discipline to EM in light of changes in residency funding. We also explore the advantages and disadvantages of transitioning to EM with previous training in another specialty. Moreover, we expand on credit equivalencies for months already completed in another training programs, as well as the difficulties to be anticipated by transitioning physicians.

Loss of Dnajc21 leads to cytopenia and altered nucleotide metabolism in zebrafish.

Mutations in the DNAJC21 gene were recently described in Shwachman-Diamond syndrome (SDS), a bone marrow failure syndrome with high predisposition for myeloid malignancies. To study the underlying biology in hematopoiesis regulation and disease, we generated the first in vivo model of Dnajc21 deficiency using the zebrafish. Zebrafish dnajc21 mutants phenocopy key SDS patient phenotypes such as cytopenia, reduced growth, and defective protein synthesis. We show that cytopenia results from impaired hematopoietic differentiation, accumulation of DNA damage, and reduced cell proliferation. The introduction of a biallelic tp53 mutation in the dnajc21 mutants leads to the development of myelodysplastic neoplasia-like features defined by abnormal erythroid morphology and expansion of hematopoietic progenitors. Using transcriptomic and metabolomic analyses, we uncover a novel role for Dnajc21 in nucleotide metabolism. Exogenous nucleoside supplementation restores neutrophil counts, revealing an association between nucleotide imbalance and neutrophil differentiation, suggesting a novel mechanism in dnajc21-mutant SDS biology.

Ability of emergency ultrasonography to detect pediatric skull fractures: a prospective, observational study.

BACKGROUND: Blunt head trauma is a common reason for medical evaluation in the pediatric Emergency Department (ED). The diagnostic work-up for skull fracture, as well as for traumatic brain injury, often involves computed tomography (CT) scanning, which may require sedation and exposes children to often-unnecessary ionizing radiation. OBJECTIVES: Our objective was to determine if bedside ED ultrasound is an accurate diagnostic tool for identifying skull fractures when compared to head CT. METHODS: We present a prospective study of bedside ultrasound for diagnosing skull fractures in head-injured pediatric patients. A consecutive series of children presenting with head trauma requiring CT scan was enrolled. Cranial bedside ultrasound imaging was performed by an emergency physician and compared to the results of the CT scan. The primary outcome was to identify the sensitivity, specificity, and predictive values of ultrasound for skull fractures when compared to head CT. RESULTS: Bedside emergency ultrasound performs with 100% sensitivity (95% confidence interval [CI] 88.2-100%) and 95% specificity (95% CI 75.0-99.9%) when compared to CT scan for the diagnosis of skull fractures. Positive and negative predictive values were 97.2% (95% CI 84.6-99.9%) and 100% (95% CI 80.2-100%), respectively. CONCLUSIONS: Compared to CT scan, bedside ultrasound may accurately diagnose pediatric skull fractures. Considering the simplicity of this examination, the minimal experience needed for an Emergency Physician to provide an accurate diagnosis and the lack of ionizing radiation, Emergency Physicians should consider this modality in the evaluation of pediatric head trauma. We believe this may be a useful tool to incorporate in minor head injury prediction rules, and warrants further investigation.

The impact of implementing the single provider model of emergency medicine in a paediatric hospital: a retrospective cohort analysis.

STUDY OBJECTIVE: The Meyer Pediatric Hospital in Florence, Italy recently implemented the single provider model of emergency medicine. Prior to these changes, patients were triaged to a paediatric surgeon or paediatrician based on the complaint. The authors assess the outcomes of patients evaluated by surgeons prior to this change and compare them with those of patients seen by emergency physicians. METHODS: A retrospective, cohort study was performed reviewing patients seen in the emergency department between 2005 and 2008 for the three most common surgical complaints encountered before the systems change: head trauma, testicular pain and abdominal pain. Outcomes include misdiagnoses, consultation rates, dispositions, imaging, interventions and surgeries. RESULTS: A total of 2415 patient visits were included. Emergency physicians saw more patients (1388 vs 1027) and obtained more consultations (25.6% vs 8.1%) than surgeons. Patients triaged directly to surgeons were more likely to be admitted to the hospital (10.3% vs 7.6%), undergo urgent interventions (9.5% vs 6.7%), undergo surgery (8.0% vs 4.8%), have more radiographic images to evaluate head trauma (12.1% vs 5.3%), be misdiagnosed (1.0% vs 0.3%) and have more plain films for abdominal pain (3.1% vs 1.3%). There is an overall trend towards fewer missed diagnoses by emergency physicians (0.3% vs 0.9%), but this difference is only statistically significant in the abdominal pain subset analysis (p=0.032, combined data p=0.052). CONCLUSIONS: The single provider model of emergency medicine where emergency physicians manage all patients presenting to the emergency department appears to be a safe and efficient model of emergency medical care.

Loss of calpain3b in Zebrafish, a Model of Limb-Girdle Muscular Dystrophy, Increases Susceptibility to Muscle Defects Due to Elevated Muscle Activity.

Limb-Girdle Muscular Dystrophy Type R1 (LGMDR1; formerly LGMD2A), characterized by progressive hip and shoulder muscle weakness, is caused by mutations in CAPN3. In zebrafish, capn3b mediates Def-dependent degradation of p53 in the liver and intestines. We show that capn3b is expressed in the muscle. To model LGMDR1 in zebrafish, we generated three deletion mutants in capn3b and a positive-control dmd mutant (Duchenne muscular dystrophy). Two partial deletion mutants showed transcript-level reduction, whereas the RNA-less mutant lacked capn3b mRNA. All capn3b homozygous mutants were developmentally-normal adult-viable animals. Mutants in dmd were homozygous-lethal. Bathing wild-type and capn3b mutants in 0.8% methylcellulose (MC) for 3 days beginning 2 days post-fertilization resulted in significantly pronounced (20-30%) birefringence-detectable muscle abnormalities in capn3b mutant embryos. Evans Blue staining for sarcolemma integrity loss was strongly positive in dmd homozygotes, negative in wild-type embryos, and negative in MC-treated capn3b mutants, suggesting membrane instability is not a primary muscle pathology determinant. Increased birefringence-detected muscle abnormalities in capn3b mutants compared to wild-type animals were observed following induced hypertonia by exposure to cholinesterase inhibitor, azinphos-methyl, reinforcing the MC results. These mutant fish represent a novel tractable model for studying the mechanisms underlying muscle repair and remodeling, and as a preclinical tool for whole-animal therapeutics and behavioral screening in LGMDR1.

phox2ba: The Potential Genetic Link behind the Overlap in the Symptomatology between CHARGE and Central Congenital Hypoventilation Syndromes.

CHARGE syndrome typically results from mutations in the gene encoding chromodomain helicase DNA-binding protein 7 (CHD7). CHD7 is involved in regulating neural crest development, which gives rise to tissues of the skull/face and the autonomic nervous system (ANS). Individuals with CHARGE syndrome are frequently born with anomalies requiring multiple surgeries and often experience adverse events post-anesthesia, including oxygen desaturations, decreased respiratory rates, and heart rate abnormalities. Central congenital hypoventilation syndrome (CCHS) affects ANS components that regulate breathing. Its hallmark feature is hypoventilation during sleep, clinically resembling observations in anesthetized CHARGE patients. Loss of PHOX2B (paired-like homeobox 2b) underlies CCHS. Employing a chd7-null zebrafish model, we investigated physiologic responses to anesthesia and compared these to loss of phox2b. Heart rates were lower in chd7 mutants compared to the wild-type. Exposure to tricaine, a zebrafish anesthetic/muscle relaxant, revealed that chd7 mutants took longer to become anesthetized, with higher respiratory rates during recovery. chd7 mutant larvae demonstrated unique phox2ba expression patterns. The knockdown of phox2ba reduced larval heart rates similar to chd7 mutants. chd7 mutant fish are a valuable preclinical model to investigate anesthesia in CHARGE syndrome and reveal a novel functional link between CHARGE syndrome and CCHS.

Developing a pediatric trauma system in Tuscany, Italy: a case study in transformative change.

BACKGROUND: Trauma is the leading cause of death and disability in Italy among patients aged between 1 year and 19 years. To transform its fragmented regional pediatric trauma system to a more organized and efficient spoke and hub model, Tuscany has developed a partnership with physicians from the United States to facilitate the process and to implement a series of strategic system changes to create the first pediatric trauma center in Italy. METHODS: After establishing a multidisciplinary and institutional task force, a rigorous needs assessment was conducted to evaluate resources and develop a strategic timeline with specific project goals. Once an intensive educational program was completed, a series of system changes were made in Tuscany to create a full-service pediatric trauma center. Once accomplished, regional changes were enacted to divert the most severely injured children to the new center. RESULTS: In <3 years, a full-service pediatric trauma center was developed and ingratiated into the adult trauma system. All these changes were achieved through relentless administrative support, creation of a strong sense of urgency, and empowerment of innovators and early adopters to effect change. CONCLUSION: Through a comprehensive process including early extensive system analysis leading to educational interventions and organizational changes, the Anna Meyer Children's Hospital has developed the first Italian pediatric trauma center.

Cervical spine injuries in children, part I: mechanism of injury, clinical presentation, and imaging.

BACKGROUND: Cervical spine injuries are difficult to diagnose in children. They tend to occur in different locations than in adults, and they are more difficult to identify based on history or physical examination. As a result, children are often subjected to radiographic examinations to rule out cervical spine injury. OBJECTIVES: This two-part series will review the classic cervical spine injuries encountered in children based on age and presentation. Part I will discuss the mechanisms of injury, clinical presentations, and the use of different imaging modalities, including X-ray studies and computed tomography (CT). Part II discusses management of these injuries and special considerations, including the role of magnetic resonance imaging, as well as injuries unique to children. DISCUSSION: Although X-ray studies have relatively low risks associated with their use, they do not identify all injuries. In contrast, CT has higher sensitivity but has greater radiation, and its use is more appropriate in children over 8 years of age. CONCLUSION: With knowledge of cervical spine anatomy and the characteristic injuries seen at different stages of development, emergency physicians can make informed decisions about the appropriate modalities for diagnosis of pediatric cervical spine injuries.

Cervical spine injuries in children, part II: management and special considerations.

BACKGROUND: The diagnosis and management of cervical spine injury is more complex in children than in adults. OBJECTIVES: Part I of this series stressed the importance of tailoring the evaluation of cervical spine injuries based on age, mechanism of injury, and physical examination findings. Part II will discuss the role of magnetic resonance imaging (MRI) as well as the management of pediatric cervical spine injuries in the emergency department. DISCUSSION: Children have several common variations in their anatomy, such as pseudosubluxation of C2-C3, widening of the atlantodens interval, and ossification centers, that can appear concerning on imaging but are normal. Physicians should be alert for signs or symptoms of atlantorotary subluxation and spinal cord injury without radiologic abnormality when treating children with spinal cord injury, as these conditions have significant morbidity. MRI can identify injuries to the spinal cord that are not apparent with other modalities, and should be used when a child presents with a neurologic deficit but normal X-ray study or CT scan. CONCLUSION: With knowledge of these variations in pediatric anatomy, emergency physicians can appropriately identify injuries to the cervical spine and determine when further imaging is needed.

The use of point-of-care bedside lung ultrasound significantly reduces the number of radiographs and computed tomography scans in critically ill patients.

BACKGROUND: Chest radiography has been reported to have low diagnostic accuracy in critically ill intensive care unit (ICU) patients, and chest computed tomography (CT) scans require patients to be transported out of the ICU, putting them at risk of adverse events. In this study we assessed the efficacy of routine bedside lung ultrasound (LUS) in the evaluation of pleural effusions (PE) in the ICU. METHODS: Three hundred seventy-six patients admitted to the ICU for major trauma (46.3%), medical pathology (41.5%), and postsurgical complications (12.2%) (May 2008 to April 2009) were included in this study. Patients were placed into either the control group (group C) or the study group (group S), on the basis of the introduction of routine LUS performed by a single group of intensivists in 1 tertiary care ICU. To reduce provider bias, the physicians conducting the LUS were not aware of the study. Collected data included patient demographics, clinical course, and number of chest radiographs and CT scans performed. As a secondary goal, we assessed the reliability of Balik's formula in PE estimation. RESULTS: No significant differences were found between the 2 groups with regard to their demographics and ICU clinical course. Group S had a significant reduction in the total number of chest radiographs obtained (-26%; P < 0.001) and CT scans (-47%; P < 0.001) in comparison with the comparison group C. A 6-month follow-up analysis of the ICU LUS protocol revealed a time-dependent decrease in the number of radiological examinations requested for patients with PE. Lastly, PE volume estimation using the LUS and Balik's formula correlates well with the effective volume drained (r = 0.65; P < 0.0001). CONCLUSIONS: Routine use of LUS in the ICU setting can be associated with a reduction of the number of chest radiographs and CT scans performed.

A qualification course in an international emergency medicine intervention.

BACKGROUND: The Tuscan Emergency Medicine Initiative is a comprehensive training program for physicians designed to create a lasting infrastructure for training in emergency medicine (EM) in a region of Italy. A "Train-the-Trainers" model was utilized to prepare physicians who were working in the emergency department (ED) to become the teachers of EM, and a master's program was created to train the next generation of emergency physicians as well as to put in place a structure into which residency training in EM will be placed. This model has been used in other projects as well; however, the dilemma of what to do with physicians who are already in practice remained an unsolved problem. OBJECTIVES: We wished to create a qualification course in EM for this important group of physicians. METHODS: Didactic lectures, workshops, simulations, and clinical rotations were utilized to standardize current emergency care delivery in the region's EDs. RESULTS: Between 2005 and 2008, 488 physicians completed the program. CONCLUSIONS: We propose this model as a way of training and including the physicians caught in the transition to specialty training in any area developing the specialty of EM.

Vacuum-assisted closure device enhances recovery of critically ill patients following emergency surgical procedures.

INTRODUCTION: Critically ill surgical patients frequently develop intra-abdominal hypertension (IAH) leading to abdominal compartment syndrome (ACS) with subsequent high mortality. We compared two temporary abdominal closure systems (Bogota bag and vacuum-assisted closure (VAC) device) in intra-abdominal pressure (IAP) control. METHODS: This prospective study with a historical control included 66 patients admitted to a medical and surgical intensive care unit (ICU) of a tertiary care referral center (Careggi Hospital, Florence, Italy) from January 2006 to April 2009. The control group included patients consecutively treated with the Bogota bag (Jan 2006-Oct 2007), whereas the prospective group was comprised of patients treated with a VAC. All patients underwent abdominal decompressive surgery. Groups were compared based upon their IAP, SOFA score, serial arterial lactates, the duration of having their abdomen open, the need for mechanical ventilation (MV) along with length of ICU and hospital stay and mortality. Data were collected from the time of abdominal decompression until the end of pressure monitoring. RESULTS: The Bogota and VAC groups were similar with regards to demography, admission diagnosis, severity of illness, and IAH grading. The VAC system was more effective in controlling IAP (P < 0.01) and normalizing serum lactates (P < 0.001) as compared to the Bogota bag during the first 24 hours after surgical decompression. There was no significant difference between the SOFA scores. When compared to the Bogota, the VAC group had a faster abdominal closure time (4.4 vs 6.6 days, P = 0.025), shorter duration of MV (7.1 vs 9.9 days, P = 0.039), decreased ICU length of stay (LOS) (13.3 vs 19.2 days, P = 0.024) and hospital LOS (28.5 vs 34.9 days; P = 0.019). Mortality rate did not differ significantly between the two groups. CONCLUSIONS: Patients with abdominal compartment syndrome who were treated with VAC decompression had a faster abdominal closure rate and earlier discharge from the ICU as compared to similar patients treated with the Bogota bag.

The Eight Cs: a guide to success in an international emergency medicine educational collaboration.

The Tuscan Emergency Medicine Initiative (TEMI) is a comprehensive emergency medicine (EM) training program designed to build an EM training infrastructure in Tuscany, Italy. The program has successfully trained a team of instructors using a train-the-trainers model, certified 350 physicians who are already practicing in emergency departments (EDs), and established a master's program as a bridge to specialty training at the region's three universities. Using lessons learned from this program, the authors identify eight factors (The Eight Cs) that can serve as a guide to implementing a collaborative EM program in other environments: collaboration, context, culture, credibility, consulting, consistency, critique, and conclusion. Each of these topics is described in detail and may be useful to other international interventions.

Evaluation of an international emergency medicine intervention in Tuscany.

OBJECTIVE: To measure the effectiveness of a 9-month emergency medicine 'train the trainers' program in Tuscany, Italy. METHODS: A total of 81 physicians with emergency department experience completed a training course in Italy. The course included 120 h of didactic lectures, 700 h of clinical rotations and 30 h of practical workshops. The effect of the training course was measured by written multiple-choice and oral case-simulation examinations, and a precourse and postcourse self-assessment instrument using a four-point Likert scale, to describe the ability to care for different types of emergency medicine patients. RESULTS: Twenty-four physicians completed the course in 2003-2004 and 57 physicians completed the course in 2004-2005. A comparison of an identical examination given as a posttest to the first group and a pretest to the second group demonstrated significant improvement on a 75-question multiple-choice examination (38.7 vs. 46.2 points, P<0.001). Improvement was also seen in oral case examinations, in pediatrics (17.8 vs. 37.3 points, P<0.001) and neurology (24.8 vs. 34.5, P<0.001). In the self-assessment survey, when asked to describe the ability to diagnose and provide initial treatment for several types of patients before and after the course, significant improvement was reported by 13 of 20 participants (65%). When asked to describe the ability to perform a variety of procedures, significant improvement was seen in seven of sixteen (44%). CONCLUSIONS: When measured by written examinations, oral examinations and physician self-assessment, a train the trainers program, designed as part of an international emergency medicine collaboration, was efficacious.

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping.

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

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.

Procedure lab used to improve confidence in the performance of rarely performed procedures.

OBJECTIVES: The objective of this study is to assess the efficacy of an animal procedure lab in improving the level of comfort in performing important emergency medicine procedures. The procedures included central venous line, chest tube, cricothyrotomy, pericardiocentesis, venous cutdown, and thoracotomy. METHODS: The students were physicians participating in the Tuscan Emergency Medicine Initiative as part of a certificate program in emergency medicine. They attended a 1-h lecture discussing the procedures to be performed. Participants filled out a questionnaire before and after the lab, which asked how many times they had performed each procedure, how comfortable they felt, on a five-point scale, performing each procedure, and whether they felt comfortable performing it by themselves, with assistance or whether they would not feel comfortable performing the procedure. Differences in rated numerical values for each procedure before and after the lab were analyzed using a two-tailed t-test. Alpha was set at 0.95. RESULTS: In all, there were 20 participants with a wide range of experience. A statistical improvement was observed in comfort level and willingness to perform the procedures independently (P<0.01). The only non-significant change was in willingness to perform central lines. CONCLUSIONS: The use of an animal lab improves the comfort level of practitioners in performing procedures. Although procedures are best learned on patients with supervision, this is not always feasible. This lab is a useful adjunct to teaching in emergency medicine and allows participants exposure to critical procedures.