Traumatic splenic laceration with delayed rupture secondary to coughing in a patient with Von Willebrand disease.

We describe the case of a 54-year-old male with Von Willebrand Disease who presented to the Emergency Department (ED) with 2 weeks of worsening abdominal pain after falling on his left flank while boating. On his initial presentation, he was found to have a Grade II splenic injury that was managed non operatively by the trauma service. Four days later, he returned to the ED when he developed severe abdominal pain after coughing and was found to have active extravasation from the splenic parenchyma with hemoperitoneum on CT angiography and a grossly positive FAST exam. Intraoperatively, he was found to have a Grade V splenic injury and subsequently underwent splenectomy.

The Structural Basis of the Binding of Various Aminopolycarboxylates by the Periplasmic EDTA-Binding Protein EppA from Chelativorans sp. BNC1.

The widespread use of synthetic aminopolycarboxylates, such as ethylenediaminetetraacetate (EDTA), as chelating agents has led to their contamination in the environment as stable metal-chelate complexes. Microorganisms can transport free EDTA, but not metal-EDTA complexes, into cells for metabolism. An ABC-type transporter for free EDTA uptake in Chelativorans sp. BNC1 was investigated to understand the mechanism of the ligand selectivity. We solved the X-ray crystal structure of the periplasmic EDTA-binding protein (EppA) and analyzed its structure-function relations through isothermal titration calorimetry, site-directed mutagenesis, molecular docking, and quantum chemical analysis. EppA had high affinities for EDTA and other aminopolycarboxylates, which agrees with structural analysis, showing that its binding pocket could accommodate free aminopolycarboxylates. Further, key amino acid residues involved in the binding were identified. Our results suggest that EppA is a general binding protein for the uptake of free aminopolycarboxylates. This finding suggests that bacterial cells import free aminopolycarboxylates, explaining why stable metal-chelate complexes are resistant to degradation, as they are not transported into the cells for degradation.

Biochemical and Structural Analysis of Substrate Specificity of a Phenylalanine Ammonia-Lyase.

Phenylalanine ammonia-lyase (PAL) is the first enzyme of the general phenylpropanoid pathway catalyzing the nonoxidative elimination of ammonia from l-phenylalanine to give trans-cinnamate. In monocots, PAL also displays tyrosine ammonia lyase (TAL) activity, leading to the formation of p-coumaric acid. The catalytic mechanism and substrate specificity of a major PAL from sorghum (Sorghum bicolor; SbPAL1), a strategic plant for bioenergy production, were deduced from crystal structures, molecular docking, site-directed mutagenesis, and kinetic and thermodynamic analyses. This first crystal structure of a monocotyledonous PAL displayed a unique conformation in its flexible inner loop of the 4-methylidene-imidazole-5-one (MIO) domain compared with that of dicotyledonous plants. The side chain of histidine-123 in the MIO domain dictated the distance between the catalytic MIO prosthetic group created from 189Ala-Ser-Gly191 residues and the bound l-phenylalanine and l-tyrosine, conferring the deamination reaction through either the Friedel-Crafts or E2 reaction mechanism. Several recombinant mutant SbPAL1 enzymes were generated via structure-guided mutagenesis, one of which, H123F-SbPAL1, has 6.2 times greater PAL activity without significant TAL activity. Additional PAL isozymes of sorghum were characterized and categorized into three groups. Taken together, this approach identified critical residues and explained substrate preferences among PAL isozymes in sorghum and other monocots, which can serve as the basis for the engineering of plants with enhanced biomass conversion properties, disease resistance, or nutritional quality.

Structural and Biochemical Characterization of Cinnamoyl-CoA Reductases.

Cinnamoyl-coenzyme A reductase (CCR) catalyzes the reduction of hydroxycinnamoyl-coenzyme A (CoA) esters using NADPH to produce hydroxycinnamyl aldehyde precursors in lignin synthesis. The catalytic mechanism and substrate specificity of cinnamoyl-CoA reductases from sorghum (Sorghum bicolor), a strategic plant for bioenergy production, were deduced from crystal structures, site-directed mutagenesis, and kinetic and thermodynamic analyses. Although SbCCR1 displayed higher affinity for caffeoyl-CoA or p-coumaroyl-CoA than for feruloyl-CoA, the enzyme showed significantly higher activity for the latter substrate. Through molecular docking and comparisons between the crystal structures of the Vitis vinifera dihydroflavonol reductase and SbCCR1, residues threonine-154 and tyrosine-310 were pinpointed as being involved in binding CoA-conjugated phenylpropanoids. Threonine-154 of SbCCR1 and other CCRs likely confers strong substrate specificity for feruloyl-CoA over other cinnamoyl-CoA thioesters, and the T154Y mutation in SbCCR1 led to broader substrate specificity and faster turnover. Through data mining using our structural and biochemical information, four additional putative CCR genes were discovered from sorghum genomic data. One of these, SbCCR2, displayed greater activity toward p-coumaroyl-CoA than did SbCCR1, which could imply a role in the synthesis of defense-related lignin. Taken together, these findings provide knowledge about critical residues and substrate preference among CCRs and provide, to our knowledge, the first three-dimensional structure information for a CCR from a monocot species.

The Structure and Catalytic Mechanism of Sorghum bicolor Caffeoyl-CoA O-Methyltransferase.

Caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT) is an S-adenosyl methionine (SAM)-dependent O-methyltransferase responsible for methylation of the meta-hydroxyl group of caffeoyl-coenzyme A (CoA) on the pathway to monolignols, with their ring methoxylation status characteristic of guaiacyl or syringyl units in lignin. In order to better understand the unique class of type 2 O-methyltransferases from monocots, we have characterized CCoAOMT from sorghum (Sorghum bicolor; SbCCoAOMT), including the SAM binary complex crystal structure and steady-state enzyme kinetics. Key amino acid residues were validated with site-directed mutagenesis. Isothermal titration calorimetry data indicated a sequential binding mechanism for SbCCoAOMT, wherein SAM binds prior to caffeoyl-CoA, and the enzyme showed allosteric behavior with respect to it. 5-Hydroxyferuloyl-CoA was not a substrate for SbCCoAOMT. We propose a catalytic mechanism in which lysine-180 acts as a catalytic base and deprotonates the reactive hydroxyl group of caffeoyl-CoA. This deprotonation is facilitated by the coordination of the reactive hydroxyl group by Ca(2+) in the active site, lowering the pKa of the 3'-OH group. Collectively, these data give a new perspective on the catalytic mechanism of CCoAOMTs and provide a basis for the functional diversity exhibited by type 2 plant OMTs that contain a unique insertion loop (residues 208-231) conferring affinity for phenylpropanoid-CoA thioesters. The structural model of SbCCoAOMT can serve as the basis for protein engineering approaches to enhance the nutritional, agronomic, and industrially relevant properties of sorghum.

Characterizations of Two Bacterial Persulfide Dioxygenases of the Metallo-ß-lactamase Superfamily.

Persulfide dioxygenases (PDOs), also known as sulfur dioxygenases (SDOs), oxidize glutathione persulfide (GSSH) to sulfite and GSH. PDOs belong to the metallo-ß-lactamase superfamily and play critical roles in animals, plants, and microorganisms, including sulfide detoxification. The structures of two PDOs from human and Arabidopsis thaliana have been reported; however, little is known about the substrate binding and catalytic mechanism. The crystal structures of two bacterial PDOs from Pseudomonas putida and Myxococcus xanthus were determined at 1.5- and 2.5-Å resolution, respectively. The structures of both PDOs were homodimers, and their metal centers and ß-lactamase folds were superimposable with those of related enzymes, especially the glyoxalases II. The PDOs share similar Fe(II) coordination and a secondary coordination sphere-based hydrogen bond network that is absent in glyoxalases II, in which the corresponding residues are involved instead in coordinating a second metal ion. The crystal structure of the complex between the Pseudomonas PDO and GSH also reveals the similarity of substrate binding between it and glyoxalases II. Further analysis implicates an identical mode of substrate binding by known PDOs. Thus, the data not only reveal the differences in metal binding and coordination between the dioxygenases and the hydrolytic enzymes in the metallo-ß-lactamase superfamily, but also provide detailed information on substrate binding by PDOs.

Double Trouble: Massive Unruptured Aortic Aneurysms.

We describe a patient who presented to the emergency department complaining of generalized weakness, dark stools, and urinary retention who was found to have two large abdominal aortic aneurysms (AAA) compressing his bilateral ureters with associated hydronephrosis and renal insufficiency. In elderly male patients presenting with signs of obstructive uropathy, AAA should be considered as a potential cause.

A Case of Palytoxin Poisoning in a Home Aquarium Enthusiast and His Family.

Inhalational exposure to palytoxin is an extremely rare cause of respiratory distress. This little-known marine toxin has the potential to cause significant morbidity and mortality. Toxicity has been best documented in cases of ingestion but has also been seen in cases of dermal exposure and inhalation of vapors. Palytoxin has been found in several coral species, some of which are favored by home aquarium enthusiasts and are commercially available. We report a case of a family who were exposed to the aerosolized toxin following the cleaning of a coral in their home aquarium. It is important that clinicians be aware of this source of toxic exposure to provide necessary care to these patients.

A Massive Overdose of Dalfampridine.

Multiple sclerosis (MS) is an immune mediated inflammatory disease that attacks myelinated axons in the central nervous system. Dalfampridine (4-aminopyridine) was approved by the Food and Drug Administration in January 2010 for treatment of MS. Our patient was a 34-year-old male with a history of MS, who was brought to the emergency department after being found unresponsive. His current medications were valacyclovir, temazepam, dalfampridine (4-AP) and a tysabri intravenous (IV) infusion. Fifteen minutes after arrival the patient seized. The seizures were refractory to benzodiazepines, barbiturates and phenytoin. The 4-AP level was 530 ng/mL (25 ng/mL and 49 ng/mL). The patient stopped seizing on hospital day 3 and was discharged 14 days later with normal mental status and neurologic exam. 4-AP is a potassium channel blocker that blocks the potassium ion current of repolarization following an action potential. The blockade of the potassium channel at the level of the membrane widens the action potential and enhances the release of acetylcholine, thus increasing post-synaptic action potentials. The treatment of patients with 4-AP overdose is supportive. Animal data suggest that patients with toxic levels of 4-AP may respond to phenytoin. Our case illustrates the highest recorded level of 4-AP in an overdose. Our patient appeared to be refractory to a combination of high doses of anticonvulsants and only improved with time.

Descriptive study of prescriptions for opioids from a suburban academic emergency department before New York's I-STOP Act.

INTRODUCTION: Controlled prescription opioid use is perceived as a national problem attributed to all specialties. Our objective was to provide a descriptive analysis of prescriptions written for controlled opioids from a database of emergency department (ED) visits prior to the enactment of the I-STOP law, which requires New York prescribers to consult the Prescription Monitoring Program (PMP) prior to prescribing Schedule II, III, and IV controlled substances for prescriptions of greater than five days duration. METHODS: We conducted a retrospective medical record review of patients 21 years of age and older, who presented to the ED between July 1, 2011 - June 30, 2012 and were given a prescription for a controlled opioid. Our primary purpose was to characterize each prescription as to the type of controlled substance, the quantity dispensed, and the duration of the prescription. We also looked at outliers, those patients who received prescriptions for longer than five days. RESULTS: A total of 9,502 prescriptions were written for opioids out of a total 63,143 prescriptions for 69,500 adult patients. Twenty-six (0.27%) of the prescriptions for controlled opioids were written for greater than five days. Most prescriptions were for five days or less (99.7%, 95% CI [99.6 to 99.8%]). CONCLUSION: The vast majority of opioid prescriptions in our ED prior to the I-STOP legislature were limited to a five-day or less supply. These new regulations were meant to reduce the ED's contribution to the rise of opioid related morbidity. This study suggests that the emergency physicians' usual prescribing practices were negligibly limited by the new restrictive regulations. The ED may not be primarily contributing to the increase in opioid-related overdoses and death. The effect of the I-STOP regulation on future prescribing patterns in the ED remains to be determined.

Epidemiology of nursemaid's elbow.

INTRODUCTION: To provide an epidemiological description of radial head subluxation, also known as nursemaid's elbow, from a database of emergency department visits. METHODS: We conducted a retrospective medical record review of patients 6 years of age and younger, who presented to the ED between January 1, 2005, and December 31, 2012, and were diagnosed with nursemaid's elbow. Inclusion criteria consisted of chart information, including date, unique account number, medical record number, weight, age, sex, and arm affected. Exclusion criteria included any charts with missing or incomplete data. RESULTS: There were 1,228 charts that met inclusion criteria. The majority of patients were female (60%). The mean age was 28.6 months (±12.6). The left arm was affected 60% of the time. Most of the included patients were over the 75(th) percentile for weight and more than one quarter were over the 95(th) percentile in each gender. CONCLUSION: The average age of children presenting with nursemaid's elbow was 28.6 months. Females were affected more than males, and the left arm was predominately affected. Most patients were above the 75(th) percentile for weight and more than one quarter were over the 95(th) percentile for weight.