Mechanistic and Structural Analysis of a Drosophila melanogaster Enzyme, Arylalkylamine N-Acetyltransferase Like 7, an Enzyme That Catalyzes the Formation of N-Acetylarylalkylamides and N-Acetylhistamine.

Arylalkylamine N-acetyltransferase like 7 (AANATL7) catalyzes the formation of N-acetylarylalkylamides and N-acetylhistamine from acetyl-CoA and the corresponding amine substrate. AANATL7 is a member of the GNAT superfamily of >10000 GCN5-related N-acetyltransferases, many members being linked to important roles in both human metabolism and disease. Drosophila melanogaster utilizes the N-acetylation of biogenic amines for the inactivation of neurotransmitters, the biosynthesis of melatonin, and the sclerotization of the cuticle. We have expressed and purified D. melanogaster AANATL7 in Escherichia coli and used the purified enzyme to define the substrate specificity for acyl-CoA and amine substrates. Information about the substrate specificity provides insight into the potential contribution made by AANATL7 to fatty acid amide biosynthesis because D. melanogaster has emerged as an important model system contributing to our understanding of fatty acid amide metabolism. Characterization of the kinetic mechanism of AANATL7 identified an ordered sequential mechanism, with acetyl-CoA binding first followed by histamine to generate an AANATL7·acetyl-CoA·histamine ternary complex prior to catalysis. Successive pH-activity profiling and site-directed mutagenesis experiments identified two ionizable groups: one with a pKa of 7.1 that is assigned to Glu-26 as a general base and a second pKa of 9.5 that is assigned to the protonation of the thiolate of the coenzyme A product. Using the data generated herein, we propose a chemical mechanism for AANATL7 and define functions for other important amino acid residues involved in substrate binding and regulation of catalysis.

Mechanistic and structural analysis of Drosophila melanogaster arylalkylamine N-acetyltransferases.

Arylalkylamine N-acetyltransferase (AANAT) catalyzes the penultimate step in the biosynthesis of melatonin and other N-acetylarylalkylamides from the corresponding arylalkylamine and acetyl-CoA. The N-acetylation of arylalkylamines is a critical step in Drosophila melanogaster for the inactivation of the bioactive amines and the sclerotization of the cuticle. Two AANAT variants (AANATA and AANATB) have been identified in D. melanogaster, in which AANATA differs from AANATB by the truncation of 35 amino acids from the N-terminus. We have expressed and purified both D. melanogaster AANAT variants (AANATA and AANATB) in Escherichia coli and used the purified enzymes to demonstrate that this N-terminal truncation does not affect the activity of the enzyme. Subsequent characterization of the kinetic and chemical mechanism of AANATA identified an ordered sequential mechanism, with acetyl-CoA binding first, followed by tyramine. We used a combination of pH-activity profiling and site-directed mutagenesis to study prospective residues believed to function in AANATA catalysis. These data led to an assignment of Glu-47 as the general base in catalysis with an apparent pKa of 7.0. Using the data generated for the kinetic mechanism, structure-function relationships, pH-rate profiles, and site-directed mutagenesis, we propose a chemical mechanism for AANATA.

Expression, purification, and characterization of mouse glycine N-acyltransferase in Escherichia coli.

Glycine N-acyltransferase (GLYAT) is a phase II metabolic detoxification enzyme for exogenous (xenobiotic) and endogenous carboxylic acids; consisting of fatty acids, benzoic acid, and salicylic acid. GLYAT catalyzes the formation of hippurate (N-benzoylglycine) from the corresponding glycine and benzoyl-CoA. Herein, we report the successful expression, purification, and characterization of recombinant mouse GLYAT (mGLYAT). A 34kDa mGLYAT protein was expressed in Escherichia coli and purified to homogeneity by nickel affinity chromatography to a final yield of 2.5mg/L culture. Characterization for both amino donors and amino acceptors were completed, with glycine serving as the best amino donor substrate, (kcat/Km)app=(5.2±0.20)×10(2)M(-1)s(-1), and benzoyl-CoA serving as the best the amino acceptor substrate, (kcat/Km)app=(4.5±0.27)×10(5)M(-1)s(-1). Our data demonstrate that mGLYAT will catalyzed the chain length specific (C2-C6) formation of N-acylglycines. The steady-state kinetic constants determined for recombinant mGLYAT for the substrates benzoyl-CoA and glycine, were shown to be consistent with other reported species (rat, human, bovine, ovine, and rhesus monkey). The successful recombinant expression and purification of mGLYAT can lead to solve unanswered questions associated with this enzyme, consisting of what is the chemical mechanism and what catalytic residues are essential for the how this phase II metabolic detoxification enzyme conjugates glycine to xenobiotic and endogenous carboxylic acids.

Obesity and Associated Outcomes for Blunt vs Penetrating Mechanism in Trauma Laparotomy Patients.

Obesity in trauma patients is an established risk factor contributing to postoperative complications, but the relationship between body mass index (BMI) and trauma patient outcomes is not well-defined, especially when stratified by mechanism of injury. We surveyed the trauma laparotomy registry at an academic level 1 trauma center over a 3-year period to identify mortality, injury severity score, and hospital length of stay (hLOS) outcome measures across BMI classes, with further stratification by mechanism of injury: blunt vs penetrating trauma. A total of 442 patients were included with mean age 44.6 (SD = 18.7) and mean BMI 28.55 (SD = 7.37). These were subdivided into blunt trauma (n = 313) and penetrating trauma (n = 129). Within the blunt trauma subgroup, the hLOS among patients who survived hospitalization significantly increased 9% for each successive BMI class (P = .022, 95% CI = 1.29-17.5). We conclude that successive increase in BMI class is associated with longer hospital stay for blunt trauma patient survivors requiring laparotomy, though additional analysis is needed to establish this relationship to other outcome measures and among penetrating trauma patients.

Pediatric intracranial aneurysms: current national trends in patient management and treatment.

PURPOSE: Pediatric intracranial aneurysms constitute a medical disease process with many unique features that present unique challenges in orchestrating their treatment. Conflicts exist in pediatric aneurysm literature as to whether endovascular therapy is equivalent to surgical therapy in assuring durable aneurysm obliteration in this population. MATERIALS AND METHODS: The national Healthcare Cost and Utilization Project (HCUP) Kid's Inpatient Dataset was queried using the HCUPnet system. Overall trends in length of stay (LOS), associated charges, and in-hospital deaths were analyzed for both subarachnoid hemorrhage (SAH) and nonruptured aneurysms from 2000 to 2009. Trends in the type of procedure, associated LOS, and charges were analyzed for SAH from 2003 to 2009. A p value <0.05 was considered statistically significant. RESULTS: Mean LOS for SAH patients was an additional 7-10 days compared to patients discharged with nonruptured aneurysms. Costs of surgery showed a slight increase, while endovascular procedures also rose 50 % from 2006 to 2009. Interestingly, mean length of stay increased for endovascular procedures from 16.5 to 17.2 days and decreased for surgical procedures from 20.4 to 14.7 days (p < 0.001). CONCLUSIONS: First, in-hospital mortality and hospital length of stay for pediatric subarachnoid hemorrhage have not significantly declined since 1997. Second, in-hospital charges for the management of both ruptured and nonruptured aneurysms rose by over 200 % from 2000 to 2009. Surgical procedures saw a 6 % increase in price, while endovascular procedures sharply rose in costs by 50 %. Finally, endovascular therapy has increased in utilization, while the frequency of surgical therapy has not changed significantly since 2003.

Obesity Associated With Increased Mortality and Hospital Length of Stay in Trauma Laparotomy Patients.

Although obesity in trauma patients is accepted as a risk factor for postoperative complications, recent literature offers conflicting evidence regarding the effect of body mass index (BMI) on mortality in trauma patients undergoing laparotomy. To address this question, we examined the patient population of a Level 1 Trauma Center during a 3-year period to compare mortality rates and other outcomes between BMI groups undergoing laparotomy. Through retrospective chart review of electronic medical records, with subsequent stratification of data based on BMI, we found that mortality, injury severity score, and hospital length of stay all increase significantly with each incremental increase in BMI class. From these data, we concluded that higher BMI class leads to greater morbidity and mortality in trauma patients undergoing laparotomy at this institution.

Pre- and Post-Portosystemic Shunt Placement Metabolomics Reveal Molecular Signatures for the Development of Hepatic Encephalopathy.

Hepatic encephalopathy (HE) is a common complication of advanced liver disease causing brain dysfunction. This is likely due to the accumulation of unfiltered toxins within the bloodstream. A known risk factor for developing or worsening HE is the placement of a transjugular intrahepatic portosystemic shunt (TIPS), which connects the pre-hepatic and post-hepatic circulation allowing some blood to bypass the dysfunctional liver and decreases portal hypertension. To better understand the pathophysiology of post-TIPS HE, we conducted a multi-center prospective cohort study employing metabolomic analyses on hepatic vein and peripheral vein blood samples from participants with cirrhosis undergoing elective TIPS placement, measuring chemical modifications and changes in concentrations of metabolites resulting from TIPS placement. In doing so, we identified numerous alterations in metabolites, including bile acids, glycerophosphocholines, and bilirubins possibly implicated in the development and severity of HE.

Racial Disparity in Liver Transplantation Listing.

BACKGROUND: Previous studies have demonstrated disparities in transplantation for women, non-Caucasians, the uninsured or publicly insured, and rural populations. We sought to correlate transplant center characteristics with patient access to the waiting list and liver transplantation. We hypothesized that liver transplant centers vary greatly in providing equitable access to the waiting list and liver transplantation. STUDY DESIGN: Center-specific, adult, deceased-donor liver transplant and waitlist data for the years 2013 to 2018 were obtained from the United Network for Organ Sharing. Waitlist race/ethnicity distributions from liver transplant centers performing ≥ 250 transplants over this period (n = 109) were compared with those of their donor service area, as calculated from 5-year US Census Bureau estimates of 2017. Center-specific characteristics correlating with disparities were analyzed using a linear regression model with a log transformed outcome. RESULTS: Non-Hispanic Blacks (NHBs) are under-represented in liver transplant listing compared with center donation service area (88/109, 81%), whereas, non-Hispanic Whites are over-represented (65/109, 58%) (p < 0.0001). Hispanics were also under-represented on the waitlist at the majority of transplant centers (68/109, 62%) (p = 0.02). Although the racial/ethnic distribution of transplantation is more reflective of the waitlist, there is a higher than expected rate of transplantation for NHBs compared to the waitlist. Predictors of disparity in listing include percentage of transplant recipients at the center who had private insurance, racial composition of the donation service area, and the distance recipients had to travel for transplant. CONCLUSIONS: Non-Hispanic Blacks are listed for liver transplantation less than would be expected. Once listed, however, racial disparities in transplantation are greatly diminished. Improvements in access to adequate health insurance appear to be essential to diminishing disparities in access to this life-saving care.

Obesity and STING1 genotype associate with 23-valent pneumococcal vaccination efficacy.

BACKGROUNDObesity has been associated with attenuated vaccine responses and an increased risk of contracting pneumococcal pneumonia, but no study to our knowledge has assessed the impact of obesity and genetics on 23-valent pneumococcal vaccine (PPSV23) efficacy. We assessed the relationship of obesity (primary analysis) and stimulator of interferon genes (STING1) genotype (secondary analysis) on PPSV23 efficacy.METHODSNonobese (BMI 22-25 kg/m2) and obese participants (BMI ≥30 kg/m2) were given a single dose of PPSV23. Blood was drawn immediately prior to and 4-6 weeks after vaccination. Serum samples were used to assess PPSV23-specific antibodies. STING1 genotypes were identified using PCR on DNA extracted from peripheral blood samples.RESULTSForty-six participants were categorized as nonobese (n = 23; 56.5% women; mean BMI 23.3 kg/m2) or obese (n = 23; 65.2% women; mean BMI 36.3 kg/m2). Obese participants had an elevated fold change in vaccine-specific responses compared with nonobese participants (P < 0.0001). The WT STING1 group (R232/R232) had a significantly higher PPSV23 response than individuals with a single copy of HAQ-STING1 regardless of BMI (P = 0.0025). When WT was assessed alone, obese participants had a higher fold serotype-specific response compared with nonobese participants (P < 0.0001), but no difference was observed between obese and nonobese individuals with 1 HAQ allele (P = 0.693).CONCLUSIONSThese observations demonstrate a positive association between obesity and PPSV23 efficacy specifically in participants with the WT STING1 genotype.TRIAL REGISTRATIONClinicalTrials.gov NCT02471014.FUNDINGThis research was supported by the NIH and the University of Florida MD-PhD Training Program.

Gender Diversity in General Surgery Residency Leadership.

OBJECTIVE: This study aimed to evaluate the proportion and characteristics of women who serve in general surgery program director (PD) and associate program director (APD) positions in the United States. DESIGN: General surgery programs (n = 276) and directors were identified using the Association for Program Directors in Surgery website; information was cross-referenced with American Medical Association FREIDA and Accreditation Council for Graduate Medical Education databases, current to July 1, 2017. Each program's website was accessed to determine the gender and academic ranking of faculty. RESULTS: Results reveal a preponderance of men in PD and APD positions. Women accounted for 18.4% (n = 51) of the 276 PD positions, with more women in APD positions (29.6%). There was no correlation between gender of PD and the corresponding APD, (χ2 = 0.68, p = 0.41; Phi coefficient = -0.0695). Of those with academic appointments, men who were PDs were more likely to be full professors when compared to women PDs (38.5% vs 24.1%, respectively). The median number of days since appointment to PD was similar in both groups (1461 days for men vs 1377 for women, p = 0.18), although more men have held PD positions longer. Programs with a higher proportion of women faculty were more likely to have a woman PD (p = 0.0397), but not those with more women residents (p = 0.225) or a woman Department Chair (p = 0.56). CONCLUSIONS: Among general surgery program directorship, men continue to hold more positions of educational leadership, although the trend appears to be shifting toward a more equal balance, particularly in those programs with proportionately more women faculty. This discrepancy may be due to academic rank or length of tenure. As more women hold academic positions in the field of general surgery, an increase in the representation of this group in leadership is anticipated. Although senior leadership (PD) positions remain disproportionately held by men, APD positions are filled by a greater percentage of women than academic surgical faculty, although the absolute percentage remains less than 50%. Educational leadership may be a viable path to academic leadership for both women and men.

A Comparison of Pathophysiology in Humans and Rodent Models of Subarachnoid Hemorrhage.

Non-traumatic subarachnoid hemorrhage (SAH) affects an estimated 30,000 people each year in the United States, with an overall mortality of ~30%. Most cases of SAH result from a ruptured intracranial aneurysm, require long hospital stays, and result in significant disability and high fatality. Early brain injury (EBI) and delayed cerebral vasospasm (CV) have been implicated as leading causes of morbidity and mortality in these patients, necessitating intense focus on developing preclinical animal models that replicate clinical SAH complete with delayed CV. Despite the variety of animal models currently available, translation of findings from rodent models to clinical trials has proven especially difficult. While the explanation for this lack of translation is unclear, possibilities include the lack of standardized practices and poor replication of human pathophysiology, such as delayed cerebral vasospasm and ischemia, in rodent models of SAH. In this review, we summarize the different approaches to simulating SAH in rodents, in particular elucidating the key pathophysiology of the various methods and models. Ultimately, we suggest the development of standardized model of rodent SAH that better replicates human pathophysiology for moving forward with translational research.

Endoscopic Dilation of Bariatric RNY Anastomotic Strictures: a Systematic Review and Meta-analysis.

Gastrojejunostomy anastomotic strictures are a complication of Roux-en-Y gastric bypass surgery without an established treatment guideline. A systematic review and meta-analysis were performed to determine the safety and efficacy of endoscopic dilation in their management. PubMed, Web of Science, and Cochrane Central (1994-2017) were searched. Data was analyzed with random effects meta-analysis and mixed effects meta-regression. Twenty-one observational studies (896 patients) were included. The stricture rate for laparoscopic patients was 6% (95% CI, 5-9%). Only 38% (95% CI, 30-47%) required greater than one dilation. Symptom improvement occurred in 97% (95% CI, 94-98%). The complication rate was 4% (95% CI, 3-6%). Endoscopic dilation of GJA strictures is safe, effective, and sustaining. This study can guide endoscopists in the treatment of a common bariatric surgical complication.

Structural and Mechanistic Analysis of Drosophila melanogaster Agmatine N-Acetyltransferase, an Enzyme that Catalyzes the Formation of N-Acetylagmatine.

Agmatine N-acetyltransferase (AgmNAT) catalyzes the formation of N-acetylagmatine from acetyl-CoA and agmatine. Herein, we provide evidence that Drosophila melanogaster AgmNAT (CG15766) catalyzes the formation of N-acetylagmatine using an ordered sequential mechanism; acetyl-CoA binds prior to agmatine to generate an AgmNAT•acetyl-CoA•agmatine ternary complex prior to catalysis. Additionally, we solved a crystal structure for the apo form of AgmNAT with an atomic resolution of 2.3 Å, which points towards specific amino acids that may function in catalysis or active site formation. Using the crystal structure, primary sequence alignment, pH-activity profiles, and site-directed mutagenesis, we evaluated a series of active site amino acids in order to assign their functional roles in AgmNAT. More specifically, pH-activity profiles identified at least one catalytically important, ionizable group with an apparent pKa of ~7.5, which corresponds to the general base in catalysis, Glu-34. Moreover, these data led to a proposed chemical mechanism, which is consistent with the structure and our biochemical analysis of AgmNAT.

Probing the chemical mechanism and critical regulatory amino acid residues of Drosophila melanogaster arylalkylamine N-acyltransferase like 2.

Arylalkylamine N-acyltransferase like 2 (AANATL2) catalyzes the formation of N-acylarylalkylamides from the corresponding acyl-CoA and arylalkylamine. The N-acylation of biogenic amines in Drosophila melanogaster is a critical step for the inactivation of neurotransmitters, cuticle sclerotization, and melatonin biosynthesis. In addition, D. melanogaster has been used as a model system to evaluate the biosynthesis of fatty acid amides: a family of potent cell signaling lipids. We have previously showed that AANATL2 catalyzes the formation of N-acylarylakylamides, including long-chain N-acylserotonins and N-acyldopamines. Herein, we define the kinetic mechanism for AANATL2 as an ordered sequential mechanism with acetyl-CoA binding first followed by tyramine to generate the ternary complex prior to catalysis. Bell shaped kcat,app - acetyl-CoA and (kcat/Km)app - acetyl-CoA pH-rate profiles identified two apparent pKa,app values of ∼7.4 and ∼8.9 that are critical to catalysis, suggesting the AANATL2-catalyzed formation of N-acetyltyramine occurs through an acid/base chemical mechanism. Site-directed mutagenesis of a conserved glutamate that corresponds to the catalytic base for other D. melanogaster AANATL enzymes did not produce a substantial depression in the kcat,app value nor did it abolish the pKa,app value attributed to the general base in catalysis (pKa ∼7.4). These data suggest that AANATL2 catalyzes the formation of N-acylarylalkylamides using either different catalytic residues or a different chemical mechanism relative to other D. melanogaster AANATL enzymes. In addition, we constructed other site-directed mutants of AANATL2 to help define the role of targeted amino acids in substrate binding and/or enzyme catalysis.

Identification of an arylalkylamine N-acyltransferase from Drosophila melanogaster that catalyzes the formation of long-chain N-acylserotonins.

Arylalkylamine N-acyltransferase-like 2(2) (AANATL2) from Drosophila melanogaster was expressed and shown to catalyze the formation of long-chain N-acylserotonins and N-acydopamines. Subsequent identification of endogenous amounts of N-acylserotonins and colocalization of these fatty acid amides and AANATL2 transcripts gives supporting evidence that AANATL2 has a role in the biosynthetic formation of these important cell signalling lipids.