Bm-iAANAT3: Expression and characterization of a novel arylalkylamine N-acyltransferase from Bombyx mori.

The arylalkylamine N-acyltransferases (AANATs) are enzymes that catalyze the acyl-CoA-dependent formation of N-acylarylalkylamides: acyl-CoA + arylalkylamine → N-acylarylalkylamides + CoA-SH. Herein, we describe our study of a previously uncharacterized AANAT from Bombyx mori: Bm-iAANAT3. Bm-iAANAT3 catalyzes the direct formation of N-acylarylalkylamides and accepts a broad range of short-chain acyl-CoA thioesters and amines as substrates. Acyl-CoA thioesters possessing an acyl chain length >10 carbon atoms are not substrates for Bm-iAANAT3. We report that Bm-iAANAT3 is a "versatile generalist", most likely, functioning in amine acetylation - a reaction in amine inactivation/excretion, cuticle sclerotization, and melanism. We propose a kinetic and chemical mechanism for Bm-iAANAT3 that is consistent with our steady-state kinetic analysis, dead-end inhibition studies, determination of the pH-rate profiles, and site-directed mutagenesis of a catalytically important amino acid in Bm-iAANAT3. These mechanistic studies of Bm-iAANAT3 will foster the development of novel compounds targeted against this enzyme and other insect AANATs for the control of insect pests.

Intraoperative Tips for Hand Surgery: A Focus on Reducing Postoperative Scarring and Tendon Rupture.

This article is a collection of intraoperative techniques performed by a single hand surgeon with literature support for these techniques for the purpose of circumventing potential limitations intraoperatively. These techniques include the use of a Beaver Blade handle (Beaver, Beaver-Visitec, Waltham, MA, US) to be used as a rasp to smooth intraosseous tunnels during tendon transfers, a Stryker (Stryker, Stryker Corporation, Kalamazoo, MI, US) or Synthes (Deputy Synthes, Johnson & Johnson, West Palm Beach, FL, US) drill as a motorized file for plate deburring, and Insorb forceps (Insorb, Incisive Surgical, Plymouth, MN, US) for skin closure. These tips serve as methods to minimize scarring and circumvent unfortunate obstacles, such as tendon rupture, and the consequential weakened repair that can occur post-operatively. These have not been previously reported in the literature but have been performed by the senior author with no resulting complications. Additionally, the common availability of the equipment allows for a potential economic benefit.

Bm-iAANAT and its potential role in fatty acid amide biosynthesis in Bombyx mori.

The purpose of this research is to unravel the substrate specificity and kinetic properties of an insect arylalkylamine N-acyltransferase from Bombyx mori (Bm-iAANAT) and to determine if this enzyme will catalyze the formation of long chain N-acylarylalkylamides in vitro. However, the determination of substrates and products for Bm-iAANAT in vitro is no guarantee that these same molecules are substrates and products for the enzyme in the organism. Therefore, RT-PCR was performed to detect the Bm-iAANAT transcripts and liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) analysis was performed on purified lipid extracts from B. mori larvae (fourth instar, Bmi4) to determine if long chain fatty acid amides are produced in B. mori. Ultimately, we found that recombinant Bm-iAANAT will utilize long-chain acyl-CoA thioesters as substrates and identified Bm-iAANAT transcripts and long-chain fatty acid amides in Bmi4. Together, these data show Bm-iAANAT will catalyze the formation of long-chain N-acylarylalkylamides in vitro and provide evidence demonstrating that Bm-iAANAT has a role in fatty acid amide biosynthesis in B. mori, as well.

Sensitivity and Specificity of Skyline and Carpal Shoot-Through Fluoroscopic Views of Volar Plate Fixation of the Distal Radius: A Cadaveric Investigation of Dorsal Cortex Screw Penetration.

BACKGROUND: Volar plate fixation of distal radius fractures can result in soft tissue injuries. Abnormal contour of the dorsal cortex of the distal radius provides difficulties in discerning screw penetration on standard radiographs. The skyline and carpal shoot-through views are additional views to improve dorsal cortex visibility. We report on the sensitivity and specificity of determining screw protrusion with these views. METHODS: Seven fresh frozen cadavers were instrumented with a distal radius volar locking plate. Initial screw length was determined by depth gauge measurement. A dorsal dissection of the wrist was performed to detect screw penetration. Protruding screws were documented and replaced with screws of the appropriate length and deemed as baseline. Screws were then sequentially lengthened by 2 and 4 mm. Skyline and carpal shoot-through views were obtained at baseline, 2 mm, and 4 mm. The images were randomized and compiled into an untimed survey asking orthopedic surgeons to determine whether screws were penetrating through the dorsal cortex. RESULTS: Based on depth gauge measurements, 4 out of 44 (9.1%) volar plate locking screws penetrated the dorsal cortex, as confirmed with dorsal dissection. Sensitivities for the skyline and carpal shoot-through views were 75% and 86% ( P ≤ .001), respectively, for 2-mm protrusions, and 76% and 89% ( P ≤ .001), respectively, for 4-mm screw protrusions. Specificities were 85% and 84% for the skyline and shoot-through views, respectively. CONCLUSIONS: We believe that the carpal shoot-through view has utility and can be implemented to augment standard intraoperative views, and may decrease the incidence of screw protrusion resulting in soft tissue injuries.

Mechanistic binding insights for 1-deoxy-D-Xylulose-5-Phosphate synthase, the enzyme catalyzing the first reaction of isoprenoid biosynthesis in the malaria-causing protists, Plasmodium falciparum and Plasmodium vivax.

We have successfully truncated and recombinantly-expressed 1-deoxy-D-xylulose-5-phosphate synthase (DXS) from both Plasmodium vivax and Plasmodium falciparum. We elucidated the order of substrate binding for both of these ThDP-dependent enzymes using steady-state kinetic analyses, dead-end inhibition, and intrinsic tryptophan fluorescence titrations. Both enzymes adhere to a random sequential mechanism with respect to binding of both substrates: pyruvate and D-glyceraldehyde-3-phosphate. These findings are in contrast to other ThDP-dependent enzymes, which exhibit classical ordered and/or ping-pong kinetic mechanisms. A better understanding of the kinetic mechanism for these two Plasmodial enzymes could aid in the development of novel DXS-specific inhibitors that might prove useful in treatment of malaria.