Targeting EYA2 tyrosine phosphatase activity in glioblastoma stem cells induces mitotic catastrophe.

Glioblastoma ranks among the most lethal of primary brain malignancies, with glioblastoma stem cells (GSCs) at the apex of tumor cellular hierarchies. Here, to discover novel therapeutic GSC targets, we interrogated gene expression profiles from GSCs, differentiated glioblastoma cells (DGCs), and neural stem cells (NSCs), revealing EYA2 as preferentially expressed by GSCs. Targeting EYA2 impaired GSC maintenance and induced cell cycle arrest, apoptosis, and loss of self-renewal. EYA2 displayed novel localization to centrosomes in GSCs, and EYA2 tyrosine (Tyr) phosphatase activity was essential for proper mitotic spindle assembly and survival of GSCs. Inhibition of the EYA2 Tyr phosphatase activity, via genetic or pharmacological means, mimicked EYA2 loss in GSCs in vitro and extended the survival of tumor-bearing mice. Supporting the clinical relevance of these findings, EYA2 portends poor patient prognosis in glioblastoma. Collectively, our data indicate that EYA2 phosphatase function plays selective critical roles in the growth and survival of GSCs, potentially offering a high therapeutic index for EYA2 inhibitors.

Characterization of Arylalkylamine N-Acyltransferase from Tribolium castaneum: An Investigation into a Potential Next-Generation Insecticide Target.

The growing issue of insecticide resistance has meant the identification of novel insecticide targets has never been more important. Arylalkylamine N-acyltransferases (AANATs) have been suggested as a potential new target. These promiscuous enzymes are involved in the N-acylation of biogenic amines to form N-acylamides. In insects, this process is a key step in melanism, hardening of the cuticle, removal of biogenic amines, and in the biosynthesis of fatty acid amides. The unique nature of each AANAT isoform characterized indicates each organism accommodates an assembly of discrete AANATs relatively exclusive to that organism. This implies a high potential for selectivity in insecticide design, while also maintaining polypharmacology. Presented here is a thorough kinetic and structural analysis of AANAT found in one of the most common secondary pests of all plant commodities in the world, Tribolium castaneum. The enzyme, named TcAANAT0, catalyzes the formation of short-chain N-acylarylalkylamines, with short-chain acyl-CoAs (C2-C10), benzoyl-CoA, and succinyl-CoA functioning in the role of acyl donor. Recombinant TcAANAT0 was expressed and purified from E. coli and was used to investigate the kinetic and chemical mechanism of catalysis. The kinetic mechanism is an ordered sequential mechanism with the acyl-CoA binding first. pH-rate profiles and site-directed mutagenesis studies identified amino acids critical to catalysis, providing insights about the chemical mechanism of TcAANAT0. A crystal structure was obtained for TcAANAT0 bound to acetyl-CoA, revealing valuable information about its active site. This combination of kinetic analysis and crystallography alongside mutagenesis and sequence analysis shines light on some approaches possible for targeting TcAANAT0 and other AANATs for novel insecticide design.

Knockdown of arylalkylamine N-acetyltransferase-like 2 in Drosophila melanogaster.

Drosophila melanogaster produces fatty acid amides, and thus, provides a model to unravel the pathways for their biosynthesis. We previously demonstrated that arylalkylamine N-acetyltransferase-like 2 (AANATL2) from D. melanogaster will catalyze the formation of long-chain N-acylserotonins and N-acyldopamines in vitro. Generating silencing RNA via the UAS/GAL4 bipartite approach for targeted gene expression effectively decreased the endogenous levels of the AANATL2 transcripts in D. melanogaster, as shown by reverse transcription quantitative polymerase chain reaction. Consistent with these data, western blot analysis of the offspring of the AANATL2 knockdown flies using an anti-AANATL2 antibody revealed a significant reduction in the expression of the AANATL2 protein. Reduced expression of AANATL2 decreased the cellular levels of N-palmitoyldopamine (PALDA), providing strong evidence that AANATL2 is responsible for the biosynthesis of PALDA in vivo. This is the first time that the expression of an AANAT has been reduced in D. melanogaster to link one of these enzymes to the in vivo production of an N-acylarylalkylamide.

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.

Ultrasound Screening for Posterior Shoulder Dislocation in Infants with Persistent Brachial Plexus Birth Palsy.

BACKGROUND: Early detection of posterior shoulder dislocation in infants with brachial plexus birth palsy (BPBP) is essential, but it may be difficult to accomplish with physical examination alone. The aim of this study was to determine the prevalence of shoulder dislocation in patients with BPBP using ultrasound and to identify which physical examination measurements correlated most with dislocation in these patients. METHODS: This study was a retrospective review of data obtained in an ultrasound screening program of infants with BPBP born from January 2011 to April 2014. Physical examination included the use of the Active Movement Scale (AMS) and measurement of passive external rotation of the shoulder. Ultrasound measurements included PHHD (percentage of the humeral head displaced posterior to the axis of the scapula) and the alpha angle (intersection of the posterior scapular margin with a line tangential to the humeral head through the glenoid). Shoulder dislocation was defined as both a PHHD of >0.5 and an alpha angle of >30°. RESULTS: Of sixty-six infants who had undergone a total of 118 ultrasound examinations (mean, 1.8; range, 1 to 5), 19 (29%) demonstrated shoulder dislocation with the shoulder positioned in internal rotation; the dislocation was first detected between 2.1 and 10.5 months of age. Infants with a dislocated shoulder demonstrated significantly less mean passive external rotation in adduction (mean, 45.8° versus 71.4°, p < 0.001) and a greater difference between internal rotation and external rotation AMS scores (mean, 5.5-point versus 3.3-point difference, p < 0.001) than those without shoulder dislocation. Passive external rotation in adduction was a better measure for discriminating between dislocation and no dislocation (area under receiver operating characteristic curve [AUC] = 0.89) than was the difference between internal and external rotation AMS scores (AUC = 0.73). A cutoff of 60° of passive external rotation in adduction (≤60° versus° >60) yielded a sensitivity of 94% and a specificity of 69%. CONCLUSIONS: Shoulder dislocation is common in infants with BPBP; 29% of the infants presenting to our tertiary care center had a dislocation during their first year of life. Ultrasound shoulder screening is appropriate for infants with BPBP. If passive external rotation in adduction is used to determine which infants should undergo ultrasound, ≤60° should be utilized as the criterion to achieve appropriate sensitivity. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

N-FATTY ACYLGLYCINES: UNDERAPPRECIATED ENDOCANNABINOID-LIKE FATTY ACID AMIDES?

Long-chain N-fatty acylglycines, R-CO-NH-CH2-COOH (where "R" refers to an unsaturated or saturated alkyl chain of at least 14 carbons) are found in mammals and insects and are structurally related to the cell-signaling, lipid-like, N-fatty acylethanolamines, R-CO-NH-CH2-CH2-OH (where "R" refers to an alkyl chain of at least 14 carbons). Accumulating evidence demonstrates that the N-fatty acylglycines have important cellular functions, but much work remains in order to fully appreciate and understand these biomolecules including: (a) more work on their functions in vivo, (b) measuring their concentrations in the cell, (c) defining the pathways for the biosynthesis and degradation, and (d) understanding the metabolic interconversion(s) between the N-fatty acylglycines and other fatty acid amides. The purpose of reviewing the current state-of-knowledge about the N-fatty acylglycines is to stimulate future research about this intriguing family of biomolecules.

Glycine N-acyltransferase-like 3 is responsible for long-chain N-acylglycine formation in N18TG2 cells.

Long-chain fatty acid amides are signaling lipids found in mammals and other organisms; however, details of the metabolic pathways for the N-acylglycines and primary fatty acid amides (PFAMs) have remained elusive. Heavy-labeled precursor and subtraction lipidomic experiments in mouse neuroblastoma N18TG2 cells, a model cell line for the study of fatty acid amide metabolism, establish the biosynthetic pathways for the N-acylglycines and the PFAMs. We provide evidence that the N-acylglycines are formed by a long-chain specific glycine-conjugating enzyme, glycine N-acyltransferase-like 3 (GLYATL3). siRNA knockdown of GLYATL3 in the N18TG2 cells resulted in a decrease in the levels of the N-acylglycines and the PFAMs. This is the first report of an enzyme responsible for long-chain N-acylglycine production in cellula. The production of the PFAMs in N18TG2 cells was reported to occur by the oxidative cleavage of the N-acylglycines, as catalyzed by peptidylglycine α-amidating monooxygenase (PAM). siRNA knockdown of PAM resulted in an accumulation of [(13)C18]N-oleoylglycine and decreased levels of [(13)C18]oleamide when the N18TG2 cells were grown in the presence of [(13)C18]oleic acid. The addition of [1-(13)C]palmitate to the N18TG2 cell growth media led to the production of a family of [1-(13)C]palmitoylated fatty acid amides, consistent with the biosynthetic pathways detailed herein.

Drosophila melanogaster as a model system to study long-chain fatty acid amide metabolism.

Long-chain fatty acid amides are cell-signaling lipids identified in mammals and, recently, in invertebrates, as well. Many details regarding fatty acid amide metabolism remain unclear. Herein, we demonstrate that Drosophila melanogaster is an excellent model system for the study long-chain fatty acid amide metabolism as we have quantified the endogenous levels of N-acylglycines, N-acyldopamines, N-acylethanolamines, and primary fatty acid amides by LC/QTOF-MS. Growth of D. melanogaster on media supplemented with [1-(13)C]-palmitate lead to a family of (13)C-palmitate-labeled fatty acid amides in the fly heads. The [1-(13)C]-palmitate feeding studies provide insight into the biosynthesis of the fatty acid amides.

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.