Mammalian brain glycoproteins exhibit diminished glycan complexity compared to other tissues

Glycosylation is essential to brain development and function, but prior studies have often been limited to a single analytical technique and excluded region- and sex-specific analyses. Here, using several methodologies, we analyze Asn-linked and Ser/Thr/Tyr-linked protein glycosylation between brain regions and sexes in mice. Brain N-glycans are less complex in sequence and variety compared to other tissues, consisting predominantly of high-mannose and fucosylated/bisected structures. Most brain O-glycans are unbranched, sialylated O-GalNAc and O-mannose structures. A consistent pattern is observed between regions, and sex differences are minimal compared to those in plasma. Brain glycans correlate with RNA expression of their synthetic enzymes, and analysis of glycosylation genes in humans show a global downregulation in the brain compared to other tissues. We hypothesize that this restricted repertoire of protein glycans arises from their tight regulation in the brain. These results provide a roadmap for future studies of glycosylation in neurodevelopment and disease.

510 Late-Breaking: Dietary Glycine Supplementation During Growing and Finishing Phases Increases Tissue Concentrations of Total Creatine and Gene Expression of Creatine-synthetic Enzymes in Low-birthweight Pigs

Dietary glycine is required for maximum growth and development in animals by stimulating muscle protein synthesis and as a component of creatine. Creatine is synthesized from glycine, arginine, and S-adenosylmethionine by arginine:glycine amidinotransferase (AGAT) and guanidinoacetate N-methyltransferase (GAMT). Sufficient creatine synthesis for growth requires adequate substrate supply. However, swine diets are deficient in glycine. Additionally, intrauterine growth restricted (IUGR) pigs have reduced glycine synthesis. This results in decreased creatine synthesis and lower total creatine content in tissues, leading to reduced cellular energy metabolism and diminished muscle protein accretion. This study was designed to test the hypothesis that dietary glycine supplementation in corn-and-soybean-meal-based diets would improve overall growth and skeletal muscle accretion in post-weaning IUGR pigs by increasing the expression of creatine-synthetic enzymes and tissue concentrations of total creatine. Fourteen IUGR pigs (birthweight = 0.98±0.03 kg, mean±SEM) and 20 normal birthweight pigs (birthweight = 1.44±0.02 kg, mean±SEM) were obtained at weaning for this study. Pigs from each birthweight group were randomly assigned to 1% glycine + 0.19% corn starch treatment group or 1.19% alanine group (isonitrogenous control) for the study (21 d to 188 d of age); tissues were collected at d 188. Data were analyzed by using 2-way ANOVA and the Duncan multiple comparison test. Glycine-supplemented IUGR pigs had greater tissue concentrations of creatine, creatinine, and creatine phosphate than control IUGR in all tissues measured (P< 0.05). Control IUGR pigs showed diminished activity and mRNA expression of creatine-synthetic enzymes (P < 0.05); this was mitigated by glycine supplementation as glycine supplemented IUGR pigs showed normal levels of enzyme activity and mRNA expression. Overall, results of this study indicate dietary glycine supplementation to IUGR pigs between weaning and market weight effectively restores creatine-synthetic enzyme activities and increase tissue concentrations of total creatine, leading to increased lean tissue growth. (Supported by USDA-NIFA)

The Natural Antimicrobial trans-Cinnamaldehyde Interferes with UDP-N-Acetylglucosamine Biosynthesis and Cell Wall Homeostasis in Listeria monocytogenes

Trans-cinnamaldehyde (t-CIN), an antimicrobial compound from cinnamon essential oil, is of interest because it inhibits various foodborne pathogens. In the present work, we investigated the antimicrobial mechanisms of t-CIN in Listeria monocytogenes using a previously isolated yvcK::Himar1 transposon mutant which shows hypersensitivity to t-CIN. Time-lapse microscopy revealed that t-CIN induces a bulging cell shape followed by lysis in the mutant. Complementation with wild-type yvcK gene completely restored the tolerance of yvcK::Himar1 strain to t-CIN and the cell morphology. Suppressor mutants which partially reversed the t-CIN sensitivity of the yvcK::Himar1 mutant were isolated from evolutionary experiments. Three out of five suppression mutations were in the glmU-prs operon and in nagR, which are linked to the biosynthesis of the peptidoglycan precursor uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc). GlmU catalyzes the last two steps of UDP-GlcNAc biosynthesis and NagR represses the uptake and utilization of N-acetylglucosamine. Feeding N-acetylglucosamine or increasing the production of UDP-GlcNAc synthetic enzymes fully or partially restored the t-CIN tolerance of the yvcK mutant. Together, these results suggest that YvcK plays a pivotal role in diverting substrates to UDP-GlcNAc biosynthesis in L. monocytogenes and that t-CIN interferes with this pathway, leading to a peptidoglycan synthesis defect.

Acetylcholine Signaling Genes are Required for Cocaine-Stimulated Egg Laying in Caenorhabditis elegans

The toxicity and addictive liability associated with cocaine abuse are well known. However, its mode of action is not completely understood, and effective pharmacotherapeutic interventions remain elusive. The cholinergic effects of cocaine on acetylcholine receptors, synthetic enzymes, and degradative enzymes have been the focus of relatively little empirical investigation. Due to its genetic tractability and anatomical simplicity, the egg laying circuit of the hermaphroditic nematode, Caenorhabditis elegans, is a powerful model system to precisely examine the genetic and molecular targets of cocaine in vivo. Here, we report a novel cocaine-induced behavioral phenotype in Caenorhabditis elegans, cocaine-stimulated egg laying. In addition, we present the results of an in vivo candidate suppression screen of synthetic enzymes, receptors, degradative enzymes, and downstream components of the intracellular signaling cascades of the main neurotransmitter systems that control Caenorhabditis elegans egg laying. Our results show that cocaine-stimulated egg laying is dependent on acetylcholine synthesis and synaptic release, functional nicotinic acetylcholine receptors, and the Caenorhabditis elegans acetylcholinesterases.

Hyperproduction of 3-hydroxypropionate by Halomonas bluephagenesis

Abstract3-Hydroxypropionic acid (3HP), an important three carbon (C3) chemical, is designated as one of the top platform chemicals with an urgent need for improved industrial production. Halomonas bluephagenesis shows the potential as a chassis for competitive bioproduction of various chemicals due to its ability to grow under an open, unsterile and continuous process. Here, we report the strategy for producing 3HP and its copolymer poly(3-hydroxybutyrate-co-3-hydroxypropionate) (P3HB3HP) by the development of H. bluephagenesis. The transcriptome analysis reveals its 3HP degradation and synthesis pathways involving endogenous synthetic enzymes from 1,3-propanediol. Combing the optimized expression of aldehyde dehydrogenase (AldDHb), an engineered H. bluephagenesis strain of whose 3HP degradation pathway is deleted and that overexpresses alcohol dehydrogenases (AdhP) on its genome under a balanced redox state, is constructed with an enhanced 1.3-propanediol-dependent 3HP biosynthetic pathway to produce 154 g L−1 of 3HP with a yield and productivity of 0.93 g g−1 1,3-propanediol and 2.4 g L−1 h−1, respectively. Moreover, the strain could also accumulate 60% poly(3-hydroxybutyrate-co-32–45% 3-hydroxypropionate) in the dry cell mass, demonstrating to be a suitable chassis for hyperproduction of 3HP and P3HB3HP.