Insights into the effects of N-glycosylation on the characteristics of the VC1 domain of the human receptor for advanced glycation end products (RAGE) secreted by Pichia pastoris

Advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs), resulting from non-enzymatic modifications of proteins, are potentially harmful to human health. They directly act on proteins, affecting structure and function, or through receptor-mediated mechanisms. RAGE, a type I transmembrane glycoprotein, was identified as a receptor for AGEs. RAGE is involved in chronic inflammation, oxidative stress-based diseases and ageing. The majority of RAGE ligands bind to the VC1 domain. This domain was successfully expressed and secreted by Pichia pastoris. Out of two N-glycosylation sites, one (Asn25) was fully occupied while the other (Asn81) was under-glycosylated, generating two VC1 variants, named p36 and p34. Analysis of N-glycans and of their influence on VC1 properties were here investigated. The highly sensitive procainamide labeling method coupled to ES-MS was used for N-glycan profiling. N-glycans released from VC1 ranged from Man9GlcNAc2- to Man15GlcNAc2- with major Man10GlcNAc2- and Man11GlcNAc2- species for p36 and p34, respectively. Circular dichroism spectra indicated that VC1 maintains the same conformation also after removal of N-glycans. Thermal denaturation curves showed that the carbohydrate moiety has a small stabilizing effect on VC1 protein conformation. The removal of the glycan moiety did not affect the binding of VC1 to sugar-derived AGE- or malondialdehyde-derived ALE-human serum albumin. Given the crucial role of RAGE in human pathologies, the features of VC1 from P. pastoris will prove useful in designing strategies for the enrichment of AGEs/ALEs from plasma, urine or tissues, and in characterizing the nature of the interaction

Multi-level analysis of on-chip optical wireless links

Networks-on-chip are being regarded as a promising solution to meet the on-going requirement for higher and higher computation capacity. In view of future kilo-cores architectures, electrical wired connections are likely to become inefficient and alternative technologies are being widely investigated. Wireless communications on chip may be therefore leveraged to overcome the bottleneck of physical interconnections. This work deals with wireless networks-on-chip at optical frequencies, which can simplify the network layout and reduce the communication latency, easing the antenna on-chip integration process at the same time. On the other end, optical wireless communication on-chip can be limited by the heavy propagation losses and the possible cross-link interference. Assessment of the optical wireless network in terms of bit error probability and maximum communication range is here investigated through a multi-level approach. Manifold aspects, concurring to the final system performance, are simultaneously taken into account, like the antenna radiation properties, the data-rate of the core-to core communication, the geometrical and electromagnetic layout of the chip and the noise and interference level. Simulations results suggest that communication up to some hundreds of \u3bcm can be pursued provided that the antenna design and/or the target data-rate are carefully tailored to the actual layout of the chip

Multi-level analysis of on-chip optical wireless links

Networks-on-chip are being regarded as a promising solution to meet the on-going requirement for higher and higher computation capacity. In view of future kilo-cores architectures, electrical wired connections are likely to become inefficient and alternative technologies are being widely investigated. Wireless communications on chip may be therefore leveraged to overcome the bottleneck of physical interconnections. This work deals with wireless networks-on-chip at optical frequencies, which can simplify the network layout and reduce the communication latency, easing the antenna on-chip integration process at the same time. On the other end, optical wireless communication on-chip can be limited by the heavy propagation losses and the possible cross-link interference. Assessment of the optical wireless network in terms of bit error probability and maximum communication range is here investigated through a multi-level approach. Manifold aspects, concurring to the final system performance, are simultaneously taken into account, like the antenna radiation properties, the data-rate of the core-to core communication, the geometrical and electromagnetic layout of the chip and the noise and interference level. Simulations results suggest that communication up to some hundreds of μm can be pursued provided that the antenna design and/or the target data-rate are carefully tailored to the actual layout of the chip

Sakacin-A antimicrobial packaging for decreasing Listeria contamination in thincut meat: preliminary assessment

BACKGROUND: Minimally processed ready-to-eat products are considered a high-risk food because of the possibility of contamination with pathogenic bacteria, including Listeria monocytogenes from the animal reservoir, and the minimal processing they undergo. In this study, a sakacin-A anti-Listeria active package was developed and tested on thin-cut veal meat slices (carpaccio). RESULTS: Enriched food-grade sakacin-A was obtained from a cell-free supernatant of a Lactobacillus sakei culture and applied (0.63 mg cm 122) onto the surface of polyethylene-coated paper sheets to obtain an active antimicrobial package. The coating retained antimicrobial features, indicating that the process did not affect sakacin-A functionality, as evidenced in tests carried out in vitro. Thin-cut veal meat slices inoculated with Listeria innocua (a surrogate of pathogenic L. monocytogenes) were laid on active paper sheets. After 48 h incubation at 4 \ub0C, the Listeria population was found to be 1.5 log units lower with respect to controls (3.05 vs 4.46 log colony-forming units (CFU) g 121). CONCLUSION: This study demonstrates the possibility of using an antimicrobial coating containing sakacin-A to inhibit or decrease the Listeria population in ready-to-eat products, thus lowering the risk of food-related diseases

Imine Deaminase Activity and Conformational Stability of UK114, the Mammalian Member of the Rid Protein Family Active in Amino Acid Metabolism

Abstract: Reactive intermediate deaminase (Rid) protein family is a recently discovered group of enzymes that is conserved in all domains of life and is proposed to play a role in the detoxification of reactive enamines/imines. UK114, the mammalian member of RidA subfamily, was identified in the early 90s as a component of perchloric acid-soluble extracts from goat liver and exhibited immunomodulatory properties. Multiple activities were attributed to this protein, but its function is still unclear. This work addressed the question of whether UK114 is a Rid enzyme. Biochemical analyses demonstrated that UK114 hydrolyzes -imino acids generated by L- or D-amino acid oxidases with a preference for those deriving from Ala > Leu = L-Met > L-Gln, whereas it was poorly active on L-Phe and L-His. Circular Dichroism (CD) analyses of UK114 conformational stability highlighted its remarkable resistance to thermal unfolding, even at high urea concentrations. The half-life of heat inactivation at 95 C, measured from CD and activity data, was about 3.5 h. The unusual conformational stability of UK114 could be relevant in the frame of a future evaluation of its immunogenic properties. In conclusion, mammalian UK114 proteins are RidA enzymes that may play an important role in metabolism homeostasis also in these organisms

Study of hypothalamic metabolism in cluster headache by proton MR spectroscopy

The authors used 1H-MRS to investigate hypothalamic metabolism in 26 patients with cluster headache (CH) and 12 healthy subjects. Hypothalamic N-acetylaspartate/creatine was reduced in patients with CH vs controls (p < 0.01). Dividing the patients into episodic CH outside- and in-cluster periods and chronic CH, the hypothalamic N-acetylaspartate/creatine in all three subgroups of patients was reduced. The reduction of the neuronal marker N-acetylaspartate is consistent with hypothalamic neuronal dysfunction in patients with CH

Two Novel Fish Paralogs Provide Insights Into the Rid Family of Imine Deaminases Active in Pre-Empting enamine/imine Metabolic Damage

Reactive Intermediate Deaminase (Rid) protein superfamily includes eight families among which the RidA is conserved in all domains of life. RidA proteins accelerate the deamination of the reactive 2-aminoacrylate (2AA), an enamine produced by some pyridoxal phosphate (PLP)-dependent enzymes. 2AA accumulation inhibits target enzymes with a detrimental impact on fitness. As a consequence of whole genome duplication, teleost fish have two ridA paralogs, while other extant vertebrates contain a single-copy gene. We investigated the biochemical properties of the products of two paralogs, identified in Salmo salar. SsRidA-1 and SsRidA-2 complemented the growth defect of a Salmonella enterica ridA mutant, an in vivo model of 2AA stress. In vitro, both proteins hydrolyzed 2-imino acids (IA) to keto-acids and ammonia. SsRidA-1 was active on IA derived from nonpolar amino acids and poorly active or inactive on IA derived from other amino acids tested. In contrast, SsRidA-2 had a generally low catalytic efficiency, but showed a relatively higher activity with IA derived from L-Glu and aromatic amino acids. The crystal structures of SsRidA-1 and SsRidA-2 provided hints of the remarkably different conformational stability and substrate specificity. Overall, SsRidA-1 is similar to the mammalian orthologs whereas SsRidA-2 displays unique properties likely generated by functional specialization of a duplicated ancestral gene

Dielectric and plasmonic vivaldi antennas for on-chip wireless communication

In this paper, different technologies enabling wireless on-chip communication are investigated. In particular, plasmonic Vivaldi antennas coupled to silicon waveguides and all-dielectric Vivaldi antennas are proposed. The design criteria and the performances of the two antenna configurations are also discussed

Antimicrobial activity of lysozyme and lactoferrin incorporated in cellulose-based food packaging

The antimicrobial proteins lysozyme and lactoferrin were incorporated into paper containing carboxymethyl cellulose, that allowed non-covalent binding of the positively charged proteins to the paper matrix. More than 60 percent of the proteins added alone or in combination during the papermaking process were released in buffered saline. The released proteins retained their structural and functional features, indicating that the papermaking process did not affect their structure. The antimicrobial activity on common food contaminants was also retained in the released protein, and a synergism between the two proteins was evident in tests carried out with paper containing both proteins against Listeria. Tests on thin meat slices laid on paper sheets containing either or both antimicrobial proteins indicated that lysozyme was most effective in preventing growth of this particular microbiota