Purified sardine and king crab trypsin stimulate IL-8 secretion and NF-κB activation, at least partly, via PAR2, but displays individual differences in transformation of the NF-κB-signal.

This article is part of Anett Kristin Larsen's doctoral thesis. Available at http://hdl.handle.net/10037/2892Respiratory symptoms occur in workers processing a great variety of seafood. Studies previously showed that salmon trypsin increases transcriptional activity of NF-κB and induces secretion of IL-8 from airway epithelial cells by activating PAR-2. The aim of this study was to explore if purified trypsins from king crab (Paralithodes camtschaticus) and sardine (Sardinops melanostictus) are able to induce similar effects in cell stimulation assays. The knowledge that crustaceans seem to display dissimilar irritant potency compared to fish inspired us to investigate if one could detect differences in intracellular signaling pathways coupled to IL-8 in human airway epithelial cells (A549). Both sardine and king crab trypsin induced secretion of IL-8 from human airway epithelial cells in a concentration-dependent manner and increased transcriptional activity of NF-κB. With the use of siRNA data indicate that these effects are both mediated, at least partly, through the activation of PAR-2. Additionally, the king crab and sardine trypsin display individual differences in transformation of the NF-κB signal into subsequent IL-8 secretion. The contribution of MEK/ERK, p38, and NF-κB to the secretion of IL-8 following stimulation with sardine and king crab trypsins were examined with the use of specific inhibitors. The results demonstrated that MEK/ERK and NF-κB are both required for sardine and king crab trypsin-induced secretion of IL-8 but via separate pathways. P38 was also found to contribute to the secretion of IL-8 seemingly by NF-κB-dependent processes

Simple Preparation of Pacific Cod Trypsin for Enzymatic Peptide Synthesis

Trypsin from the pyloric caeca of Pacific cod (Gadus macrocephalus) was easily prepared by affinity chromatography on Benzamidine Sepharose 6B and gel filtration on Superdex 75. Pacific cod trypsin was composed of three isozymes, and their molecular masses were estimated 23,756.34 Da, 23,939.62 Da, and 24,114.81 Da by desorption/ionization time-of-flight mass spectroscopy (MALDI/TOF-MS) and their isoelectric points (pIs) were approximately 5.1, 6.0, and 6.2, respectively. The isolated Pacific cod trypsin showed high similarity to other frigid-zone fish trypsins. The kinetic behavior of tryptic hydrolysis toward N-p-tosyl-L-arginine methyl ester hydrochloride (TAME), N-benzoyl-L-arginine p-nitroanilide hydrochloride (BAPA), and p-amidinophenyl ester were also analyzed. In addition, the cod trypsin-catalyzed dipeptide synthesis was investigated using twelve series of “inverse subdtrates” that is p- and m-isomer of amidinophenyl, guanidinophenyl, (amidinomethyl)phenyl, (guanidinomethyl)phenyl, and four position isomers of guanidinonaphtyl esters derived from N-(tert-butoxycarbonyl)amino acid as acyl donor components. They were found to couple with an acyl acceptor such as L-alanine p-nitroanilide to produce dipeptide in the presence of the trypsin. All inverse substrates tested in this study undergo less enantioselective coupling reaction. The p-guanidinophenyl ester was most practical substrate in twelve series tested. The enzymatic hydrolysis of the resulting products was negligible

Exploiting metallophilicity for the assembly of inorganic nanocrystals and conjugated organic molecules

The accurate engineering of interfaces between inorganic nanocrystals and semiconducting organic molecules is currently viewed as key for further developments in critical fields such as photovoltaics and photocatalysis. In this work, a new and unconventional source of interface interaction based on metal–metal bonds is presented. With this aim, an AuI organometallic gelator was exploited for the formation of hydrogel-like nanocomposites containing inorganic nanoparticles and conjugated organic molecules. Noteworthy, the establishment of metallophilic interactions at the interface between the two moieties greatly enhances interparticle coupling in the composites. Thus, we believe that this new hybrid system might represent a promising alternative in several fields, such as in the fabrication of improved light-harvesting devices.Peer ReviewedPostprint (author's final draft

Poly(amidoamine)s synthesis, characterisation and interaction with BSA

Cationic poly(amidoamine)s (PAAs) were synthesised and characterised by NMR and gel permeation chromatography. Their thermal properties were investigated using thermogravimetric analysis and differential scanning calorimetry. Although poly(amidoamine)s have been used as endosomolytic polymers for protein intracellular delivery, the interaction of the polymers with the proteins still need to be investigated. BSA was used as a model protein and complexation with the different poly(amidoamine) s was investigated using gel retardation assays, fluorescence spectroscopy and high sensitivity differential scanning calorimetry. Our results indicate that the thermal stability of BSA was affected upon interaction and complexation with the poly(amidoamine)s, however these interactions did not seem to modify the structure of the protein. Polymer flexibility seemed to favour polymer/protein complexation and promoted thermal stability

A multi-redox responsive cyanometalate-based metallogel

A tetrathiafulvalene (TTF) based tridentate ligand (-(4’-methyl-4,5-di-n-dodecylthylthiotetrathiafulvalene-5’-ylthio)-’-[tris-2,2,2-(1-pyrazolyl)ethoxy]-p-xylene) (L) with long-chain alkyl moieties was prepared in order to obtain a new multi-redox active gelator based on a mixed-metal octanuclear complex [FeIII4NiII4(CN)12(tp)4(L)4](BF4)4 (1). The magnetism, electrochemistry and gelation behaviour of 1 were studied and 1,2-dichlorobenzene solutions of 1 are shown to display thermoreversible gelation behaviour at room temperature. Furthermore, the gel phase of 1 is shown to undergo room-temperature gel-to-sol transformations induced by both the oxidation and reduction of the gelator complex by F4TCNQ or [FeII(Cp*)2], respectively

Polymeric Core–Shell Assemblies Mediated by Host–Guest Interactions: Versatile Nanocarriers for Drug Delivery

No AbstractPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/61526/1/982_ftp.pd

Tuning the Size of Nanoassembles: A Hierarchical Transfer of Information from Dendrimers to Polyion Complexes

This is the peer reviewed version of the following article: Amaral, S., Tawara, M., Fernandez-Villamarin, M., Borrajo, E., Martínez-Costas, J., & Vidal, A. et al. (2018). Tuning the Size of Nanoassembles: A Hierarchical Transfer of Information from Dendrimers to Polyion Complexes. Angew. Chem. Int. Ed., 57(19), 5273-5277, which has been published in final form at https://doi.org/10.1002/anie.201712244. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived VersionsThe generation of dendrimers is a powerful tool in the control of the size and biodistribution of polyion complexes (PIC). Using a combinatorial screening of six dendrimers (18–243 terminal groups) and five oppositely charged PEGylated copolymers, a dendrimer‐to‐PIC hierarchical transfer of structural information was revealed with PIC diameters that increased from 80 to 500 nm on decreasing the dendrimer generation. This rise in size, which was also accompanied by a micelle‐to‐vesicle transition, is interpreted according to a cone‐ to rod‐shaped progression in the architecture of the unit PIC (uPIC). This precise size tuning enabled dendritic PICs to act as nanorulers for controlled biodistribution. Overall, a domino‐like control of the size and biological properties of PIC that is not attainable with linear polymers is feasible through dendrimer generationMinisterio de Economía y Competitividad. Grant Numbers: CTQ2015-69021-R, CTQ2014-61470-EXP, BFU2013-43513-R Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia. Grant Numbers: GRC2014/040, Centro Singular de Investigación de Galicia Accreditation 2016-2019, ED431G/09 European Regional Development FundS

Incorporation of proteins into complex coacervates

Complex coacervates have found a renewed interest in the past few decades in various fields such as food and personal care products, membraneless cellular compartments, the origin of life, and, most notably, as a mode of transport and stabilization of drugs. Here, we describe general methods for characterizing the phase behavior of complex coacervates and quantifying the incorporation of proteins into these phase separated materials