UV-induced ligand exchange in MHC class I protein crystals

High-throughput structure determination of protein−ligand complexes is central in drug development and structural proteomics. To facilitate such high-throughput structure determination we designed an induced replacement strategy. Crystals of a protein complex bound to a photosensitive ligand are exposed to UV light, inducing the departure of the bound ligand, allowing a new ligand to soak in. We exemplify the approach for a class of protein complexes that is especially recalcitrant to high-throughput strategies: the MHC class I proteins. We developed a UV-sensitive, “conditional”, peptide ligand whose UV-induced cleavage in the crystals leads to the exchange of the low-affinity lytic fragments for full-length peptides introduced in the crystallant solution. This “in crystallo” exchange is monitored by the loss of seleno-methionine anomalous diffraction signal of the conditional peptide compared to the signal of labeled MHC β2m subunit. This method has the potential to facilitate high-throughput crystallography in various protein families

Class I major histocompatibility complexes loaded by a periodate trigger

Class I major histocompatibility complexes (MHCs) present peptide ligands on the cell surface for recognition by appropriate cytotoxic T cells. The unstable nature of unliganded MHC necessitates the production of recombinant class I complexes through in vitro refolding reactions in the presence of an added excess of peptides. This strategy is not amenable to high-throughput production of vast collections of class I complexes. To address this issue, we recently designed photocaged MHC ligands that can be cleaved by a UV light trigger in the MHC bound state under conditions that do not affect the integrity of the MHC structure. The results obtained with photocaged MHC ligands demonstrate that conditional MHC ligands can form a generally applicable concept for the creation of defined peptide−MHCs. However, the use of UV exposure to mediate ligand exchange is unsuited for a number of applications, due to the lack of UV penetration through cell culture systems and due to the transfer of heat upon UV irradiation, which can induce evaporation. To overcome these limitations, here, we provide proof-of-concept for the generation of defined peptide−MHCs by chemical trigger-induced ligand exchange. The crystal structure of the MHC with the novel chemosensitive ligand showcases that the ligand occupies the expected binding site, in a conformation where the hydroxyl groups should be reactive to periodate. We proceed to validate this technology by producing peptide−MHCs that can be used for T cell detection. The methodology that we describe here should allow loading of MHCs with defined peptides in cell culture devices, thereby permitting antigen-specific T cell expansion and purification for cell therapy. In addition, this technology will be useful to develop miniaturized assay systems for performing high-throughput screens for natural and unnatural MHC ligands

Food regulation as fundamental ingredient in health law

In deze bijdrage richten wij ons op de regulering van één van deze gedragsrisico’s, namelijkongezond voedsel en met name fastfood. Ongeveer 60% van de volwassenen en één op dedrie kinderen in de Europese Unie (EU) kampt met overgewicht of obesitas. In de afgelopendecennia is het aantal mensen met overgewicht en obesitas alleen maar gestegen, en geenenkel land in de EU is op weg om de doelstellingen voor het verminderen van overgewichttegen 2025 te halen.7 We beginnen met een normatieve vraag: is er zoiets als een recht opgezond voedsel, en, – zo ja – hoe ziet dat recht eruit? Vervolgens bespreken wij deverschillende reguleringsopties die de overheid ter beschikking heeft om ongezond voedsel tereguleren, zoals prijsmaatregelen en het nader reguleren van de marketing. Tot slot richten wijons specifiek op een van de dimensies van voedselregulering, te weten het reguleren van degezonde voedselomgeving

Food regulation as fundamental ingredient in health law

In deze bijdrage richten wij ons op de regulering van één van deze gedragsrisico’s, namelijkongezond voedsel en met name fastfood. Ongeveer 60% van de volwassenen en één op dedrie kinderen in de Europese Unie (EU) kampt met overgewicht of obesitas. In de afgelopendecennia is het aantal mensen met overgewicht en obesitas alleen maar gestegen, en geenenkel land in de EU is op weg om de doelstellingen voor het verminderen van overgewichttegen 2025 te halen.7 We beginnen met een normatieve vraag: is er zoiets als een recht opgezond voedsel, en, – zo ja – hoe ziet dat recht eruit? Vervolgens bespreken wij deverschillende reguleringsopties die de overheid ter beschikking heeft om ongezond voedsel tereguleren, zoals prijsmaatregelen en het nader reguleren van de marketing. Tot slot richten wijons specifiek op een van de dimensies van voedselregulering, te weten het reguleren van degezonde voedselomgeving

Screening Libraries of Amphiphilic Janus Dendrimers Based on Natural Phenolic Acids to Discover Monodisperse Unilamellar Dendrimersomes

Natural, including plant, and synthetic phenolic acids are employed as building blocks for the synthesis of constitutional isomeric libraries of self-assembling dendrons and dendrimers that are the simplest examples of programmed synthetic macromolecules. Amphiphilic Janus dendrimers are synthesized from a diversity of building blocks including natural phenolic acids. They self-assemble in water or buffer into vesicular dendrimersomes employed as biological membrane mimics, hybrid and synthetic cells. These dendrimersomes are predominantly uni- or multilamellar vesicles with size and polydispersity that is predicted by their primary structure. However, in numerous cases, unilamellar dendrimersomes completely free of multilamellar assemblies are desirable. Here, we report the synthesis and structural analysis of a library containing 13 amphiphilic Janus dendrimers containing linear and branched alkyl chains on their hydrophobic part. They were prepared by an optimized iterative modular synthesis starting from natural phenolic acids. Monodisperse dendrimersomes were prepared by injection and giant polydisperse by hydration. Both were structurally characterized to select the molecular design principles that provide unilamellar dendrimersomes in higher yields and shorter reaction times than under previously used reaction conditions. These dendrimersomes are expected to provide important tools for synthetic cell biology, encapsulation, and delivery

The relationship between Europeanisation and policy styles: a study of agricultural and public health policymaking in three EU Member States

© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDer-ivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distri-bution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.The role of policy styles in policymaking has attracted renewed scholarly interestin recent years. One of the central debates in this literature revolves around thequestion of how to reconcile archetype national policy styles with considerabledifferences in modus operandi across policy sectors. A sector-specific featurethat is considered a key determinant of the manifestation of archetypenational policy styles in the European Union is the degree of Europeanisationof policy sectors. This paper picks up this suggestion by addressing thequestion of whether and how Europeanisation affects the degree to whichfeatures of an archetype national policy style are manifest within a sector. Weaddress this question by exploring sectoral policy styles in agricultural andfood-related public health policymaking across three EU Member States: TheNetherlands, the United Kingdom (England), and France. Our findings suggestthat the degree of Europeanisation of a policy sector does prove an importantcondition that helps to understand the relationship between national andsectoral policy styles. More specifically, Europeanisation has the strongesteffect when sectors face a higher adaptation pressure, i.e., when there is alarger misfit between sectoral regimes and EU-induced institutional demands.We suggest various promising avenues of future research on this relationship.Peer reviewe

Tissue patrol by resident memory CD8+ T cells in human skin

Emerging data show that tissue-resident memory T (TRM) cells play an important protective role at murine and human barrier sites. TRM cells in the epidermis of mouse skin patrol their surroundings and rapidly respond when antigens are encountered. However, whether a similar migratory behavior is performed by human TRM cells is unclear, as technology to longitudinally follow them in situ has been lacking. To address this issue, we developed an ex vivo culture system to label and track T cells in fresh skin samples. We validated this system by comparing in vivo and ex vivo properties of murine TRM cells. Using nanobody labeling, we subsequently demonstrated in human ex vivo skin that CD8+ TRM cells migrated through the papillary dermis and the epidermis, below sessile Langerhans cells. Collectively, this work allows the dynamic study of resident immune cells in human skin and provides evidence of tissue patrol by human CD8+ TRM cells.Toxicolog