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Cellular interactions are essential for tissue organization and functionality. In particular, immune cells rely on direct and usually transient interactions with other immune and non-immune populations to specify and regulate their function. To study these “kiss-and-run” interactions directlyin vivo, we previously developed LIPSTIC (Labeling Immune Partnerships by SorTagging Intercellular Contacts), an approach that uses enzymatic transfer of a labeled substrate between the molecular partners CD40L and CD40 to label interacting cells. Reliance on this pathway limited the use of LIPSTIC to measuring interactions between CD4+helper T cells and antigen presenting cells, however. Here, we report the development of a universal version of LIPSTIC (uLIPSTIC), which can record physical interactions both among immune cells and between immune and non-immune populations irrespective of the receptors and ligands involved. We show that uLIPSTIC can be used, among other things, to monitor the priming of CD8+T cells by dendritic cells, reveal the cellular partners of regulatory T cells in steady state, and identify germinal center (GC)-resident T follicular helper (Tfh) cells based on their ability to interact cognately with GC B cells. By coupling uLIPSTIC with single-cell transcriptomics, we build a catalog of the immune populations that physically interact with intestinal epithelial cells (IECs) and find evidence of stepwise acquisition of the ability to interact with IECs as CD4+T cells adapt to residence in the intestinal tissue. Thus, uLIPSTIC provides a broadly useful technology for measuring and understanding cell–cell interactions across multiple biological systems.

Germinal centers (GCs) are the site of antibody affinity maturation, a fundamental immunological process that increases the potency of antibodies and thereby their ability to protect against infection. GC biology is highly dynamic in both time and space, making it ideally suited for intravital imaging. Using multiphoton laser scanning microscopy (MPLSM), the field has gained insight into the molecular, cellular, and structural changes and movements that coordinate affinity maturation in real time in their native environment. On the other hand, several limitations of MPLSM have had to be overcome to allow full appreciation of GC events taking place across different timescales. Here, we review the technical advances afforded by intravital imaging and their contributions to our understanding of GC biology.

Maintaining high, or even sufficient, solubility of every peptide segment in chemical protein synthesis (CPS) remains a critical challenge; insolubility of just a single peptide segment can thwart a total synthesis venture. Multiple approaches have been used to address this challenge, most commonly by employing a chemical tool to temporarily improve peptide solubility. In this chapter, we discuss chemical tools for introducing semipermanent solubilizing sequences (termed helping hands) at the side chains of Lys and Glu residues. We describe the synthesis, incorporation by Fmoc-SPPS, and cleavage conditions for utilizing these two tools. For Lys sites, we discuss the Fmoc-Ddap-OH dimedone-based linker, which is achiral, synthesized in one step, can be introduced directly at primary amines, and is removed using hydroxylamine (or hydrazine). For Glu sites, we detail the new Fmoc-SPPS building block, Fmoc-Glu(AlHx)-OH, which can be prepared in an efficient process over two purifications. Solubilizing sequences are introduced directly on-resin and later cleaved with palladium-catalyzed transfer under aqueous conditions to restore a native Glu side chain. These two chemical tools are straightforward to prepare and implement, and we anticipate continued usage in "difficult" peptide segments following the protocols described herein.

As a response to the Russian invasion of Crimea, NATO returned to the core military concepts of deterrence and collective defence. This NATO adaption has recently come to include attempts to integrate offensive cyber effects into NATO force structure and response doctrine. The article argues—counter to what is publicly declared—that such an integration does little to strengthen NATO's deterrence posture and is unlikely to deter non-military, hybrid cyber activity below the threshold of collective defence. The article identifies several practical challenges to the current integration effort, which include the temporal dimension of developing exploits, battle damage assessment and deconfliction. With these challenges in mind, the article suggests that deploying minor and less resourceful cyber effects that cause persistent ‘cyber annoyances’ holds an unappreciated potential as they can drain opponent resources, disturb vital IT-systems and complicate decision-making. The article ends by arguing that NATO should not adapt its collective defence clause to cyberspace. A more active NATO in cyberspace risks undermining the cyber-intelligence norm that so far has prevented escalation and thereby increasing the likelihood that Russia misinterprets intelligence and active cyber defence activities as military preparation, armament or an attack in the making.

Native chemical ligation (NCL) enables the total chemical synthesis of proteins. However, poor peptide segment solubility remains a frequently encountered challenge. Here we introduce a traceless linker that can be temporarily attached to Glu side chains to overcome this problem. This strategy employs a new tool, Fmoc-Glu(AlHx)-OH, which can be directly installed using standard Fmoc-based solid-phase peptide synthesis. The incorporated residue, Glu(AlHx), is stable to a wide range of chemical protein synthesis conditions and is removed through palladium-catalyzed transfer under aqueous conditions. General handling characteristics, such as efficient incorporation, stability and rapid removal were demonstrated through a model peptide modified with Glu(AlHx) and a Lys(6) solubilizing tag. Glu(AlHx) was incorporated into a highly insoluble peptide segment during the total synthesis of the bacteriocin AS-48. This challenging peptide was successfully synthesized and folded, and it has comparable antimicrobial activity to the native AS-48. We anticipate widespread use of this easy-to-use, robust linker for the preparation of challenging synthetic peptides and proteins.

Cucurbit[7]uril (CB[7]) is a supramolecular binding host for peptides and proteins with N-terminal Phe. However, the low occurrence of such peptides and proteins limits broader applications of this unique host-guest interaction. Here, we report a strategy to expand the scope of CB[7]-peptide interaction by site-specifically introducing N-terminal substitutions (e.g. benzyl groups) using reductive alkylation. N-terminal benzylated peptides have similar affinity to CB[7] as native peptides with N-terminal Phe and even stronger interactions can be achieved using better ligands. We further expanded this host-guest interaction to be stimuli responsive. By introducing benzyl carboxylate substituents, the CB[7]-peptide interaction shows pH-dependent binding. Furthermore, benzyl boronate substituents led to saccharide-dependent CB[7]-peptide interactions. We demonstrated that using this strategy to introduce stronger CB[7] binders to the N-terminus of human calcitonin (hCT) results in increased aggregation stability in the presence of CB[7]. This strategy to expand CB[7]-peptide interaction scope opens opportunities for future applications in peptides and proteins.

Germinal centers (GCs) form in secondary lymphoid organs in response to infection and immunization and are the source of affinity-matured B cells. The duration of GC reactions spans a wide range, and long-lasting GCs (LLGCs) are potentially a source of highly mutated B cells. We show that rather than consisting of continuously evolving B cell clones, LLGCs elicited by influenza virus or SARS-CoV-2 infection in mice are sustained by progressive replacement of founder clones by naive-derived invader B cells that do not detectably bind viral antigens. Rare founder clones that resist replacement for long periods are enriched in clones with heavily mutated immunoglobulins, including some with very high affinity for antigen, that can be recalled by boosting. Our findings reveal underappreciated aspects of the biology of LLGCs generated by respiratory virus infection and identify clonal replacement as a potential constraint on the development of highly mutated antibodies within these structures.

Germinal centers (GCs) are microanatomical structures in secondary lymphoid organs where B cells undergo affinity maturation for antigen during the course of an immune response. This process is driven by a subset of T cells termed T follicular helper cells (Tfh) that through a multistep process gain access to the GC niche within the B cell follicle. This protocol details how to study Tfh behavior in vivo, on a single cell level, using two-photon intravital microscopy of the murine popliteal lymph node.

Germinal centers (GCs) are microanatomical structures in secondary lymphoid organs where B cells undergo affinity maturation for antigen during the course of an immune response. This process is driven by a subset of T cells termed T follicular helper cells (Tfh) that through a multistep process gain access to the GC niche within the B cell follicle. This protocol details how to study Tfh behavior in vivo, on a single cell level, using two-photon intravital microscopy of the murine popliteal lymph node.

While social support is generally considered a helpful resource for employees, it can also serve as a job stressor. Unhelpful workplace social support (UWSS) is any action taken by a supervisor and...

The application of solid-phase peptide synthesis and native chemical ligation in chemical protein synthesis (CPS) has enabled access to synthetic proteins that cannot be produced recombinantly, such as site-specific post-translationally modified or mirror-image proteins (D-proteins). However, CPS is commonly hampered by aggregation and insolubility of peptide segments and assembly intermediates. Installation of a solubilizing tag consisting of basic Lys or Arg amino acids can overcome these issues. Through the introduction of a traceless cleavable linker, the solubilizing tag can be selectively removed to generate native peptide. Here we describe the synthesis of a next-generation amine-reactive linker N-Fmoc-2-(7-amino-1-hydroxyheptylidene)-5,5-dimethylcyclohexane-1,3-dione (Fmoc-Ddap-OH) that can be used to selectively introduce semi-permanent solubilizing tags ("helping hands") onto Lys side chains of difficult peptides. This linker has improved stability compared to its predecessor, a property that can increase yields for multi-step syntheses with longer handling times. We also introduce a new linker cleavage protocol using hydroxylamine that greatly accelerates removal of the linker. The utility of this linker in CPS was demonstrated by the preparation of the synthetically challenging Shiga toxin subunit B (StxB) protein. This robust and easy-to-use linker is a valuable addition to the CPS toolbox for the production of challenging synthetic proteins.

Regulating germinal center contractionGerminal centers (GCs) in secondary lymphoid organs are where mature B cells expand and differentiate. Although GC formation is well studied, the control of GC duration and contraction is less well understood. Using intravital imaging of mouse GCs and single-cell RNA sequencing, Jacobsenet al.report that T follicular helper (TFH) cells are a critical player in this process. They found that some late-GC TFHcells upregulate the transcription factor FOXP3 and acquire a regulatory T cell–like phenotype. These cells are distinct from T follicular regulatory (TFR) cells and, unlike TFRcells, are needed to shut down the GC reaction. Tweaking this process may be key to extending GC lifetimes and enhancing antibody responses in the context of vaccination.Science, abe5146, this issue p.eabe5146

We employ time- and angle-resolved photoemission spectroscopy to study the spin- and valley-selective photoexcitation and dynamics of free carriers at the $\overline{\mathit{K}}$ and ${\overline{\mathit{K}}}^{\ensuremath{'}}$ points in singly oriented single-layer ${\mathrm{WS}}_{2}/\mathrm{Au}(111)$. Our results reveal that in the valence band maximum an ultimate valley polarization of free holes of 84% can be achieved upon excitation with circularly polarized light at room temperature. Notably, we observe a significantly smaller valley polarization for the photoexcited free electrons in the conduction band minimum. Clear differences in the carrier dynamics between electrons and holes imply intervalley scattering processes into dark states being responsible for the efficient depolarization of the excited electron population.

Einen Ansatz zur Unterstützung manueller Tätigkeiten stellen Exoskelette dar. Durch den Einsatz dieser Systeme lassen sich verschiedene biomechanische sowie arbeitsphysiologische Effekte erzielen. Verschiedene Faktoren haben hierauf Einfluss. In Studien mit einem Unterstützungssystem „Lucy“ hat sich gezeigt, dass mit physischer Unterstützung eine subjektive und quantitative Entlastung erzielt sowie die Konzentrationsleistung gesteigert werden kann. One approach to support manual activities are exoskeletons. Through the use of these systems, various biomechanical and work physiological effects can be achieved. Various factors influence this. In studies with a support system Lucy has been shown that physical support can provide subjective and quantitative relief and increase concentration.

The immune system plays a crucial role in many diseases. Activation or suppression of immunity is often related to clinical outcome. Methods to explore the dynamics of immune responses are important to elucidate their role in conditions characterized by inflammation, such as infectious disease, cancer, or autoimmunity. Immuno-PET is a noninvasive method by which disease and immune cell infiltration can be explored simultaneously. Using radiolabeled antibodies or fragments derived from them, it is possible to image disease-specific antigens and immune cell subsets. Methods: We developed a method to noninvasively image human immune responses in a relevant humanized mouse model. We generated a camelid-derived single-domain antibody specific for human class II major histocompatibility complex products and used it to noninvasively image human immune cell reconstitution in nonobese diabetic severe combined immune deficiency gamma-/- mice reconstituted with human fetal thymus, liver, and liver-derived hematopoietic stem cells (BLT mice). Results: We showed imaging of infiltrating immunocytes in BLT mice that spontaneously developed a graft-versus-host-like condition, characterized by alopecia and blepharitis. In diseased animals, we showed an increased PET signal in the liver, attributable to infiltration of activated class II major histocompatibility complex(+) T cells. Conclusion: Noninvasive imaging of immune infiltration and activation could thus be of importance for diagnosis and evaluation of treatment of graft-versus-host disease and holds promise for other diseases characterized by inflammation.