Tumor Necrosis Factor (TNF) is a key pro-inflammatory cytokine whose sensing by TNFR1 triggers gene activation or cell death induction. While TNF cytotoxicity can be beneficial during infections by supporting effective immune responses, its chronic or excessive induction is harmful and promotes inflammatory pathologies. Protective brakes, known as cell death checkpoints, normally repress TNF cytotoxicity and therefore constitute crucial safeguards against these diseases. Death by TNF mainly proceeds upon inactivation of a checkpoint by microbial effector proteins or pathological mutations. We previously identified lysosomal turnover of TNFR1 Complex II by TAX1BP1-mediated selective macro-autophagy as a brake on TNF cytotoxicity. Here, we propose an alternative mechanism that prevents TNF-induced RIPK1 kinase-independent apoptosis. We found that inhibiting the ESCRT machinery, HSC70 or TAX1BP1 interferes with the TNF-dependent targeting of activated CASPASE-8 into endosomal intralumenal vesicles (ILVs) and is associated with apoptosis induction. Furthermore, we identified TAX1BP1 and TNFR1 Complex II components as TNF-induced cargoes of extracellular vesicles, suggesting that exosomal release of TNFR1 Complex II serves as a parallel detoxification pathway to lysosomal turnover. Finally, we show that Salmonella Typhimurium and Mycobacterium tuberculosis effector proteins activate TNF cytotoxicity by inhibiting components of the ESCRT machinery involved in this detoxification process. The ESCRT machinery and the chaperone HSC70 package activated CASP8 into intralumenal vesicles preventing TNF-induced apoptosis. Microbial effector proteins interfere with this detoxification mechanism and sensitize cells to TNF cytotoxicity.

This is a multidisciplinary re-evaluation of the Guda Homiliary, a medieval manuscript copied and illuminated by a scribe who identified herself as 'a sinful woman'. We combine historical and palaeographical detective work with non-destructive Raman micro-spectroscopy to offer a multidisciplinary reassessment of this important witness to the presence of highly skilled female scribes in medieval Europe. We take a multi-scalar approach, beginning with a fresh analysis of the self-portrait initial itself, and then zooming out, first to its context within the broader manuscript, and then into the book's place within the spiritual and economic landscapes of medieval Germany and beyond.

Direct targeting of the downstream mitogen-activated protein kinase (MAPK) pathway to suppress extracellular-regulated kinase (ERK) activation in KRAS and BRAF mutant colorectal cancer (CRC) has proven clinically unsuccessful, but promising results have been obtained with combination therapies including epidermal growth factor receptor (EGFR) inhibition. To elucidate the interplay between EGF signalling and ERK activation in tumours, we used patient-derived organoids (PDOs) from KRAS and BRAF mutant CRCs. PDOs resemble in vivo tumours, model treatment response and are compatible with live-cell microscopy. We established real-time, quantitative drug response assessment in PDOs with single-cell resolution, using our improved fluorescence resonance energy transfer (FRET)-based ERK biosensor EKAREN5. We show that oncogene-driven signalling is strikingly limited without EGFR activity and insufficient to sustain full proliferative potential. In PDOs and in vivo, upstream EGFR activity rigorously amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways. Our data provide a mechanistic understanding of the effectivity of EGFR inhibitors within combination therapies against KRAS and BRAF mutant CRC.

Cryopyrin-associated periodic syndromes (CAPS) are autoinflammatory disorders caused by gain-of-function NLRP3 variants. Although NLRP3 inflammasomes mediate IL-1β secretion through Gasdermin D (GSDMD), we show that GSDMD deletion did not prevent autoinflammation in mice ubiquitously expressing the Nlrp3A350V variant. Inflamed skin of Nlrp3A350V-expressing GSDMD-deficient mice displayed citrullinated histone 3-containing neutrophil extracellular traps (CitH3-NETs). CitH3-NETs induced IL-1β secretion from murine Nlrp3A350V-expressing GSDMD-deficient macrophages as well as from human CAPS patient monocytes and macrophages. Blocking protein arginine deiminase-4 (PAD4) prevented CitH3 release and disabled the IL-1β-inducing NET effects, identifying CitH3 as crucial trigger. Mechanistically, CitH3-NETs activated GSDME in GSDMD-deficient Nlrp3A350V macrophages, and GSDME deletion prevented pathology in Nlrp3A350V-expressing GSDMD-deficient mice. In addition to this GSDME-dependent autoinflammation axis, PAD4 deletion also prevented autoinflammation in mice with neutrophil-specific Nlrp3A350V expression that develop CAPS in a GSDMD-dependent manner. These observations support a CAPS model in which PAD4-mediated CitH3-NET release can trigger both GSDMD-dependent and GSDME-dependent autoinflammation.

Cellular stress, infection, and inflammation lead to various forms of cell death. Depending on the stimulus, cell type, and cellular conditions, different modes of regulated cell death might be engaged. These include apoptosis, necroptosis, and pyroptosis, which are driven by genetically programmed mechanisms, and ferroptosis, a type of metabolic cell death. The outcome of these distinct cell death modalities is the activation of specific pore-forming mechanisms: caspase-3-mediated cleavage of gasdermin E in secondary necrosis following apoptosis (also classified as pyroptosis), RIPK3-mediated phosphorylation of MLKL in necroptosis, and caspase-1/11/4/5-mediated cleavage of GSDMD during pyroptosis. In the case of ferroptosis, a metabolic cell death modality driven by imbalances in iron, lipid, and redox metabolism, the plasma membrane also becomes permeabilized due to oxidative modifications of acyl chains in phospholipids. On top of the pore-forming mechanisms, NINJ1 detects cellular swelling ("oncosis") and triggers a massive plasma membrane rupture as a final stage of the cellular cataclysm, releasing large molecules and intracellular contents. Understanding the mechanisms of regulated necrotic cell death through signaling pathways or by disrupting metabolic networks offers tangible targeting strategies to enhance or reduce cell death processes and associated subroutines in various diseases, including cancer, ischemia/reperfusion conditions, inflammation, and degenerative diseases. Besides the molecular biology, we will concentrate this chapter on the effects of necroptosis, pyroptosis, and ferroptosis in cancer and inflammatory pathologies in the brain, intestine, and skin.

RIPK1 is a crucial regulator of cell survival, inflammation and cell death. Human RIPK1 deficiency leads to early-onset intestinal inflammation and peripheral T cell imbalance, though its role in αβT cell-mediated intestinal homeostasis remains unclear. In this study, we demonstrate that mice with RIPK1 ablation in conventional αβT cells (Ripk1ΔCD4) developed a severe small intestinal pathology characterized by small intestinal elongation, crypt hyperplasia, and duodenum-specific villus atrophy. Using mixed bone marrow chimeras reveals a survival disadvantage of αβT cells compared to γδT cells in the small intestine. Broad-spectrum antibiotic treatment ameliorates crypt hyperplasia and prevents intestinal elongation, though villus atrophy persists. Conversely, crossing Ripk1ΔCD4 with TNF receptor 1 Tnfr1-/- knockout mice rescues villus atrophy but not intestinal elongation. Finally, combined ablation of Ripk1∆CD4 and Casp8∆CD4 fully rescues intestinal pathology, revealing that αβT cell apoptosis in Ripk1∆CD4 drives the enteropathy. These findings demonstrate that RIPK1-mediated survival of αβT cells is essential for proximal small intestinal homeostasis. In Ripk1∆CD4 mice, the imbalanced T cell compartment drives microbiome-mediated intestinal elongation and TNF-driven villus atrophy.

Positive selection of thymocytes is essential for laying the foundations of the mammalian immune system that include the T cell repertoire, self-tolerance, and prevention of autoimmunity. Themis, the archetypal member of a metazoan protein family featuring distinctive CABIT domains, crucially regulates thymocyte positive selection by linking signalling by the T cell receptor (TCR) to the linker of activation of TCR (LAT). Intriguingly, Themis has been proposed to function via a constitutive complex with the multifunctional adaptor Grb2. Although poised to represent a paradigm shift in our understanding of TCR signalling, the structural and mechanistic basis of such an assembly has remained enigmatic. Here, we present the cryo-EM structure of Themis in complex with Grb2, which reveals how the tandem CABIT domains of Themis engulf the C-terminal SH3 domain of Grb2 (Grb2SH3C) to enable its latching onto the proline rich sequence of Themis. The remaining two domains of Grb2 adopt at least three conformational poses set to interact with other binding partners such as Sos1. Structural insights from unbound Themis unmask the pronounced flexibility of the CABIT domains of Themis, which becomes ordered upon binding to Grb2 to create a binding hotspot for their constitutive complex. Indeed, Themis variants that abrogate interactions with Grb2 also fail to activate the tyrosine phosphatase SHP-1 after TCR stimulation, analogous to the functional phenotype of Themis-deficient cells. Collectively, our study draws the blueprint of the Themis-Grb2 complex as a dynamic structural hub in T cell development. ### Competing Interest Statement The authors have declared no competing interest.

ABSTRACT Macro‐Raman mapping has been proposed as a novel technique to visualise the molecular distribution over a surface of typically several tens of cm 2 . By combining large stages with a fibre‐optic probe head of a Raman spectrometer and appropriate data processing, it is possible to obtain large Raman maps. Therefore, a data cube containing thousands of spectra is processed in such a way that the relevant spectral information is represented as different colours in the image. A mapping experiment consists of the alignment, the recording of the spectra and postprocessing. In the current work, it is aimed to deliver a proof of concept of the macro‐Raman mapping of a three‐dimensional (3‐D) object, such as a statue. When Raman mapping experiments are performed using microscope objectives, the surface should be relatively flat, and the distance between the surface and the spectrometer cannot display abrupt changes. Therefore, the macro‐Raman set‐up needs to be equipped with a long working distance lens. By measuring for each point the distance to the surface, and by knowing the positions of the stage, the recorded spectral information is related to the 3‐D position of the measurement point. Thus, a 3‐D point cloud is obtained, and the linked spectral information is colour‐coded to obtain a 3‐D macro‐Raman map. To be able to record information from all areas of the painted statuette, including from points that are on planes parallel to the laser beam, several maps have been recorded, and the point clouds were merged, resulting in a complete reconstruction of the 3‐D artefact.

Background and aimsCell death plays a central role in atheroma plaque progression and aggravation. This study investigates the role of caspase-8 in regulating macrophage cell death modalities, specifically apoptosis and necroptosis, within atheroma plaques.MethodsBone marrow from caspase-8-deficient (Casp8komac) and cohoused wildtype littermates were transplanted in atherosclerosis-prone Ldlr-/- recipient mice fed with a proatherogenic diet. Aortic plaque development, necrotic core formation, and cell death were analyzed through histological and biochemical assays. In vitro investigation of macrophages exposed to atherogenic stimuli assessed the effects of caspase-8 inhibition on apoptotic and necroptotic pathways.ResultsDespite lower plasma cholesterol levels and reduced number of inflammatory monocytes, caspase-8-deficient mice exhibited more pronounced atherosclerotic lesions with enlarged necrotic cores and an increased number of dead cells. In vitro, in macrophages exposed to oxidized LDL or oxysterols, the inhibition of caspase-8 revealed a shift from apoptosis to necroptosis as confirmed by increased phosphorylation of MLKL along with decreased cleavage of caspase-3 and -7.Discussion and perspectivesThe study highlights the role of caspase-8 in atherosclerosis in tuning the balance between apoptosis and necroptosis. Caspase-8 inhibition leads to a switch towards necroptosis and accumulation of dead cell corpses that contributes to enhanced plaque severity. These findings suggest that reducing caspase-8-regulated necroptosis and necrosis in macrophages could represent a therapeutic strategy to stabilize plaques and reduce cardiovascular risk.

Epithelial to Mesenchymal transitions (EMT) drive cell plasticity and are associated with cell features such as invasiveness, migration and stemness. They are orchestrated by select families of EMT-associated transcription factors, which exhibit pleiotropic roles in the malignant progression of various cancer types, such as breast and colorectal cancer (CRC). This has spurred interest in EMT as a promising target for the development of novel therapeutic strategies. In this study, we developed a phenotypic dual EMT Sensor screening assay, amendable to efficient high-throughput identification of small molecules interfering with EMT. In a proof-of-concept screening we identified anti-EMT repurposing drugs. From these, we validated RepSox, a selective inhibitor of the TGF-β type I receptor ALK5, and demonstrated that it is potently blocking EMT in both breast and colorectal cancer cell lines in vitro. In addition, utilizing a Drosophila melanogaster metastatic CRC model we confirmed the ability of the identified anti-EMT hits to suppress metastatic behavior in vivo.

Advancements in Raman spectroscopy have broadened the utilization possibilities for food applications. The present review covers the working principle and methodology of the emerging technique in the context of wheat (flour) as a bakery ingredient. Special attention is paid to the primary constituents of wheat flour, starch and gluten proteins, both in their isolated forms and within complex matrices such as flour, dough, and various end products. This review examines how compositional and structural variations in these components are reflected in their Raman spectra and imaging characteristics and how this can be interpreted in terms of quality and functionality. The review concludes by outlining prospective research directions and future opportunities for advancing Raman-based analysis in cereal and bakery science.

The efficacy of conventional chemotherapy does not only rely on the cytotoxic action of the drug compound itself. Indeed, proper drug-induced immunogenic cell death (ICD) can stimulate immunosurveillance and mount a systemic anti-tumor response. We aimed to further amplify the therapeutic activity of oxaliplatin (OxPt) chemotherapy-induced ICD by combining this with an imidazoquinoline (IMDQ) TLR7/8 agonist. We hypothesized that innate immune activation by TLR7/8 activation primes the immune system against tumor neoantigens, thereby mounting tumor-specific T cell responses that contribute to killing primary tumor cells and distal metastases. To this end, we initially synthesized a covalent conjugate of OxPt, an imidazoquinoline TLR7/8 agonist (i.e., IMDQ), and an alkyl lipid. We hypothesized that such a lipidated conjugate would, upon intratumoral injection, increase the residence time in the tumor and reduce systemic dissemination and, hence, off-target toxicity. Whereas combination therapy with OxPt and IMDQ in native form improved, relative to single treatment, the anti-tumor efficacy against the primary treated tumor and a secondary distal tumor, this was not the case for OxPt-IMDQ-lipid conjugate therapy. We then altered the molecular design of the combination therapy and synthesized amphiphilic OxPt and IMDQ conjugates, comprising a cholesteryl motif and a hydrophilic poly(ethylene glycol) (PEG) chain. Intratumoral combination therapy with OxPt-PEG-cholesteryl and IMDQ-PEG-cholesteryl reduced, compared to native drug compounds, systemic innate inflammatory responses, and more efficiently eradicated primary and distal tumors. Furthermore, we found that combination therapy with OxPt-PEG-cholesteryl and IMDQ-PEG-cholesteryl induced antigen-specific anti-tumor responses and high infiltration levels of CD8+ T cells into the tumor.

Cytotoxic chemotherapies have devastating side effects, particularly within the gastrointestinal tract. Gastrointestinal toxicity includes the death and damage of the epithelium and an imbalance in the intestinal microbiota, otherwise known as dysbiosis. Whether dysbiosis is a direct contributor to tissue toxicity is a key area of focus. Here, from both mammalian and bacterial perspectives, we uncover an intestinal epithelial cell death-Enterobacteriaceae signaling axis that fuels dysbiosis. Specifically, our data demonstrate that chemotherapy-induced epithelial cell apoptosis and the purine-containing metabolites released from dying cells drive the inter-kingdom transcriptional re-wiring of the Enterobacteriaceae, including fundamental shifts in bacterial respiration and promotion of purine utilization-dependent expansion, which in turn delays the recovery of the intestinal tract. Inhibition of epithelial cell death or restriction of the Enterobacteriaceae to homeostatic levels reverses dysbiosis and improves intestinal recovery. These findings suggest that supportive therapies that maintain homeostatic levels of Enterobacteriaceae may be useful in resolving intestinal disease.

The thymus plays a pivotal role in generating a highly-diverse repertoire of T lymphocytes while preventing autoimmunity. Thymus seeding progenitors (TSPs) are a heterogeneous group of multipotent progenitors that migrate to the thymus via CCR7 and CCR9 receptors. While NOTCH guides thymus progenitors toward T cell fate, the absence or disruption of NOTCH signaling renders the thymus microenvironment permissive to other cell fates. Following T cell commitment, developing T cells undergo multiple selection checkpoints by engaging with the extracellular matrix, and interacting with thymic epithelial cells (TECs) and other immune subsets across the different compartments of the thymus. The different selection checkpoints assess the T cell receptor (TCR) performance, with failure resulting in either repurposing (agonist selection), or cell death. Additionally, environmental cues such as inflammation and endocrine signaling induce acute thymus atrophy, contributing to the demise of most developing T cells during thymic selection. We discuss the occurrence of acute thymus atrophy in response to systemic inflammation. The thymus demonstrates high plasticity, shaping inflammation by abrogating T cell development and undergoing profound structural changes, and facilitating regeneration and restoration of T cell development once inflammation is resolved. Despite the challenges, thymic selection ensures a highly diverse T cell repertoire capable of discerning between self and non-self antigens, ultimately egressing to secondary lymphoid organs where they complete their maturation and exert their functions.

Acute systemic inflammation critically alters the function of the immune system, often promoting myelopoiesis at the expense of lymphopoiesis. In the thymus, systemic inflammation results in acute thymic atrophy and, consequently, impaired T-lymphopoiesis. The mechanism by which systemic inflammation impacts the thymus beyond suppressing T-cell development is still unclear. Here, we describe how the synergism between TL1A and IL-18 suppresses T-lymphopoiesis to promote thymic myelopoiesis. The protein levels of these two cytokines were elevated in the thymus during viral-induced thymus atrophy infection with murine cytomegalovirus (MCMV) or pneumonia virus of mice (PVM). In vivo administration of TL1A and IL-18 induced acute thymic atrophy, while thymic neutrophils expanded. Fate mapping with Ms4a3-Cre mice demonstrated that thymic neutrophils emerge from thymic granulocyte-monocyte progenitors (GMPs), while Rag1-Cre fate mapping revealed a common developmental path with lymphocytes. These effects could be modeled ex vivo using neonatal thymic organ cultures (NTOCs), where TL1A and IL-18 synergistically enhanced neutrophil production and egress. NOTCH blockade by the LY411575 inhibitor increased the number of neutrophils in the culture, indicating that NOTCH restricted steady-state thymic granulopoiesis. To promote myelopoiesis, TL1A, and IL-18 synergistically increased GM-CSF levels in the NTOC, which was mainly produced by thymic ILC1s. In support, TL1A- and IL-18-induced granulopoiesis was completely prevented in NTOCs derived from Csf2rb-/- mice and by GM-CSFR antibody blockade, revealing that GM-CSF is the essential factor driving thymic granulopoiesis. Taken together, our findings reveal that TL1A and IL-18 synergism induce acute thymus atrophy while promoting extramedullary thymic granulopoiesis in a NOTCH and GM-CSF-controlled manner.

Macro-Raman mapping is an approach that is well suited to study the distribution of molecules over a surface of typically several cm2, such as the pigment distribution on a painting. During such an experiment, thousands Raman spectra are recorded in a predefined array of points. As there can be a huge difference in Raman sensitivity between different molecules, an alternative approach is proposed, where measurement times are adapted according to the quality of the Raman scatterer. Therefore, an iterative process is implemented, involving recording short accumulations, followed by a quick evaluation. As soon as the accumulated spectrum reaches a quality criterium, measurement of the next point is started, while in the other case further accumulations are added. The influence of using multiple accumulations opposite to a single measurement for a prolonged period has been assessed by comparing the overall measurement time and the spectral intensity of the main Raman band (520.5 cm-1) of silicon. Another prerequisite for this algorithm to be successful is the need for a quick and reliable evaluation of the spectral quality. This was examined by applying this function on the accumulations of Raman spectra of 49 paint samples from a colour chart. Finally, the variable-accumulation algorithm is applied during a macro-Raman mapping experiment of a watercolour painting on paper. Despite a significant gain in overall measuring time, this novel approach yields a qualitative data set that can be used for creating clear Raman maps, using different data processing methods. A new Macro-Raman mapping algorithm is proposed, where for each spectrum the number of accumulations is adapted according to the Raman sensitivity of the molecules present. image

Macro-Raman mapping is an approach that allows to obtain high-resolution molecular maps of artefacts, over an area of several square centimetres. The method is based on recording thousands of spectra in a grid. The main drawback of this method is that it is very time consuming. A straightforward approach to reduce the measurement time is achieved by reducing the number of points that are measured. Not all points of the map are equally informative: Pixels that are surrounded by similar points might be less interesting to measure. Therefore, an algorithm is proposed to select where to measure and which points to omit. The missing pixels can be filled in a posteriori, by using a suitable interpolation algorithm. The selection of the omitted pixels can be performed based on information that is available from other analytical techniques. In this case, a selection was made based on the local variances in a colour picture. The approach is evaluated by recording macro-Raman maps of details of a Neptune watercolour painting on paper. In a first stage, the Raman intensities of the scaled and baseline corrected spectra at specific band positions were colour coded and plotted as Raman maps. The interpolation of the Raman maps yielded satisfying results. The image outline could clearly be identified in the maps, and the differently coloured zones were distinguished. Next to this univariate approach, it was demonstrated that also a multivariate data extraction method (principal component analysis) is compatible with the proposed algorithm to measure 25% less datapoints. By selecting carefully some points with a lower information content, it is possible to reduce the sample points and thus to enhance the speed of the macro-Raman mapping experiment. image

The T cell population size is stringently controlled before, during, and after immune responses, as improper cell death regulation can result in autoimmunity and immunodeficiency. RIPK1 is an important regulator of peripheral T cell survival and homeostasis. However, whether different peripheral T cell subsets show a differential requirement for RIPK1 and which programmed cell death pathway they engage in vivo remains unclear. In this study, we demonstrate that conditional ablation of Ripk1 in conventional T cells (Ripk1ΔCD4) causes peripheral T cell lymphopenia, as witnessed by a profound loss of naive CD4+, naive CD8+, and FoxP3+ regulatory T cells. Interestingly, peripheral naive CD8+ T cells in Ripk1ΔCD4 mice appear to undergo a selective pressure to retain RIPK1 expression following activation. Mixed bone marrow chimeras revealed a competitive survival disadvantage for naive, effector, and memory T cells lacking RIPK1. Additionally, tamoxifen-induced deletion of RIPK1 in CD4-expressing cells in adult life confirmed the importance of RIPK1 in post-thymic survival of CD4+ T cells. Ripk1K45A mice showed no change in peripheral T cell subsets, demonstrating that the T cell lymphopenia was due to the scaffold function of RIPK1 rather than to its kinase activity. Enhanced numbers of Ripk1ΔCD4 naive T cells expressed the proliferation marker Ki-67+ despite the peripheral lymphopenia and single-cell RNA sequencing revealed T cell-specific transcriptomic alterations that were reverted by additional caspase-8 deficiency. Furthermore, Ripk1ΔCD4Casp8 ΔCD4 and Ripk1ΔCD4Tnfr1−/− double-knockout mice rescued the peripheral T cell lymphopenia, revealing that RIPK1-deficient naive CD4+ and CD8+ cells and FoxP3+ regulatory T cells specifically die from TNF- and caspase-8-mediated apoptosis in vivo. Altogether, our findings emphasize the essential role of RIPK1 as a scaffold in maintaining the peripheral T cell compartment and preventing TNFR1-induced apoptosis.