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Background: The tumour microenvironment significantly influences the clinical response of patients to therapeutic immune checkpoint inhibition (ICI), but a comprehensive understanding of the underlying immune-regulatory proteome is still lacking., Objectives: To decipher targetable biologic processes that determine tumour-infiltrating lymphocytes (TiLs) as a cellular equivalent of clinical response to ICI., Methods: We mapped the spatial distribution of proteins in TiL-enriched vs. TiL-low compartments in melanoma by combining microscopy, matrix-assisted laser desorption mass spectrometry imaging and liquid chromatography-mass spectrometry, as well as computational data mining. Pharmacological modulation of sirtuin 1 (SIRT1) activity in syngeneic mouse models was used to evaluate the efficacy of pharmacological SIRT1 activation in two syngeneic melanoma mouse models, one known to be α-programmed cell death protein 1 (PD-1) sensitive and the other α-PD-1 resistant., Results: Spatial proteomics and gene ontology-based enrichment analysis identified > 145 proteins enriched in CD8high tumour compartments, including negative regulators of mammalian target of rapamycin signalling such as SIRT1. Multiplexed immunohistochemistry confirmed that SIRT1 protein was expressed more in CD8high than in CD8low compartments. Further analysis of bulk and single-cell RNA sequencing data from melanoma tissue samples suggested the expression of SIRT1 by different lymphocyte subpopulations (CD8+ T cells, CD4+ T cells and B cells). Furthermore, we showed in vivo that pharmacological SIRT1 activation increased the immunological effect of α-PD-1 ICI against melanoma cells in mice, which was accompanied by an increase in T-cell infiltration and T-cell-related cytokines, including interferon (IFN)-γ, CCL4, CXCL9, CXCL10 and tumour necrosis factor-α. In silico analysis of large transcriptional data cohorts showed that SIRT1 was positively associated with the proinflammatory T-cell chemokines CXCL9, CXCL10 and IFN-γ, and prolonged overall survival of patients with melanoma., Conclusions: Our study deciphers the proteomics landscape in human melanoma, providing important information on the tumour microenvironment and identifying SIRT1 as having important prognostic and therapeutic implications., Competing Interests: Conflicts of interest J.M.-P. has served as a consultant for and/or has received honoraria from Bristol Myers Squibb, Novartis and Sanofi; and has received travel support from Bristol Myers Squibb, Novartis, Pierre Fabre and Therakos. S.U. declares research support from Bristol Myers Squibb and Merck Serono; speakers and advisory board honoraria from Bristol Myers Squibb, Merck Sharp & Dohme, Merck Serono, and Novartis; and meeting and travel support from Almirall, Bristol Myers Squibb, IGEA Clinical Biophysics, Merck Sharp & Dohme, Novartis, Pierre Fabre and Sun Pharma, outside the submitted work. D. Schadendorf has received grants and other support from Bristol Myers Squibb; personal fees from Bristol Myers Squibb during the conduct of the study; personal fees from Amgen; personal fees from Boehringer Ingelheim; personal fees from InFlarX; personal fees and other support from Roche; grants, personal fees and other support from Novartis; personal fees from Incyte; personal fees and other support from Regeneron; personal fees from 4SC; personal fees from Sanofi; personal fees from Neracare; personal fees from Pierre-Fabre; personal fees and other support from Merck-EMD; personal fees from Pfizer; personal fees and other support from Philiogen; personal fees from Array; and personal fees and other support from MSD Sharp & Dohme, outside the submitted work. A.R. reports grants from Novartis, Bristol Myers Squibb and Adtec; personal fees from Merck Sharp & Dohme; and nonfinancial support from Amgen, Roche, Merck Sharp & Dohme, Novartis, Bristol Myers Squibb and Teva., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Association of Dermatologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Skin microbiome dysbiosis with a Staphylococcus overabundance is a feature of actinic keratosis (AK) and squamous skin carcinoma (SCC) patients. The impact of lesion-directed treatments for AK lesions such as diclofenac (DIC) and cold atmospheric plasma (CAP) on the lesional microbiome is not established. We studied 321 skin microbiome samples of 59 AK patients treated with DIC 3% gel versus CAP. Microbial DNA from skin swabs taken before start of treatment (week 0), at the end of the treatment period (week 24), and 3 months after end of treatment (week 36) was analyzed after sequencing the V3/V4 region of the 16S rRNA gene. The relative abundance of S. aureus was scrutinized by a tuf gene specific TaqMan PCR assay. The total bacterial load and both, relative and absolute abundance of Staphylococcus genus was reduced upon both therapies at week 24 and 36 compared to week 0. Notably, the lesional microbiome of patients responding to CAP therapy at week 24 was characterized by an increased relative abundance of Corynebacterium genus compared to nonresponders. A higher relative abundance of Staphylococcus aureus at week 36 was a feature of patients classified as nonresponders for both treatments 12 weeks after therapy completion. The reduction of the Staphylococcus abundance after treatment of AK lesions and alterations linked to treatment response encourage further studies for investigation of the role of the skin microbiome for both, the carcinogenesis of epithelial skin cancer and its function as predictive therapeutic biomarker in AK. IMPORTANCE The relevance of the skin microbiome for development of actinic keratosis (AK), its progression into squamous skin cancer, and for field-directed treatment response is unknown. An overabundance of staphylococci characterizes the skin microbiome of AK lesions. In this study, analyses of the lesional microbiome from 321 samples of 59 AK patients treated with diclophenac gel versus cold atmospheric plasma (CAP) revealed a reduced total bacterial load and reduced relative and absolute Staphylococcus genus abundance upon both treatments. A higher relative Corynebacterium abundance was a feature of patients classified as responders at the end of CAP-treatment period (week 24) compared with nonresponders and the Staphylococcus aureus abundance of patients classified as responders 3 months after treatment completion was significantly lower than in nonresponders. The alterations of the skin microbiome upon AK treatment encourage further investigations for establishing its role for carcinogenesis and its function as predictive biomarker in AK., Competing Interests: The authors declare no conflict of interest.

Both targeted and untargeted mass spectrometry-based metabolomics approaches are used to understand the metabolic processes taking place in various organisms, from prokaryotes, plants, fungi to animals and humans. Untargeted approaches allow to detect as many metabolites as possible at once, identify unexpected metabolic changes, and characterize novel metabolites in biological samples. However, the identification of metabolites and the biological interpretation of such large and complex datasets remain challenging. One approach to address these challenges is considering that metabolites are connected through informative relationships. Such relationships can be formalized as networks, where the nodes correspond to the metabolites or features (when there is no or only partial identification), and edges connect nodes if the corresponding metabolites are related. Several networks can be built from a single dataset (or a list of metabolites), where each network represents different relationships, such as statistical (correlated metabolites), biochemical (known or putative substrates and products of reactions), or chemical (structural similarities, ontological relations). Once these networks are built, they can subsequently be mined using algorithms from network (or graph) theory to gain insights into metabolism. For instance, we can connect metabolites based on prior knowledge on enzymatic reactions, then provide suggestions for potential metabolite identifications, or detect clusters of co-regulated metabolites. In this review, we first aim at settling a nomenclature and formalism to avoid confusion when referring to different networks used in the field of metabolomics. Then, we present the state of the art of network-based methods for mass spectrometry-based metabolomics data analysis, as well as future developments expected in this area. We cover the use of networks applications using biochemical reactions, mass spectrometry features, chemical structural similarities, and correlations between metabolites. We also describe the application of knowledge networks such as metabolic reaction networks. Finally, we discuss the possibility of combining different networks to analyze and interpret them simultaneously., Competing Interests: Author RS is employed by Bruker BioSpin GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Amara, Frainay, Jourdan, Naake, Neumann, Novoa-del-Toro, Salek, Salzer, Scharfenberg and Witting.)

Concurrent suboptimal supply of several nutrients requires the coordination of nutrient-specific transcriptional, phenotypic, and metabolic changes in plants in order to optimize growth and development in most agricultural and natural ecosystems. Phosphate (P i ) and iron (Fe) deficiency induce overlapping but mostly opposing transcriptional and root growth responses in Arabidopsis thaliana . On the metabolite level, P i deficiency negatively modulates Fe deficiency-induced coumarin accumulation, which is controlled by Fe as well as P i deficiency response regulators. Here, we report the impact of Fe availability on seedling growth under P i limiting conditions and on P i deficiency-induced accumulation of amino acids and organic acids, which play important roles in P i use efficiency. Fe deficiency in P i replete conditions hardly changed growth and metabolite profiles in roots and shoots of Arabidopsis thaliana , but partially rescued growth under conditions of P i starvation and severely modulated P i deficiency-induced metabolic adjustments. Analysis of T-DNA insertion lines revealed the concerted coordination of metabolic profiles by regulators of Fe (FIT, bHLH104, BRUTUS, PYE) as well as of P i (SPX1, PHR1, PHL1, bHLH32) starvation responses. The results show the interdependency of P i and Fe availability and the interplay between P i and Fe starvation signaling on the generation of plant metabolite profiles.

Hypericin is a molecule of high pharmaceutical importance that is synthesized and stored in dark glands (DGs) of St. John's Wort (Hypericum perforatum). Understanding which genes are involved in dark gland development and hypericin biosynthesis is important for the development of new Hypericum extracts that are highly demanded for medical applications. We identified two transcription factors whose expression is strictly synchronized with the differentiation of DGs. We correlated the content of hypericin, pseudohypericin, endocrocin, skyrin glycosides and several flavonoids with gene expression and DG development to obtain a revised model for hypericin biosynthesis. Here, we report for the first time genotypes which are polymorphic for the presence/total absence (G+/G-) of DGs in their placental tissues (PTs). DG development was characterized in PTs using several microscopy techniques. Fourier transform infrared microscopy was established as a novel method to precisely locate polyaromatic compounds, such as hypericin, in plant tissues. In addition, we obtained transcriptome and metabolome profiles of unprecedented resolution in Hypericum. This study addresses for the first time the development of dark glands and identifies genes that constitute strong building blocks for the further elucidation of hypericin synthesis, its manipulation in plants, its engineering in microbial systems and its applications in medical research., (© 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Plant-specific EFFECTORS OF TRANSCRIPTION (ET) are characterised by a variable number of highly conserved ET repeats, which are involved in zinc and DNA binding. In addition, ETs share a GIY-YIG domain, involved in DNA nicking activity. It was hypothesised that ETs might act as epigenetic regulators. Here, methylome, transcriptome and phenotypic analyses were performed to investigate the role of ET factors and their involvement in DNA methylation in Arabidopsis thaliana. Comparative DNA methylation and transcriptome analyses in flowers and seedlings of et mutants revealed ET-specific differentially expressed genes and mostly independently characteristic, ET-specific differentially methylated regions. Loss of ET function results in pleiotropic developmental defects. The accumulation of cyclobutane pyrimidine dimers after ultraviolet stress in et mutants suggests an ET function in DNA repair., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Secondary metabolites are involved in the plant stress response. Among these are scopolin and its active form scopoletin, which are coumarin derivatives associated with reactive oxygen species scavenging and pathogen defence. Here we show that scopolin accumulation can be induced in the root by osmotic stress and in the leaf by low-temperature stress in Arabidopsis thaliana. A genetic screen for altered scopolin levels in A. thaliana revealed a mutant compromised in scopolin accumulation in response to stress; the lesion was present in a homologue of THO1 coding for a subunit of the THO/TREX complex. The THO/TREX complex contributes to RNA silencing, supposedly by trafficking precursors of small RNAs. Mutants defective in THO, AGO1, SDS3 and RDR6 were impaired with respect to scopolin accumulation in response to stress, suggesting a mechanism based on RNA silencing such as the trans-acting small interfering RNA pathway, which requires THO/TREX function., (© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.)