Adipose tissue homeostasis plays a critical role in metabolic disease but the metabolic circuitry regulating adipose tissue dynamics remains unclear. In this study, polyamine metabolism emerges as an important regulator of adipose tissue pathophysiology. We identify AZIN2 (Antizyme inhibitor 2), a protein promoting polyamine synthesis and acetylation, as a major regulator of total acetyl-CoA in adipocyte progenitors (APs). AZIN2 deficient APs demonstrate increased H3K27 acetylation marks in genes related to lipid metabolism, cell cycle arrest and cellular senescence, and enhanced adipogenesis compared to wild-type counterparts. Upon high-fat diet (HFD)-induced obesity, AZIN2 deficiency in mice provokes enhanced AP adipogenic commitment, adipose tissue hypertrophy, lipid storage perturbations, inflammation and insulin resistance. IL4 promotes Azin2 expression in APs but not mature adipocytes due to diminished IL4 receptor expression in the latter. In human visceral and subcutaneous adipose tissue, AZIN2 expression positively correlates with expression of early progenitor markers and genes associated with protection against insulin resistance, while it negatively correlates with markers of lipogenesis. In sum, AZIN2-driven polyamine metabolism preserves adipose tissue health, a finding that could be therapeutically harnessed for the management of obesity-associated metabolic disease. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND Despite its promise, cardiac regenerative therapy remains clinically elusive due to the difficulty of spatio-temporal control of proliferative induction, and the need to coordinately reprogram multiple regulatory pathways to overcome the strict post-mitotic state of human adult cardiomyocytes. The present study was designed to identify a novel combinatorial miRNA therapy to address this unmet therapeutic need.METHODS We performed a combinatorial miRNA interference screen specifically targeting cardiac-predominant miRNAs regulating key aspects of cardiomyocyte mitotic induction to cell-cycle completion, including sarcomerogenesis, metabolic and cell-cycle control pathways. Cardiomyocyte proliferation and cardiac function were assessed in human cardiac biopsies, human cardiac tissue mimetics and in mouse disease models.RESULTS We identified combinatorial interference of miR-1a and miR-15b (LNA-1a/15b) as drivers of adult cardiomyocyte proliferation. Due to miR-1a/15b function on multiple processes modulating adult cardiomyocyte mitosis, its inhibition augmented adult cardiomyocyte cell-cycle completion and daughter cell formation, and improved contractility in in vitro 2D and 3D ischemic models, and in a mouse model of ST-segment elevation myocardial infarction (STEMI). Due to the cardiac-restricted pattern of miR-1a/15b expression, this strategy provides a feasible strategy for specific cardiomyocyte proliferative induction with minimal risk of neoplasm formation and off-target toxicity.CONCLUSIONS Combinatorial miR-1a/15b inhibition drives mitotic re-entry in adult cardiomyocytes and improves cardiac function in response to myocardial infarction. Our data provides a novel and clinically feasible LNA-based anti-miR-1a/15b strategy to attenuate heart failure and highlights an underutilized therapeutic strategy for simultaneous co-regulation of multiple disease pathways through combinatorial miRNA interference.### Competing Interest StatementT.Y., S.D. and J.K. are inventors on a patent application pertaining to the inhibition of miR-1a and miR-15b for the treatment of heart disease.### Funding StatementThis work was supported by the Messer Foundation, LOEWE Center for Cell and Gene Therapy and the European Innovation Council (GA: 822455) to J.K., and the SFB-TRR 267 (Non-coding RNA in the cardiovascular system) and the German Research Foundation (DFG) (Exc2026) to S.D. and J.K., and instrument grant support (INST 515/28-1 FUGG) to M.P., the European Research Council (Angiolnc) to S.D., and the Foundation for Pathobiochemistry and Molecular Diagnostics to P.M. Y.W. was supported by the China Scholarship Council (CSC) Grant #202108080020.### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Ethics committee of Goethe University Frankfurt Research Services gave ethical approval for this work. All necessary patient/participant consent has been obtained and patient/participant/sample identifiers cannot be used to identify individuals. Ethics committee of Regierungsprasidium Darmstadt gave ethical approval for the animal studies this work.I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors.

BackgroundReduced and oxidized glutathione play an important role for the intracellular detoxification of reactive oxygen species. The iron-dependent formation of such reactive oxygen species in conjunction with the inhibition of the redox-balancing enzyme glutathione peroxidase 4 underlie an imbalance in the cellular redox state, thereby resulting in a non-apoptotic form of cell death, defined as ferroptosis, which is relevant in several pathologies.MethodsHere we present a rapid ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) based method providing the accurate quantification of 12 glutathione pathway metabolites after in situ derivatization with N-Ethylmaleimide (NEM). The method was validated regards linearity, recovery and accuracy as well as precision. The assay includes glutathione and its oxidized form glutathione disulfide. Furthermore, the related precursors cysteine, cystine, glutamic acid, γ-glutamylcysteine and cysteinylglycine, biomarkers of protein crosslinking such as cystathionine and lanthionine, as well as metabolites of the transsulfuration pathway, methionine, homocysteine and serine are simultaneously determined.ResultsTwelve glutathione pathway metabolites were simultaneously analyzed in four different human cell line extracts within a total LC run time of 5.5 min. Interday coefficients of variation (1.7 % to 12.0 %), the mean observed accuracy (100.0 % ± 5.2 %), linear quantification ranges over three orders of magnitude for all analytes and sufficient metabolite stability after NEM-derivatization demonstrate method reliability. Immediate derivatization with NEM at cell harvesting prevents autooxidation of glutathione, ensures accurate results for the GSH/GSSG redox ratio and thereby allows interpretation of cellular redox state.ConclusionThe described UPLC-MS/MS method provides a sensitive and selective tool for a fast and simultaneous analysis of glutathione pathway metabolites, its direct precursors and related compounds. Assay performance characteristics demonstrate the suitability of the method for applications in different cell cultures. Therefore, by providing glutathione related functional metabolic readouts, the method enables investigations in mechanisms of ferroptosis and alterations in oxidative stress levels in several pathophysiologies.

Summary Background and objectives Outdoor workers are at increased risk of developing non‐melanoma skin cancer. We aimed to address the lack of validated German‐language measurement instruments for outdoor workers’ sun safety behavior and knowledge by compiling and validating two questionnaires. Participants and methods By expert consensus, items for the assessment of protective behavior (OccuSun) were compiled based on existing instruments. For knowledge, a translation of the Skin Cancer and Sun Knowledge (SCSK) scale was selected. After a pre‐test, a validation study including 68 outdoor workers (62% female) was conducted in 2020. Results The retest reliability was r = 0.93 (95% confidence interval: 0.86–0.96) for the protection score and r s = 0.78 (0.67–0.86) for the knowledge score. Protective behaviors were correlated with respective diary data (0.38 ≤ r s ≤ 0.74, p < 0.001) and skin pigmentation changes (–0.23 ≥ r s ≥ –0.42, 0.007 ≤ p ≤ 0.165) but not with self‐reported sunburn frequency (0.21 ≥ r s ≥ –0.04). Conclusions Among German outdoor workers, two questionnaires for the assessment of sun protection behavior (OccuSun) and knowledge (SCSK) demonstrated good reliability. The OccuSun had generally good validity. Both instruments are fit for subsequent validation to determine their sensitivity to change.

Zusammenfassung Hintergrund und Ziele Beschäftigte im Freien haben ein erhöhtes Risiko, an Nicht‐Melanom‐Hautkrebs zu erkranken. Ziel dieser Studie war es, den Mangel an validierten deutschsprachigen Messinstrumenten für das Sonnenschutzverhalten und ‐wissen von Außenbeschäftigten durch das Zusammenstellen und Validieren von zwei Fragebögen zu beheben. Teilnehmer und Methoden Per Expertenkonsens wurden auf der Grundlage bestehender Messinstrumente Items für die Bewertung des Schutzverhaltens zusammengestellt (OccuSun). Für das Wissen wurde eine Übersetzung der Skin‐Cancer‐and‐Sun‐Knowledge ‐Skala (SCSK) ausgewählt. Nach einem Pretest wurde im Jahr 2020 eine Validierungsstudie mit 68 im Freien arbeitenden Personen (62% weiblich) durchgeführt. Ergebnisse Die Retest‐Reliabilität betrug r = 0,93 (95%‐Konfidenzintervall: 0,86–0,96) für den Schutzscore und r s = 0,78 (0,67–0,86) für den Wissensscore. Das Schutzverhalten korrelierte mit den entsprechenden Tagebuchdaten (0,38 ≤ r s ≤ 0,74, p < 0,001) und den Veränderungen der Hautpigmentierung (–0,23 ≥ r s ≥ –0,42, 0,007 ≤ p ≤ 0,165), nicht aber mit der selbstberichteten Häufigkeit von Sonnenbränden (0,21 ≥ r s ≥ –0,04). Schlussfolgerungen Zwei Fragebögen zur Erfassung des Sonnenschutzverhaltens (OccuSun) und Sonnenschutzwissens (SCSK) zeigten bei deutschen Außenbeschäftigten eine gute Reliabilität. Der OccuSun hatte allgemein eine gute Validität. Beide Instrumente eignen sich für eine anschließende Validierung, um ihre Änderungssensitivität zu bestimmen.

The spurge hawkmoth Hyles euphorbiae (Lepidoptera, Sphingidae) is an insect herbivore feeding exclusively on spurges (Euphorbia). These are toxic due to their secondary metabolites including diterpene esters with numerous harmful biological activities, preventing their use by most herbivores. Extracts of frass from H. euphorbiae and of its main larval food plant in Central Europe, the cypress spurge (Euphorbia cyparissias L.), were analyzed via liquid chromatography using a triple-quadrupole (MS/MS) and a high-resolution–quadrupole time-of-flight mass spectrometer (QTOF-MS). We report for the first time the finding of the tigliane (phorbol ester) phorbol-13-acetate and two 12-O-tetradecanoylphorbol-13-acetate (TPA) isomers of unknown structure (compounds 2 and 3) in E. cyparissias. Our data support the assignment of these two compounds as tiglianes and are therefore referred to by us as “putative tiglianes”. Additionally, the distribution of the three reported compounds within the plant parts (leaves, stem and root) was analyzed to gain first insights into the metabolite conditions H. euphorbiae copes with in nature. Phorbol-13-acetate was detected in the plant’s root only, while the other compounds were observed in all three plant parts, although in different distributions. Moreover, our results indicate that the occurrence of compound 3 rises upon larval feeding. Finally, the presence of the plant-derived putative tiglianes in frass from E. cyparissias leaves feeding larvae is confirmed, corroborating the close plant–herbivore interaction.

BACKGROUND:Kynurenine and respective metabolites exhibit bioactivity as well as tryptophan, an essential amino acid, and the neurotransmitter serotonin. Dysregulations in the kynurenine pathway are involved in neurodegenerative/neuropsychiatric disorders and diabetes mellitus type 2 but also in cancer. Therefore, measurements of kynurenine-related metabolites will improve the general understanding for kynurenine pathway relevance in disease pathogenesis. METHODS:Tryptophan, serotonin, picolinic acid, quinolinic acid, 3-OH-kynurenine, kynurenine, 3-OH-anthranilic acid, kynurenic acid, anthranilic acid as well as nicotinic acid and the redox cofactor NAD+ were analyzed in heterogeneous matrices by ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). After validation, the described method was applied for measurements of native metabolite concentrations in murine tissues and cellular systems including pathway-shift monitoring after treatment with the tryptophan-2,3-dioxygenase-inhibitor 680C91. In addition, the method was evaluated for its ability for integration into multi-omics approaches using a single sample metabolite extraction procedure. RESULTS:A simple and sensitive UPLC-MS/MS method for simultaneous quantification of up to 10 kynurenine-related metabolites in four biological matrices was developed. Within a run time of 6.5 min, chromatographic separation of kynurenine-related metabolites, including the isomers nicotinic acid and picolinic acid, was achieved without derivatization. Validation parameters, including interday precision (<14.8%), mean accuracy (102.4% ± 12.9%) and linear detection ranges of more than three orders of magnitude, indicate method reliability. Depending the investigated sample matrix, the majority of metabolites were successfully detected and quantified in native murine and cell culture derived sample materials. Furthermore, the method allowed to monitor the impact of a tryptophan-2,3-dioxygenase-inhibitor on kynurenine pathway in a cellular system and is suitable for multi-assay analyses using aliquots from the same cell extract. CONCLUSION:The described UPLC-MS/MS method provides a simple tool for the simultaneous quantification of kynurenine pathway metabolites. Due to its suitability for many physiological matrices, the method provides wide application for disease-related experimental settings.

The conditionally essential amino acid arginine and its metabolic products play an important role in different biological processes, such as metabolic regulation of the immune response, including macrophage activation and polarization and regulation of T cell function. Furthermore, the polyamine spermidine has a role in aging and age-related diseases. Additionally, altered polyamine metabolism may be associated with neurodegenerative diseases, while polyamine levels may present useful biomarkers associated with severity of Parkinson's disease or with progression of non-alcoholic fatty liver disease. In the present study, a simple, derivatization-free hydrophilic interaction liquid chromatography based tandem mass spectrometry (LC-MS/MS) method is described, that allows the accurate quantification of arginine and related amine, polyamine and acetylated polyamine metabolites in different experimental sample matrices, such as cell lysates, cell culture supernatants and tissues. Ten arginine metabolites, including citrulline, agmatine, ornithine, putrescine, spermidine, spermine, N1-acetylspermidine, N1-acetylspermine, N1,N12diacetylspermine and arginine in conjunction with the metabolic cofactors S-adenosylhomocysteine and S-adenosylmethionine are simultaneously analyzed within a total LC-MS/MS run time of 9.5 min. The assay is suitable to quantify concentration ranges over multiple orders of magnitude for all metabolites with averaged accuracies observed at 103.2% +/- 6.8%, 99.0% +/- 4.2% and 100.4% +/- 4.3% in cell lysates, cell culture supernatant and tissue extracts, respectively. Inter-day coefficients of variation ranged from 5.9 to 14.8% in cell lysates, 6.7 to 14.6% in cell culture supernatants and 5.3 to 12.0% in tissue extracts. The method was successfully applied to cell culture systems of different origin as well as different murine tissues and organs. The herein described LC-MS/MS method provides a simple tool for a fast and simultaneous analysis of arginine metabolites, including polyamines and their respective metabolic cofactors. Assay performance characteristics demonstrate suitability for applications in different experimental and preclinical settings.(c) 2022 Elsevier B.V. All rights reserved.

Background: Severe anaphylaxis (SA) in Hymenoptera venom allergy has been associated with a number of risk factors. However, the effect of several of those risk factors on the severity of anaphylaxis is poorly defined. Objective: To evaluate risk factors for SA in Hymenoptera venom allergy. Methods: We evaluated data from 500 patients who were referred to our department for the diagnosis of Hymenoptera venom allergy during a period of 11 years to identify risk factors for SA. Results: Six significant risk factors for SA were identified (P <.05): short interval from sting to reaction, absence of urticaria or angioedema (U/A) during anaphylaxis, older age, male sex, elevation of baseline serum tryptase (BST) level, and diagnosis of systemic mastocytosis. Moreover, elevation in BST level was significantly associated with the absence of U/A and older age. No association could be established between SA and comorbidities, concurrent cardiovascular medication, or the severity of the systemic reaction during the initiation of venom immunotherapy. Conclusion: Apart from BST and older age, male sex, short interval from sting to reaction, and absence of U/A are also risk factors for SA. The association between elevated BST level and SA was largely confined to those who had an absence of U/A after field sting, possibly because of the higher risk of concurrent systemic mastocytosis. Patients with an SA after a field sting do not have an elevated risk of systemic reactions during the initiation of venom immunotherapy compared with patients with mild anaphylaxis; therefore, additional preventive measures are not necessary. (C) 2020 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.