Regulatory myeloid cells paralyze T cells through cell-cell transfer of the metabolite methylglyoxal.

Regulatory myeloid immune cells, such as myeloid-derived suppressor cells (MDSCs), populate inflamed or cancerous tissue and block immune cell effector functions. The lack of mechanistic insight into MDSC suppressive activity and a marker for their identification has hampered attempts to overcome T cell inhibition and unleash anti-cancer immunity. Here, we report that human MDSCs were characterized by strongly reduced metabolism and conferred this compromised metabolic state to CD8+ T cells, thereby paralyzing their effector functions. We identified accumulation of the dicarbonyl radical methylglyoxal, generated by semicarbazide-sensitive amine oxidase, to cause the metabolic phenotype of MDSCs and MDSC-mediated paralysis of CD8+ T cells. In a murine cancer model, neutralization of dicarbonyl activity overcame MDSC-mediated T cell suppression and, together with checkpoint inhibition, improved the efficacy of cancer immune therapy. Our results identify the dicarbonyl methylglyoxal as a marker metabolite for MDSCs that mediates T cell paralysis and can serve as a target to improve cancer immune therapy.

Mass-spectrometry-based draft of the Arabidopsis proteome.

Plants are essential for life and are extremely diverse organisms with unique molecular capabilities1. Here we present a quantitative atlas of the transcriptomes, proteomes and phosphoproteomes of 30 tissues of the model plant Arabidopsis thaliana. Our analysis provides initial answers to how many genes exist as proteins (more than 18,000), where they are expressed, in which approximate quantities (a dynamic range of more than six orders of magnitude) and to what extent they are phosphorylated (over 43,000 sites). We present examples of how the data may be used, such as to discover proteins that are translated from short open-reading frames, to uncover sequence motifs that are involved in the regulation of protein production, and to identify tissue-specific protein complexes or phosphorylation-mediated signalling events. Interactive access to this resource for the plant community is provided by the ProteomicsDB and ATHENA databases, which include powerful bioinformatics tools to explore and characterize Arabidopsis proteins, their modifications and interactions.

The p21CIP1-CDK4-DREAM axis is a master regulator of genotoxic stress-induced cellular senescence.

Cellular senescence, a major driver of aging, can be stimulated by DNA damage, and is counteracted by the DNA repair machinery. Here we show that in p16INK4a-deficient cells, senescence induction by the environmental genotoxin B[a]P or ionizing radiation (IR) completely depends on p21CIP1. Immunoprecipitation-based mass spectrometry interactomics data revealed that during senescence induction and maintenance, p21CIP1 specifically inhibits CDK4 and thereby activates the DREAM complex. Genome-wide transcriptomics revealed striking similarities in the response induced by B[a]P and IR. Among the top 100 repressed genes 78 were identical between B[a]P and IR and 76 were DREAM targets. The DREAM complex transcriptionally silences the main proliferation-associated transcription factors E2F1, FOXM1 and B-Myb as well as multiple DNA repair factors. Knockdown of p21CIP1, E2F4 or E2F5 diminished both, repression of these factors and senescence. The transcriptional profiles evoked by B[a]P and IR largely overlapped with the profile induced by pharmacological CDK4 inhibition, further illustrating the role of CDK4 inhibition in genotoxic stress-induced senescence. Moreover, data obtained by live-cell time-lapse microscopy suggest the inhibition of CDK4 by p21CIP1 is especially important for arresting cells which slip through mitosis. Overall, we identified the p21CIP1/CDK4/DREAM axis as a master regulator of genotoxic stress-induced senescence.

Multi-layered chromatin proteomics identifies cell vulnerabilities in DNA repair.

The DNA damage response (DDR) is essential to maintain genome stability, and its deregulation predisposes to carcinogenesis while encompassing attractive targets for cancer therapy. Chromatin governs the DDR via the concerted interplay among different layers, including DNA, histone post-translational modifications (hPTMs) and chromatin-associated proteins. Here, we employ multi-layered proteomics to characterize chromatin-mediated functional interactions of repair proteins, signatures of hPTMs and the DNA-bound proteome during DNA double-strand break (DSB) repair at high temporal resolution. Our data illuminate the dynamics of known and novel DDR-associated factors both at chromatin and at DSBs. We functionally attribute novel chromatin-associated proteins to repair by non-homologous end-joining (NHEJ), homologous recombination (HR) and DSB repair pathway choice. We reveal histone reader ATAD2, microtubule organizer TPX2 and histone methyltransferase G9A as regulators of HR and involved in poly-ADP-ribose polymerase-inhibitor sensitivity. Furthermore, we distinguish hPTMs that are globally induced by DNA damage from those specifically acquired at sites flanking DSBs (γH2AX foci-specific) and profiled their dynamics during the DDR. Integration of complementary chromatin layers implicates G9A-mediated monomethylation of H3K56 in DSBs repair via HR. Our data provide a dynamic chromatin-centered view of the DDR that can be further mined to identify novel mechanistic links and cell vulnerabilities in DSB repair.

Bacterial rhamnolipids and their 3-hydroxyalkanoate precursors activate Arabidopsis innate immunity through two independent mechanisms.

Plant innate immunity is activated upon perception of invasion pattern molecules by plant cell-surface immune receptors. Several bacteria of the genera Pseudomonas and Burkholderia produce rhamnolipids (RLs) from l-rhamnose and (R)-3-hydroxyalkanoate precursors (HAAs). RL and HAA secretion is required to modulate bacterial surface motility, biofilm development, and thus successful colonization of hosts. Here, we show that the lipidic secretome from the opportunistic pathogen Pseudomonas aeruginosa, mainly comprising RLs and HAAs, stimulates Arabidopsis immunity. We demonstrate that HAAs are sensed by the bulb-type lectin receptor kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION/S-DOMAIN-1-29 (LORE/SD1-29), which also mediates medium-chain 3-hydroxy fatty acid (mc-3-OH-FA) perception, in the plant Arabidopsis thaliana HAA sensing induces canonical immune signaling and local resistance to plant pathogenic Pseudomonas infection. By contrast, RLs trigger an atypical immune response and resistance to Pseudomonas infection independent of LORE. Thus, the glycosyl moieties of RLs, although abolishing sensing by LORE, do not impair their ability to trigger plant defense. Moreover, our results show that the immune response triggered by RLs is affected by the sphingolipid composition of the plasma membrane. In conclusion, RLs and their precursors released by bacteria can both be perceived by plants but through distinct mechanisms.

Simultaneous Nbs1 and p53 inactivation in neural progenitors triggers high-grade gliomas.

AIMS: Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder caused by hypomorphic mutations of NBS1. NBS1 is a member of the MRE11-RAD50-NBS1 (MRN) complex that binds to DNA double-strand breaks and activates the DNA damage response (DDR). Nbs1 inactivation in neural progenitor cells leads to microcephaly and premature death. Interestingly, p53 homozygous deletion rescues the NBS1-deficient phenotype allowing long-term survival. The objective of this work was to determine whether simultaneous inactivation of Nbs1 and p53 in neural progenitors triggered brain tumorigenesis and if so in which category this tumour could be classified. METHODS: We generated a mouse model with simultaneous genetic inactivation of Nbs1 and p53 in embryonic neural stem cells and analysed the arising tumours with in-depth molecular analyses including immunohistochemistry, array comparative genomic hybridisation (aCGH), whole exome-sequencing and RNA-sequencing. RESULTS: NBS1/P53-deficient mice develop high-grade gliomas (HGG) arising in the olfactory bulbs and in the cortex along the rostral migratory stream. In-depth molecular analyses using immunohistochemistry, aCGH, whole exome-sequencing and RNA-sequencing revealed striking similarities to paediatric human HGG with shared features with radiation-induced gliomas (RIGs). CONCLUSIONS: Our findings show that concomitant inactivation of Nbs1 and p53 in mice promotes HGG with RIG features. This model could be useful for preclinical studies to improve the prognosis of these deadly tumours, but it also highlights the singularity of NBS1 among the other DNA damage response proteins in the aetiology of brain tumours.

Adult thymus contains FoxN1(-) epithelial stem cells that are bipotent for medullary and cortical thymic epithelial lineages.

Within the thymus, two major thymic epithelial cell (TEC) subsets-cortical and medullary TECs-provide unique structural and functional niches for T cell development and establishment of central tolerance. Both lineages are believed to originate from a common progenitor cell, yet the cellular and molecular identity of these bipotent TEC progenitors/stem cells remains ill defined. Here we identify rare stromal cells in the murine adult thymus, which under low-attachment conditions formed spheres (termed "thymospheres"). These thymosphere-forming cells (TSFCs) displayed the stemness features of being slow cycling, self-renewing, and bipotent. TSFCs could be significantly enriched based on their distinct surface antigen phenotype. The FoxN1 transcription factor was dispensable for TSFCs maintenance in situ and for commitment to the medullary and cortical TEC lineages. In summary, this study presents the characterization of the adult thymic epithelial stem cells and demonstrates the dispensability of FoxN1 function for their stemness.

Modeling the Orthosteric Binding Site of the G Protein-Coupled Odorant Receptor OR5K1.

With approximately 400 encoding genes in humans, odorant receptors (ORs) are the largest subfamily of class A G protein-coupled receptors (GPCRs). Despite its high relevance and representation, the odorant-GPCRome is structurally poorly characterized: no experimental structures are available, and the low sequence identity of ORs to experimentally solved GPCRs is a significant challenge for their modeling. Moreover, the receptive range of most ORs is unknown. The odorant receptor OR5K1 was recently and comprehensively characterized in terms of cognate agonists. Here, we report two additional agonists and functional data of the most potent compound on two mutants, L1043.32 and L2556.51. Experimental data was used to guide the investigation of the binding modes of OR5K1 ligands into the orthosteric binding site using structural information from AI-driven modeling, as recently released in the AlphaFold Protein Structure Database, and from homology modeling. Induced-fit docking simulations were used to sample the binding site conformational space for ensemble docking. Mutagenesis data guided side chain residue sampling and model selection. We obtained models that could better rationalize the different activity of active (agonist) versus inactive molecules with respect to starting models and also capture differences in activity related to minor structural differences. Therefore, we provide a model refinement protocol that can be applied to model the orthosteric binding site of ORs as well as that of GPCRs with low sequence identity to available templates.

DAZAP2 acts as specifier of the p53 response to DNA damage.

The DNA damage-responsive tumor suppressors p53 and HIPK2 are well established regulators of cell fate decision-making and regulate the cellular sensitivity to DNA-damaging drugs. Here, we identify Deleted in Azoospermia-associated protein 2 (DAZAP2), a small adaptor protein, as a novel regulator of HIPK2 and specifier of the DNA damage-induced p53 response. Knock-down or genetic deletion of DAZAP2 strongly potentiates cancer cell chemosensitivity both in cells and in vivo using a mouse tumour xenograft model. In unstressed cells, DAZAP2 stimulates HIPK2 polyubiquitination and degradation through interplay with the ubiquitin ligase SIAH1. Upon DNA damage, HIPK2 site-specifically phosphorylates DAZAP2, which terminates its HIPK2-degrading function and triggers its re-localization to the cell nucleus. Interestingly, nuclear DAZAP2 interacts with p53 and specifies target gene expression through modulating a defined subset of p53 target genes. Furthermore, our results suggest that DAZAP2 co-occupies p53 response elements to specify target gene expression. Collectively, our findings propose DAZAP2 as novel regulator of the DNA damage-induced p53 response that controls cancer cell chemosensitivity.

[Incapacity to work in prehospital German emergency medical services staff]. / Arbeitsunfähigkeit bei präklinischen Rettungskräften in Deutschland: Ergebnisse der EMS-Health-Studie.

Background: The determination of incapacity to work is a central approach for analyses of absence due to sickness. Nevertheless, no data are yet available for incapacity to work and associated factors in the German prehospital emergency medical services (EMS) staff. Objective: The aim of this analysis was to identify the proportion of EMS staff with at least one incapacity for work (AU) in the previous 12 months and associated factors. Material and methods: This was a nationwide survey study with rescue workers. Factors associated with work disability were identified using multivariable logistic regression, calculating odds ratios (OR) and associated 95% confidence intervals (95% CI). Results: Included in this analysis were 2298 employees of the German emergency medical services (female 42.6%, male 57.2%). Overall, 60.10% of female participants and 58.98% of male participants reported an incapacity for work in the previous 12 months. Incapacity for work was significantly associated with having a high school diploma (high school diploma: OR: 0.51, 95% CI 0.30; 0.88, p = 0.016; reference: secondary school diploma), working in a rural environment (OR: 0.65, 95% CI 0.50; 0.86, p = 0.003) or urban environment (OR: 0.72, 95% CI: 0.53; 0.98, p = 0.037). Furthermore, hours worked per week (OR: 1.01, 95% CI: 1.00; 1.02, p = 0.003) and 5-< 10 years of service (OR: 1.40, 95% CI: 1.04; 1.89, p = 0.025) were associated with higher odds of work disability. Neck and back pain, depression, osteoarthritis, and asthma in the previous 12 months also showed a significant association with work disability in the same time period. Conclusion: This analysis shows that chronic diseases, educational attainment, area of assignment, years of service, and hours worked per week, among others, were associated with incapacity for work in the previous 12 months in German EMS staff.

Recommendations on dose level selection for repeat dose toxicity studies.

Prior to registering and marketing any new pharmaceutical, (agro)chemical or food ingredient product manufacturers must, by law, generate data to ensure human safety. Safety testing requirements vary depending on sector, but generally repeat-dose testing in animals form the basis for human health risk assessments. Dose level selection is an important consideration when designing such studies, to ensure that exposure levels that lead to relevant hazards are identified. Advice on dose level selection is provided in test guidelines and allied guidance documents, but it is not well harmonised, particularly for selection of the highest dose tested. This paper further builds on concepts developed in a technical report by the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) which recommends pragmatic approaches to dose selection considering regulatory requirements, animal welfare and state of the art scientific approaches. Industry sectors have differing degrees of freedom to operate regarding dose level selection, depending on the purpose of the studies and the regulatory requirements/legislation, and this is reflected in the overall recommended approaches. An understanding of systemic exposure should be utilised where possible (e.g., through toxicokinetic approaches) and used together with apical endpoints from existing toxicity studies to guide more appropriate dose level selection. The highest dose should be limited to a reasonable level, causing minimal but evident toxicity to the test animals without significantly compromising their well-being. As the science of predictive human exposure further develops and matures, this will provide exciting and novel opportunities for more human-relevant approaches to dose level selection.

Benzo[a]pyrene represses DNA repair through altered E2F1/E2F4 function marking an early event in DNA damage-induced cellular senescence.

Transcriptional regulation of DNA repair is of outmost importance for the restoration of DNA integrity upon genotoxic stress. Here we report that the potent environmental carcinogen benzo[a]pyrene (B[a]P) activates a cellular DNA damage response resulting in transcriptional repression of mismatch repair (MMR) genes (MSH2, MSH6, EXO1) and of RAD51, the central homologous recombination repair (HR) component, ultimately leading to downregulation of MMR and HR. B[a]P-induced gene repression is caused by abrogated E2F1 signalling. This occurs through proteasomal degradation of E2F1 in G2-arrested cells and downregulation of E2F1 mRNA expression in G1-arrested cells. Repression of E2F1-mediated transcription and silencing of repair genes is further mediated by the p21-dependent E2F4/DREAM complex. Notably, repression of DNA repair is also observed following exposure to the active B[a]P metabolite BPDE and upon ionizing radiation and occurs in response to a p53/p21-triggered, irreversible cell cycle arrest marking the onset of cellular senescence. Overall, our results suggest that repression of MMR and HR is an early event during genotoxic-stress induced senescence. We propose that persistent downregulation of DNA repair might play a role in the maintenance of the senescence phenotype, which is associated with an accumulation of unrepairable DNA lesions.

Bacillus cereus Toxin Repertoire: Diversity of (Iso)cereulide(s).

The emetic Bacillus cereus toxin cereulide (1) poses a significant safety risk in the food industry, causing emesis and nausea after consumption of contaminated foods. Analogously to cereulide, the structures of various isocereulides, namely, isocereulides A-G, have been recently reported and could also be identified in B. cereus-contaminated food samples. The HPLC fractionation of B. cereus extracts allows us to isolate additional isocereulides. By applying MSn sequencing, post-hydrolytic dipeptide, amino acid and α-hydroxy acid analyses using UPLC-ESI-TOF-MS to purify the analytes, seven new isocereulides H-N (2-8) could be elucidated in their chemical structures. The structure elucidation was supported by one-dimensional and two-dimensional NMR spectra of the isocereulides H (2), K (5), L and N (6 + 8) and M (7). The toxicity of 2-8 was investigated in a HEp-2 cell assay to determine their respective 50% effective concentration (EC50). Thus, 2-8 exhibited EC50 values ranging from a 0.4- to 1.4-fold value compared to cereulide (1). Missing structure-activity correlations indicate the necessity to determine the toxic potential of all naturally present isocereulides as single compounds to be able to perform a thorough toxicity evaluation of B. cereus-contaminated foods in the future.

Intensive care unit occupancy predictions in the COVID-19 pandemic based on age-structured modelling and differential flatness.

The COVID-19 pandemic confronts governments and their health systems with great challenges for disease management. In many countries, hospitalization and in particular ICU occupancy is the primary measure for policy makers to decide on possible non-pharmaceutical interventions. In this paper a combined methodology for the prediction of COVID-19 case numbers, case-specific hospitalization and ICU admission rates as well as hospital and ICU occupancies is proposed. To this end, we employ differential flatness to provide estimates of the states of an epidemiological compartmental model and estimates of the unknown exogenous inputs driving its nonlinear dynamics. A main advantage of this method is that it requires the reported infection cases as the only data source. As vaccination rates and case-specific ICU rates are both strongly age-dependent, specifically an age-structured compartmental model is proposed to estimate and predict the spread of the epidemic across different age groups. By utilizing these predictions, case-specific hospitalization and case-specific ICU rates are subsequently estimated using deconvolution techniques. In an analysis of various countries we demonstrate how the methodology is able to produce real-time state estimates and hospital/ICU occupancy predictions for several weeks thus providing a sound basis for policy makers.

Multiplexed DNA Methylation Analysis in Colorectal Cancer Using Liquid Biopsy and Its Diagnostic and Predictive Value.

Early diagnosis of colorectal cancer (CRC) is of high importance as prognosis depends on tumour stage at the time of diagnosis. Detection of tumour-specific DNA methylation marks in cfDNA has several advantages over other approaches and has great potential for solving diagnostic needs. We report here the identification of DNA methylation biomarkers for CRC and give insights in our methylation-sensitive restriction enzyme coupled qPCR (MSRE-qPCR) system. Targeted microarrays were used to investigate the DNA methylation status of 360 cancer-associated genes. Validation was done by qPCR-based approaches. A focus was on investigating marker performance in cfDNA from 88 patients (44 CRC, 44 controls). Finally, the workflow was scaled-up to perform 180plex analysis on 110 cfDNA samples, to identify a DNA methylation signature for advanced colonic adenomas (AA). A DNA methylation signature (n = 44) was deduced from microarray experiments and confirmed by quantitative methylation-specific PCR (qMSP) and by MSRE-qPCR, providing for six genes' single areas under the curve (AUC) values of >0.85 (WT1, PENK, SPARC, GDNF, TMEFF2, DCC). A subset of the signatures can be used for patient stratification and therapy monitoring for progressed CRC with liver metastasis using cfDNA. Furthermore, we identified a 35-plex classifier for the identification of AAs with an AUC of 0.80.

Systematic Evaluation of Liquid Chromatography (LC) Column Combinations for Application in Two-Dimensional LC Metabolomic Studies.

In comparison to proteomics, the application of two-dimensional liquid chromatography (2D LC) in the field of metabolomics is still premature. One reason might be the elevated chemical complexity and the associated challenge of selecting proper separation conditions in each dimension. As orthogonality of dimensions is a major issue, the present study aimed for the identification of successful stationary phase combinations. To determine the degree of orthogonality, first, six different metrics, namely, Pearson's correlation coefficient (1 - |R|), the nearest-neighbor distances (H̅NND), the "asterisk equations" (AO), and surface coverage by bins (SCG), convex hulls (SCCH), and α-convex hulls (SCαH), were critically assessed by 15 artificial 2D data sets, and a systematic parameter optimization of α-convex hulls was conducted. SGG, SCαH with α = 0.1, and H̅NND generated valid results with sensitivity toward space utilization and data distribution and, therefore, were applied to pairs of experimental retention time sets obtained for >350 metabolites, selected to represent the chemical space of human urine. Normalized retention data were obtained for 23 chromatographic setups, comprising reversed-phase (RP), hydrophilic interaction liquid chromatography (HILIC), and mixed-mode separation systems with an ion exchange (IEX) contribution. As expected, no single LC setting provided separation of all considered analytes, but while conventional RP×HILIC combinations appeared rather complementary than orthogonal, the incorporation of IEX properties into the RP dimension substantially increased the 2D potential. Eventually, one of the most promising column combinations was implemented for an offline 2D LC time-of-flight mass spectrometry analysis of a lyophilized urine sample. Targeted screening resulted in a total of 164 detected metabolites and confirmed the outstanding coverage of the 2D retention space.

Analysis of microplastics in drinking water and other clean water samples with micro-Raman and micro-infrared spectroscopy: minimum requirements and best practice guidelines.

Microplastics are a widespread contaminant found not only in various natural habitats but also in drinking waters. With spectroscopic methods, the polymer type, number, size, and size distribution as well as the shape of microplastic particles in waters can be determined, which is of great relevance to toxicological studies. Methods used in studies so far show a huge diversity regarding experimental setups and often a lack of certain quality assurance aspects. To overcome these problems, this critical review and consensus paper of 12 European analytical laboratories and institutions, dealing with microplastic particle identification and quantification with spectroscopic methods, gives guidance toward harmonized microplastic particle analysis in clean waters. The aims of this paper are to (i) improve the reliability of microplastic analysis, (ii) facilitate and improve the planning of sample preparation and microplastic detection, and (iii) provide a better understanding regarding the evaluation of already existing studies. With these aims, we hope to make an important step toward harmonization of microplastic particle analysis in clean water samples and, thus, allow the comparability of results obtained in different studies by using similar or harmonized methods. Clean water samples, for the purpose of this paper, are considered to comprise all water samples with low matrix content, in particular drinking, tap, and bottled water, but also other water types such as clean freshwater.

Cell Fate Regulation upon DNA Damage: p53 Serine 46 Kinases Pave the Cell Death Road.

Mild and massive DNA damage are differentially integrated into the cellular signaling networks and, in consequence, provoke different cell fate decisions. After mild damage, the tumor suppressor p53 directs the cellular response to cell cycle arrest, DNA repair, and cell survival, whereas upon severe damage, p53 drives the cell death response. One posttranslational modification of p53, phosphorylation at Serine 46, selectively occurs after severe DNA damage and is envisioned as a marker of the cell death response. However, the molecular mechanism of action of the p53 Ser46 phospho-isomer, the molecular timing of this phosphorylation event, and its activating effects on apoptosis and ferroptosis still await exploration. In this essay, the current body of evidence on the molecular function of this deadly p53 mark, its evolutionary conservation, and the regulation of the key players of this response, the p53 Serine 46 kinases, are reviewed and dissected.

Using historical control data in bioassays for regulatory toxicology.

Historical control data (HCD) consist of pooled control group responses from bioassays. These data must be collected and are often used or reported in regulatory toxicology studies for multiple purposes: as quality assurance for the test system, to help identify toxicological effects and their effect-size relevance and to address the statistical multiple comparison problem. The current manuscript reviews the various classical and potential new approaches for using HCD. Issues in current practice are identified and recommendations for improved use and discussion are provided. Furthermore, stakeholders are invited to discuss whether it is necessary to consider uncertainty when using HCD formally and statistically in toxicological discussions and whether binary inclusion/exclusion criteria for HCD should be revised to a tiered information contribution to assessments. Overall, the critical value of HCD in toxicological bioassays is highlighted when used in a weight-of-evidence assessment.

Identification of Salicylates in Willow Bark (Salix Cortex) for Targeting Peripheral Inflammation.

Salix cortex-containing medicine is used against pain conditions, fever, headaches, and inflammation, which are partly mediated via arachidonic acid-derived prostaglandins (PGs). We used an activity-guided fractionation strategy, followed by structure elucidation experiments using LC-MS/MS, CD-spectroscopy, and 1D/2D NMR techniques, to identify the compounds relevant for the inhibition of PGE2 release from activated human peripheral blood mononuclear cells. Subsequent compound purification by means of preparative and semipreparative HPLC revealed 2'-O-acetylsalicortin (1), 3'-O-acetylsalicortin (2), 2'-O-acetylsalicin (3), 2',6'-O-diacetylsalicortin (4), lasiandrin (5), tremulacin (6), and cinnamrutinose A (7). In contrast to 3 and 7, compounds 1, 2, 4, 5, and 6 showed inhibitory activity against PGE2 release with different potencies. Polyphenols were not relevant for the bioactivity of the Salix extract but salicylates, which degrade to, e.g., catechol, salicylic acid, salicin, and/or 1-hydroxy-6-oxo-2-cycohexenecarboxylate. Inflammation presents an important therapeutic target for pharmacological interventions; thus, the identification of relevant key drugs in Salix could provide new prospects for the improvement and standardization of existing clinical medicine.