Mastoparan M Suppressed NLRP3 Inflammasome Activation by Inhibiting MAPK/NF-κB and Oxidative Stress in Gouty Arthritis.

Background: Gouty arthritis is characterized by the accumulation of monosodium urate crystals (MSU) in the synovial joints and surrounding tissues. Mastoparan M (Mast-M) is a biologically active peptide composed of 14 amino acids, extracted from wasp venom. This study aims to assess the impact of Mast-M on in vitro and in vivo gouty arthritis induced by lipolyaccharide (LPS) plus MSU crystal stimulation. Methods: PMA-differentiated THP-1 macrophages were pre-treated with Mast-M or left untreated, followed by stimulation with LPS and MSU crystals. Cell lysates were collected to assess the expression of the NLRP3 inflammasome, inflammatory signaling pathways, and oxidative stress. Furthermore, to evaluate the in vivo anti-inflammatory effect of Mast-M, an experimental acute gouty arthritis mouse model was established through intra-articular injection of MSU crystals. Results: Mast-M treatment demonstrated significant inhibition of the phosphorylation of MAPKs/NF-κB signaling pathways and reduction in oxidative stress expression in LPS and MSU-induced THP-1 macrophages. This resulted in the suppression of downstream NLRP3 inflammasome activation and IL-1ß release. In vivo, Mast-M effectively attenuated the inflammation induced by MSU in mice with gouty arthritis. Specifically, Mast-M reduced swelling in the paws, inhibited the infiltration of neutrophils and macrophages into periarticular tissue, and decreased the activation of the NLRP3 inflammasome and IL-1ß production. Conclusion: Mast-M significantly improves gouty arthritis, and its potential mechanism may be achieved by inhibiting the MAPK/NF-κB pathway and alleviating oxidative stress, thus suppressing the activation of NLRP3 inflammasomes.

Response mechanisms of 3 typical plants nitrogen and phosphorus nutrient cycling to nitrogen deposition in temperate meadow grasslands.

The increase of nitrogen (N) deposition and the diversity of its components lead to significant changes in the structure and function of temperate meadow steppe, which could affect plant nutrient uptake, nutrient resorption and litter decomposition, thus affecting the biogeochemical cycle process. The distribution and metabolism of nitrogen and phosphorus in plants determine the growth process and productivity of plants. Plant nutrient uptake, nutrient resorption and litter decomposition play an important role in the nutrient cycling process of ecosystem. This study closely combined these three processes to carry out experiments with different nitrogen dosages and types, and systematically explored the response of nitrogen and phosphorus nutrient cycling to nitrogen deposition. The results showed that nitrogen deposition can greatly affect ecosystem nutrient cycle of nitrogen and phosphorus. Firstly, Nitrogen deposition has significant effect on plant nutrient uptake. Nitrogen uptake of stems and leaves increased with the increase of nitrogen addition dosage, while phosphorus uptake of stems and leaves showed a downward trend or no significant effect. Besides, nitrogen addition type had a significant effect on nitrogen and phosphorus content of stems. Secondly, Nitrogen addition dosage had a significant effect on plant nutrient resorption, while nitrogen addition type had no significant effect on it. Thirdly, nitrogen deposition has significant effect on litter decomposition. With the increase of nitrogen addition dosage, the initial nitrogen content of litters increased and the decomposition rate of litters accelerated. Nitrogen application type had significant effect on stem litter decomposition. These results indicated that nitrogen deposition significantly affects plant nutrient cycling, and thus affects the structure and function of grassland ecosystem.

Impacts of climate change and human activities on vegetation coverage variation in mountainous and hilly areas in Central South of Shandong Province based on tree-ring.

Introduction: It is of great significance to understand the characteristics and influencing factors of vegetation coverage variation in the warm temperate zone. As a typical region of the warm temperate zone in eastern China, the mountainous and hilly region in central-south Shandong Province has fragile ecological environment and soil erosion problem. Studying on vegetation dynamics and its influencing factors in this region will help to better understand the relationship between climate change and vegetation cover change in the warm temperate zone of eastern China, and the influence of human activities on vegetation cover dynamics. Methods: Based on dendrochronology, a standard tree-ring width chronology was established in the mountainous and hilly region of central-south Shandong Province, and the vegetation coverage from 1905 to 2020 was reconstructed to reveal the dynamic change characteristics of vegetation cover in this region. Secondly, the influence of climate factors and human activities on the dynamic change of vegetation cover was discussed through correlation analysis and residual analysis. Results and discussion: In the reconstructed sequence, 23 years had high vegetation coverage and 15 years had low vegetation coverage. After low-pass filtering, the vegetation coverage of 1911-1913, 1945-1951, 1958-1962, 1994-1996, and 2007-2011 was relatively high, while the vegetation coverage of 1925-1927, 1936-1942, 2001-2003, and 2019-2020 was relatively low. Although precipitation determined the variation of vegetation coverage in this study area, the impacts of human activities on the change of vegetation coverage in the past decades cannot be ignored. With the development of social economy and the acceleration of urbanization, the vegetation coverage declined. Since the beginning of the 21st century, ecological projects such as Grain-for-Green have increased the vegetation coverage.

Activation of p62-NRF2 Axis Protects against Doxorubicin-Induced Ferroptosis in Cardiomyocytes: A Novel Role and Molecular Mechanism of Resveratrol.

Doxorubicin (DOX) is a most common anthracycline chemotherapeutic agent; however, its clinical efficacy is limited due to its severe and irreversible cardiotoxicity. Ferroptosis, characterized by iron overload and lipid peroxidation, plays a pivotal role in DOX-induced cardiotoxicity. Resveratrol (RSV) displays cardioprotective and anticancer effects, owing to its antioxidative and anti-inflammatory properties. However, the role and mechanism of RSV in DOX-mediated ferroptosis in cardiomyocytes is unclear. This study showed that DOX decreased cell viability, increased iron accumulation and lipid peroxidation in H9c2 cells; however, these effects were reversed by RSV and ferroptosis inhibitor ferrostatin-1 (Fer-1) pre-treatment. Additionally, RSV significantly increased the cell viability of H9c2 cells treated with ferroptosis inducers Erastin (Era) and RSL3. Mechanistically, RSV inhibited mitochondrial reactive oxygen species (mtROS) overproduction and upregulated the p62-NRF2/HO-1 pathway. RSV-induced NRF2 activation was partially dependent on p62, and the selective inhibition of p62 (using p62-siRNA interference) or NRF2 (using NRF2 specific inhibitor, ML385) significantly abolished the anti-ferroptosis function of RSV. Furthermore, RSV treatment protected mice against DOX-induced cardiotoxicity, including significantly improving left ventricular function, ameliorating myocardial fibrosis and suppressing ferroptosis. Consistent with in vitro results, RSV also upregulated the p62-NRF2/HO-1 expression, which was inhibited by DOX, in the myocardium. Notably, the protective effect of RSV in DOX-mediated ferroptosis was similar to that of Fer-1 in vitro and in vivo. Thus, the p62-NRF2 axis plays a critical role in regulating DOX-induced ferroptosis in cardiomyocytes. RSV as a potent p62 activator has potential as a therapeutic target in preventing DOX-induced cardiotoxicity via ferroptosis modulation.

Hyperuricemia contributes to glucose intolerance of hepatic inflammatory macrophages and impairs the insulin signaling pathway via IRS2-proteasome degradation.

Aim: Numerous reports have demonstrated the key importance of macrophage-elicited metabolic inflammation in insulin resistance (IR). Our previous studies confirmed that hyperuricemia or high uric acid (HUA) treatment induced an IR state in several peripheral tissues to promote the development of type 2 diabetes mellitus (T2DM). However, the effect of HUA on glucose uptake and the insulin sensitivity of macrophages and its mechanism is unclear. Methods: To assess systemic IR, we generated hyperuricemic mice by urate oxidase knockout (UOX-KO). Then, glucose/insulin tolerance, the tissue uptake of 18F-fluorodeoxyglucose, body composition, and energy balance were assessed. Glucose uptake of circulating infiltrated macrophages in the liver was evaluated by glucose transporter type 4 (GLUT-4) staining. Insulin sensitivity and the insulin signaling pathway of macrophages were demonstrated using the 2-NBDG kit, immunoblotting, and immunofluorescence assays. The immunoprecipitation assay and LC-MS analysis were used to determine insulin receptor substrate 2 (IRS2) levels and its interacting protein enrichment under HUA conditions. Results: Compared to WT mice (10 weeks old), serum uric acid levels were higher in UOX-KO mice (WT, 182.3 ± 5.091 µM versus KO, 421.9 ± 45.47 µM). Hyperuricemic mice with metabolic disorders and systemic IR showed inflammatory macrophage recruitment and increased levels of circulating proinflammatory cytokines. HUA inhibited the nuclear translocation of GLUT-4 in hepatic macrophages, restrained insulin-induced glucose uptake and glucose tolerance, and blocked insulin IRS2/PI3K/AKT signaling. Meanwhile, HUA mediated the IRS2 protein degradation pathway and activated AMPK/mTOR in macrophages. LC-MS analysis showed that ubiquitination degradation could be involved in IRS2 and its interacting proteins to contribute to IR under HUA conditions. Conclusion: The data suggest that HUA-induced glucose intolerance in hepatic macrophages contributed to insulin resistance and impaired the insulin signaling pathway via IRS2-proteasome degradation.

Inorganic quantum dots - anammox consortia hybrid for stable nitrogen elimination under high-intensity solar-simulated irradiation.

External stimulus such as light irradiation is able to deteriorate intracellular redox homeostasis and induce photooxidative damage to non-photogenic bacteria. Exploiting effective strategies to help bacteria resisting infaust stress is meaningful for achieving a stable operation of biological treatment system. In this work, selenium-doped carbon quantum dots (Se-CQDs) were blended into anaerobic ammonia oxidation (anammox) bacteria and an inorganic nanoparticle-microbe hybrid was successfully fabricated to evaluate its nitrogen removal performance under solar-simulated irradiation. It was found that the specific anammox activity decreased by 29.7 ± 5.2% and reactive oxygen species (ROS) content increased by 134.8 ± 4.1% under 50,000 lux light. Sludge activity could be completely recovered under the optimum dosage of 0.42 mL·(g volatile suspended solid) -1 Se-CQDs. Hydroxyl radical (·OH) and superoxide anion radical (·O2-) were identified as the leading ROS inducing lipid peroxidation and antioxidase function detriment. Also, the structure of ladderane lipids located on anammoxosome was destroyed by ROS and functional genes abundances declined accordingly. Although cell surface coated Se-CQDs could absorb ultraviolet light and partially mitigated the photoinhibition, the direct scavenging of ROS by intracellular Se-CQDs primarily contributed to the cellular redox homeostasis, antioxidase activity recovery and sludge activity improvement. The findings of this work provide in-depth understanding the metabolic response mechanism of anammox consortia to light irradiation and might be valuable for a more stable and sustainable nitrogen removal technology, i.e., algal-bacterial symbiotic system, development.

SLL-627 Is a Highly Selective and Potent κ Opioid Receptor (KOR) Agonist with an Unexpected Nonreduction in Locomotor Activity.

Undue central nervous system (CNS) side effects including dysphoria and sedation remain to be a challenge for the development of κ opioid receptor (KOR) agonists as effective and safe analgesics. On the basis of our previous work on morphinan-based KOR agonists, a series of 7α-methyl-7ß-substituted northebaine derivatives were designed, synthesized, and biologically assayed. Among others, compound 4a (SLL-627) has been identified as a highly selective and potent KOR agonist both in vitro and in vivo, and its molecular basis was also examined and discussed. Besides low liability to conditioned place aversion (CPA) test, treatment of SLL-627 was associated with a nonreduction in locomotor activity, compared to most of the other arylacetamide- or morphinan-based KOR agonists which generally exhibited apparently sedative effects. This unexpected finding provides new insights to dissociate analgesia from sedation for future discovery of innovative KOR agonists.

Coagulants put phosphate-accumulating organisms at a competitive disadvantage with glycogen-accumulating organisms in enhanced biological phosphorus removal system.

Enhanced biological phosphorus removal (EBPR) process is susceptible to the changed operation condition, which results in an unstable treatment performance. In this work, long-term effect of coagulants addition, aluminum salt for the reactor R1 and iron salt for the reactor R2, on EBPR systems was comprehensively evaluated. Results showed that during the initial 30 days' coagulant addition, effluent chemical oxygen demand and phosphorus can be reduced below 25 and 0.5 mg·L-1, respectively. Further supply of metal salts would stimulate microbial extracellular polymeric substance excretion and induce reactive oxygen species accumulation, which destroyed the cell membrane integrity and deteriorated the phosphorus removal performance. Moreover, coagulants would decrease the relative abundance of Candidatus Accumulibacter while increase the relative abundance of Candidatus Competibacter, leading phosphors accumulating organisms in a disadvantage position. The results of this work might be valuable for the operation of chemical assisted biological phosphorus removal bioreactor.

Surface oxygen vacancies formation on Zn2SnO4 for bisphenol-A degradation under visible light: The tuning effect by peroxymonosulfate.

Visible light catalysis has been widely coupled with persulfate activation for refractory pollutants removal, while the exact role of persulfate played in such composite system is still questionable. In this work, the relation between peroxymonosulfate (PMS) induced structure change and visible light responsive activity of inverse spinel: i.e., Zn2SnO4, was deciphered. Under the visible light illumination (λ> 420nm) PMS addition would endow the composite system with pollutant removal performance. Batch test revealed that 60% of bisphenol-A (5 mg L-1) was mineralized within 3 h reaction time, by dosing 0.81 mM PMS and 0.1 g L-1 catalyst. The above oxidative system was also effective for other refractory pollutants elimination. Further analysis indicated that PMS could reduce the band gap of spinel from 2.75 to 2.52 eV and thereby enabling its visible light activity. Photogenerated h+ induced •OH and e- mediated •O2- contributed to the pollutant removal while h+ played a leading role. Density functional theory revealed that PMS would capture oxygen atom of spinel and induce surface oxygen vacancy defect structure formation. Also, three-oxygen atom coordinated Zn was identified as the possible catalyze site. This work is valuable for deep understanding the exact role of persulfate in photocatalytic system.

Discovery of an M-Substituted N-Cyclopropylmethyl-7α-phenyl-6,14-endoethanotetrahydronorthebaine as a Selective, Potent, and Orally Active κ-Opioid Receptor Agonist with an Improved Central Nervous System Safety Profile.

The search for selective kappa opioid receptor (κOR) agonists with an improved safety profile is an area of interest in opioid research. In this work, a series of m-substituted analogs were designed, synthesized, and assayed, resulting in the identification of compound 6c (SLL-1206) as a κOR agonist with single-digit nanomolar activities. The subtype selectivity of compound 6c appeared to be a consequence of an enormous decrease in the affinity for µOR and δOR, rather than a significant increase in the affinity for κOR, which was not the case for SLL-039, another selective and potent κOR agonist identified in our previous work. Besides reduced central nervous system effects, SLL-1206 exhibited substantially improved physicochemical and pharmacokinetic properties compared with SLL-039, with increases of over 20-fold in aqueous solubility and approximately 40-fold in oral bioavailability in rats.

Plant Size Plays an Important Role in Plant Responses to Low Water Availability and Defoliation in Two Woody Leguminosae Species.

Plant size influences plant responses to combined environmental factors under climate change. However, their roles in plant ecophysiological responses are not fully understood. Two rapidly growing Leguminosae species (Robinia pseudoacacia and Amorpha fruticosa) were used to examine plant responses to combined drought and defoliation treatments (two levels of both treatments). Both 1.5 month-old seedlings and 3 month-old seedlings were grown in a greenhouse, and seedling growth, leaf gas exchanges, stem hydraulics, and concentrations of non-structural carbohydrates were determined after 60 days of treatment. Our results indicated defoliation had no significant effect on plant height, basal diameter, and total biomass whatever plant sizes and species. Under the low water availability treatment, the defoliated seedlings significantly increased by 24% in stem water potential compared with non-defoliated seedlings in large R. pseudoacacia. Compared with the high water availability in large non-defoliated R. pseudoacacia seedlings, the low water availability significantly reduced by 26% in stem starch concentration to maintain the stem soluble sugar concentration stable, but not in small R. pseudoacacia seedlings. We also found a negative correlation between leaf and root soluble sugar concentration under low water availability in A. fruticosa. The results demonstrate defoliation could relieve the effect of low water availability in large seedlings. Large seedlings had more compensatory mechanisms in response to defoliation and drought treatments than small seedlings, thus species with large carbon reserves are more recommended for vegetation restoration under combined drought and defoliation conditions. Future studies with more species are crucial for obtaining more rigorous conclusions.

Universal Method to Fabricate Transition Metal Single-Atom-Anchored Carbon with Excellent Oxygen Reduction Reaction Activity.

Single-atom catalysts (SACs) have attracted great attention due to their high atom-utilization and catalytic efficiency. However, a universal synthetic route is still lacking, which restricts the SAC-related investigation and application. Here, we report a simple and cost-effective method to fabricate transition metal SACs through ion exchange and annealing procedures. Benefiting from the "egg-box" structure property of alginate, the metal ion can be effectively anchored into the organic center. Using CuCl2 as a representative transition metal ion, the Cu SAC structure was synthesized and identified by aberration-corrected high-angle annular dark-field scanning transmission electron microscopy and X-ray absorption fine structure spectroscopy. Through optimizing CuCl2 concentration, the obtained Cu SAC exhibited a good oxygen reduction reaction activity, whose onset potential, half wave potential, and limiting current density are all comparable to those of 20 wt % Pt/C. Cu-N4 was identified as the responsible catalytic site. More importantly, other transition metal SACs can be easily synthesized via altering metallic solution, which proves the universality of our proposed method. This work may be valuable for the cost-effective and universal SAC synthetic method development.

Conversion of municipal wastewater-derived waste to an adsorbent for phosphorus recovery from secondary effluent.

The sustainable management and recirculation of phosphorus resources are essential to our human lives. In this work, phosphorus removal and recovery from secondary effluent were achieved using municipal wastewater-derived materials as adsorbents. Through modification with 0.5 M NaOH for 30 min, iron containing sludge that originated from the coagulation pretreatment of municipal wastewater was successfully converted to phosphorus adsorbent. The maximal adsorption capacity of the prepared adsorbent was estimated to be 22 mg-P/g, and the adsorption performance remained stable in the pH range of 5-8. FeO(OH) was identified as the key adsorption site, and the ligand exchange mediated chemical adsorption was the main mechanism for phosphorus removal by the prepared material. Moreover, a laboratory-scale continuous-flow adsorption column experiment showed that the surplus phosphorus in secondary effluent could be readily reduced to <0.1 mg/L. By pyrolysis of P-laden alkali-treated iron sludge under oxygen limited conditions, the phosphorus was recovered and successfully applied to support wheat growth. This work provides valuable information for both the sustainable management of phosphorus streams in wastewater and cyclic utilization of waste sludge.

Discovery of a Highly Selective and Potent κ Opioid Receptor Agonist from N-Cyclopropylmethyl-7α-phenyl-6,14-endoethanotetrahydronorthebaines with Reduced Central Nervous System (CNS) Side Effects Navigated by the Message-Address Concept.

Effective and safe analgesics represent an unmet medical need for the treatment of acute and chronic pain. A series of N-cyclopropylmethyl-7α-phenyl-6,14-endoethanotetrahydronorthebaines were designed, synthesized, and assayed, leading to the discovery of a benzylamine derivative (compound 4, SLL-039) as a highly selective and potent κ opioid agonist (κ, Ki = 0.47 nM, κ/µ = 682, κ/δ = 283), which was confirmed by functional assays in vitro and antinociceptive assays in vivo. The in vivo effect could be blocked by pretreatment with the selective κ antagonist nor-BNI. Moreover, this compound did not induce sedation, a common dose limiting effect of κ opioid receptor agonists, at its analgesic dose compared to U50,488H. The dissociation of sedation/antinociception found in SLL-039 was assumed to be correlated with the occupation of its benzamide motif in a unique subsite involving V1182.63, W124EL1, and E209EL2.

Discovery, cocrystallization and biological evaluation of novel piperidine derivatives as high affinity Ls-AChBP ligands possessing α7 nAChR activities.

A series of novel pyridine-substituted piperidine derivatives were discovered as low nanomolar Ls-AChBP ligands with α7 nAChR partial agonism or antagonism activities. A high-resolution antagonist-bound Ls-AChBP complex was successfully resolved with a classic Loop C opening phenomenon and unique sulfur-π interactions which deviated from our previous docking mode to a large extent. With the knowledge of the co-complex, 27 novel piperidine derivatives were designed and synthesized. The structure-activity relationships (SARs) of the aromatic and pyridine regions were well established and binding modes were illustrated with the help of molecular docking which indicated that interactions with Trp 143 and the "water bridge" are essential for the high binding affinities. Halogen bonding as well as the space around 5'- or 6'- position of the pyridine ring was also proposed to influence the binding conformation of the compounds. Notably, two enantiomers of compound 2 showed opposite functions toward α7 nAChR and compound (S)-2 showed sub-nanomolar affinity (Ki = 0.86 nM) on Ls-AChBP and partial agonism (pEC50 = 4.69 ±â€¯0.11,Emax = 36.1%) on α7 nAChR with reasonable pharmacokinetics (PK) properties and fine ability of blood-brain-barrier (BBB) penetration. This study provided promising hits to develop candidates targeting nAChR-related CNS diseases.

7ß-Methyl substituent is a structural locus associated with activity cliff for nepenthone analogues.

With the purpose of identifying novel selective κ opioid receptor (KOR) antagonists as potential antidepressants from nepenthone analogues, starting from N-nor-N-cyclopropylmethyl-nepenthone (SLL-020ACP), a highly selective and potent KOR agonist, a series of 7ß-methyl-nepenthone analogues was conceived, synthesized and assayed on opioid receptors based on the concept of hybridization. According to the pharmacological results, the functional reversal observed in orvinol analogues by introduction of 7ß-methyl substituent could not be reproduced in nepenthone analogues. Alternatively, introduction of 7ß-methyl substituent was associated with substantial loss of both subtype selectivity and potency but not efficacy for nepenthone analogues, which was not found in 7ß-methyl orvinol analogues. Surprisingly, SLL-603, a 7ß-methyl analogue of SLL-020ACP, was identified to be a KOR full agonist. The possible molecular mechanism for the heterogeneity in activity cliff was also investigated. In conclusion, 7ß-methyl substituent was a structural locus associated with activity cliff and demonstrated as a pharmacological heterogeneity between nepenthone and orvinol analogues that warrants further investigations.

Synthetic access to isoxazoline-functionalized isoquinolines via microwave-assisted iminoxyl radical-participated cascade cyclization of vinyl isocyanides.

A convenient microwave-assisted method for the synthesis of isoxazoline-functionalized isoquinolines via the n-Bu4NI (TBAI) catalyzed radical cascade cyclization of vinyl isocyanides with ß,γ-unsaturated ketoximes has been described. The methodology presented here achieves a wide substrate scope and good to excellent yields. A radical mechanism is proposed, which is supported by the intermolecular competing kinetic isotope effect (KIE) experiment and the radical trapping experiment.

Discovery of novel 20S proteasome inhibitors by rational topology-based scaffold hopping of bortezomib.

A series of structurally novel proteasome inhibitors 1-12 have been developed based rational topology-based scaffold hopping of bortezomib. Among these novel proteasome inhibitors, compound 10 represents an important advance due to the comparable proteasome-inhibitory activity (IC50 = 9.7 nM) to bortezomib (IC50 = 8.3 nM), the remarkably higher BEI and SEI values and the effectiveness in metabolic stability. Therefore, compound 10 provides an excellent lead suitable for further optimization.

Microwave-assisted synthesis of hydroxyl-containing isoquinolines by metal-free radical cyclization of vinyl isocyanides with alcohols.

A convenient microwave-assisted protocol for the synthesis of hydroxyl-containing isoquinolines from a metal-free radical cyclization reaction of vinyl isonitriles with alcohols was developed with moderate-to-excellent yields. Vinyl isonitriles are coupled with alkyl radicals through direct catalytic functionalization of the α sp3 C-H bond of alcohols. The methodology demonstrates a broad substrate scope, shows excellent functional group tolerance, is highly atom-economical and highly efficient, thus enabling the preparation of diverse potentially valuable hydroxyl-containing isoquinolines.

An Ursolic Acid Derived Small Molecule Triggers Cancer Cell Death through Hyperstimulation of Macropinocytosis.

Macropinocytosis is a transient endocytosis that internalizes extracellular fluid and particles into vacuoles. Recent studies suggest that hyperstimulation of macropinocytosis can induce a novel nonapoptotic cell death, methuosis. In this report, we describe the identification of an ursolic acid derived small molecule (compound 17), which induces cancer cell death through hyperstimulation of macropinocytosis. 17 causes the accumulation of vacuoles derived from macropinosomes based on transmission electron microscopy, time-lapse microscopy, and labeling with extracellular fluid phase tracers. The vacuoles induced by 17 separate from other cytoplasmic compartments but acquire some characteristics of late endosomes and lysosomes. Inhibiting hyperstimulation of macropinocytosis with the specific inhibitor amiloride blocks cell death, implicating that 17 leads to cell death via macropinocytosis, which is coincident with methuosis. Our results uncovered a novel cell death pathway involved in the activity of 17, which may provide a basis for further development of natural-product-derived scaffolds for drugs that trigger cancer cell death by methuosis.