Caspase-1 Engages Full-Length Gasdermin D through Two Distinct Interfaces That Mediate Caspase Recruitment and Substrate Cleavage.

The recognition and cleavage of gasdermin D (GSDMD) by inflammatory caspases-1, 4, 5, and 11 are essential steps in initiating pyroptosis after inflammasome activation. Previous work has identified cleavage site signatures in substrates such as GSDMD, but it is unclear whether these are the sole determinants for caspase engagement. Here we report the crystal structure of a complex between human caspase-1 and the full-length murine GSDMD. In addition to engagement of the GSDMD N- and C-domain linker by the caspase-1 active site, an anti-parallel ß sheet at the caspase-1 L2 and L2' loops bound a hydrophobic pocket within the GSDMD C-terminal domain distal to its N-terminal domain. This "exosite" interface endows an additional function for the GSDMD C-terminal domain as a caspase-recruitment module besides its role in autoinhibition. Our study thus reveals dual-interface engagement of GSDMD by caspase-1, which may be applicable to other physiological substrates of caspases.

Crystal Structures of the Full-Length Murine and Human Gasdermin D Reveal Mechanisms of Autoinhibition, Lipid Binding, and Oligomerization.

Gasdermin D (GSDMD) is an effector molecule for pyroptosis downstream of canonical and noncanonical inflammasome signaling pathways. Cleavage of GSDMD by inflammatory caspases triggers the oligomerization and lipid binding by its N-terminal domain, which assembles membrane pores, whereas its C-terminal domain binds the N-terminal domain to inhibit pyroptosis. Despite recent progress in our understanding of the structure and function of the murine gasdermin A3 (mGSDMA3), the molecular mechanisms of GSDMD activation and regulation remain poorly characterized. Here, we report the crystal structures of the full-length murine and human GSDMDs, which reveal the architecture of the GSDMD N-terminal domains and demonstrate distinct and common features of autoinhibition among gasdermin family members utilizing their ß1-ß2 loops. Disruption of the intramolecular domain interface enhanced pyroptosis, whereas mutations at the predicted lipid-binding or oligomerization surface reduced cytolysis. Our study provides a framework for understanding the autoinhibition, lipid binding, and oligomerization of GSDMD by using overlapping interfaces.

Chemical modulation of gasdermin D activity: Therapeutic implications and consequences.

The gasdermin family of proteins are central effectors of the inflammatory, lytic cell death modality known as pyroptosis. Characterized in 2015, the most well-studied member gasdermin D can be proteolyzed, typically by caspases, to generate an active pore-forming N-terminal domain. At least well-studied three pharmacological inhibitors (necrosulfonamide, disulfiram, dimethyl fumarate) since 2018 have been shown to affect gasdermin D activity either through modulation of processing or interference with pore formation. A multitude of murine in vivo studies have since followed. Here, we discuss the current state of research surrounding these three inhibitors, caveats to their use, and a set of guiding principles that researchers should consider when pursuing further studies of gasdermin D inhibition.

Endogenous Interfacial Mo-C/N-Mo-S Bonding Regulates the Active Mo Sites for Maximized Li+ Storage Areal Capacity.

Active sites, mass loading, and Li-ion diffusion coefficient are the benchmarks for boosting the areal capacity and storage capability of electrode materials for lithium-ion batteries. However, simultaneously modulating these criteria to achieve high areal capacity in LIBs remains challenging. Herein, MoS2 is considered as a suitable electroactive host material for reversible Li-ion storage and establish an endogenous multi-heterojunction strategy with interfacial Mo-C/N-Mo-S coordination bonding that enables the concurrent regulation of these benchmarks. This strategy involves architecting 3D integrated conductive nanostructured frameworks composed of Mo2 C-MoN@MoS2 on carbon cloth (denoted as C/MMMS) and refining the sluggish kinetics in the MoS2 -based anodes. Benefiting from the rich hetero-interface active sites, optimized Li adsorption energy, and low diffusion barrier, C/MMMS reaches a mass loading of 12.11 mg cm-2 and showcases high areal capacity and remarkable rate capability of 9.6 mAh cm-2 @0.4 mA cm-2 and 2.7 mAh cm-2 @6.0 mA cm-2 , respectively, alongside excellent stability after 500 electrochemical cycles. Moreover, this work not only affirms the outstanding performance of the optimized C/MMMS as an anode material for supercapacitors, underscoring its bifunctionality but also offers valuable insight into developing endogenous transition metal compound electrodes with high mass loading for the next-generation high areal capacity energy storage devices.

The role of macrophage polarization and cellular crosstalk in the pulmonary fibrotic microenvironment: a review.

Pulmonary fibrosis (PF) is a progressive interstitial inflammatory disease with a high mortality rate. Patients with PF commonly experience a chronic dry cough and progressive dyspnoea for years without effective mitigation. The pathogenesis of PF is believed to be associated with dysfunctional macrophage polarization, fibroblast proliferation, and the loss of epithelial cells. Thus, it is of great importance and necessity to explore the interactions among macrophages, fibroblasts, and alveolar epithelial cells in lung fibrosis, as well as in the pro-fibrotic microenvironment. In this review, we discuss the latest studies that have investigated macrophage polarization and activation of non-immune cells in the context of PF pathogenesis and progression. Next, we discuss how profibrotic cellular crosstalk is promoted in the PF microenvironment by multiple cytokines, chemokines, and signalling pathways. And finally, we discuss the potential mechanisms of fibrogenesis development and efficient therapeutic strategies for the disease. Herein, we provide a comprehensive summary of the vital role of macrophage polarization in PF and its profibrotic crosstalk with fibroblasts and alveolar epithelial cells and suggest potential treatment strategies to target their cellular communication in the microenvironment.

Urban Stormwater Phosphorus Export Control: Comparing Traditional and Low-impact Development Best Management Practices.

Data from the International Stormwater Best Management Practices (BMP) Database were used to compare the phosphorus (P) control performance of six categories of stormwater BMPs representing traditional systems (stormwater pond, wetland basin, and detention basin) and low-impact development (LID) systems (bioretention cell, grass swale, and grass strip). Machine learning (ML) models were trained to predict the reduction or enrichment factors of surface runoff concentrations and loadings of total P (TP) and soluble reactive P (SRP) for the different categories of BMP systems. Relative to traditional BMPs, LIDs generally enriched TP and SRP concentrations in stormwater surface outflow and yielded poorer P runoff load control. The SRP concentration reduction and enrichment factors of LIDs also tended to be more sensitive to variations in climate and watershed characteristics. That is, LIDs were more likely to enrich surface runoff SRP concentrations in drier climates, when inflow SRP concentrations were low, and for watersheds exhibiting high impervious land cover. Overall, our results imply that stormwater BMPs do not universally attenuate urban P export and that preferentially implementing LIDs over traditional BMPs may increase TP and SRP export to receiving freshwater bodies, hence magnifying eutrophication risks.

Effect of Self-developed Ye'an Analgetic Decoction/Jiawei Shaoyao Gancao Decoction Combined with Tramadol on TCM Symptom Scores and RLS Severity of patients with Restless Legs Syndrome.

Objective: To investigate the effect of self-developed Ye'an Analgetic Decoction/Jiawei Shaoyao Gancao Decoction on Traditional Chinese Medicine (TCM) symptom scores and RLS Severity of patients with restless legs syndrome (RLS). Methods: This was a clinical comparative study. Eighty patients with RLS admitted to Baoding No.1 Central Hospital from January 2022 to December 2022 were randomly divided into observation group and control group(n=40). Patients in the control group were given basic and oral tramadol treatment, while those in the observation group were given self-developed Ye'an Analgetic Decoction/Jiawei Shaoyao Gancao Decoction based on the treatment in the control group. The differences of TCM symptom scores, RLS severity (IRLS), quality of life (QOL-RLS), sleep quality (PSQI) and clinical efficacy between the two groups were compared. Results: Before treatment, no statistically significant differences were observed in the TCM symptom scores, IRLS scores, QOL-RLS scores and PSQI scores between the two groups (p>0.05). After treatment, the above scores decreased significantly in both groups, with a higher degree of decrease in the observation group than in the control group, indicating statistically significant differences (p<0.05). The QOL-RLS scores were significantly higher in the observation group than in the control group, with a statistically significant difference (p<0.05). The overall response rate in the observation group was 95.00%, which was higher than that in the control group (80.00%), with a statistically significant difference (p<0.05). Conclusion: Self-developed Ye'an Analgetic Decoction/Jiawei Shaoyao Gancao Decoction leads to numerous benefits in the treatment of RLS, such as obviously ameliorating patients' clinical symptoms, reducing RLS severity, and improving their quality of life and sleep quality.

Type 2 diabetes aggravates periodontitis-induced pathological changes in the dental pulp.

OBJECTIVES: The inner mechanism of how diabetes affects dental pulp of patients with periodontitis has seldom been reported. We collected clinical samples and explored the influence of diabetes and periodontitis on the pathological change of dental pulp. METHODS: Dental pulp from healthy individuals and patients with periodontitis with or without diabetes were collected based on strict inclusion and exclusion criteria. Dental pulp was morphologically observed; advanced glycation end products (AGEs) and lectin-like oxidized low-density lipoprotein receptor-1 (LOX1) were examined. Oxidative stress (OS), inflammatory indices, and apoptotic levels were assessed. RESULTS: Morphologically, fibrous structure in the dental pulp of patients with diabetic periodontitis (DP) group was sparse and disordered, and the blood vessel wall was thickened. Diabetes related indexes as AGEs and LOX1 were upregulated. Superoxide dismutase 2 expression was decreased, and OS level was increased. Matrix metalloproteinase 3 and other relevant proinflammatory cytokines levels were increased. The elevated OS and inflammation contributed to upregulation of apoptotic levels in DP group. CONCLUSIONS: Diabetes aggravates the pathological changes in the dental pulp of periodontitis patients possibly due to upregulated AGEs and LOX1. Our results highlight the importance of early oral intervention in patients with DP.

Transcriptomic profiling of human granulosa cells between women with advanced maternal age with different ovarian reserve.

BACKGROUND: Age-related diminished ovarian reserve (DOR) is not absolute. Some advanced maternal age (AMA) still have normal ovarian reserve (NOR) and often show better pregnancy outcomes. Exploring the transcriptomic profile of granulosa cells (GCs) in AMA could lead to new ideas for mitigating age-related diminished ovarian reserve. AIM: This study aimed to analyze the transcriptomic profile of GCs in AMA with different ovarian reserve. RESULTS: In total, 6273 statistically significant differential expression genes (DEGs) (|log2fc|> 1, q < 0.05) were screened from the two groups, among which 3436 genes were upregulated, and 2837 genes were downregulated in the DOR group. Through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, the potential functions of dysregulated genes in AMA with DOR or NOR were predicted. The GO enrichment analysis revealed that the DEGs were mainly enriched in obsolete oxidation-reduction process, mitochondrion, metal ion binding, ATP binding, etc. The KEGG pathway enrichment analysis revealed that the above-mentioned DEGs were mainly enriched in ferroptosis, regulation of actin cytoskeleton, oxidative phosphorylation, etc. Meanwhile, verification of the mRNA expression levels of DEGs revealed the possible involvement of "ferroptosis" in age-related diminished ovarian reserve. CONCLUSIONS: From a new clinical perspective, we presented the first data showing the transcriptomic profile in GCs between AMA with different ovarian reserve. At the same time, we identified the role of ferroptosis in the GCs of AMA, providing a new biological basis for studying ovarian aging and improving pregnancy outcomes of AMA.

Chloramine-T-Enabled Mass Spectrometric Analysis of C═C Isomers of Unsaturated Fatty Acids and Phosphatidylcholines in Human Thyroids.

Specific locations of carbon-carbon double bonds (C═C) in lipids often play an essential role in biological processes, and there has been a booming development in C═C composition analysis by mass spectrometry. However, a universal derivatization and fragmentation pattern for the annotation of C═C positions in lipids is still challenging and attractive. To expand this field in lipidomics, a flexible and convenient N-tosylaziridination method was developed, with high derivatization efficiency, sensitivity, and specificity. The derivatization was very fast (15 s), and C═C numbers as well as locations could be pinpointed specifically in tandem mass spectra. By qualitative and quantitative studies of paratumor and tumor thyroid tissues of human beings, the total content of unsaturated fatty acids was suggested to be increased in tumor tissues, and good correlations in and between lysophosphatidylcholines and phosphatidylcholines were revealed by Spearman analysis. Further studies of C═C isomers showed that n-6/n-3 ratios were closely associated with human thyroid tumorigenesis, and high ratios of n-6/n-3 isomers seemed to suffer a high risk of carcinogenesis. Other isomers were not very representative; however, C═C in n-9/n-7 could also be significant for oncology research. Generally, it is supposed that both total amounts and C═C isomer ratios were related to cancer, and N-tosylaziridine derivatization could provide an alternative strategy for the C═C isomer study of disease models.

Sirtuin 2 Prevents Liver Steatosis and Metabolic Disorders by Deacetylation of Hepatocyte Nuclear Factor 4α.

BACKGROUND AND AIMS: Sirtuin 2 (SIRT2), an NAD+ -dependent deacetylase, is involved in various cellular processes regulating metabolic homeostasis and inflammatory responses; however, its role in hepatic steatosis and related metabolic disorders is unknown. APPROACH AND RESULTS: Integrating the published genomic data on NAFLD samples from humans and rodents available in the Gene Expression Omnibus, we found that SIRT2 was significantly down-regulated in livers from patients with advanced NAFLD and high-fat diet (HFD)-induced NAFLD mice. This study further revealed that SIRT2 was markedly decreased in obese (ob/ob) mice and in palmitate-treated HepG2 cells. Restoration of hepatic SIRT2 expression in ob/ob or HFD-fed mice largely alleviated insulin resistance, hepatic steatosis, and systematic inflammation, whereas SIRT2 liver-specific ablation exacerbated these metabolic dysfunctions in HFD-fed C57BL/6J mice. Mechanistically, SIRT2 stabilized the hepatocyte nuclear factor 4α (HNF4α) protein by binding to and deacetylating HNF4α on lysine 458. Furthermore, HNF4α was sufficient to mediate SIRT2 function, and SIRT2-HNF4α interaction was required for SIRT2 function both in vivo and in vitro. CONCLUSIONS: Collectively, the present study provided evidence that SIRT2 functions as a crucial negative regulator in NAFLD and related metabolic disorders and that targeting the SIRT2-HNF4α pathway may be a promising strategy for NAFLD treatment.

Chiral Analysis of Lactate during Direct Contact Coculture by Single-Cell On-Probe Enzymatic Dehydrogenation Derivatization: Unraveling Metabolic Changes Caused by d-Lactate.

In vitro noncontact cell-based coculture models are frequently employed to study cell-to-cell communication. However, these models cannot accurately represent the complexity of in vivo signaling. d-Lactate is an unusual metabolite produced and released by cancer cells. The characterization of d-lactate is challenging as it shares the same mass but has much lower amounts compared with l-lactate. Herein, d-α-hydroxy acids were specifically recognized and dehydrogenated by d-α-hydroxy acid dehydrogenase. The dehydrogenation products were rapidly quaternized for enhancement of mass signals. An on-probe enzymatic dehydrogenation-derivatization method was proposed for chiral analysis of α-hydroxy acids at the single-cell level. It is a promising amplification methodology and affords over 3 orders of magnitude signal enhancement. Furthermore, direct contact coculture models were used to precisely mimic the tumor microenvironment and explore the communication between cancer and normal cells. Single-cell mass spectrometry (SCMS) was further applied to easily sample cell extracts and study the differences of the aspects of small molecule metabolism in cocultured cells. On the basis of direct contact coculture SCMS, several differential small molecule metabolites and differences of oxidative stress between cocultured and monocultured normal cells were successfully detected. Additionally, d-lactate was discovered as a valuable differential metabolite with application of the two developed methods. It may account for the cancer-associated metabolic behavior of normal cells. These changes could be relieved after d-lactate metabolism-related drug treatment. This discovery may promote the investigation of d-lactate metabolism, which may provide a novel direction for cancer therapy.

Powerful Steroid-Based Chiral Selector for High-Throughput Enantiomeric Separation of α-Amino Acids Utilizing Ion Mobility-Mass Spectrometry.

Stereospecific recognition of amino acids (AAs) plays a crucial role in chiral biomarker-based diagnosis and prognosis. Separation of AA enantiomers is a long and tedious task due to the requirement of AA derivatization prior to the chromatographic or electrophoretic steps which are also time-consuming. Here, a mass-tagged chiral selector named [d0]/[d5]-estradiol-3-benzoate-17ß-chloroformate ([d0]/[d5]-17ß-EBC) with high reactivity and good enantiomeric resolution in regard to AAs was developed. After a quick and easy chemical derivatization step of AAs using 17ß-EBC as the single chiral selector before ion mobility-mass spectrometry analysis, good enantiomer separation was achieved for 19 chiral proteinogenic AAs in a single analytical run (∼2 s). A linear calibration curve of enantiomeric excess was also established using [d0]/[d5]-17ß-EBC. It was demonstrated to be capable of determining enantiomeric ratios down to 0.5% in the nanomolar range. 17ß-EBC was successfully applied to investigate the absolute configuration of AAs among peptide drugs and detect trace levels of d-AAs in complex biological samples. These results indicated that [d0]/[d5]-17ß-EBC may contribute to entail a valuable step forward in peptide drug quality control and discovering chiral disease biomarkers.

Rapid Characterization of Polymer Materials Using Arc Plasma-Based Dissociation-Mass Spectrometry.

Fingerprinting spectra of polymer materials containing information of monomers' molecular weight and detailed structure, constituents, and sequences were obtained by a direct analytical process using arc plasma-based dissociation (APD)-mass spectrometry. The thermal arc plasma generated using a simple arc discharge device induces the dissociation of the polymeric backbone, producing mass spectra with strong regularity within seconds. The molecular weight of the repeating unit was revealed by equal intervals between peak series and protonated monomer ions in the mass spectra. Meanwhile, lots of secondary fragment ions were produced to provide abundant structural information. For polyethers, it is even possible to decipher (read) the "sequence" directly from their spectra. Polymers composed of isomers or only differing in their initiator moieties were easily distinguished with their characteristic APD mass spectra. The spectra were highly reproducible according to the results of similarity calculation. Unlike pyrolysis mass spectrometry, in the APD device, polymers in liquid, solid, powder, and crude samples can be analyzed directly without any pretreatment, and the regular spectra are easier to interpret. Compared with other direct analytical methods, more structural informative spectra can be acquired owing to the high energy, high temperature, and unique chemical reactivity of arc plasma. Thus, this technique is promising to be a valuable tool in rapid elucidation of polymer materials.

High-Pressure Synthesis and Magnetism of the 4H-BaMnO3 Single Crystal and Its 6H-Type Polymorph.

A 4H-type BaMnO3 single crystal was prepared by combining the floating zone method with high-pressure treatment at 5 GPa and 1023 K. The crystal crystallizes to a hexagonal structure with space group P63/mmc and lattice parameters a = 5.63723(5) Å and c = 9.22355(8) Å. In this structure, face-sharing MnO6 octahedral dimers connect with each other by corner O atoms along the c-axis direction, forming an -A-B-A-C-type 4H arrangement. A long-range antiferromagnetic (AFM) phase transition is found to occur at TN ≈ 263 K. When the synthesis pressure increases to 20 GPa, a new polymorphic phase is obtained. This higher-pressure phase still possesses the hexagonal P63/mmc symmetry, but the lattice parameters change to be a = 5.61349(2) Å and c = 13.66690(9) Å with a unit cell volume reduction of 2.05%. In this new phase, the c-axis MnO6 dimers are separated by MnO6 octahedral layers in the ab plane, forming an -A-B-C-A-C-B-type 6H structure. The 6H phase exhibits two long-range AFM orderings at TN1 ≈ 220 K and TN2 ≈ 25 K, respectively. The different magnetic properties are discussed on the basis of the detailed structural constitutions of 4H- and 6H-BaMnO3.

Os Doping Suppressed Cu-Fe Charge Transfer and Induced Structural and Magnetic Phase Transitions in LaCu3Fe4-xOsxO12 (x = 1 and 2).

B-site Os-doped quadruple perovskite oxides LaCu3Fe4-xOsxO12 (x = 1 and 2) were prepared under high-pressure and high-temperature conditions. Although parent compound LaCu3Fe4O12 experiences Cu-Fe intermetallic charge transfer that changes the Cu3+/Fe3+ charge combination to Cu2+/Fe3.75+ at 393 K, in the Os-doped samples, the Cu and Fe charge states are found to be constant 2+ and 3+, respectively, indicating the complete suppression of charge transfer. Correspondingly, Os6+ and mixed Os4.5+ valence states are determined by X-ray absorption spectroscopy for x = 1 and x = 2 compositions, respectively. The x = 1 sample crystallizes in an Fe/Os disordered structure with the Im3̅ space group. It experiences a spin-glass transition around 480 K. With further Os substitution up to x = 2, the crystal symmetry changes to Pn3̅, where Fe and Os are orderly distributed in a rocksalt-type fashion at the B site. Moreover, this composition shows a long-range Cu2+(↑)Fe3+(↑)Os4.5+(↓) ferrimagnetic ordering near 520 K. This work provides a rare example for 5d substitution-suppressed intermetallic charge transfer as well as induced structural and magnetic phase transitions with high spin ordering temperature.

Life Cycle Assessment of Urine Diversion and Conversion to Fertilizer Products at the City Scale.

Urine diversion has been proposed as an approach for producing renewable fertilizers and reducing nutrient loads to wastewater treatment plants. Life cycle assessment was used to compare environmental impacts of the operations phase of urine diversion and fertilizer processing systems [via (1) a urine concentration alternative and (2) a struvite precipitation and ion exchange alternative] at a city scale to conventional systems. Scenarios in Vermont, Michigan, and Virginia were modeled, along with additional sensitivity analyses to understand the importance of key parameters, such as the electricity grid and wastewater treatment method. Both urine diversion technologies had better environmental performance than the conventional system and led to reductions of 29-47% in greenhouse gas emissions, 26-41% in energy consumption, approximately half the freshwater use, and 25-64% in eutrophication potential, while acidification potential ranged between a 24% decrease to a 90% increase. In some situations, wastewater treatment chemical requirements were eliminated. The environmental performance improvement was usually dependent on offsetting the production of synthetic fertilizers. This study suggests that urine diversion could be applied broadly as a strategy for both improving wastewater management and decarbonization.

Non-targeted screening of pyranosides in Rhodiola crenulata using an all ion fragmentation-exact neutral loss strategy combined with liquid chromatography-quadrupole time-of-flight mass spectrometry.

INTRODUCTION: Pyranosides as one kind of natural glycosides contain a pyran ring linked to an aglycone in the structure. They occur widely in plants and possess diverse biological activities. The discovery of new pyranosides not only contributes to research on natural products but also may promote pharmaceutical development. OBJECTIVES: A non-targeted liquid chromatography-quadrupole time-of-flight mass spectrometry method coupled with an all ion fragmentation-exact neutral loss (AIF-ENL) strategy was developed for the screening of pyranosides in plants. METHODS: Pyranosides in various types were collected as a model. The AIF-ENL strategy comprised three steps: AIF spectrum acquisition and generation, ENL-based searching and identification, and confirmation of structural type using target second-stage mass spectrometry (MS/MS). The strategy was systematically evaluated based on the matrix effects, fragmentation stability, scan rate and screening efficiency and finally applied to Rhodiola crenulata (Hook. f. et Thoms) H. Ohba. RESULTS: The method was proved to be an efficient tool for the screening of pyranosides. When it was applied to R. crenulata, a total of 24 pyranoside candidates were detected. Among them, six were tentatively identified on the basis of the agreement of their elemental composition with the reported. The other 18 were detected in R. crenulata for the first time. CONCLUSION: The method offers a new platform for discovering pyranosides. In addition, the developed non-targeted strategy can also be used for other natural products, such as flavonoids and coumarins, as long as there is a common fragmentation behaviour in their MS/MS to generate characteristic neutral losses or fragments.

Rapid Discrimination of Citrus reticulata 'Chachi' by Electrospray Ionization-Ion Mobility-High-Resolution Mass Spectrometry.

A common idea is that some dishonest businessmen often disguise Citrus reticulata Blanco varieties as Citrus reticulata 'Chachi', which places consumers at risk of economic losses. In this work, we combined high-resolution ion mobility (U-shaped mobility analyzer) with high-resolution mass spectrometry to rapidly distinguish Citrus reticulata 'Chachi' from other Citrus species. The samples were analyzed directly through simple extraction and the analytes were separated in one second. It only took about 1 min to perform a cycle of sample analysis and data acquisition. The results showed that polymethoxylated flavones and their isomers were separated easily by the ion mobility analyzer and preliminarily identified according to the accurate mass. Moreover, the collision cross-section values of all analytes, which could be used as auxiliary parameters to characterize and identify the compounds in the samples, were measured. Twenty-four samples were grouped as two clusters by multivariate analysis, which meant that Citrus reticulata 'Chachi' could be effectively differentiated. It was confirmed that the developed method had the potential to rapidly separate polymethoxylated flavones and distinguish between Citrus reticulata 'Chachi' and other Citrus reticulata Blanco varieties.

Mass Spectrometry-Based Discovery of New Chemical Scaffold Rearrangement Ions: Aza-biphenylene as a Novel Potent Biradical Agent in Cancer Chemotherapy.

Discovery of a new drug is time-consuming, laborious, and expensive. Herein, a novel integrative strategy for discovering potential new lead compounds has been developed, which was based on the characteristics of mass spectrometry (MS). MS was used to predict the potential forced degradation products (DPs) and metabolites of drugs by electrospray ionization and collision-induced dissociation (CID). Special rearrangement ions representing unique predicted DPs and metabolites were identified. The consistency between the predicted and the measured results was proven by in vitro metabolism and forced degradation of a commercial drug, respectively. From this, new chemical scaffold rearrangement ions named (aza)-biphenylenes, as potent anticancer agents, were discovered. As a representative aza-biphenylene analogue, 2-azabiphenylene was proven in vitro to induce apoptosis and inhibit the growth of various human cancer cells in a dose-dependent manner. Surprisingly, 2-azabiphenylene exhibited the best comparable bioactivity with the positive control sorafenib, but showed significantly lower in vitro cytotoxicity than sorafenib (at least a 5-fold decrease in cytotoxicity) because it could be targeted to the tumor microenvironment at low pH. A biradical mechanism accompanied by a mitochondrion-dependent oxidative stress mechanism was proposed to explore its anticancer mechanism. The highly reactive intermediate aza-biphenylenediyl worked as an active pharmaceutical ingredient and induced apoptosis of cancer cells. This provided the basis for the potential applications of CID-induced special rearrangement ions in developing new lead compounds.