Mechanistic Insights Research Articles

A Systematic Review of Herbal Interventions for the Management of Cardiovascular Diseases.

Cardiovascular diseases represent a significant global health burden, necessitating diverse approaches for effective management. Herbal interventions have gained attention as potential adjuncts or alternatives to conventional therapies due to their perceived safety and therapeutic potential. This structured abstract provides a comprehensive review of herbal interventions for the management of CVDs, summarising key findings, mechanisms of action, and clinical implications. This systematic review aims to evaluate the impact of various herbal interventions employed for managing cardiovascular diseases. We conducted an extensive literature search across electronic databases, including PubMed, Scopus, and Web of Science, from inception to 2022. Studies were included if they investigated the use of herbal remedies for preventing or treating CVDs. Data extraction and synthesis focused on botanical sources, active compounds, mechanisms of action, and clinical outcomes. Numerous herbal interventions have demonstrated promising cardiovascular benefits. A number of medicinal herbs well identified to treat CVD are Moringaoleifera, Ginseng, Ginkgo biloba, Celosia argentea, Gongronematrifolium, Gynostemmapentaphyllum, Bombaxceiba, Gentianalutea, Allium sativum, Crataegusspp, Curcuma longa, Camellia sinensis, and Zingiberofficinale. Mechanistic insights reveal that herbal interventions often target multiple pathways involved in CVD pathogenesis. These mechanisms encompass anti-inflammatory, antioxidant, anti-thrombotic, anti-hypertensive, and lipid-lowering effects. Additionally, some herbs enhance endothelial function, promote nitric oxide production, and exert vasodilatory effects, contributing to improved cardiovascular health. Clinical studies have provided evidence of the efficacy of certain herbal interventions in reducing CVD risk factors and improving patient outcomes. However, more rigorous, large-scale clinical trials are needed to establish their long-term safety and effectiveness. It is crucial to consider potential herb-drug interactions and standardise dosages for reliable therapeutic outcomes. This comprehensive review highlights the potential of herbal interventions as valuable adjuncts or alternatives for managing cardiovascular diseases. Herbal remedies offer diverse mechanisms of action, targeting key CVD risk factors and pathways. While promising, their clinical utility warrants further investigation through well-designed trials to establish their safety and efficacy, paving the way for integrated approaches to cardiovascular disease management. Healthcare providers and patients should engage in informed discussions about the use of herbal interventions alongside conventional therapies in the context of CVD prevention and treatment.

Chemical diversity and antimelanoma potential of rosemary essential oils: Unveiling mechanistic insights through quantitative proteomics

Rosemary essential oil (REO) is widely recognized as a popular natural product because of its antioxidant, antibacterial, and anti-inflammatory properties, but its role in cancer treatment remains relatively understudied. In light of melanoma’s status as one of the most hazardous tumors, this study employed GCMS to analyze three REOs, focusing on their antiproliferative effects and the underlying mechanisms in melanoma cells. Through multivariate analysis, active components associated with cytotoxicity were identified, with the ‘Dutch Mill’ REO showing the strongest antiproliferative effect and selected for further investigation. The impacts on the cell cycle and death mechanisms were explored in B16-F10 cells, and quantitative proteomics revealed 165 differentially regulated proteins, some of which were significantly associated with ferroptosis. REO notably increased reactive oxygen species levels, decreased mitochondrial membrane potential, and elevated lipid peroxidation in B16-F10 cells. The qRTPCR results confirmed significant alterations in the ferroptosis pathway. Furthermore, the antiproliferative and ROS-promoting effects of REO were reversed by ferroptosis inhibitors. These findings highlight the promising potential of REO in health care and pharmaceuticals.

Rv2231c, a unique histidinol phosphate aminotransferase from Mycobacterium tuberculosis, supports virulence by inhibiting host-directed defense

Nitrogen metabolism of M. tuberculosis is critical for its survival in infected host cells. M. tuberculosis has evolved sophisticated strategies to switch between de novo synthesis and uptake of various amino acids from host cells for metabolic demands. Pyridoxal phosphate-dependent histidinol phosphate aminotransferase-HspAT enzyme is critically required for histidine biosynthesis. HspAT is involved in metabolic synthesis of histidine, phenylalanine, tyrosine, tryptophan, and novobiocin. We showed that M. tuberculosis Rv2231c is a conserved enzyme with HspAT activity. Rv2231c is a monomeric globular protein that contains α-helices and β-sheets. It is a secretory and cell wall-localized protein that regulates critical pathogenic attributes. Rv2231c enhances the survival and virulence of recombinant M. smegmatis in infected RAW264.7 macrophage cells. Rv2231c is recognized by the TLR4 innate immune receptor and modulates the host immune response by suppressing the secretion of the antibacterial pro-inflammatory cytokines TNF, IL-12, and IL-6. It also inhibits the expression of co-stimulatory molecules CD80 and CD86 along with antigen presenting molecule MHC-I on macrophage and suppresses reactive nitrogen species formation, thereby promoting M2 macrophage polarization. Recombinant M. smegmatis expressing Rv2231c inhibited apoptosis in macrophages, promoting efficient bacterial survival and proliferation, thereby increasing virulence. Our results indicate that Rv2231c is a moonlighting protein that regulates multiple functions of M. tuberculosis pathophysiology to increase its virulence. These mechanistic insights can be used to better understand the pathogenesis of M. tuberculosis and to design strategies for tuberculosis mitigation.

Base‐ and Visible‐Light‐Promoted Formation of 3‐Benzyl‐3‐Methoxyisoindolin‐1‐one

AbstractAryl diazonium salts are versatile compounds known for their reactivity in various transformations. In this study, we explored the arylation of 3‐methylene isoindolinones using aryl diazonium salts. The objective was to develop a method for accessing 3‐benzyl‐3‐methoxyisoindolin‐1‐ones, a class of compounds with diverse biological activities and synthetic importance. Optimization of the reaction conditions revealed the significance of light irradiation for improved yields. The substrate scope investigation demonstrated the compatibility of various diazonium salts and N‐substituents on the 3‐methylene isoindolinone scaffold, yielding the corresponding methoxy lactams with high yields. Mechanistic insights were obtained by observing the effect of base and light irradiation. Two initiation mechanisms were proposed: in the presence of a base, the reaction could proceed even without light irradiation, while light irradiation was necessary in the absence of a base. DFT calculations were performed to elucidate the mechanism of the reaction and provide energetic considerations

The Enigma of Tau Protein Aggregation: Mechanistic Insights and Future Challenges.

Tau protein misfolding and aggregation are pathological hallmarks of Alzheimer's disease and over twenty neurodegenerative disorders. However, the molecular mechanisms of tau aggregation in vivo remain incompletely understood. There are two types of tau aggregates in the brain: soluble aggregates (oligomers and protofibrils) and insoluble filaments (fibrils). Compared to filamentous aggregates, soluble aggregates are more toxic and exhibit prion-like transmission, providing seeds for templated misfolding. Curiously, in its native state, tau is a highly soluble, heat-stable protein that does not form fibrils by itself, not even when hyperphosphorylated. In vitro studies have found that negatively charged molecules such as heparin, RNA, or arachidonic acid are generally required to induce tau aggregation. Two recent breakthroughs have provided new insights into tau aggregation mechanisms. First, as an intrinsically disordered protein, tau is found to undergo liquid-liquid phase separation (LLPS) both in vitro and inside cells. Second, cryo-electron microscopy has revealed diverse fibrillar tau conformations associated with different neurodegenerative disorders. Nonetheless, only the fibrillar core is structurally resolved, and the remainder of the protein appears as a "fuzzy coat". From this review, it appears that further studies are required (1) to clarify the role of LLPS in tau aggregation; (2) to unveil the structural features of soluble tau aggregates; (3) to understand the involvement of fuzzy coat regions in oligomer and fibril formation.

Mechanistic Insights into 1,2-bis(2,4,6-tribromophenoxy)ethane-Induced Male Reproductive Toxicity in Zebrafish.

The novel brominated flame retardant, 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE), has increasingly been detected in environmental and biota samples. However, limited information is available regarding its toxicity, especially at environmentally relevant concentrations. In the present study, adult male zebrafish were exposed to varying concentrations of BTBPE (0, 0.01, 0.1, 1, and 10 μg/L) for 28 days. The results demonstrated underperformance in mating behavior and reproductive success of male zebrafish when paired with unexposed females. Additionally, a decline in sperm quality was confirmed in BTBPE-exposed male zebrafish, characterized by decreased total motility, decreased progressive motility, and increased morphological malformations. To elucidate the underlying mechanism, an integrated proteomic and phosphoproteomic analysis was performed, revealing a predominant impact on mitochondrial functions at the protein level and a universal response across different cellular compartments at the phosphorylation level. Ultrastructural damage, increased expression of apoptosis-inducing factor, and disordered respiratory chain confirmed the involvement of mitochondrial impairment in zebrafish testes. These findings not only provide valuable insights for future evaluations of the potential risks posed by BTBPE and similar chemicals but also underscore the need for further research into the impact of mitochondrial dysfunction on reproductive health.

Abstract PO3-23-10: Continued treatment with CDK4/6 inhibitors slow tumor progression by extending G1-phase duration in drug resistant populations

Abstract CDK4/6 inhibitors (CDK4/6i) have significantly improved the prognosis of patients with metastatic hormone receptor-positive breast cancer. However, the development of drug resistance leads to the discontinuation of CDK4/6i treatment. The potential benefits of maintaining CDK4/6i treatment in the context of resistance and their underlying molecular mechanisms remain elusive. Here we show that despite tumor progression, continued treatment with CDK4/6i notably extends the G1 phase in drug-resistant cells, thereby slowing their growth. Our single-cell data reveal that the duration of the G1 phase in these resistant cells is approximately tripled under continuous CDK4/6i treatment compared to conditions with CDK4/6i withdrawal, while the durations of the S and G2 phases remain comparable. The maintenance of CDK4/6i induces an alternative inactivation of the retinoblastoma (Rb) protein in drug-resistant cells through its post-translational degradation. However, this alternative form of Rb inactivation is ineffective and thus induces slow activation kinetics of E2F transcription factors, significantly delaying G1 progression. The beneficial effect of maintaining CDK4/6i treatment was further validated in breast cancer xenograft models. Furthermore, our findings suggest that given the spectrum of various CDK4/6i, switching between different CDK4/6i types after the emergence of drug resistance may further delay tumor progression. This study provides new mechanistic insights into the maintenance of CDK4/6i treatment despite the development of drug resistance. Citation Format: Jessica Armand, Sungsoo Kim, Hee Won Yang. Continued treatment with CDK4/6 inhibitors slow tumor progression by extending G1-phase duration in drug resistant populations [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-23-10.

Boosted photocatalytic removal of NO using direct Z-scheme UiO-66-NH2/Bi2MoO6 nanoflowers heterojunction: mechanism insight and humidity effect

Boosted photocatalytic removal of NO using direct Z-scheme UiO-66-NH2/Bi2MoO6 nanoflowers heterojunction: mechanism insight and humidity effect

Abstract PO4-14-10: Circulating Tumor DNA as a Biomarker for ADCs in Metastatic Breast Cancer

Abstract Background: Breast cancer is a complex disease characterized by heterogeneity, and the analysis of circulating tumor DNA (ctDNA) holds promise for capturing this heterogeneity. Recently, ctDNA testing has been employed in clinical practice to guide targeted therapies for metastatic breast cancer, particularly in cases of hormone receptor-positive disease with mutations in PIK3CA and ESR1. This approach becomes necessary when traditional tumor biopsies are inadequate for next-generation sequencing (NGS) testing or fail to capture the full extent of the cancer's heterogeneity. Moreover, the utilization of antibody-drug conjugates (ADC) in addressing tumor heterogeneity continues to expand. However, there has been no biomarker, outside of HER2 protein expression, to predict ADC response. In this project, we test the hypothesis that ctDNA can be utilized as a biomarker to predict response to novel therapies such as ADCs. Methods: We analyzed a subset of patients from the Dallas Metastatic Breast Cancer Study comprised of patients with metastatic breast cancer (n= 109) who underwent ctDNA testing using the Tempus xF liquid biopsy ctDNA sequencing panel. This panel detects 105-genes that are known oncogenic drivers and resistance mutations. The data was collected from a single academic medical center between the initial year of ctDNA collection in 2019 and 2023. Results: Among these patients, 85 (77.9%) had hormone receptor-positive disease, 13 (11.9%) had triple-negative breast cancer, and 11 (10.0%) had HER2-positive disease (including 3 (2.75%) patients with triple-positive disease). Only 22 of these patients had tissue biopsies that underwent NGS testing. Analysis of all patients revealed that the most common gene alteration was PIK3CA, which was identified in 70 patients. Specifically, the most frequently observed alteration was PIK3CA p.545K, found in 25 patients. Table 1 further details the frequency of each gene in this subset of patients. Among the patients, 34 out of 109 (31%) received an ADC, while only 8 out of 109 (7%) received immunotherapy. Patients with CDKN2A achieved the longest progression-free survival (PFS) on an ADC, with a median PFS of 7 months, whereas patients carrying mutations in TP53, PIK3R1, PIK3CA, PTEN, NF1, BRAF, RHOA, FGFR4, KRAS, TERT, GATA3, HNF1A, FB1, STOP had median PFSs of 2 months or less. Table 2 provides additional information on the response of patients with specific mutations to ADCs. Notably, only one patient received sequential ctDNA testing, and in that case, there was a gain of TP53 p.N239S following sacituzumab govitecan administration compared to the baseline ctDNA test, followed by a gain of PIK3CA p.P449_L455del after trastuzumab deruxtecan administration. Conclusions: As we observe changes in subtypes following treatment progression, researchers are actively seeking biomarkers to better characterize real-time changes in tumor biology that can predict response and resistance to treatments. Further studies are needed to identify additional genes present in ctDNA that can provide mechanistic insights into why certain patients respond favorably to ADCs while others do not. We are currently enrolling patients for a prospective study that aims to assess baseline ctDNA levels and monitor changes in variant allele frequency following treatment with ADCs and immunotherapy. Table 2 Median PFS on ADC by Gene Citation Format: Hannah Chang, Isabela Anawate, Alyssa Low, Shao-Po Huang, Julia Maues, Christine Hodgdon, Isaac Chan. Circulating Tumor DNA as a Biomarker for ADCs in Metastatic Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-14-10.

Unveiling the Mechanism of Plasma-Catalyzed Oxidation of Methane to C2+ Oxygenates over Cu/UiO-66-NH2.

Nonthermal plasma (NTP) offers the potential for converting CH4 with CO2 into liquid products under mild conditions, but controlling liquid selectivity and manipulating intermediate species remain significant challenges. Here, we demonstrate the effectiveness of the Cu/UiO-66-NH2 catalyst in promising the conversion of CH4 and CO2 into oxygenates within a dielectric barrier discharge NTP reactor under ambient conditions. The 10% Cu/UiO-66-NH2 catalyst achieved an impressive 53.4% overall liquid selectivity, with C2+ oxygenates accounting for ∼60.8% of the total liquid products. In situ plasma-coupled Fourier-transform infrared spectroscopy (FTIR) suggests that Cu facilitates the cleavage of surface adsorbed COOH species (*COOH), generating *CO and enabling its migration to the surface of Cu particles. This surface-bound *CO then undergoes C-C coupling and hydrogenation, leading to ethanol production. Further analysis using CO diffuse reflection FTIR and 1H nuclear magnetic resonance spectroscopy indicates that in situ generated surface *CO is more effective than gas-phase CO (g) in promoting C-C coupling and C2+ liquid formation. This work provides valuable mechanistic insights into C-C coupling and C2+ liquid production during plasma-catalytic CO2 oxidation of CH4 under ambient conditions. These findings hold broader implications for the rational design of more efficient catalysts for this reaction, paving the way for advancements in sustainable fuel and chemical production.

Abstract PO4-14-12: Predicting Response to HER2 Tyrosine Kinase Inhibitors and Antibody Drug Conjugates in HER2 Mutant Invasive Lobular Carcinoma Using CRISPR/Cas9 Knock-in Cell lines and Patient-derived Organoids

Abstract Background: Activating mutations in ERBB2 (HER2) are enriched >4-fold in invasive lobular carcinoma (ILC) with a rate of up to 19% in metastatic ILC. ILC is a histologic subtype of breast cancer characterized by loss of E-cadherin (CDH1), suggesting a potential interaction between loss of CDH1 and mutations in ERBB2. Recent trials have demonstrated promising single agent efficacy using the irreversible pan-HER tyrosine kinase inhibitor (TKI), neratinib, in patients with metastatic ERBB2 mutant ILC. However, further studies on combination therapies with other anticancer agents are needed to increase response rate and progression free survival for these patients. HER2-targeted antibody drug conjugates (ADC), particularly trastuzumab deruxtecan (T-DXd), have shown great promise in HER2-low metastatic breast cancer. Yet, their efficacy as a single agent or with HER2 TKIs in HER2-low and -mutant ILC is unknown and warrants investigation. Methods: Past studies analyzing ERBB2 mutations used overexpression of ERBB2 mutant cDNA, but this approach does not faithfully recapitulate the human disease. To model ERBB2 missense mutations as found in human breast cancers, we used CRISPR-based prime editing to generate a panel of isogenic ILC cell lines and patient-derived organoids (PDO) harboring ERBB2 wild-type (WT) or ERBB2 mutations (S310F or V777L). Both of these ERBB2 mutations have been previously characterized and are known to be activating mutations. We then used them to test neratinib and other TKIs with ADCs, including T-DXd and trastuzumab emtansine (T-DM1). Results: We successfully introduced single copy, heterozygous activating ERBB2 mutations (S310F or V777L) into two ERBB2-nonamplified metastatic ILC cell lines (MDA-MB-134 and SUM44PE) and one ERBB2-nonamplified metastatic ILC PDO (IPM-BO-053). Positive clones carrying the mutations were verified by Sanger sequencing and droplet digital PCR and subsequently pooled together. We further demonstrated that these mutations hyperactivated HER2 and downstream signaling pathways. ILC cell lines harboring these mutations showed enhanced sensitivity to HER2 TKIs but not ADCs. In contrary, ERBB2 mutations did not alter responses of IPM-BO-053 PDOs to HER2 TKIs but significantly increased responses to ADCs. Interestingly, we also observed accelerated HER2 protein degradation upon heregulin stimulation in ERBB2 mutant ILC PDOs, suggesting activating ERBB2 mutations may enhance ADC/HER2 complex internalization, degradation, and release of payloads. Lastly, we explored drug synergy between HER2 TKIs and ADCs in isogenic IPM-BO-053 PDOs and found that combination of T-DXd and neratinib or afatinib showed synergy (Combination Index < 1). Conclusions: Although the reason for the discrepancies in drug response between ILC cell lines and PDOs is not clear, we hypothesize that response in 3D PDOs might be more faithfully representing response seen in patients. We will generate additional ILC PDOs with knock-in ERBB2 mutations to validate our findings. Irreversible HER2 TKIs, such as neratinib and afatinib, showed synergy with T-DXd in ERBB2 mutant ILC PDOs. This holds important therapeutic implications in light of current treatment options for ILC. In future experiments, we will test if neratinib or afatinib increases endocytic uptake of T-DXd using a fluorescently labeled endocytosis tracker. Further, an in-depth molecular characterization of our isogenic cell lines and PDOs models is ongoing to gain mechanistic insights into how activating ERBB2 mutations increase HER2 internalization and degradation. Our in vitro studies provide a strong foundation for in vivo testing of neratinib or afatinib with T-DXd for ERBB2 mutant ILC. Citation Format: Jie Bin Liu, Danielle Tseng, Jagmohan Hooda, Daniel Brown, Adrian Lee, Steffi Oesterreich. Predicting Response to HER2 Tyrosine Kinase Inhibitors and Antibody Drug Conjugates in HER2 Mutant Invasive Lobular Carcinoma Using CRISPR/Cas9 Knock-in Cell lines and Patient-derived Organoids [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-14-12.

NHC-CDI Ligands Boost Multicarbon Production in Electrocatalytic CO2 Reduction by Increasing Accumulated Charged Intermediates and Promoting *CO Dimerization on Cu.

Copper-based materials exhibit significant potential as catalysts for electrochemical CO2 reduction, owing to their capacity to generate multicarbon hydrocarbons. The molecular functionalization of Cu electrodes represents a simple yet powerful strategy for improving the intrinsic activity of these materials by favoring specific reaction pathways through the creation of tailored microenvironments around the surface active sites. However, despite its success, comprehensive mechanistic insights derived from experimental techniques are often limited, leaving the active role of surface modifiers inconclusive. In this work, we show that N-heterocyclic carbene-carbodiimide-functionalized Cu catalysts display a remarkable activity for multicarbon product formation, surpassing bare Cu electrodes by more than an order of magnitude. These hybrid catalysts operate efficiently using an electrolyzer equipped with a gas diffusion electrode, achieving a multicarbon product selectivity of 58% with a partial current density of ca. -80 mA cm-2. We found that the activity for multicarbon product formation is closely linked to the surface charge that accumulates during electrocatalysis, stemming from surface intermediate buildup. Through X-ray photoelectron spectroscopy, we elucidated the role of the molecular additives in altering the electronic structure of the Cu electrodes, promoting the stabilization of surface CO. Additionally, in situ Raman measurements established the identity of the reaction intermediates that accumulate during electrocatalysis, indicating preferential CO binding on Cu step sites, known for facilitating C-C coupling. This study underscores the significant potential of molecular surface modifications in developing efficient electrocatalysts for CO2 reduction, highlighting surface charge as a pivotal descriptor of multicarbon product activity.

Abstract PO1-14-07: Programme of mast cell subsets to potentiate breast cancer immunotherapy: from bed to bench to bed (the phase 2 platform RENAISSANCE trial)

Abstract Background: Immune checkpoint inhibitors (ICIs) have heralded a new era in breast cancer treatment; however, response rates remain limited, making precision immune-oncology a major unmet need. In addition to T cells, effective immune responses to ICIs rely on coordinated interactions between innate and adaptive immune cells. Mast cells are evolutionarily conserved, tissue-resident cells of importance to human health. Specific subsets of mast cells might be endowed with opposite roles in cancer treatment, yet the extent of mast cell heterogeneity and its clinical merit in immunotherapy remain undefined. Objective: We sought to comprehensively characterize mast cells in breast cancer, investigate their association with immunotherapy response with in-depth mechanistic insights, and identify actionable strategies to modulate mast cell functional states, thereby optimizing immunotherapy efficacy. Methods: We employed single-cell profiling on longitudinal breast cancer samples from three independent clinical trials (NCT04613674, NCT03197389 and GSE169246) to delineate mast cell heterogeneity in anti-PD-(L)1 therapy. By integrating multi-omic analyses, tissue characterization, preclinical experiments, transgenic mice, and high-throughput drug screening, we outlined the molecular features, underlying mechanisms, and clinical relevance of distinct mast cells to elicit ICI-responsive microenvironments. Subsequently, we launched RENAISSANCE (NCT05076682), a proof-of-concept, Bayesian adaptive, phase 2 platform trial, to evaluate the efficacy and safety of combining mast cell therapeutics with anti-PD-1 backbone therapy in metastatic triple-negative breast cancer (TNBC) patients who progressed after immunotherapy. The primary endpoint was the objective response rate (ORR) assessed using RECIST v1.1 criteria. Results: We identified a distinct population of mast cells termed antigen-presenting mast cells (APMCs), constituting approximately 30% of intratumoral mast cells and correlating with improved clinical benefit of anti-PD-(L)1 therapy in TNBC. APMCs displayed MHC-II and costimulatory molecules, and indicated the presence of tumor-reactive T cells and tertiary lymphoid structures. Using three immunocompetent mouse models, we confirmed the immunomodulatory capacity of APMCs in immunotherapy. Mechanistically, by employing Cpa3CreERT2Cd74fl/fl mice, we demonstrated that APMCs potentiate anti-PD-1 efficacy and antitumor T cell immunity through their antigen-presentation machinery. Interestingly, we identified cromolyn, an FDA-approved drug for allergy, as a potential therapeutic agent that elicited APMC-dependent CD8+ T cell cytotoxicity to synergize with anti-PD-1 therapy. Between February 2022 and March 2023, 10 patients with immunotherapy-refractory metastatic TNBC were enrolled to receive cromolyn plus camrelizumab backbone treatment. Given Bayesian predictive probability, this arm was “graduated” due to meeting the pre-specified efficacy boundary, with an ORR of 40.0% (4/10). The treatment was well tolerated with similar safety profiles of relevant drugs. Conclusions: Our findings provide crucial insights into the impact of mast cell heterogeneity on the clinical response to ICIs at a single-cell level, and pave the way for APMC-directed therapeutic interventions in cancer treatment. To our knowledge, this is the first prospective study in breast cancer of cromolyn plus anti-PD-1 backbone regimen after anti-PD-(L)1 immunotherapy failure, demonstrating significant antitumor activity and commendable tolerability. Consequently, we suggest a phase 3 randomized study to consolidate this finding, which might be an effective treatment in patients for whom there are few effective treatment options. Citation Format: Song-Yang Wu, Xi Jin, Yin Liu, Wen-Jia Zuo, Li Chen, Xiyu Liu, Lei Fan, Zhong-Hua Wang, Yan-Fei Liu, Yi-Zhou Jiang, Zhi-Ming Shao. Programme of mast cell subsets to potentiate breast cancer immunotherapy: from bed to bench to bed (the phase 2 platform RENAISSANCE trial) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-14-07.

Statins Do Not Significantly Affect Oxidative Nitrosative Stress Biomarkers in the PREVENT Randomized Clinical Trial.

Preventing Anthracycline Cardiovascular Toxicity with Statins (PREVENT) (NCT01988571) randomized breast cancer or lymphoma patients receiving anthracyclines to atorvastatin 40 mg daily or placebo. We evaluated the effects of atorvastatin on oxidative and nitrosative stress biomarkers, and explored whether these biomarkers could explain the lack of effect of atorvastatin on LVEF in PREVENT. Blood samples were collected and cardiac magnetic resonance imaging was performed prior to doxorubicin initiation and at 6 and 24 months. Thirteen biomarkers (arginine-nitric oxide metabolites, paraoxonase-1 [PON-1] activity, and myeloperoxidase) were measured. Dimensionality reduction using principal component analysis was used to define biomarker clusters. Linear mixed-effects models determined the changes in biomarkers over time according to treatment group. Mediation analysis determined if biomarker clusters explained the lack of effect of atorvastatin on LVEF. Among 202 participants with available biomarkers, median age was 53 years; 86.6% had breast cancer; median LVEF was 62%. Cluster 1 levels, reflecting arginine methylation metabolites, were lower over time with atorvastatin, although this was not statistically significant (p=0.081); cluster 2 levels, reflecting PON-1 activity, were significantly lower with atorvastatin (p=0.024). There were no significant changes in other biomarker clusters (p>0.05). Biomarker clusters did not mediate an effect of atorvastatin on LVEF (p>0.05) Conclusions: Atorvastatin demonstrated very modest effects on oxidative/nitrosative stress biomarkers in this low cardiovascular risk population. Our findings provide potential mechanistic insight into the lack of effect of atorvastatin on LVEF in the PREVENT trial.

Novel imidazolone derivatives as potential dual inhibitors of checkpoint kinases 1 and 2: Design, synthesis, cytotoxicity evaluation, and mechanistic insights

Applying various drug design strategies including ring variation, substituents variation, and ring fusion, two series of 2-(alkylthio)-5-(arylidene/heteroarylidene)imidazolones and imidazo[1,2-a]thieno[2,3-d]pyrimidines were designed and prepared as dual potential Chk1 and Chk2 inhibitors. The newly synthesized hybrids were screened in NCI 60 cell line panel where the most active derivatives 4b, d-f, and 6a were further estimated for their five dose antiproliferative activity against the most sensitive tumor cells including breast MCF-7 and MDA-MB-468 and non-small cell lung cancer EKVX as well as normal WI-38 cell. Noticeably, increasing the carbon chain attached to thiol moiety at C-2 of imidazolone scaffold elevated the cytotoxic activity. Hence, compounds 4e and 4f, containing S-butyl fragment, exhibited the most antiproliferative activity against the tested cells where 4f showed extremely potent selectivity toward them. As well, compound 6a, containing imidazothienopyrimidine core, exerted significant cytotoxic activity and selectivity toward the examined cells. The mechanistic investigation of the most active cytotoxic analogs was achieved through the evaluation of their inhibitory activity against Chk1 and Chk2. Results revealed that 4f displayed potent dual inhibition of both Chk1 and Chk2 with IC50 equal 0.137 and 0.25 μM, respectively. It also promoted its antiproliferative and Chk suppression activity via EKVX cell cycle arrest at S phase through stimulating the apoptotic approach. The apoptosis induction was also emphasized by elevating the expression of Caspases-3 and Bax, that are accompanied by Bcl-2 diminution. The in silico molecular docking and ADMET profiles of the most active analogs have been carried out to evaluate their potential as significant anticancer drug candidates.

Structural and Mechanistic Insights into the Transport of Aristolochic Acids and their Active Metabolites by Human Serum Albumin

Aristolochic acids I and II (AA-I/II) are carcinogenic principles of Aristolochia plants, which have been employed in traditional medicinal practices and discovered as food contaminants. While the deleterious effects of AAs are broadly acknowledged, there is a dearth of information to define the mechanisms underlying their carcinogenicity. Following bioactivation in the liver, N-hydroxyaristolactam and N-sulfonyloxyaristolactam metabolites are transported via circulation and elicit carcinogenic effects by reacting with cellular DNA. In this study, we apply DNA adduct analysis, X-ray crystallography, isothermal titration calorimetry (ITC) and fluorescence quenching to investigate the role of human serum albumin (HSA) in modulating AA carcinogenicity. We find that HSA extends the half-life and reactivity of N-sulfonyloxyaristolactam-I with DNA, thereby protecting activated AAs from heterolysis. Applying novel pooled plasma HSA crystallization methods, we report high-resolution structures of myristic acid-enriched HSA (HSAMYR) and its AA complexes (HSAMYR/AA-I and HSAMYR/AA-II) at 1.9 Å resolution. Whereas AA-I is located within HSA subdomain IB, AA-II occupies subdomains IIA and IB. ITC binding profiles reveal two distinct AA sites in both complexes with association constants of 1.5 and 0.5 · 106 M-1 for HSA/AA-I versus 8.4 and 9.0 · 105 M-1 for HSA/AA-II. Fluorescence quenching of the HSA Trp214 suggests variable impacts of fatty acids on ligand binding affinities. Collectively, our structural and thermodynamic characterizations yield significant insights into AA binding, transport, toxicity, and potential allostery, critical determinants for elucidating the mechanistic roles of HSA in modulating AA carcinogenicity.

Microstructural and mechanistic insights into the Tension-Compression asymmetry of rapidly solidified Fe-Cr alloys: A phase field and strain gradient plasticity study

Rapid solidification in Additively Manufactured (AM) metallic materials results in the development of significant microscale internal stresses, which are attributed to the printing induced dislocation substructures. The resulting backstress due to the Geometrically Necessary Dislocations (GNDs) is responsible for the observed Tension-Compression (TC) asymmetry. We propose a combined Phase Field (PF)-Strain Gradient J2 Plasticity (SGP) framework to investigate the TC asymmetry in such microstructures. The proposed PF model is an extension of Kobayashi’s dendritic growth framework, modified to account for the orientation-based anisotropy and multi-grain interaction effects. The SGP model has consideration for anisotropic temperature-dependent elasticity, dislocation strengthening, solid solution strengthening, along with GND-induced directional backstress. This model is employed to predict the solute segregation, dislocation substructure and backstress development during solidification and the post-solidification anisotropic mechanical properties in terms of the TC asymmetry of rapidly solidified Fe-Cr alloys. It is observed that higher thermal gradients (and hence, cooling rates) lead to higher magnitudes of solute segregation, GND density, and backstress. This also correlates with a corresponding increase in the predicted TC asymmetry. The results presented in this study point to the microstructural factors, such as dislocation substructure and solute segregation, and mechanistic factors, such as backstress, which may contribute to the development of TC asymmetry in rapidly solidified microstructures.

Self-discharge in flowless Zn-Br2 batteries and its mitigation

Several decades after the invention of the flow Zn-Br2 systems, persistent attempts have been made to develop stationary Zn-Br2 batteries. Such development should increase the energy density of the system simultaneously significantly reducing their cost and opening new challenges associated with the cell design and its performance. One of the major concerns is the rapid self-discharge of stationary systems leading to spontaneous charge loss during battery storage time. While self-discharge in flow cells is generally attributed to the chemical oxidation of the Zn anode, we show that the origin of self-discharge in a static configuration is completely different. By systematic investigations of activated carbon with different surface areas under varied charging conditions, mechanistic insights into this phenomenon were provided. Based on this understanding, we proposed herein an effective way to suppress the cathode's self-discharge by encapsulation of a bromine complexing agent inside the electrode's pore matrix. The modified electrodes unveil unique chemical kinetics of deaggregation of the stable fuse salt phase composed of bromine complexing agent and Br3− anions upon discharging. To the best of our knowledge, such a phenomenon has not been reported yet. The proposed system demonstrates high capacity (up to 300 mAh/g) and impressive long-term stability.

Mechanistic Insights into the Palladium-Catalyzed Perfluoroalkylative Carbonylation of Unactivated Alkenes to β-Perfluoroalkyl Esters: A DFT Study.

Transition metal-catalyzed multicomponent carbonylation is an efficient synthetic strategy to access multifunctional esters in high yields with broad functional group tolerance and good chemoselectivity. Considering the development of highly efficient synthetic methods for esters, it remains significant to grasp the mechanism of constructing multifunctional esters. Herein, density functional theoretical calculations were carried out to acquire mechanistic insight into the synthesis of β-perfluoroalkyl esters from a specific palladium-catalyzed perfluoroalkylative carbonylation of unactivated alkenes using carbon monoxide. A detailed mechanistic understanding of this reaction route includes (1) multistep radical reaction process, (2) C-C coupling and CO insertion, (3) ligand exchange, and (4) Pd-based intermediate oxidation and reductive elimination. The multistep radical process was fundamentally rationalized, including Rf· formation and radicals A and E from unactivated alkene and CO oxidation, respectively. The potential energy calculation indicated that the CO insertion into the perfluorinated alkyl radicals preceded Pd-catalyzed oxidation in the competitively multistep free radical reaction process. In addition, the I-/PhO- exchange step was predicted to be spontaneous to products. The IGMH analysis further attested to the reductive elimination process involved in the rate-determining step. Thus, a simple and valid density functional theory (DFT) approach was developed to reveal the multistep radical mechanism for the Pd-catalyzed perfluoroalkylative carbonylation of unactivated alkenes to access functional β-perfluoroalkyl esters.

An experimental and theoretical study on mechanistic insights into urolithin-based ratiometric fluorescent probe for instant quantitative detection of fluoride ions

Fluoride detection has been playing an important role in chemical, biological, and medicinal field, especially for keeping physical health and resisting environmental pollution. Herein, a urolithin B fluorescent probe has been successfully developed with good sensitivity, selectivity, anti-interference ability. The low limit of detection (LOD) refers to 0.156 μM, and the instant response time to F− is less than 1 s. The probe is suitable for quantitatively and qualitatively ratiometric detection for F− in solution with two distinct emission bands at 425 (blue) and 566 nm (orange), with the coordinate change of CIE from (0.38, 0.41) to (0.22, 0.11). Urolithin B displayed a remarkable ratiometric fluorescence response towards F−. The detection mechanistic was further proposed by NMR and electronic spectroscopic experiments combining with time-dependent density functional theoretical calculation.