Although many chemotherapeutic agents induce neutrophil extracellular trap (NET) formation, how NETs affect therapeutic efficacy across different cancer types remains poorly understood. Here, we report that irinotecan-induced NETs exhibit differential cytotoxicity against cancer cells through the proteolytic activity of cathepsin G (CTSG), with colon cancer cells exhibiting high sensitivity while liver cancer cells demonstrating marked resistance. Through bioinformatic analysis and siRNA-mediated knockdown validation, we identified SERPINA3, a serine protease inhibitor, as a key factor in this resistance. Mechanistically, SERPINA3 inhibits CTSG-mediated cleavage of the anti-apoptotic protein 14-3-3ε, thereby protecting cells from NET-induced apoptosis. Interestingly, the NF-κB signaling pathway governs SERPINA3 expression in liver cancer cells, with activated p65 directly binding to its promoter. Targeting SERPINA3 with antisense oligonucleotides successfully sensitized liver cancer cells to irinotecan therapy. These findings elucidate a critical mechanism of chemoresistance in liver cancer and propose targeting SERPINA3 as a promising therapeutic strategy to enhance chemotherapy efficacy.

Colorectal cancer is the second most common cause of cancer-related death worldwide, with a rising incidence, highlighting an urgent need for novel therapeutics. In this study, we developed several polyamine conjugates. Compound 6 (C6) was selected as the lead compound and was evaluated for anticancer activity in vitro and in vivo. In vitro experiments showed that C6 inhibited cell proliferation, colony formation, migration, and invasion of colorectal cancer cells while inducing apoptosis, pyroptosis, and autophagosome formation. Genetic or pharmacological inhibition of autophagy weakened C6-induced apoptosis and gasdermin E (GSDME)-dependent pyroptosis. Inactivation of caspase 3 activity by AC-DEVD-CHO decreased the levels of N-terminal GSDME induced by C6. Furthermore, animal models exhibited suppressed tumor growth and dissemination after treatment with C6. Taken together, our findings highlight C6 as a potential drug against colorectal cancer.

BACKGROUND:Neuronal pyroptosis is involved in neuronal cell death and neurological damage after cerebral ischemia-reperfusion. 14,15-Epoxyeicosatrienoic acid (14,15-EET) can reduce neuronal loss induced by cerebral ischemia-reperfusion by regulating mitochondrial biological processes. However, it remains unclear how 14,15-EET regulates mitochondrial homeostasis, inhibits neuronal pyroptosis, and promotes neurological functional recovery after cerebral ischemia-reperfusion. METHODS:Mice with middle cerebral artery occlusion and reperfusion were used as an animal model to study the cerebral ischemia-reperfusion disease. The neurological function of mice was performed at 1, 3, and 5 days to test the therapeutic effects of 14,15-EET. Transmission electron microscope imaging and Nissl staining were used to analyze neuronal morphological structure, mitophagy, and neuronal pyroptosis. Western blot and transcriptome were used to detect the levels of mitophagy and neuronal pyroptosis signaling pathway-related molecules. HT22 cells were used in in vitro studies to detect the mechanism by which 14,15-EET reduces neuronal pyroptosis after oxygen-glucose deprivation/reoxygenation treatment. RESULTS:14,15-EET treatment reduced cerebral infarct volumes and improved neurological functional recovery in mice after cerebral ischemia-reperfusion. 14,15-EET treatment maintained the morphological structure of neurons in the ischemic penumbra area as well as the dendritic spine density in mice after cerebral ischemia-reperfusion. The upregulation of NLRP1 (NOD-like receptor thermal protein domain associated protein 1), IL (interleukin)-1β, caspase-1, and GSDMD (gasdermin D) induced by cerebral ischemia-reperfusion was inhibited, and the expression of mitophagy proteins Parkin and LC3B (microtubule-associated protein 1 light chain 3 B) was increased by 14,15-EET treatment. Transcriptome profiling found that 14,15-EET exerts a neuroprotection role in promoting neural function recovery by activating the WNT (wingless-type mouse mammary tumor virus integration site family) signaling pathway. We found that 14,15-EET upregulated the WNT pathway proteins such as WNT1, WNT3A, β-catenin, and p-GSK-3β (phosphorylation of glycogen synthase kinase 3β) in vivo and in vitro. The WNT signaling pathway inhibitor XAV-939 reduced the expression of mitophagy protein Parkin and upregulated the expression of caspase-1 and GSDMD in HT22 cells with oxygen-glucose deprivation/reoxygenation and 14,15-EET treatment. CONCLUSIONS:14,15-EET regulates mitochondrial homeostasis to inhibit neuronal pyroptosis, thereby promoting the recovery of neurological function in mice after cerebral ischemia-reperfusion. These results provide new ideas for maintaining mitochondrial homeostasis and inhibiting neuronal pyroptosis after cerebral ischemia-reperfusion.

The blastema is key to forming complete tissues in regenerating Dugesia japonica (D. japonica). However, the dynamic changes in cellular compositions and transcription landscapes in blastema during regeneration are understudied. Here, through genome reannotation, 3D spatial transcriptome construction, single-cell RNA sequencing (scRNA-seq), and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) analyses of changes in gene expression and chromatin structures, we delineate key transcription factors regulating the developmental trajectories of major cell clusters in the regenerating head. Importantly, we find that the T cell factor 4 (DjTcf4)-positive cells highly accumulate at wound areas, and its gene network is critical for the proper timing of development during regeneration in multiple progenitor cells. Depletion of DjTcf4 and its target genes leads to singular eye and/or dull tail phenotypes and delays regeneration. Taken together, we build multi-omics atlases in D. japonica and reveal the noncanonical function of the DjTcf4 network in developmental pattern formation, laying a foundation for studies of regeneration in D. japonica.

Lipid nanoparticles (LNPs) hold great potential for delivery of macromolecular antimicrobials. Herein, we designed a series of anionic LNPs capable of delivering cationic polymyxin B (PMB) for effective and safe treatment of Gram-negative bacterial infection. The use of anionic lipid induced self-assembly of PMB, encapsulating cationic PMB molecules into LNPs via electrostatic interactions (PMB-LNPs). Anionic lipid architecture and lipid:PMB mixing ratios had a substantial influence on particle size, surface charge, and drug release properties of PMB-LNPs. Following in vitro screening assays, C14-Glu5/PMB (1:4) LNP was identified as the lead formulation due to its potent bactericidal activity against a panel of tested Gram-negative bacteria strains. Confocal and SEM imaging studies validated the importance of fractionated PMB release in mediating the enhanced binding of C14-Glu5/PMB (1:4) LNPs on the bacterial outer membrane. Additionally, PMB-LNPs conferred prolonged blood circulation, preferential liver accumulation, and superior tolerability over free PMB. In a murine neutropenic A. baumannii infection model, administration of C14-Glu5/PMB LNPs eliminated bacteria in the liver and suppressed bacterial regrowth in the kidney more effectively than free PMB, resulting in an improved body weight status and histological appearance. This study may provide insights into the de novo design of LNP-based nanomedicines for antimicrobial therapies.

The nucleosome remodeling and deacetylase (NuRD) complex, well known for its ATP-dependent chromatin remodeling and histone deacetylation activities combined in one multi-subunit complex, plays an evolutionarily conserved role in chromatin structures and gene regulation during cell growth, proliferation, and development. However, the composition and function of the NuRD complex in planarians remain incompletely unknown. Here, we identified six core components within the NuRD complex and characterized their biological roles in planarians. RNA interference (RNAi) mediated knockdown of these genes resulted in similar perturbations to both tissue homeostasis and regeneration, and the overlapping downstream genes regulated upon depletion of MBD2/3 or CHD4 showed similar expression alterations to that after knockdown of other NuRD complex genes, suggesting that NuRD core members may act in one complex. Additionally, the overlapping upregulated genes after depletion of NuRD complex members were expressed in neoblast and progenitor cells, among which NuRD complex core genes were enriched, suggesting transcriptional correlation between the overlapping upregulated genes and NuRD core members. Furthermore, upstream regulatory sites of the upregulated genes exhibited significant enrichment of H3K27ac, indicating the NuRD complex may deacetylate histone to modulate these genes. Notably, depletion of either MBD2/3 or CHD4 in planarians significantly upregulated multiple progenitor marker genes while reducing the number of somatic cells in the epidermis and intestine and downregulating multiple somatic cell marker genes, indicating that the NuRD complex may drive differentiation into somatic lineages in planarians. Collectively, our work provides a foundation to understand the essential roles of the NuRD complex in orchestrating cell differentiation, tissue homeostasis and regeneration in planarian.

Peptidyl arginine deiminase 4 (PAD4) is an important biocatalytic enzymes involved in the conversion of protein arginine to citrulline, its dysregulation has a great impact on many physiological processes. Recently, PAD4 has emerged as a potential therapeutic target for the treatment of various diseases including rheumatoid arthritis (RA). Traditional Chinese Medicines (TCMs), also known as herbal plants, have gained great attention by the scientific community due to their good therapeutic performance and far fewer side effects observed in the clinical treatment. However, limited researches have been reported to screen natural PAD4 inhibitors from herbal plants. The color developing reagent (COLDER) or fluorescence based methods have been widely used in PAD4 activity assay and inhibitor screening. However, both methods measure the overall absorbance or fluorescence in the reaction solution, which are easy to be affected by the background interference due to colorful extracts from herbal plants. In this study, a simple, and robust high-performance liquid chromatography ultraviolet-visible (HPLC-UV) based method was developed to determine PAD4 activity. The proposed strategy was established based on COLDER principle, while used hydrophilic l-arginine instead of hydrophobic N-benzoyl-l-arginine ethyl ester (BAEE) as a new substrate to determine PAD4 inhibition activity of herbal extracts. The herbal extracts and PAD4 generated hydrophobic l-citrulline were successfully separated by the HPLC, and the developed method was optimized and validated with a known PAD4 inhibitor (GSK484) in comparison with COLDER assay. The IC50 value of GSK484 measured by HPLC-UV method was 153 nM, and the detection limit of the citrulline was 0.5 nmol, respectively, with a linear range of 0.5 nmol to 20 nmol. The IC50 value of the HPLC-UV method was improved by nearly three times compared with COLDER assay (527 nM), and the results indicated the reliability of PAD4 inhibition via HPLC-UV method. The inhibitory effect against PAD4 were fast and accurately screened for the twenty-four extracts from eight herbs. Among them, Ephedra Herba extracts showed significant inhibitory activity against the PAD4 with the IC50 values of three extracts (ethanol, ethyl acetate and water) ranging from 29.11 mu g/mL to 41.36 mu g/mL, which may help researchers to discover novel natural compounds holding high PAD4 inhibition activity.

Triple-negative breast cancer is a highly aggressive and heterogeneous breast cancer subtype characterized by early metastasis, poor prognosis, and high recurrence. Targeting histone citrullination-mediated chromatin dysregulation to induce epigenetic alterations shows great promise in TNBC therapy. We report the synthesis, optimization, and evaluation of a novel series of β-carboline-derived peptidyl arginine deiminase 4 inhibitors that exhibited potent inhibition of TNBC cell proliferation. The most outstanding PAD4 inhibitor, compound 28, hindered the PAD4-H3cit-NET signaling pathway and inhibited the growth of solid tumors and pulmonary metastatic nodules in the 4T1 in situ mouse model. Furthermore, 28 improved the tumor immune microenvironment by reshaping neutrophil phenotype, upregulating the proportions of dendritic cells and M1 macrophages, and reducing the amount of myeloid-derived suppressor cells. In conclusion, our work offered 28 as an efficacious PAD4 inhibitor that exerts a combination of conventional chemotherapy and immune-boosting effects, which represents a potential therapy strategy for TNBC.

Esophageal squamous cell carcinoma (ESCC) is one of the most common forms of malignancy worldwide. However, there is currently a lack of effective chemotherapeutic drugs for ESCC. Ivermectin is a broad-spectrum antiparasitic drug with notable antitumor activity. However, the cellular and molecular mechanisms by which ivermectin inhibits cancer growth remain unclear. In this study, we elucidate the role of ivermectin in ESCC suppression by activating the endoplasmic reticulum (ER) stress and autophagy pathways. In transcriptome analyses, we find that activating transcription factor 4 (ATF4) and DNA damage inducible transcript 3 (DDIT3) are involved in the activation of ER stress by ivermectin. Moreover, ivermectin treatment suppresses the growth of ESCC xenograft tumors in nude mice. Taken together, our results establish the antitumor molecular role of ivermectin in targeting the ER stress-autophagy pathway and suggest that ivermectin is a potential drug candidate for the treatment of ESCC.

The worldwide overall 5-year survival rate of esophageal squamous cell carcinoma (ESCC) patients is less than 20%, and novel therapeutic strategies for these patients are urgently needed. Harmine is a natural β-carboline alkaloid, which received great interest in cancer research because of its biological and anti-tumor activities. The aim of this study is to examine the effects of harmine on ESCC and its mechanism. We investigated the effects of harmine on proliferation, cell cycle, apoptosis, and tumor growth in vivo. RNA sequencing (RNA-seq), real-time PCR, and western blotting were used to detect the mechanism. Harmine inhibited ESCC cell growth in vitro and tumor growth in vivo. Differentially expressed genes in harmine-treated ESCC cells were mainly involved in protein processing in the endoplasmic reticulum (ER). Real-time PCR and western blotting confirmed harmine-induced cellular ER stress. CRISPR-Cas9 knockout of C/EBP homologous protein (CHOP) abolished harmine-induced expression of death receptor 5 and apoptosis. Harmine also induced the expression of CHOP-mediated sestrin-2, which in turn contributes to autophagosome formation via suppressing the AMP-activated protein kinase-protein kinase B-mammalian target of rapamycin signaling pathway. In conclusion, our results demonstrate that harmine inhibits the growth of ESCC through its regulation of ER stress, suggesting that it is a promising candidate for ESCC treatment.

The deimination or citrullination of arginine residues in the polypeptide chain by peptidylarginine deiminase 4 alters the charge state of the polypeptide chain and affects the function of proteins. It is one of the main ways of protein post-translational modifications to regulate its function. Peptidylarginine deiminase 4 is widely expressed in multiple tissues and organs of the body, especially the central nervous system, and regulates the normal development of organisms. The abnormal expression and activation of peptidylarginine deiminase 4 is an important pathological mechanism for the occurrence and development of central nervous system diseases such as multiple sclerosis, Alzheimer’s disease, cerebral ischemia reperfusion injury, and glioblastoma.

Animal-derived basement-membrane matrices such as Geltrex are used to grow cells and tissues. Particularly, these are commonly applied to support tumor growth in animals for cancer research. However, a material derived from an animal source has an undefined composition, and may thus have unavoidable batch-to-batch variation in properties. To overcome these issues, a series of synthetic short peptides to form hydrogels is designed in combination with gelatin to promote cell adhesion and growth. The peptides have sequences of (X1Y1X2Y2)2 , where X1 and X2 are hydrophobic residues, while Y1 and Y2 are hydrophilic residues. The peptides spontaneously fold and self-assemble into a β-sheet secondary structure upon contact with salts, and then aggregate to form hydrophilic networks of hydrogels. Hybrid hydrogels formed by mixing the peptide IEVEIRVK (IVK8) with gelatin are injectable and enzymatically degradable. The hybrid hydrogels at optimal compositions support SW480 and HepG2 tumor spheroid growth in vitro as effectively as Geltrex. More importantly, the peptide/gelatin hydrogels support tumor growth in a SW480 human colorectal adenocarcinoma xenograft mouse model. Altogether, the results illustrate that the synthetic peptide/gelatin hybrid hydrogel is a promising scaffold that can be used to support cell and tissue growth both in vitro and in vivo.

Exhaustion of chimeric antigen receptor (CAR) T cells is one of the limitations for CAR T efficacy in solid tumors and for tumor recurrence after initial CAR T treatment. Tumor treatment with a combination of programmed cell death receptor-1 (PD-1)/programmed cell death ligand-1 (PD-L1) blockage and CD28-based CAR T cells has been intensively studied. However, it remains largely unclear whether autocrine single-chain variable fragments (scFv) PD-L1 antibody can improve 4-1BB-based CAR T cell anti-tumor activity and revert CAR T cell exhaustion. Here, we studied T cells engineered with autocrine PD-L1 scFv and 4-1BB-containing CAR. The antitumor activity and exhaustion of CAR T cells were investigated in vitro and in a xenograft cancer model using NCG mice. CAR T cells with autocrine PD-L1 scFv antibody demonstrate enhanced anti-tumor activity in solid tumors and hematologic malignancies by blocking the PD-1/PD-L1 signaling. Importantly, we found that CAR T exhaustion was largely diminished by autocrine PD-L1 scFv antibody in vivo. As such, 4-1BB CAR T with autocrine PD-L1 scFv antibody combined the power of CAR T cells and the immune checkpoint inhibitor, thereby increasing the anti-tumor immune function and CAR T persistence, providing a cell therapy solution for a better clinical outcome.

The supplementary data contain Supplementary Materials and Methods, Supplementary Figures 1 through 8 with legends, and Supplementary Tables 1 to 5. Supplementary Fig. 1. YW3-56 inhibits growth of p53 wild type and mutant cells. Supplementary Fig. 2. PAD inhibitor YW3-56 inhibits PAD4 in HL-60 derived granulocytic cells. Supplementary Fig. 3. Effects of YW3-56 on the ATF4 and AMPK gene networks. Supplementary Fig. 4. ATF4 directly binds to the SESN2 and DDIT4 promoters. Supplementary Fig. 5. ChIP-exo analyses of ATF4 binding to its target sites. Supplementary Figure 6. mTOR inhibition following thapsigargin induced ER stress. Supplementary Figure 7. Effects of ATF4, SESN2, and DDIT4 depletion on cell growth and YW3-56 mediated cell killing. Supplementary Figure 8. Features of YW3-56 induced cell death. Supplementary Table 1. List of qRT-PCR and ChIP-qPCR primers used in this research work. Supplementary Table 2. Genes with altered expression after YW3-56 treatment. (see Excel spreadsheet) Supplementary Table 3. List of ATF4 associated genes. (see Excel spreadsheet) Supplementary Table 4. List of CEBPB associated genes. (see Excel spreadsheet) Supplementary Table 5. Expression changes of ATF4 and CEBPB candidate genes after YW3-56 treatment.

The global pandemic due to the severe acute respiratorysyndromecoronavirus 2 (SARS-CoV-2) has led to more than 661 million infectionsand over 6.6 million deaths worldwide since its inception in 2019.There is an urgent need for safe and effective antiviral solutions,especially those with rapid inactivation kinetics, to help preventviral transmissions. In this study, we report the design and synthesisof amphiphilic sulfonated polycarbonates and investigate the effectof polycarbonate compositions on their antiviral activity. A polycarbonatewith an optimal balance of the hydrophilic anionic sulfonate (& SIM;30%)and the hydrophobic n-butyl (& SIM;70%) pendentgroups inactivates various types of coronaviruses in solution, includinghuCoV-OC43, ancestral wild-type SARS-CoV-2, and the delta variant,within seconds, while exhibiting low cytotoxicity to mammalian cells(i.e., Vero E6) at the virucidal concentration. This amphiphilic sulfonatedpolycarbonate may be a promising solution to inactivate viruses assprays for prevention of viral infection.

AIM:To explore whether 14, 15-EET regulates mitochondrial dynamics to exert neuroprotective effects after cerebral ischemia-reperfusion and its underlying mechanisms.METHODS:The mouse middle cerebral artery occlusion reperfusion model was used to observe brain infarct volume and neuronal apoptosis by TTC staining and Tunel assay, modified neurological severity score to detect neurological impairment, HE staining and Nissl staining to observe neuron damage, western blot and immunofluorescence methods to detect the expression of mitochondrial dynamics-related proteins, transmission electron microscopy, and Golgi-Cox staining to detect mitochondrial morphology and neuronal dendritic spines.RESULTS:14, 15-EET reduced the neuronal apoptosis and cerebral infarction volume induced by middle cerebral artery occlusion reperfusion (MCAO/R), inhibited the degradation of dendritic spines, maintained the structural integrity of neurons, and alleviated neurological impairment. Cerebral ischemia-reperfusion induces mitochondrial dynamics disorders, upregulates the expression of the mitochondrial division protein Fis 1, and inhibits the expression of mitochondrial fusion proteins MFN1, MFN2, and OPA1, while 14, 15-EET treatment reverses this process. Mechanistic studies have shown that 14, 15-EET promotes the phosphorylation of AMPK, upregulates the expression of SIRT1 and phosphorylation of FoxO1, thereby inhibiting mitochondrial division and promoting mitochondrial fusion, preserving mitochondrial dynamics, maintaining neuronal morphological and structural integrity, and alleviating neurological impairment induced by middle cerebral artery occlusion reperfusion. Compound C treatment diminishes the neuroprotective effect of 14, 15-EET following MCAO/R in mice.CONCLUSION:This study elucidates the novel neuroprotective mechanism of 14, 15-EET, providing a novel approach for the development of drugs based on mitochondrial dynamics.

Infectious pathogens, such as SARS-CoV-2, can remain viable on common fabric surfaces for days, posing a significant risk of fomite transmission. Antimicrobial coatings are a widely employed approach for pathogen eradication upon direct contact. However, fabricating such coatings on fabric substrates mostly necessitates toxic organic solvents and complex equipment/procedures. Most coatings also require a long contact time for complete disinfection, which may compromise their usefulness in mitigating the spread of highly infectious pathogens. Herein, we report a sustainable and scalable water-mediated method to prepare a copper iodide (CuI) coating on flexible cotton fabrics, attaining highly potent antimicrobial efficacy and rapid germicidal kinetics. Only water is required as the processing solvent for the in situ formation of CuI nanoparticles on the substrate, and the unconsumed reagents can be fully recycled, making the reported method a green, economical, and zero-waste technology promising for industrial scale-up. Within just 2 min of contact, the coated cotton fabric containing 5.1 wt % CuI nanoparticles exhibits near-complete inactivation of murine hepatitis coronavirus (>99.9%) and Salmonella bacteriophage P22 (>99.9999%) as models for the enveloped and nonenveloped viral species, respectively. It is also able to eliminate a variety of bacteria and fungi with 3.5-7.4 log reductions in 2-5 min. Furthermore, in view of the robust durability and skin compatibility of the coating, this simple yet powerful approach holds great promise for practical applications, especially in future infectious disease outbreaks.

Ovarian cancer recurrence and chemotherapy resistance are still urgent issues, and exploring the mechanisms of metastasis and chemotherapy resistance is beneficial to the development of therapeutic methods. Caspase recruitment domain family member 9 (CARD9) and homeobox B5 (HOXB5) are related and both are upregulated in ovarian cancer. This study aimed to define their functions in ovarian cancer cell proliferation, migration, and cisplatin sensitivity. The levels of CARD9 were detected in acquired ovarian cancer tissues and cell lines. CARD9 was indeed abnormally upregulated in them. CARD9 knockdown significantly suppressed cell proliferation, colony formation, migration, cycle arrest, and cisplatin sensitivity. HOXB5 bound to the CARD9 promoter, and HOXB5 overexpression reversed the regulation by CARD9 knockdown in cells, as well as the activation of NF-κB signaling. This indicated that CARD9 was positively regulated by HOXB5 in ovarian cancer cells. Together, CARD9 is involved in ovarian cancer cell proliferation, migration, and cisplatin sensitivity via NF-κB signaling after transcriptional activation by HOXB5.