Anoctamin-1 is induced by TGF-ß and contributes to lung myofibroblast differentiation.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by progressive scarring of the lungs and resulting in deterioration in lung function. Transforming growth factor-ß (TGF-ß) is one of the most established drivers of fibrotic processes. TGF-ß promotes the transformation of tissue fibroblasts to myofibroblasts, a key finding in the pathogenesis of pulmonary fibrosis. We report here that TGF-ß robustly upregulates the expression of the calcium-activated chloride channel anoctamin-1 (ANO1) in human lung fibroblasts (HLFs) at mRNA and protein levels. ANO1 is readily detected in fibrotic areas of IPF lungs in the same area with smooth muscle α-actin (SMA)-positive myofibroblasts. TGF-ß-induced myofibroblast differentiation (determined by the expression of SMA, collagen-1, and fibronectin) is significantly inhibited by a specific ANO1 inhibitor, T16Ainh-A01, or by siRNA-mediated ANO1 knockdown. T16Ainh-A01 and ANO1 siRNA attenuate profibrotic TGF-ß signaling, including activation of RhoA pathway and AKT, without affecting initial Smad2 phosphorylation. Mechanistically, TGF-ß treatment of HLFs results in a significant increase in intracellular chloride levels, which is prevented by T16Ainh-A01 or by ANO1 knockdown. The downstream mechanism involves the chloride-sensing "with-no-lysine (K)" kinase (WNK1). WNK1 siRNA significantly attenuates TGF-ß-induced myofibroblast differentiation and signaling (RhoA pathway and AKT), whereas the WNK1 kinase inhibitor WNK463 is largely ineffective. Together, these data demonstrate that 1) ANO1 is a TGF-ß-inducible chloride channel that contributes to increased intracellular chloride concentration in response to TGF-ß; and 2) ANO1 mediates TGF-ß-induced myofibroblast differentiation and fibrotic signaling in a manner dependent on WNK1 protein but independent of WNK1 kinase activity.NEW & NOTEWORTHY This study describes a novel mechanism of differentiation of human lung fibroblasts (HLFs) to myofibroblasts: the key process in the pathogenesis of pulmonary fibrosis. Transforming growth factor-ß (TGF-ß) drives the expression of calcium-activated chloride channel anoctmin-1 (ANO1) leading to an increase in intracellular levels of chloride. The latter recruits chloride-sensitive with-no-lysine (K) kinase (WNK1) to activate profibrotic RhoA and AKT signaling pathways, possibly through activation of mammalian target of rapamycin complex-2 (mTORC2), altogether promoting myofibroblast differentiation.

Growth of Size-Tunable Ag2O Polyhedra and Revelation of Their Bulk and Surface Lattices.

By primarily adjusting the reagent amounts, particularly the volume of AgNO3 solution introduced, Ag2O cubes with decreasing sizes from 440 to 79 nm, octahedra from 714 to 106 nm, and rhombic dodecahedra from 644 to 168 nm are synthesized. 733 nm cuboctahedra are also prepared for structural analysis. With in-house X-ray diffraction (XRD) peak calibration, shape-related peak shifts are recognizable. Synchrotron XRD measurements at 100 K reveal the presence of bulk and surface layer lattices. Bulk cell constants also deviate slightly. They show a negative thermal expansion behavior with shrinking cell constants at higher temperatures. The Ag2O crystals exhibit size- and facet-dependent optical properties. Bandgaps red-shift continuously with increasing particle sizes. Optical facet effect is also observable. Moreover, synchrotron XRD peaks of a mixture of Cu2O rhombicuboctahedra and edge- and corner-truncated cubes exposing all three crystal faces can be deconvoluted into three components with the bulk and the [111] microstrain phase as the major component. Interestingly, while the unheated Cu2O sample shows clear diffraction peak asymmetry, annealing the sample to 450 K yields nearly symmetric peaks even when returning the sample to room temperature, meaning even moderately high temperatures can permanently change the crystal lattice.

PEDOT:PSS Nanoparticle Membranes for Organic Solvent Nanofiltration.

Recycling of valuable solutes and recovery of organic solvents via organic solvent nanofiltration (OSN) are important for sustainable development. However, the trade-off between solvent permeability and solute rejection hampers the application of OSN membranes. To address this issue, the poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) nanoparticle membrane with hierarchical pores is constructed for OSN via vacuum filtration. The small pores (the free volume of the polymer chain) charge for the solute rejection (high rejection efficiency for low molecule weight solute) and allow solvent passing while the large pores (the void between two PEDOT:PSS nanoparticles) promote the solvent transport. Owing to the lack of connectivity among the large pores, the fabricated PEDOT:PSS nanoparticle membrane enhanced solvent permeance while maintaining a high solute rejection efficiency. The optimized PEDOT:PSS membrane affords a MeOH permeance of 7.2 L m-2 h-1 bar-1 with over 90% rejection of organic dyes, food additives, and photocatalysts. Moreover, the rigidity of PEDOT endows the membrane with distinctive stability under high-pressure conditions. The membrane is used to recycle the valuable catalysts in a methanol solution for 150 h, maintaining good separation performance. Considering its high separation performance and stability, the proposed PEDOT:PSS membrane has great potential for industrial applications.

Shape-Tunable BaTiO3 Crystals Presenting Facet-Dependent Optical and Piezoelectric Properties.

BaTiO3 octahedra, edge-, and corner-truncated cubes, and cubes with four tunable sizes from 132 to 438 nm are synthesized by a solvothermal growth approach. Acetic acid treatment can cleanly remove BaCO3 impurity. Rietveld refinement of X-ray diffraction patterns and Raman spectra help to confirm the particles have a tetragonal crystal structure. The crystals also exhibit size- and facet-dependent bandgap shifts. BaTiO3 octahedra show larger piezoelectric, ferroelectric, and pyroelectric effects than truncated cubes and cubes. The measured dielectric constant differences should be associated with their various facet-dependent behaviors. Piezoelectric nanogenerators fabricated from BaTiO3 octahedra consistently show the best performance than those containing truncated cubes and cubes. In particular, a nanogenerator with 30 wt.%-incorporated octahedra displays an open-circuit voltage of 23 V and short-circuit current of 324 nA. The device performance is also highly stable. The maximum output power reaches 3.9 µW at 60 MΩ. The fabricated nanogenerator can provide sufficient electricity to power light-emitting diodes. This work further demonstrates that various physical properties of semiconductor crystals show surface dependence.

Experimental Revelation of Surface and Bulk Lattices in Faceted Cu2 O Crystals.

Semiconductor crystals have generally shown facet-dependent electrical, photocatalytic, and optical properties. These phenomena have been proposed to result from the presence of a surface layer with bond-level deviations. To provide experimental evidence of this structural feature, synchrotron X-ray sources are used to obtain X-ray diffraction (XRD) patterns of polyhedral cuprous oxide crystals. Cu2 O rhombic dodecahedra display two distinct cell constants from peak splitting. Peak disappearance during slow Cu2 O reduction to Cu with ammonia borane differentiates bulk and surface layer lattices. Cubes and octahedra also show two peak components, while diffraction peaks of cuboctahedra are comprised of three components. Temperature-varying lattice changes in the bulk and surface regions also show shape dependence. From transmission electron microscopy (TEM) images, slight plane spacing deviations in surface and inner crystal regions are measured. Image processing provides visualization of the surface layer with depths of about 1.5-4 nm giving dashed lattice points instead of dots from atomic position deviations. Close TEM examination reveals considerable variation in lattice spot size and shape for different particle morphologies, explaining why facet-dependent properties are emerged. Raman spectrum reflects the large bulk and surface lattice difference in rhombic dodecahedra. Surface lattice difference can change the particle bandgap.

Tumor cell-released LC3-positive EVs promote lung metastasis of breast cancer through enhancing premetastatic niche formation.

Most breast cancer-related deaths are caused by metastasis in vital organs including the lungs. Development of supportive metastatic microenvironments, referred to as premetastatic niches (PMNs), in certain distant organs before arrival of metastatic cells, is critical in metastasis. However, the mechanisms of PMN formation are not fully clear. Here, we demonstrated that chemoattractant C-C motif chemokine ligand 2 (CCL2) could be stimulated by heat shock protein 60 (HSP60) on the surface of murine 4 T1 breast cancer cell-released LC3+ extracellular vesicles (LC3+ EVs) via the TLR2-MyD88-NF-κB signal cascade in lung fibroblasts, which subsequently promoted lung PMN formation through recruiting monocytes and suppressing T cell function. Consistently, reduction of LC3+ EV release or HSP60 level or neutralization of CCL2 markedly attenuated PMN formation and lung metastasis. Furthermore, the number of circulating LC3+ EVs and HSP60 level on LC3+ EVs in the plasma of breast cancer patients were positively correlated with disease progression and lung metastasis, which might have potential value as biomarkers of lung metastasis in breast cancer patients (AUC = 0.898, 0.694, respectively). These findings illuminate a novel mechanism of PMN formation and might provide therapeutic targets for anti-metastasis therapy for patients with breast cancer.

Human attention-inspired volume reconstruction method on serial section electron microscopy images.

The alignment of a 2D microscopic image stack to create a 3D image volume is an indispensable aspect of serial section electron microscopy (EM) technology, which could restore the original 3D integrity of biological tissues destroyed by chemical fixation and physical dissection. However, due to the similar texture intrasection and complex variations intersections of neural images, previous registration methods usually failed to yield reliable correspondences. And this also led to misalignment and impeded restoring the z-axis anatomical continuity of the neuron volume. In this article, inspired by human behaviors in finding correspondences, which use the topological relationship of image contents, we developed a spatial attention-based registration method for serial EM images to improve registration accuracy. Our approach combined the U-Net framework with spatial transformer networks (STN) to regress corresponding transformation maps in an unsupervised training fashion. The spatial attention (SA) module was incorporated into the U-Net architecture to increase the distinctiveness of image features by modeling its topological relationship. Experiments are conducted on both simulated and real data sets (MAS and RegCremi). Quantitative and qualitative comparisons demonstrate that our approach results in state of art accuracy (using the evaluation index of NCC, SSIM, Dice, Landmark error) and providing smooth and reliable transformation with less texture blur and unclear boundary than existing techniques. Our method is able to restore image stacks for visualization and quantitative analysis of EM image sequences.

Anti-fibrotic effects of tannic acid through regulation of a sustained TGF-beta receptor signaling.

BACKGROUND: Pulmonary fibrosis is a progressive disease characterized by structural distortion of the lungs. Transforming growth factor-beta (TGF-beta) is a key cytokine implicated in the pathogenesis of pulmonary fibrosis. TGF-beta-induced myofibroblast differentiation characterized by expression of smooth muscle alpha-actin and extracellular matrix proteins is a key process in pathogenesis of fibrotic disease. Tannic acid is a natural polyphenol with diverse applications. In this study, we investigated the effect of tannic acid on myofibroblast differentiation and pulmonary fibrosis in cultured cells and in bleomycin model of the disease. METHODS: Primary cultured human lung fibroblasts (HLF) were used. The relative levels of proteins were determined by Western blotting. HLF contraction was measured by traction microscopy. Bleomycin-induced pulmonary fibrosis in mice was used as the disease model. RESULTS: Tannic acid inhibited TGF-beta-induced expression of collagen-1 and smooth muscle alpha-actin (SMA) as well as force generation by HLF. Tannic acid did not affect initial phosphorylation of Smad2 in response to TGF-beta, but significantly inhibited sustained Smad2 phosphorylation, which we recently described to be critical for TGF-beta-induced myofibroblast differentiation. Accordingly, tannic acid inhibited Smad-dependent gene transcription in response to TGF-beta, as assessed using luciferase reporter for the activity of Smad-binding elements. Finally, in mouse model of bleomycin-induced pulmonary fibrosis, therapeutic application of tannic acid resulted in a significant reduction of lung fibrosis, decrease in collagen-1 content and of Smad2 phosphorylation in the lungs. CONCLUSIONS: This study demonstrates the anti-fibrotic effect of tannic acid in vitro and in vivo through a regulation of sustained Smad2 phosphorylation.

Broad temperature range of cubic blue phase present in simple binary mixture systems containing rodlike Schiff base mesogens with tolane moiety.

Four simple rodlike Schiff base mesogens with tolane moiety were synthesized and applied to stabilize cubic blue phases (BPs) in simple binary mixture systems for the first time. When the chiral additive or was added into a chiral salicylaldimine-based compound, the temperature range of the cubic BP could be extended by more than 20 °C. However, when the chiral Schiff base mesogen was blended with chiral dopant possessing opposite handedness, , BPs could not be observed. Interestingly, the widest temperature range of the cubic BPs (∼35 °C) could be induced by adding the rodlike chiral dopant or into the rodlike racemic Schiff base mesogen with hydroxyl group. On the basis of our experimental results and molecular modeling, the appearance and temperature range of the BPs are affected by the dipole moment and the biaxiality of the molecular geometry. Accordingly, we demonstrated that the hydroxyl group and the methyl branch in this type of Schiff base mesogen play an important role in the stabilization of BPs.

Gata5 deficiency causes airway constrictor hyperresponsiveness in mice.

Gata5 is a transcription factor expressed in the lung, but its physiological role is unknown. To test whether and how Gata5 regulates airway constrictor responsiveness, we studied Gata5(-/-), Gata5(+/-), and wild-type mice on the C57BL/6J background. Cholinergic airway constrictor responsiveness was assessed invasively in mice without and with induction of allergic airway inflammation through ovalbumin sensitization and aerosol exposure. Gata5-deficient mice displayed native airway constrictor hyperresponsiveness (AHR) in the absence of allergen-induced inflammation. Gata5-deficient mice retained their relatively greater constrictor responsiveness even in ovalbumin-induced experimental asthma. Gata5 deficiency did not alter the distribution of cell types in bronchoalveolar lavage fluid, but bronchial epithelial mucus metaplasia was more prominent in Gata5(-/-) mice after allergen challenge. Gene expression profiles revealed that apolipoprotein E (apoE) was the fifth most down-regulated transcript in Gata5-deficient lungs, and quantitative RT-PCR and immunostaining confirmed reduced apoE expression in Gata5(-/-) mice. Quantitative RT-PCR also revealed increased IL-13 mRNA in the lungs of Gata5-deficient mice. These findings for the first time show that Gata5 regulates apoE and IL-13 expression in vivo and that its deletion causes AHR. Gata5-deficient mice exhibit an airway phenotype that closely resembles that previously reported for apoE(-/-) mice: both exhibit cholinergic AHR in native and experimental asthma states, and there is excessive goblet cell metaplasia after allergen sensitization and challenge. The Gata5-deficient phenotype also shares features that were previously reported for IL-13-treated mice. Together, these results indicate that Gata5 deficiency induces AHR, at least in part, by blunting apoE and increasing IL-13 expression.

Pulmonary neuroendocrine cells: crucial players in respiratory function and airway-nerve communication.

Pulmonary neuroendocrine cells (PNECs) are unique airway epithelial cells that blend neuronal and endocrine functions, acting as key sensors in the lung. They respond to environmental stimuli like allergens by releasing neuropeptides and neurotransmitters. PNECs stand out as the only lung epithelial cells innervated by neurons, suggesting a significant role in airway-nerve communication via direct neural pathways and hormone release. Pathological conditions such as asthma are linked to increased PNECs counts and elevated calcitonin gene-related peptide (CGRP) production, which may affect neuroprotection and brain function. CGRP is also associated with neurodegenerative diseases, including Parkinson's and Alzheimer's, potentially due to its influence on inflammation and cholinergic activity. Despite their low numbers, PNECs are crucial for a wide range of functions, highlighting the importance of further research. Advances in technology for producing and culturing human PNECs enable the exploration of new mechanisms and cell-specific responses to targeted therapies for PNEC-focused treatments.

Enhancing the efficacy of vaccinia-based oncolytic virotherapy by inhibiting CXCR2-mediated MDSC trafficking.

Oncolytic virotherapy is an innovative approach for cancer treatment. However, recruitment of myeloid-derived suppressor cells (MDSCs) into the tumor microenvironment (TME) after oncolysis-mediated local inflammation leads to tumor resistance to the therapy. Using the murine malignant mesothelioma model, we demonstrated that the in situ vaccinia virotherapy recruited primarily polymorphonuclear MDSCs (PMN-MDSCs) into the TME, where they exhibited strong suppression of cytotoxic T lymphocytes in a reactive oxygen species-dependent way. Single-cell RNA sequencing analysis confirmed the suppressive profile of PMN-MDSCs at the transcriptomic level and identified CXCR2 as a therapeutic target expressed on PMN-MDSCs. Abrogating PMN-MDSC trafficking by CXCR2-specific small molecule inhibitor during the vaccinia virotherapy exhibited enhanced antitumor efficacy in 3 syngeneic cancer models, through increasing CD8+/MDSC ratios in the TME, activating cytotoxic T lymphocytes, and skewing suppressive TME into an antitumor environment. Our results warrant clinical development of CXCR2 inhibitor in combination with oncolytic virotherapy.

Cardamonin attenuates iron overload-induced osteoblast oxidative stress through the HIF-1α/ROS pathway.

BACKGROUND: Osteoporosis(OP) is a bone disease under research. Iron overload is a significant risk factor. Iron balance is crucial for bone metabolism and biochemical processes. When there is an excess of iron in the body, it tends to produce reactive oxygen species (ROS) which can cause oxidative damage to cells. The flavonoid compound, Cardamonin (CAR), possesses potent anti-inflammatory and anti-iron overload properties that can be beneficial in mitigating the risk of OP. PURPOSE: This study investigates the potential therapeutic interventions and underlying mechanisms of CAR for treating OP in individuals with iron overload. METHODS: The model of iron-overloaded mice was established by intraperitoneally injecting iron dextran(ID) into the mice. OP severity was evaluated with micro-CT and Hematoxylin-Eosin (HE) staining in vivo. In vitro, the iron-overloaded osteoblast model was induced by ferric ammonium citrate. Cell counting kit 8 assay to evaluate cell viability, Annexin V-FITC/PI assay to detect cell apoptosis. A range of cellular markers were detected, including the variation in mitochondrial membrane potential (MMP), levels of malondialdehyde (MDA), ROS, and lipid hydroperoxide (LPO). RESULTS: CAR can reverse bone loss in iron overload-induced OP mouse models in vivo. CAR attenuates the impairment of iron overload on the activity and apoptosis of MC3T3-E1 cells as well as the accumulation of ROS and LPO activation via HIF-1α/ROS pathways. CONCLUSION: CAR downregulating HIF-1α pathways prevents inhibition of iron overload-induced osteoblasts dysfunctional by attenuating ROS accumulation, reducing oxidative stress, promotes bone formation, and alleviates OP.

Curcumenol regulates Histone H3K27me3 demethylases KDM6B affecting Succinic acid metabolism to alleviate cartilage degeneration in knee osteoarthritis.

BACKGROUND: Cartilage metabolism dysregulation is a crucial driver in knee osteoarthritis (KOA). Modulating the homeostasis can mitigate the cartilage degeneration in KOA. Curcumenol, derived from traditional Chinese medicine Curcuma Longa L., has demonstrated potential in enhancing chondrocyte proliferation and reducing apoptosis. However, the specific mechanism of Curcumenol in treating KOA remains unclear. This study aimed to demonstrate the molecular mechanism of Curcumenol in treating KOA based on the transcriptomics and metabolomics, and both in vivo and in vitro experimental validations. MATERIALS AND METHODS: In this study, a destabilization medial meniscus (DMM)-induced KOA mouse model was established. And the mice were intraperitoneally injected with Curcumenol at 4 and 8 mg/kg concentrations. The effects of Curcumenol on KOA cartilage and subchondral was evaluated using micro-CT, histopathology, and immunohistochemistry (IHC). In vitro, OA chondrocytes were induced with 10 µg/mL lipopolysaccharide (LPS) and treated with Curcumenol to evaluate the proliferation, apoptosis, and extracellular matrix (ECM) metabolism through CCK8 assay, flow cytometry, and chondrocyte staining. Furthermore, transcriptomics and metabolomics were utilized to identify differentially expressed genes (DEGs) and metabolites. Finally, integrating multi-omics analysis, virtual molecular docking (VMD), and molecular dynamics simulation (MDS), IHC, immunofluorescence (IF), PCR, and Western blot (WB) validation were conducted to elucidate the mechanism by which Curcumenol ameliorates KOA cartilage degeneration. RESULTS: Curcumenol ameliorated cartilage destruction and subchondral bone loss in KOA mice, promoted cartilage repair, upregulated the expression of COL2 while downregulated MMP3, and improved ECM synthesis metabolism. Additionally, Curcumenol also alleviated the damage of LPS on the proliferation activity and suppressed apoptosis, promoted ECM synthesis. Transcriptomic analysis combined with weighted gene co-expression network analysis (WGCNA) identified a significant downregulation of 19 key genes in KOA. Metabolomic profiling showed that Curcumenol downregulates the expression of d-Alanyl-d-alanine, 17a-Estradiol, Glutathione, and Succinic acid, while upregulating Sterculic acid and Azelaic acid. The integrated multi-omics analysis suggested that Curcumenol targeted KDM6B to regulate downstream protein H3K27me3 expression, which inhibited methylation at the histone H3K27, consequently reducing Succinic acid levels and improving KOA cartilage metabolism homeostasis. Finally, both in vivo and in vitro findings indicated that Curcumenol upregulated KDM6B, suppressed H3K27me3 expression, and stimulated collagen II expression and ECM synthesis, thus maintaining cartilage metabolism homeostasis and alleviating KOA cartilage degeneration. CONCLUSION: Curcumenol promotes cartilage repair and ameliorates cartilage degeneration in KOA by upregulating KDM6B expression, thereby reducing H3K27 methylation and downregulating Succinic Acid, restoring metabolic stability and ECM synthesis.

An herbal formulation "Shugan Xiaozhi decoction" ameliorates methionine/choline deficiency-induced nonalcoholic steatohepatitis through regulating inflammation and apoptosis-related pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Shugan Xiaozhi (SGXZ) decoction is a traditional Chinese medicine used for treating nonalcoholic steatohepatitis (NASH). It has been used clinically for over 20 years and proved to be effective; however, the molecular mechanism underlying the effects of SGXZ decoction remains unclear. AIM OF THE STUDY: We analyzed the chemical components, core targets, and molecular mechanisms of SGXZ decoction to improve NASH through network pharmacology and in vivo experiments. MATERIALS AND METHODS: The chemical components, core targets, and related signaling pathways of SGXZ decoction intervention in NASH were predicted using network pharmacology. Molecular docking was performed to verify chemical components and their core targets. The results were validated in the NASH model treated with SGXZ decoction. Mouse liver function was assessed by measuring ALT and AST levels. TC and TG levels were determined to evaluate lipid metabolism, and lipid deposition was assessed via oil red O staining. Mouse liver damage was determined via microscopy following hematoxylin and eosin staining. Liver fibrosis was assessed via Masson staining. Western blot (WB) and immunohistochemical (IHC) analyses were performed to detect inflammation and the expression of apoptosis-related proteins, including IL-1ß, IL-6, IL-18, TNF-α, MCP1, p53, FAS, Caspase-8, Caspase-3, Caspase-9, Bax, Bid, Cytochrome c, Bcl-2, and Bcl-XL. In addition, WB and IHC were used to assess protein expression associated with the TLR4/MyD88/NF-κB pathway. RESULTS: Quercetin, luteolin, kaempferol, naringenin, and nobiletin in SGXZ decoction were effective chemical components in improving NASH, and TNF-α, IL-6, and IL-1ß were the major core targets. Molecular docking indicated that these chemical components and major core targets might interact. KEGG pathway analysis showed that the pathways affected by SGXZ decoction, primarily including apoptosis and TLR4/NF-κB signaling pathways, interfere with NASH. In vivo experiments indicated that SGXZ decoction considerably ameliorated liver damage, fibrosis, and lipid metabolism disorder in MCD-induced NASH mouse models. In addition, WB and IHC verified the underlying molecular mechanisms of SGXZ decoction as predicted via network pharmacology. SGXZ decoction inhibited the activation of apoptosis-related pathways in MCD-induced NASH mice. Moreover, SGXZ decoction suppressed the activation of TLR4/MyD88/NF-κB pathway in MCD-induced NASH mice. CONCLUSION: SGXZ decoction can treat NASH through multiple targets and pathways. These findings provide new insights into the effective treatment of NASH using SGXZ decoction.

Tumor cell-released autophagosomes (TRAPs) induce PD-L1-decorated NETs that suppress T-cell function to promote breast cancer pulmonary metastasis.

BACKGROUND: Lung metastasis is the primary cause of breast cancer-related mortality. Neutrophil extracellular traps (NETs) are involved in the progression of breast cancer. However, the mechanism of NET formation is not fully understood. This study posits that tumor cell-released autophagosomes (TRAPs) play a crucial role in this process. METHODS: TRAPs were isolated from breast cancer cell lines to analyze their impact on NET formation in both human and mouse neutrophils. The study used both in vitro and in vivo models, including Toll-like receptor 4 (TLR4-/-) mice and engineered breast cancer cell lines. Immunofluorescence, ELISA, Western blotting, RNA sequencing, and flow cytometry were employed to dissect the signaling pathways leading to NET production and to explore their immunosuppressive effects, particularly focusing on the impact of NETs on T-cell function. The therapeutic potential of targeting TRAP-induced NETs and their immunosuppressive functions was evaluated using DNase I and αPD-L1 antibodies. Clinical relevance was assessed by correlating circulating levels of TRAPs and NETs with lung metastasis in patients with breast cancer. RESULTS: This study showed that TRAPs induced the formation of NETs in both human and mouse neutrophils by using the high mobility group box 1 and activating the TLR4-Myd88-ERK/p38 signaling axis. More importantly, PD-L1 carried by TRAP-induced NETs inhibited T-cell function in vitro and in vivo, thereby contributing to the formation of lung premetastatic niche (PMN) immunosuppression. In contrast, Becn1 KD-4T1 breast tumors with decreased circulating TRAPs in vivo reduced the formation of NETs, which in turn attenuated the immunosuppressive effects in PMN and resulted in a reduction of breast cancer pulmonary metastasis in murine models. Moreover, treatment with αPD-L1 in combination with DNase I that degraded NETs restored T-cell function and significantly reduced tumor metastasis. TRAP levels in the peripheral blood positively correlated with NET levels and lung metastasis in patients with breast cancer. CONCLUSIONS: Our results demonstrate a novel role of TRAPs in the formation of PD-L1-decorated NETs, which may provide a new strategy for early detection and treatment of pulmonary metastasis in patients with breast cancer.

Structural Diversity and Dimensionality of Three Cu(II)-dpds-C5O5 2- Coordination Polymers Controlled by the Coordination Sphere of Cu(II) Centers and the Coordination Modes of C5O5 2- (dpds = 4,4'-Dipyridyldisulfide).

Three supramolecular architectures, [Cu2(dpds)2(C5O5)2(H2O)4]·3H2O (1), [Cu(dpds)(C5O5)]·3H2O (2), and [Cu2(dpds)2(C5O5)2]·9H2O·C2H5OH (3) (dpds = 4,4'-dipyridyldisulfide and C5O5 2- (croconate) = dianion of 4,5-dihydroxycyclopent-4-ene-1,2,3-trione), have been synthesized and structurally characterized. Compound 1 contains two crystallographically independent Cu(II) ions, which are both distorted octahedral geometry with elongation along the croconate- and H2O-bound axial positions and bonded with two N atoms of two dpds, two O atoms of one C5O5 2-, and two H2O molecules. Two crystallographically independent dpds ligands, both adopting the bis-monodentate bridging mode, connect two Cu(II) ions to form a one-dimensional zigzag chain-like coordination polymer. In 2 and 3, there are two and three crystallographically independent Cu(II) ions, respectively, which are all distorted octahedral geometries with elongation along the croconate-bound axial positions six-coordinated and bonded with two N atoms of two dpds ligands in cis- or/and trans-forms and four O atoms of two C5O5 2- ligands. The dpds ligands in 2 and 3 all adopt the bis-monodentate bridging mode, and the C5O5 2- ligands act as bridging ligands with bridging bis-bidentate through three C5O5 2- oxygen atoms in 2 and bridging bis-bidentate through four adjacent C5O5 2- oxygen atoms in 3, respectively, linking the Cu(II) ions to generate a two-dimensional layered and a three-dimensional metal-organic framework, respectively. The structural diversity and dimensionality observed in 1-3 may be attributed to the cis- or/and trans-coordination sphere of Cu(II) centers with two dpds ligands and the coordination modes of croconate ligands. Thermal stability and in situ temperature-dependent structural variations of 1-3 have been verified by thermogravimetric analysis and powder X-ray diffraction measurements. Compounds 1 and 3 both exhibit water vapor capture behaviors with hysteresis isotherms.

A Prime-Boost Vaccination Approach Induces Lung Resident Memory CD8+ T Cells Derived from Central Memory T Cells That Prevent Tumor Lung Metastasis.

Memory T cells play a key role in immune protection against cancer. Vaccine-induced tissue-resident memory T (TRM) cells in the lung have been shown to protect against lung metastasis. Identifying the source of lung TRM cells can help to improve strategies, preventing tumor metastasis. Here, we found that a prime-boost vaccination approach using intramuscular DNA vaccine priming, followed by intranasal live-attenuated influenza-vectored vaccine (LAIV) boosting induced higher frequencies of lung CD8+ TRM cells compared with other vaccination regimens. Vaccine-induced lung CD8+ TRM cells, but not circulating memory T cells, conferred significant protection against metastatic melanoma and mesothelioma. Central memory T (TCM) cells induced by the DNA vaccination were major precursors of lung TRM cells established after the intranasal LAIV boost. Single-cell RNA sequencing analysis indicated that transcriptional reprogramming of TCM cells for differentiation into TRM cells in the lungs started as early as day 2 post the LAIV boost. Intranasal LAIV altered the mucosal microenvironment to recruit TCM cells via CXCR3-dependent chemotaxis and induced CD8+ TRM-associated transcriptional programs. These results identified TCM cells as the source of vaccine-induced CD8+ TRM cells that protect against lung metastasis. Significance: Prime-boost vaccination shapes the mucosal microenvironment and reprograms central memory T cells to generate lung resident memory T cells that protect against lung metastasis, providing insights for the optimization of vaccine strategies.

Ultrapotent class I neutralizing antibodies post Omicron breakthrough infection overcome broad SARS-CoV-2 escape variants.

BACKGROUND: The spread of emerging SARS-CoV-2 immune escape sublineages, especially JN.1 and KP.2, has resulted in new waves of COVID-19 globally. The evolving memory B cell responses elicited by the parental Omicron variants to subvariants with substantial antigenic drift remain incompletely investigated. METHODS: Using the single B cell antibody cloning technology, we isolated single memory B cells, delineated the B cell receptor repertoire and conducted the pseudovirus-based assay for recovered neutralizing antibodies (NAb) screening. We analyzed the cryo-EM structures of top broadly NAbs (bnAbs) and evaluated their in vivo efficacy (golden Syrian hamster model). FINDINGS: By investigating the evolution of human B cell immunity, we discovered a new panel of bnAbs arising from vaccinees after Omicron BA.2/BA.5 breakthrough infections. Two lead bnAbs neutralized major Omicron subvariants including JN.1 and KP.2 with IC50 values less than 10 ng/mL, representing ultrapotent receptor binding domain (RBD)-specific class I bnAbs. They belonged to the IGHV3-53/3-66 clonotypes instead of evolving from the pre-existing vaccine-induced IGHV1-58/IGKV3-20 bnAb ZCB11. Despite sequence diversity, they targeted previously unrecognized, highly conserved conformational epitopes in the receptor binding motif (RBM) for ultrapotent ACE2 blockade. The lead bnAb ZCP3B4 not only protected the lungs of hamsters intranasally challenged with BA.5.2, BQ.1.1 and XBB.1.5 but also prevented their contact transmission. INTERPRETATION: Our findings demonstrated that class I bnAbs have evolved an ultrapotent mode of action protecting against highly transmissible and broad Omicron escape variants, and their epitopes are potential targets for novel bnAbs and vaccine development. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.