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.

Changes in posterior corneal elevation after small incision lenticule extraction for different myopic diopters.

AIM: To study the changes and effect factors of posterior corneal surface after small incision lenticule extraction (SMILE) with different myopic diopters. METHODS: Ninety eyes of 90 patients who underwent SMILE were included in this retrospective study. Patients were allocated into three groups based on the preoperative spherical equivalent (SE): low myopia (SE≥-3.00 D), moderate myopia (-3.00 D>SE>-6.00 D) and high myopia (SE≤-6.00 D). Posterior corneal surfaces were measured by a Scheimpflug camera preoperatively and different postoperative times (1wk, 1, 3, 6mo, and 1y). Posterior mean elevation (PME) at 25 predetermined points of 3 concentric circles (2-, 4-, and 6-mm diameter) above the best fit sphere was analyzed. RESULTS: All surgeries were completed uneventfully and no ectasia was found through the observation. The difference of myopia group was significant at the 2-mm ring at 1 and 3mo postoperatively (1mo: P=0.017; 3mo: P=0.018). The effect of time on ΔPME was statistically significant (2-mm ring: P=0.001; 4-mm ring: P<0.001; 6-mm ring: P<0.001). The effect of different corneal locations on ΔPME was significant except 1wk postoperatively (1mo: P=0.000; 3mo: P=0.000; 6mo: P=0.001; 1y: P=0.001). Posterior corneal stability was linearly correlated with SE, central corneal thickness, ablation depth, residual bed thickness, percent ablation depth and percent stromal bed thickness. CONCLUSION: The posterior corneal surface changes dynamically after SMILE. No protrusion is observed on the posterior corneal surface in patients with different degrees of myopia within one year after surgery. SMILE has good stability, accuracy, safety and predictability.

Neutrophil extracellular traps promoting fibroblast activation and aggravating limb ischemia through Wnt5a pathway.

Although the formation of NETs contributes to cancer cell invasion and distant metastasis, its role in the pathological progression of limb ischemia remains unknown. This study investigated the functional significance of NETs in cell-cell crosstalk during limb ischemia. The changes of cell subsets in lower limb ischemia samples were detected by single-cell RNA sequencing. The expression of neutrophil extracellular traps (NETs) related markers in lower limb ischemia samples was detected by immunohistochemistry and Western blotting. The signaling pathway of NETs activation in fibroblasts was verified by immunofluorescence, PCR and Western blotting. Through single-cell RNA sequencing (scRNA-seq), we identified 9 distinct cell clusters, with significantly upregulated activation levels in fibroblasts and neutrophils and phenotypic transformation of smooth muscle cells (SMCs) into a proliferative state in ischemic tissue. At the same time, the interaction between fibroblasts and smooth muscle cells was significantly enhanced in ischemic tissue. NETs levels rise and fibroblast activation is induced in ischemic conditions. Mechanistically, activated fibroblasts promote smooth muscle cell proliferation through the Wnt5a pathway. In ischemic mice, inhibition of Wnt5a mitigated vascular remodeling and subsequent ischemia. These findings highlighting the role of cell-cell crosstalk in ischemia and vascular remodeling. We found that the NETs-initiated fibroblast-SMC interaction is a critical regulator of limb ischemia via Wnt5a pathway, a potential therapeutic target for the treatment.

Eucommia granules activate Wnt/ß-catenin pathway, and improve oxidative stress, inflammation, and endothelial injury in preeclampsia rats.

PURPOSE: Pre-eclampsia (PE) is a pregnancy-related complication. Eucommia is effective in the treatment of hypertensive disorders in pregnancy, but the specific effects and possible mechanisms of Eucommia granules (EG) in PE remain unknown. The aim of this study was to investigate the effects and possible mechanisms of EG in PE rats. METHODS: Pregnant Sprague Dawley rats were divided into five groups (n = 6): the control group, the model group, the low-dose group, the medium-dose group, and the high-dose group of EG. The PE model was established by subcutaneous injection of levonitroarginine methyl ester. Saline was given to the blank and model groups, and the Eucommia granules were given by gavage to the remaining groups. Blood pressure and urinary protein were detected. The body length and weight of the pups and the weight of the placenta were recorded. Superoxide dismutase (SOD) activity and levels of malondialdehyde (MDA), placental growth factor (PIGF), and soluble vascular endothelial growth factor receptor-1 (sFIt-1) were measured in the placenta. Pathological changes were observed by hematoxylin-eosin staining. Wnt/ß-catenin pathway-related protein expression was detected using Western blot. RESULTS: Compared with the model group, the PE rats treated with EG had lower blood pressure and urinary protein. The length and weight of the pups and placental weight were increased. Inflammation and necrosis in the placental tissue was improved. SOD level increased, MDA content and sFIt-1/PIGF ratio decreased, and Wnt/ß-catenin pathway-related protein expression level increased. Moreover, the results of EG on PE rats increased with higher doses of EG. CONCLUSIONS: EG may activate the Wnt/ß-catenin pathway and inhibit oxidative stress, inflammation, and vascular endothelial injury in PE rats, thereby improving the perinatal prognosis of preeclamptic rats. EG may inhibit oxidative stress, inflammation, and vascular endothelial injury through activation of the Wnt/ß-catenin pathway in preeclampsia rats, thereby improving perinatal outcomes in PE rats.

Intra-nanoparticle plasmonic nanogap based spatial-confinement SERS analysis of polypeptides.

Sensing and characterizing water-soluble polypeptides are essential in various biological applications. However, detecting polypeptides using Surface-Enhanced Raman Scattering (SERS) remains a challenge due to the dominance of aromatic amino acid residues and backbones in the signal, which hinders the detection of non-aromatic amino acid residues. Herein, intra-nanoparticle plasmonic nanogap were designed by etching the Ag shell in Au@AgNPs (i.e., obtaining AuAg cores) with chlorauric acid under mild conditions, at the same time forming the outermost Au shell and the void between the AuAg cores and the Au shell (AuAg@void@Au). By varying the Ag to added chloroauric acid molar ratios, we pioneered a simple, controllable, and general synthetic strategy to form interlayer-free nanoparticles with tunable Au shell thickness, achieving precise regulation of electric field enhancement within the intra-nanogap. As validation, two polypeptide molecules, bacitracin and insulin B, were successfully synchronously encapsulated and spatial-confined in the intra-nanogap for sensing. Compared with concentrated 50 nm AuNPs and Au@AgNPs as SERS substrates, our simultaneous detection method improved the sensitivity of the assay while benefiting to obtain more comprehensive characteristic peaks of polypeptides. The synthetic strategy of confining analytes while fabricating plasmonic nanostructures enables the diffusion of target molecules into the nanogap in a highly specific and sensitive manner, providing the majority of the functionality required to achieve peptide detection or sequencing without the hassle of labeling.

Eucommia granules activate Wnt/β-catenin pathway, and improve oxidative stress, inflammation, and endothelial injury in preeclampsia rats

ABSTRACT Purpose: Pre-eclampsia (PE) is a pregnancy-related complication. Eucommia is effective in the treatment of hypertensive disorders in pregnancy, but the specific effects and possible mechanisms of Eucommia granules (EG) in PE remain unknown. The aim of this study was to investigate the effects and possible mechanisms of EG in PE rats. Methods: Pregnant Sprague Dawley rats were divided into five groups (n = 6): the control group, the model group, the low-dose group, the medium-dose group, and the high-dose group of EG. The PE model was established by subcutaneous injection of levonitroarginine methyl ester. Saline was given to the blank and model groups, and the Eucommia granules were given by gavage to the remaining groups. Blood pressure and urinary protein were detected. The body length and weight of the pups and the weight of the placenta were recorded. Superoxide dismutase (SOD) activity and levels of malondialdehyde (MDA), placental growth factor (PIGF), and soluble vascular endothelial growth factor receptor-1 (sFIt-1) were measured in the placenta. Pathological changes were observed by hematoxylin-eosin staining. Wnt/β-catenin pathway-related protein expression was detected using Western blot. Results: Compared with the model group, the PE rats treated with EG had lower blood pressure and urinary protein. The length and weight of the pups and placental weight were increased. Inflammation and necrosis in the placental tissue was improved. SOD level increased, MDA content and sFIt-1/PIGF ratio decreased, and Wnt/β-catenin pathway-related protein expression level increased. Moreover, the results of EG on PE rats increased with higher doses of EG. Conclusions: EG may activate the Wnt/β-catenin pathway and inhibit oxidative stress, inflammation, and vascular endothelial injury in PE rats, thereby improving the perinatal prognosis of preeclamptic rats. EG may inhibit oxidative stress, inflammation, and vascular endothelial injury through activation of the Wnt/β-catenin pathway in preeclampsia rats, thereby improving perinatal outcomes in PE rats.

Role of IL-34 and its receptors in inflammatory diseases.

In recent years, IL-34 has been widely discussed as a novel cytokine. IL-34 is a pro-inflammatory cytokine binding four distinct receptors, namely CSF-1R, syndecan-1, PTP-ζ and TREM2. Previous studies have shown that IL-34 and its receptors play important roles in the development and progression of various inflammatory diseases. Therefore, IL-34 has the potential to be a biomarker and therapeutic target for inflammatory diseases. However, further study is still needed to identify the specific mechanism through which IL-34 contributes to illness. In this article, we review the recent advances in the biological roles of IL-34 and its receptors as well as their roles in the development and therapeutic application of inflammatory diseases.

Host CD34+ cells are replacing donor endothelium of transplanted heart.

BACKGROUND: Endothelium dysfunction is a central problem for early rejection due to the host alloimmune response and the late status of arteriosclerosis in heart transplantation. However, reliable pieces of evidence are still limited concerning the source of the regenerated endothelium within the transplanted heart. METHODS: We analyzed single-cell RNA sequencing data and constructed an inducible lineage tracing mouse, combined heart transplantation with bone marrow transplantation and a parabiosis model, cellular components, and endothelial cell populations in cardiac graft lesions. RESULTS: Our single-cell RNA sequencing analysis of a transplanted heart allowed for the establishment of an endothelial cell atlas with a heterogeneous population, including arterial, venous, capillary, and lymphatic endothelial cells. Along with genetic cell lineage tracing, we demonstrated that the donor cells were mostly replaced by recipient cells in the cardiac allograft, up to 83.29% 2 weeks after transplantation. Furthermore, recipient nonbone marrow CD34+ endothelial progenitors contributed significantly to extracellular matrix organization and immune regulation, with higher apoptotic ability in the transplanted hearts. Mechanistically, peripheral blood-derived human endothelial progenitor cells differentiate into endocardial cells via Vascular endothelial growth factor receptor-mediated pathways. Host circulating CD34+ endothelial progenitors could repair the damaged donor endothelium presumably through CCL3-CCR5 chemotaxis. Partial depletion of host CD34+ cells resulted in delayed endothelial regeneration. CONCLUSIONS: We created an annotated fate map of endothelial cells in cardiac allografts, indicating how recipient CD34+ cells could replace the donor endothelium via chemokine CCL3-CCR5 interactions. The mechanisms we discovered could have a potential therapeutic effect on the long-term outcomes of heart transplantation.

Synthesis and anticancer activity of podophyllotoxin derivatives with nitrogen-containing heterocycles.

Three series of podophyllotoxin derivatives with various nitrogen-containing heterocycles were designed and synthesized. The antitumor activity of these podophyllotoxin derivatives was evaluated in vitro against a panel of human tumor cell lines. The results showed that podophyllotoxin-imidazolium salts and podophyllotoxin-1,2,4-triazolium salts a1-a20 exhibited excellent cytotoxic activity. Among them, a6 was the most potent cytotoxic compound with IC50 values of 0.04-0.29 µM. Podophyllotoxin-1,2,3-triazole derivatives b1-b5 displayed medium cytotoxic activity, and podophyllotoxin-amine compounds c1-c3 has good cytotoxic activity with IC50 value of 0.04-0.58 µM. Furthermore, cell cycle and apoptosis experiments of compound a6 were carried out and the results exhibited that a6 could induce G2/M cell cycle arrest and apoptosis in HCT-116 cells.

CD34+ cell-derived fibroblast-macrophage cross-talk drives limb ischemia recovery through the OSM-ANGPTL signaling axis.

CD34+ cells improve the perfusion and function of ischemic limbs in humans and mice. However, there is no direct evidence of the differentiation potential and functional role of these cells in the ischemic muscle microenvironment. Here, we combined the single-cell RNA sequencing and genetic lineage tracing technology, then provided exact single-cell atlases of normal and ischemic limb tissues in human and mouse, and consequently found that bone marrow (BM)-derived macrophages with antigen-presenting function migrated to the ischemic site, while resident macrophages underwent apoptosis. The macrophage oncostatin M (OSM) regulatory pathway was specifically turned on by ischemia. Simultaneously, BM CD34+-derived proregenerative fibroblasts were recruited to the ischemia niche, where they received macrophage-released OSM and promoted angiopoietin-like protein-associated angiogenesis. These findings provided mechanisms on the cellular events and cell-cell communications during tissue ischemia and regeneration and provided evidence that CD34+ cells serve as fibroblast progenitors promoting tissue regeneration.

The role of spexin in energy metabolism.

Spexin, also identified as neuropeptide Q (NPQ), is a 14 amino acid peptide discovered by bioinformatic methods. It has a conserved structure in many species and is widely expressed in the central nervous system and peripheral tissues. It has an associated receptor, galanin receptor 2/3 (GALR2/3). Mature spexin peptides can exert various functions by activating GALR2/3, such as inhibiting food intake, inhibiting lipid absorption, reducing body weight, and improving insulin resistance. Spexin is expressed in the adrenal gland, pancreas, visceral fat, and thyroid, with the highest expression in the adrenal gland, followed by the pancreas. Physiologically, spexin and insulin interact in pancreatic islets. Spexin may be one of the regulators of endocrine function in the pancreas. Spexin is a possible indicator of insulin resistance and it has a variety of functional properties, here we review its role in energy metabolism.

Single cell and lineage tracing studies reveal the impact of CD34+ cells on myocardial fibrosis during heart failure.

BACKGROUND: CD34+ cells have been used to treat the patients with heart failure, but the outcome is variable. It is of great significance to scrutinize the fate and the mechanism of CD34+ cell differentiation in vivo during heart failure and explore its intervention strategy. METHODS: We performed single-cell RNA sequencing (scRNA-seq) of the total non-cardiomyocytes and enriched Cd34-tdTomato+ lineage cells in the murine (male Cd34-CreERT2; Rosa26-tdTomato mice) pressure overload model (transverse aortic constriction, TAC), and total non-cardiomyocytes from human adult hearts. Then, in order to determine the origin of CD34+ cell that plays a role in myocardial fibrosis, bone marrow transplantation model was performed. Furthermore, to further clarify the role of CD34 + cells in myocardial remodeling in response to TAC injury, we generated Cd34-CreERT2; Rosa26-eGFP-DTA (Cre/DTA) mice. RESULTS: By analyzing the transcriptomes of 59,505 single cells from the mouse heart and 22,537 single cells from the human heart, we illustrated the dynamics of cell landscape during the progression of heart hypertrophy, including CD34+ cells, fibroblasts, endothelial and immune cells. By combining genetic lineage tracing and bone marrow transplantation models, we demonstrated that non-bone-marrow-derived CD34+ cells give rise to fibroblasts and endothelial cells, while bone-marrow-derived CD34+ cell turned into immune cells only in response to pressure overload. Interestingly, partial depletion of CD34+ cells alleviated the severity of myocardial fibrosis with a significant improvement of cardiac function in Cd34-CreERT2; Rosa26-eGFP-DTA model. Similar changes of non-cardiomyocyte composition and cellular heterogeneity of heart failure were also observed in human patient with heart failure. Furthermore, immunostaining showed a double labeling of CD34 and fibroblast markers in human heart tissue. Mechanistically, our single-cell pseudotime analysis of scRNA-seq data and in vitro cell culture study revealed that Wnt-ß-catenin and TGFß1/Smad pathways are critical in regulating CD34+ cell differentiation toward fibroblasts. CONCLUSIONS: Our study provides a cellular landscape of CD34+ cell-derived cells in the hypertrophy heart of human and animal models, indicating that non-bone-marrow-derived CD34+ cells differentiating into fibroblasts largely account for cardiac fibrosis. These findings may provide novel insights for the pathogenesis of cardiac fibrosis and have further potential therapeutic implications for the heart failure.

Therapeutic Effects of Salvianolic Acid B on Angiotensin II-Induced Atrial Fibrosis by Regulating Atrium Metabolism via Targeting AMPK/FoxO1/miR-148a-3p Axis.

The present study highlights the effects of salvianolic acid B (Sal B) on angiotensin II (Ang II)-activated atrial fibroblasts as well as the associated potential mechanism from the metabonomics perspective. Metabolic profile analysis performed an optimal separation of the Ang II and control group, indicating a recovery impact of Sal B on Ang II-activated fibroblasts (FBs). We found that metabolite levels in the Ang II + Sal B group were reversed to normal. Moreover, 23 significant metabolites were identified. Metabolic network analysis indicated that these metabolites participated in purine metabolism and FoxO signaling pathway. We found that Sal B activated AMP-activated protein kinase (AMPK) phosphorylation, which further promoted FoxO1 activation and increased miR-148a-3p level. We further verified that Sal B modulate the abnormal AMP, phosphocreatine, glutathione (GSH), and reactive oxygen species (ROS) production in Ang II-stimulated FBs. Collectively, Sal B can protect the Ang II-activated FBs from fibrosis and oxidative stress via AMPK/FoxO1/miRNA-148a-3p axis.

ESM1 Interacts with c-Met to Promote Gastric Cancer Peritoneal Metastasis by Inducing Angiogenesis.

The peritoneum is the most common metastatic site of advanced gastric cancer and is associated with extremely poor prognosis. Endothelial-specific molecule 1 (ESM1) was found to be significantly associated with gastric cancer peritoneal metastasis (GCPM); however, the biological functions and molecular mechanisms of ESM1 in regulating GCPM remain unclear. Herein, we demonstrated that ESM1 expression was significantly upregulated in gastric cancer tissues and positively correlated with platelet endothelial cell adhesion molecule-1 (CD31) levels. Moreover, clinical validation, in in vitro and in vivo experiments, confirmed that ESM1 promoted gastric cancer angiogenesis, eventually promoting gastric cancer peritoneal metastasis. Mechanistically, ESM1 promoted tumor angiogenesis by binding to c-Met on the vascular endothelial cell membrane. In addition, our results confirmed that ESM1 upregulated VEGFA, HIF1α, and MMP9 expression and induced angiogenesis by activating the MAPK/ERK pathway. In conclusion, our findings identified the role of ESM1 in gastric cancer angiogenesis and GCPM, thus providing insights into the diagnosis and treatment of advanced gastric cancer.

Deep Learning-Based Multicapturer SERS Platform on Plasmonic Nanocube Metasurfaces for Multiplex Detection of Organophosphorus Pesticides in Environmental Water.

In situ rapid detection of contaminants in environmental water is crucial for protecting the ecological environment and human health; however, it is always hindered by the complexity of sample matrices, trace content, and unknown species. Herein, we demonstrate a deep learning-based multicapturer surface-enhanced Raman scattering (SERS) platform on plasmonic nanocube metasurfaces for multiplex determination of organophosphorus pesticides (OPPs) residues. Poly(vinylpyrrolidone), 4-mercaptobenzoic acid, and l-cysteine are assembled on Ag nanocubes (AgNCs) and act as capturers to chemically define OPPs. Meanwhile, the OPPs-captured AgNCs efficiently close the interparticle distance and generate plasmonic metasurfaces, guaranteeing ultrasensitive and reproducible SERS analysis. Furthermore, by strategically combining all capturer-OPP SERS spectra, comprehensive "combined-SERS spectra" are reconstructed to enhance spectral variations of each OPP. Based on the combined-SERS spectra, a deep learning model is trained to predict OPPs, which significantly improve the qualitative and quantitative analysis accuracy. We successfully identified multiple OPPs in farmland, river, and fishpond water using this strategy. The whole detection procedure requires only 30 min, including sampling, SERS measurements, and deep learning analyses. This combination of a multicapturer SERS platform with the deep learning algorithm creates a rapid and reliable analytical strategy for multiplex detection of target molecules, providing a potential paradigm shift for environment-related research.

Fatty acid palmitate suppresses FoxO1 expression via PERK and IRE1 unfolded protein response in C2C12 myotubes.

Forkhead Box O1 (FoxO1) is a transcription factor with a unique fork head domain that indirectly participates in a variety of physiological processes and plays an important role in type 2 diabetes. Palmitate as the most abundant free fatty acid, accounting for 28-32% of total free fatty acids in human plasma. There is a direct relationship between palmitate and insulin resistance-induced type 2 diabetes. In addition, palmitate can activate the unfolded protein response signaling pathway induced by endoplasmic reticulum (ER) stress. This study aimed to investigate the response of FoxO1 to palmitate and the relationship with ER stress in C2C12 myotubes. Treatment of palmitate or tunicamycin promoted ER stress-related genes expression but suppressed FoxO1 expression, while 4-phenylbutyrate presented the opposite activity in palmitate-pretreated C2C12 myotubes, indicating that ER stress might be closely associated with FoxO1 expression. Moreover, palmitate-suppressed FoxO1 expression was reversed in C2C12 cells when the PERK and IRE-1 signaling pathway was inhibited by treatment with GSK2656157 or 4µ8C. However, no differences were observed when the ATF6 signaling pathway was suppressed by knockout of the ATF6 gene. These findings suggest that palmitate suppressed FoxO1 expression via the PERK and IRE1 signaling pathways.

The cross-talk of cancer-associated fibroblasts assist in prognosis and immunotherapy in patients with breast carcinoma.

The association between cancer-associated fibroblasts (CAFs) and tumor microenvironment (TME) is a key factor in promoting tumor progression. However, the correlation between CAFs and TME in breast carcinoma has not been elucidated. Thus, further study about the cross-effect between CAFs and TME can provide novel strategies for breast carcinoma treatment, particularly targeted immunotherapy. First, we systematically analyzed cell communication in a single-cell dataset and identified the interacted genes between CAFs and TME components. Then, a robust fibroblast-related score (FRS) model was developed using the LASSO algorithm. The FRS can be a reliable adverse prognostic factor in three cohorts with breast carcinoma. Functional enrichment analysis and single-sample Gene Set Enrichment Analysis showed that patients with a high FRS had cold tumors with active proliferation and immunosuppression. Patients with a low FRS presented with hot tumors with active immune and cell-killing functions. Genomic variation analysis revealed that patients with a low FRS had a higher somatic mutation load and copy number variation burden. Finally, patients with a low FRS were more sensitive to chemotherapy and immunotherapy, particularly anti-PD-1 therapy. In conclusion, a reliable FRS model was constructed not only reliable for predicting prognosis but also competent to estimate clinical immunotherapy and chemotherapy response for patients with BRCA, which might provide significant clinical implications for guiding clinical decision-making for patients with BRCA.

Deep Learning-Based Label-Free Surface-Enhanced Raman Scattering Screening and Recognition of Small-Molecule Binding Sites in Proteins.

Identification of small-molecule binding sites in proteins is of great significance in analysis of protein function and drug design. Modified sites can be recognized via proteolytic cleavage followed by liquid chromatography-mass spectrometry (LC-MS); however, this has always been impeded by the complexity of peptide mixtures and the elaborate synthetic design for tags. Here, we demonstrate a novel technique for identifying protein binding sites using a deep learning-based label-free surface-enhanced Raman scattering (SERS) screening (DLSS) strategy. In DLSS, the deep learning model that was trained with large SERS signals could detect signal features of small molecules with high accuracy (>99%). Without any secondary tag, the small molecules are directly complexed with proteins. After proteolysis and LC, SERS signals of all LC fractions are collected and input into the model, whereby the fractions containing the small-molecule-modified peptides can be recognized by the model and sent to MS/MS to identify the binding site(s). By using an automated DLSS system, we successfully identified the modification sites of fomepizole in alcohol dehydrogenase, which is coordinated with zinc along with three peptides. We also showed that the DLSS strategy works for identification of amino-acid residues that covalently bond with ibrutinib in Bruton tyrosine kinase. These results suggest that the DLSS strategy, which provides high molecular recognition capability to LC-MS analysis, has potential in drug discovery, proteomics, and metabolomics.

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.