A Rapid, Scalable Laser-Scribing Process to Prepare Si/Graphene Composites for Lithium-Ion Batteries.

Silicon has gained significant attention as a lithium-ion battery anode material due to its high theoretical capacity compared to conventional graphite. Unfortunately, silicon anodes suffer from poor cycling performance caused by their extreme volume change during lithiation and de-lithiation. Compositing silicon particles with 2D carbon materials, such as graphene, can help mitigate this problem. However, an unaddressed challenge remains: a simple, inexpensive synthesis of Si/graphene composites. Here, a one-step laser-scribing method is proposed as a straightforward, rapid (≈3 min), scalable, and less-energy-consuming (≈5 W for a few minutes under air) process to prepare Si/laser-scribed graphene (LSG) composites. In this research, two types of Si particles, Si nanoparticles (SiNPs) and Si microparticles (SiMPs), are used. The rate performance is improved after laser scribing: SiNP/LSG retains 827.6 mAh g-1 at 2.0 A gSi+C -1 , while SiNP/GO (before laser scribing) retains only 463.8 mAh g-1 . This can be attributed to the fast ion transport within the well-exfoliated 3D graphene network formed by laser scribing. The cyclability is also improved: SiNP/LSG retains 88.3% capacity after 100 cycles at 2.0 A gSi+C -1 , while SiNP/GO retains only 57.0%. The same trend is found for SiMPs: the SiMP/LSG shows better rate and cycling performance than SiMP/GO composites.

Understanding the Degradation Mechanisms of Conducting Polymer Supercapacitors.

Conducting polymers like polyaniline (PANI) are promising pseudocapacitive electrode materials, yet experience instability in cycling performance. Since polymers often degrade into oligomers, short chain length anilines have been developed to improve the cycling stability of PANI-based supercapacitors. However, the capacitance degradation mechanisms of aniline oligomer-based materials have not been systematically investigated and are little understood. Herein, two composite electrodes based on aniline trimers (AT) and carbon nanotubes (CNTs) are studied as model systems and evaluated at both pre-cycling and post-cycling states through physicochemical and electrochemical characterizations. The favorable effect of covalent bonding between AT and CNTs is confirmed to enhance cycling stability by preventing the detachment of aniline trimer and preserving the electrode microstructure throughout the charge/discharge cycling process. In addition, higher porosity has a positive effect on electron/ion transfer and the adaptation to volumetric changes, resulting in higher conductivity and extended cycle life. This work provides insights into the mechanism of enhanced cycling stability of aniline oligomers, indicating design features for aniline oligomer electrode materials to improve their electrochemical performance.

Molecular Engineering of Hierarchical Conducting Polymer Composites for Highly Stable Supercapacitors.

Long cycle life and high energy/power density are imperative to energy storage systems. Polyaniline (PANI) has shown great potential as an electrode material but is limited by poor cycling and rate performance. We present a molecular design approach of binding short-chain aniline trimers (ATs) and carbon nanotubes (CNTs) through the formation of amide covalent linkages enabled by a simple laser scribing technique. The covalently coupled AT/CNT (cc-AT/CNT) composite retains 80% of its original capacitance after 20 000 charge/discharge cycles, which readily outperforms long-chain PANI/CNT composites without covalent connections. The compact AT/CNT heterointerfaces produce fast charge/discharge kinetics and excellent rate capability. The flexible symmetric quasi-solid-state devices can be stably cycled beyond 50 000 cycles, at least 5 times longer than most PANI/CNT-based symmetric supercapacitors reported to date. This molecular design of durable conducting polymer-based electrode materials enabled by laser irradiation presents a feasible approach toward robust advanced energy storage devices.

Are current wound classifications valid for predicting prognosis in people treated for limb-threatening diabetic foot ulcers?

This study aims to investigate whether the current wound classifications were valid for the treatment prognosis of subjects treated for limb-threatening diabetic foot ulcers (LTDFU). A total of 1548 patients with LTDFU and infection were studied, with wounds recorded using the Wagner, Texas, PEDIS and WIfI classifications while major lower extremity amputations (LEAs) or in-hospital mortality incidences were defined as poor outcomes. Among them, 153 (9.9%) patients received major LEAs and 38 (2.5%) patients died. After adjustments, the Wagner classification and Texas stage as well as clinical factors such as comorbidity with major adverse cardiac events (MACE), being under dialysis and having serum levels of C-reactive protein (CRP) and albumin were independent factors for prognosis. For patients without dialysis, Wagner and Texas stage stood out independently for prognosis. For patients on dialysis, only levels of CRP (odds ratio [OR] = 2.2 in Wagner, OR = 2.0 in WIfI, OR = 2.2 in Texas, OR = 2.3 in PEDIS) and albumin (OR = 0.4 in four classifications) were valid predictors. The Wagner system and Texas stage were valid for predicting prognosis in treatment for LTDFUs, suggesting a role of vascular perfusion. MACE history, levels of CRP and albumin level should assist in prediction; more significantly, only levels of CRP and albumin appeared valid for those subjects undergoing dialysis.

Obesity-mediated upregulation of the YAP/IL33 signaling axis promotes aggressiveness and induces an immunosuppressive tumor microenvironment in breast cancer.

Obesity is a well-known risk factor for breast cancer formation and is associated with elevated mortality and a poor prognosis. An obesity-mediated inflammatory microenvironment is conducive to the malignant progression of tumors. However, the detailed molecular mechanism is still needed to be clarified. Herein, we identified that breast cancer cells from mice with diet-induced obesity exhibited increased growth, invasiveness, and stemness capacities. A transcriptome analysis revealed that expressions of interleukin 33 (IL33) signaling pathway-related genes were elevated in obesity-associated breast cancer cells. Importantly, IL33 expression was significantly associated with the yes-associated protein (YAP) signature, and IL33 was transcriptionally regulated by YAP. Suppression of IL33 reduced tumor migration and invasion, while the addition of IL33 activated nuclear factor (NF)-κB signaling and revived tumor mobility in YAP-silenced cells. Furthermore, suppression of YAP attenuated IL33 expression which was accompanied by relief of obesity-mediated immunosuppression. Clinical analyses showed that IL33 expression was markedly associated with macrophage and regulatory T cell infiltration. These findings reveal a crucial role of the YAP/IL33 axis in promoting aggressiveness and immunosuppression of obesity-associated breast cancer progression.

A Low-Cost, Sensitive Reporter System Using Membrane-Tethered Horseradish Peroxidase for Efficient Gene Expression Analysis.

Reporter gene assays are essential for high-throughput analysis, such as drug screening or determining downstream signaling activation/inhibition. However, use of this technology has been hampered by the high cost of the substrate (e.g., d-Luciferin (d-Luc)) in the most common firefly luciferase (FLuc) reporter gene assay. Although alternate luciferase is available worldwide, its substrate has remained expensive, and a more affordable option is still in demand. Here, we present a membrane-tethered horseradish peroxidase (mHRP), a new reporter system composed of a cell membrane expressing HRP that can preserve its enzymatic function on the cell surface, facilitates contact with HRP substrates (e.g., ABTS and TMB), and avoids the cell lysis process and the use of the high-priced luciferase substrate. An evaluation of the light signal sensitivity of mHRP compared to FLuc showed that both had comparable signal sensitivity. We also identified an extended substrate half-life of more than 5-fold that of d-Luc. Of note, this strategy provided a more stable detection signal, and the cell lysis process is not mandatory. Furthermore, with this strategy, we decreased the total amount of time taken for analysis and increased the time of detection limit of the reporter assay. Pricing analysis showed a one-third to one twenty-eighth price drop per single test of reporter assay. Given the convenience and stability of the mHRP reporter system, we believe that our strategy is suitable for use as an alternative to the luciferase reporter assay.

Downregulation of TAZ elicits a mitochondrial redox imbalance and ferroptosis in lung epithelial cells exposed to diesel exhaust particles.

Mitochondrial dysfunction was reported to be involved in the development of lung diseases including chronic obstructive pulmonary disease (COPD). However, molecular regulation underlying metabolic disorders in the airway epithelia exposed to air pollution remains unclear. In the present study, lung bronchial epithelial BEAS-2B and alveolar epithelial A549 cells were treated with diesel exhaust particles (DEPs), the primary representative of ambient particle matter. This treatment elicited cell death accompanied by induction of lipid reactive oxygen species (ROS) production and ferroptosis. Lipidomics analyses revealed that DEPs increased glycerophospholipid contents. Accordingly, DEPs upregulated expression of the electron transport chain (ETC) complex and induced mitochondrial ROS production. Mechanistically, DEP exposure downregulated the Hippo transducer transcriptional co-activator with PDZ-binding motif (TAZ), which was further identified to be crucial for the ferroptosis-associated antioxidant system, including glutathione peroxidase 4 (GPX4), the glutamate-cysteine ligase catalytic subunit (GCLC), and glutathione-disulfide reductase (GSR). Moreover, immunohistochemistry confirmed downregulation of GPX4 and upregulation of lipid peroxidation in the bronchial epithelium of COPD patients and Sprague-Dawley rats exposed to air pollution. Finally, proteomics analyses confirmed alterations of ETC-related proteins in bronchoalveolar lavage from COPD patients compared to healthy subjects. Together, our study discovered that involvement of mitochondrial redox dysregulation plays a vital role in pulmonary epithelial cell destruction after exposure to air pollution.

Thin-Film Composite Membranes with a Hybrid Dimensional Titania Interlayer for Ultrapermeable Nanofiltration.

The interfacial properties within a composite structure of membranes play a vital role in the separation properties and application performances. Building an interlayer can facilitate the formation of a highly selective layer as well as improve the interfacial properties of the composite membrane. However, it is difficult for a nanomaterial-based interlayer to increase the flux and retention of nanofiltration membranes simultaneously. Here, we report a nanofiltration membrane with a hybrid dimensional titania interlayer that exhibits excellent separation performance. The interlayer, composed of Fe-doped titania nanosheets and titania nanoparticles, helps the formation of an ultrathin (∼30 nm thick) and defect-free polyamide selective layer with an ideal nanostructure. The hybrid dimensional interlayer endows the membrane with a superior permeability and alleviates flux decline. In addition, the rigid interlayer framework on a PVDF support drastically improves the pressure resistance of nanofiltration membranes and shows negligible flux loss up to 1.5 MPa of pressure.

Mechanistic Insights into IscU Conformation Regulation for Fe-S Cluster Biogenesis Revealed by Variable Temperature Electrospray Ionization Native Ion Mobility Mass Spectrometry.

Iron-sulfur (Fe-S) cluster (ISC) cofactors are required for the function of many critical cellular processes. In the ISC Fe-S cluster biosynthetic pathway, IscU assembles Fe-S cluster intermediates from iron, electrons, and inorganic sulfur, which is provided by the cysteine desulfurase enzyme IscS. IscU also binds to Zn, which mimics and competes for binding with the Fe-S cluster. Crystallographic and nuclear magnetic resonance spectroscopic studies reveal that IscU is a metamorphic protein that exists in multiple conformational states, which include at least a structured form and a disordered form. The structured form of IscU is favored by metal binding and is stable in a narrow temperature range, undergoing both cold and hot denaturation. Interestingly, the form of IscU that binds IscS and functions in Fe-S cluster assembly remains controversial. Here, results from variable temperature electrospray ionization (vT-ESI) native ion mobility mass spectrometry (nIM-MS) establish that IscU exists in structured, intermediate, and disordered forms that rearrange to more extended conformations at higher temperatures. A comparison of Zn-IscU and apo-IscU reveals that Zn(II) binding attenuates the cold/heat denaturation of IscU, promotes refolding of IscU, favors the structured and intermediate conformations, and inhibits the disordered high charge states. Overall, these findings provide a structural rationalization for the role of Zn(II) in stabilizing IscU conformations and IscS in altering the IscU active site to prepare for Zn(II) release and cluster synthesis. This work highlights how vT-ESI-nIM-MS can be applied as a powerful tool in mechanistic enzymology by providing details of relationships among temperature, protein conformations, and ligand/protein binding.

Lnc-IL7R alleviates PM2.5-mediated cellular senescence and apoptosis through EZH2 recruitment in chronic obstructive pulmonary disease.

BACKGROUND: Long-term exposure to PM2.5 (particulate matter with an aerodynamic diameter of ≤ 2.5 µm) is associated with pulmonary injury and emphysema in patients with chronic obstructive pulmonary disease (COPD). We investigated mechanisms through which the long noncoding RNA lnc-IL7R contributes to cellular damage by inducing oxidative stress in COPD patients exposed to PM2.5. METHODS: Associations of serum lnc-IL7R levels with lung function, emphysema, and previous PM2.5 exposure in COPD patients were analyzed. Reactive oxygen species and lnc-IL7R levels were measured in PM2.5-treated cells. The levels of lnc-IL7R and cellular senescence-associated genes, namely p16INK4a and p21CIP1/WAF1, were determined through lung tissue section staining. The effects of p16INK4a or p21CIP1/WAF1 regulation were examined by performing lnc-IL7R overexpression and knockdown assays. The functions of lnc-IL7R-mediated cell proliferation, cell cycle, senescence, colony formation, and apoptosis were examined in cells treated with PM2.5. Chromatin immunoprecipitation assays were conducted to investigate the epigenetic regulation of p21CIP1/WAF1. RESULTS: Lnc-IL7R levels decreased in COPD patients and were negatively correlated with emphysema or PM2.5 exposure. Lnc-IL7R levels were upregulated in normal lung epithelial cells but not in COPD cells exposed to PM2.5. Lower lnc-IL7R expression in PM2.5-treated cells induced p16INK4a and p21CIP1/WAF1 expression by increasing oxidative stress. Higher lnc-IL7R expression protected against cellular senescence and apoptosis, whereas lower lnc-IL7R expression augmented injury in PM2.5-treated cells. Lnc-IL7R and the enhancer of zeste homolog 2 (EZH2) synergistically suppressed p21CIP1/WAF1 expression through epigenetic modulation. CONCLUSION: Lnc-IL7R attenuates PM2.5-mediated p21CIP1/WAF1 expression through EZH2 recruitment, and its dysfunction may augment cellular injury in COPD.

3-Nitrobenzanthrone promotes malignant transformation in human lung epithelial cells through the epiregulin-signaling pathway.

Exposure to environmental and occupational contaminants leads to lung cancer. 3-Nitrobenzanthrone (3-nitro-7H-benz[de]anthracen-7-one, 3-NBA) is a potential carcinogen in ambient air or diesel particulate matter. Studies have revealed that short-term exposure to 3-NBA induces cell death, reactive oxygen species activation, and DNA adduct formation and damage. However, details of the mechanism by which chronic exposure to 3-NBA influences lung carcinogenesis remain largely unknown. In this study, human lung epithelial BEAS-2B cells were continuously exposed to 0-10-µM 3-NBA for 6 months. NanoString analysis was conducted to evaluate gene expression in the cells, revealing that 3-NBA-mediated transformation results in a distinct gene expression signature including carbon cancer metabolism, metastasis, and angiogenesis. Alterations in tumor-promoting genes such as EREG (epiregulin), SOX9, E-cadherin, TWIST, and IL-6 were involved in epithelial cell aggressiveness. Kaplan-Meier plotter analyses indicated that increased EREG and IL-6 expressions in early-stage lung cancer cells are correlated with poor survival. In vivo xenografts on 3-NBA-transformed cells exhibited prominent tumor formation and metastasis. EREG knockout cells exposed to 3-NBA for a short period exhibited high apoptosis and low colony formation. By contrast, overexpression of EREG in 3-NBA-transformed cells markedly activated the PI3K/AKT and MEK/ERK signaling pathways, resulting in tumorigenicity. Furthermore, elevated IL-6 and EREG expressions synergistically led to STAT3 signaling activation, resulting in clonogenic cell survival and migration. Taken together, chronic exposure of human lung epithelial cells to 3-NBA leads to malignant transformation, in which the EREG signaling pathway plays a pivotal mediating role. • Short-term exposure of lung epithelial cells to 3-NBA can lead to ROS production and cell apoptosis. • Long-term chronic exposure to 3-NBA upregulates the levels of tumor-promoting genes such as EREG and IL-6. • Increased EREG expression in 3-NBA-transformed cells markedly contributes to tumorigenesis through PI3K/AKT and MEK/ERK activation and synergistically enhances the IL-6/STAT3 signaling pathway, which promotes tumorigenicity.

Conducting Polyaniline for Antifouling Ultrafiltration Membranes: Solutions and Challenges.

Conjugated polyaniline can impact the field of water filtration membranes due to its hydrophilic and antibacterial nature, facile and inexpensive synthesis procedure, heat and acid tolerance, and unique doping/dedoping chemistry. However, the gelation effect, its rigid backbone, and the limited hydrophilicity of polyaniline severely restrict the adaptability to membranes and their antifouling performance. This Mini Review summarizes important works of polyaniline-related ultrafiltration membranes, highlighting solutions to conquer engineering obstacles in processing and challenges in enhancing surface hydrophilicity with an emphasis on chemistry. As a pH-responsive polymer convertible to a conductive salt, this classic material should continue to bring unconventional advances into the realm of water filtration membranes.

The Feasibility of a Machine Learning Approach in Predicting Successful Ventilator Mode Shifting for Adult Patients in the Medical Intensive Care Unit.

Background and Objectives: Traditional assessment of the readiness for the weaning from the mechanical ventilator (MV) needs respiratory parameters in a spontaneous breath. Exempted from the MV disconnecting and manual measurements of weaning parameters, a prediction model based on parameters from MV and electronic medical records (EMRs) may help the assessment before spontaneous breath trials. The study aimed to develop prediction models using machine learning techniques with parameters from the ventilator and EMRs for predicting successful ventilator mode shifting in the medical intensive care unit. Materials and Methods: A retrospective analysis of 1483 adult patients with mechanical ventilators for acute respiratory failure in three medical intensive care units between April 2015 and October 2017 was conducted by machine learning techniques to establish the predicting models. The input candidate parameters included ventilator setting and measurements, patients' demographics, arterial blood gas, laboratory results, and vital signs. Several classification algorithms were evaluated to fit the models, including Lasso Regression, Ridge Regression, Elastic Net, Random Forest, Extreme Gradient Boosting (XGBoost), Support Vector Machine, and Artificial Neural Network according to the area under the Receiver Operating Characteristic curves (AUROC). Results: Two models were built to predict the success shifting from full to partial support ventilation (WPMV model) or from partial support to the T-piece trial (sSBT model). In total, 3 MV and 13 nonpulmonary features were selected for the WPMV model with the XGBoost algorithm. The sSBT model was built with 8 MV and 4 nonpulmonary features with the Random Forest algorithm. The AUROC of the WPMV model and sSBT model were 0.76 and 0.79, respectively. Conclusions: The weaning predictions using machine learning and parameters from MV and EMRs have acceptable performance. Without manual measurements, a decision-making system would be feasible for the continuous prediction of mode shifting when the novel models process real-time data from MV and EMRs.

Overexpression of GLUT3 promotes metastasis of triple-negative breast cancer by modulating the inflammatory tumor microenvironment.

Triple-negative breast cancer (TNBC) exhibits a higher level of glycolytic capacity and are commonly associated with an inflammatory microenvironment, but the regulatory mechanism and metabolic crosstalk between the tumor and tumor microenvironment (TME) are largely unresolved. Here, we show that glucose transporter 3 (GLUT3) is particularly elevated in TNBC and associated with metastatic progression and poor prognosis in breast cancer patients. Expression of GLUT3 is crucial for promoting the epithelial-to-mesenchymal transition and enhancing invasiveness and distant metastasis of TNBC cells. Notably, GLUT3 is correlated with inflammatory gene expressions and is associated with M1 tumor-associated macrophages (TAMs), at least in part by C-X-C Motif Chemokine Ligand 8 (CXCL8). We found that expression of GLUT3 regulates CXCL8 production in TNBC cells. Secretion of CXCL8 participates in GLUT3-overexpressing TNBC cells-elicited activation of inflammatory TAMs, which further enhances GLUT3 expression and mobility of TNBC cells. Our findings demonstrate that aerobic glycolysis in TNBC not only promotes aggressiveness of tumor cells but also initiates a positive regulatory loop for enhancing tumor progression by modulating the inflammatory TME.

Variable-Temperature Electrospray Ionization for Temperature-Dependent Folding/Refolding Reactions of Proteins and Ligand Binding.

Stabilities and structure(s) of proteins are directly coupled to their local environment or Gibbs free energy landscape as defined by solvent, temperature, pressure, and concentration. Solution pH, ionic strength, cofactors, chemical chaperones, and osmolytes perturb the chemical potential and induce further changes in structure, stability, and function. At present, no single analytical technique can monitor these effects in a single measurement. Mass spectrometry and ion mobility-mass spectrometry play increasingly essential roles in studies of proteins, protein complexes, and even membrane protein complexes; however, with few exceptions, the effects of the solution temperature on the stability and structure(s) of analytes have not been thoroughly investigated. Here, we describe a new variable-temperature electrospray ionization (vT-ESI) source that utilizes a thermoelectric chip to cool and heat the solution contained within the static ESI emitter. This design allows for solution temperatures to be varied from ∼5 to 98 °C with short equilibration times (<2 min) between precisely controlled temperature changes. The performance of the apparatus for vT-ESI-mass spectrometry and vT-ESI-ion mobility-mass spectrometry studies of cold- and heat-folding reactions is demonstrated using ubiquitin and frataxin. Instrument performance for studies on temperature-dependent ligand binding is shown using the chaperonin GroEL.

The analysis for time of referral to a medical center among patients with diabetic foot infection.

BACKGROUND: Diabetic foot infection (DFI) is a limb- and life-threatening complication for diabetic patients needing immediate and comprehensive treatment. Early referral of DFI patients to a diabetic foot center is recommended but there appears limited validated evidence, with the association between referral time and clinical outcomes of limb- preservation or in-hospital mortality still lacking. METHODS: This retrospective research studied consecutive type 2 diabetic patients with DFI treated at the major diabetic foot center in Taiwan from 2014 to 2017. Six hundred and sixty-eight patients presented with limb-threatening DFI. After stratifying their referral days into quartiles, the demographic information and clinical outcomes were analyzed. RESULTS: One hundred and seventy-two patients were placed in the first quartile (Q1) with less than 9 days of referral time; 164 in the second quartile (Q2) with 9-21 days; 167 in the third quartile (Q3) with 21-59 days; and 165 in the fourth quartile (Q4) with >59 days. End-stage renal disease (ESRD), major adverse cardiac events (MACE) and peripheral arterial disease (PAD) were noted as being higher in the Q4 group compared with the Q1 group (25.45% vs 20.35% in ESRD, 47.27% vs 26.16% in MACE and 78.79% vs 52.33% in PAD respectively). The Q1 group had more patients presenting with systemic inflammatory responsive syndrome (SIRS) (29.07% in Q1 vs 25.45% in Q4 respectively, P=0.019). Regarding poor outcome (major lower-extremity amputation (LEA) or in-hospital mortality), the Q4 group had 21.21% of patients in this category and the Q1 group had 10.47%. The odds ratio of each increased referral day on poor prognosis was 1.006 with 95% confidence interval 1.003-1.010 (P=<0.001). In subgroups, the impact on poor prognosis by day was most obvious in patients with SIRS (OR 1.011, 95% CI 1.004-1.018, P=0.003) and those with PAD (OR 1.004, 95% CI 1.001-1.008, P=0.028). CONCLUSIONS: The deferred referral of DFI patients to the diabetic foot center might be associated with poor treatment outcome either in major LEA or mortality, particularly in patients with SIRS or PAD. Both physician and patient awareness of disease severity and overcoming the referral barrier is suggested. TRIAL REGISTRATION: Not applicable.

Nanostructured Graphene Oxide Composite Membranes with Ultrapermeability and Mechanical Robustness.

Graphene oxide (GO) membranes have great potential for separation applications due to their low-friction water permeation combined with unique molecular sieving ability. However, the practical use of deposited GO membranes is limited by the inferior mechanical robustness of the membrane composite structure derived from conventional deposition methods. Here, we report a nanostructured GO membrane that possesses great permeability and mechanical robustness. This composite membrane consists of an ultrathin selective GO nanofilm (as low as 32 nm thick) and a postsynthesized macroporous support layer that exhibits excellent stability in water and under practical permeability testing. By utilizing thin-film lift off (T-FLO) to fabricate membranes with precise optimizations in both selective and support layers, unprecedented water permeability (47 L·m-2·hr-1·bar-1) and high retention (>98% of solutes with hydrated radii larger than 4.9 Å) were obtained.

Melatonin Downregulates PD-L1 Expression and Modulates Tumor Immunity in KRAS-Mutant Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) patients harboring a KRAS mutation have unfavorable therapeutic outcomes with chemotherapies, and the mutation also renders tolerance to immunotherapies. There is an unmet need for a new strategy for overcoming immunosuppression in KRAS-mutant NSCLC. The recently discovered role of melatonin demonstrates a wide spectrum of anticancer impacts; however, the effect of melatonin on modulating tumor immunity is largely unknown. In the present study, melatonin treatment significantly reduced cell viability accompanied by inducing cell apoptosis in KRAS-mutant NSCLC cell lines including A549, H460, and LLC1 cells. Mechanistically, we found that lung cancer cells harboring the KRAS mutation exhibited a higher level of programmed death ligand 1 (PD-L1). However, treatment with melatonin substantially downregulated PD-L1 expressions in both the presence and absence of interferon (IFN)-γ stimulation. Moreover, KRAS-mutant lung cancer cells exhibited higher Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) levels, and PD-L1 expression was positively correlated with YAP and TAZ in lung cancer cells. Treatment with melatonin effectively suppressed YAP and TAZ, which was accompanied by downregulation of YAP/TAZ downstream gene expressions. The combination of melatonin and an inhibitor of YAP/TAZ robustly decreased YAP and PD-L1 expressions. Clinical analysis using public databases revealed that PD-L1 expression was positively correlated with YAP and TAZ in patients with lung cancer, and PD-L1 overexpression suggested poor survival probability. An animal study further revealed that administration of melatonin significantly inhibited tumor growth and modulated tumor immunity in a syngeneic mouse model. Together, our data revealed a novel antitumor mechanism of melatonin in modulating the immunosuppressive tumor microenvironment by suppressing the YAP/PD-L1 axis and suggest the therapeutic potential of melatonin for treating NSCLC.

Molecular Mechanism of ISC Iron-Sulfur Cluster Biogenesis Revealed by High-Resolution Native Mass Spectrometry.

Iron-sulfur (Fe-S) clusters are ubiquitous protein cofactors that are required for many important biological processes including oxidative respiration, nitrogen fixation, and photosynthesis. Biosynthetic pathways assemble Fe-S clusters with different iron-to-sulfur stoichiometries and distribute these clusters to appropriate apoproteins. In the ISC pathway, the pyridoxal 5'-phosphate-dependent cysteine desulfurase enzyme IscS provides sulfur to the scaffold protein IscU, which templates the Fe-S cluster assembly. Despite their functional importance, mechanistic details for cluster synthesis have remained elusive. Recent advances in native mass spectrometry (MS) have allowed proteins to be preserved in native-like structures and support applications in the investigation of protein structure, dynamics, ligand interactions, and the identification of protein-associated intermediates. Here, we prepared samples under anaerobic conditions and then applied native MS to investigate the molecular mechanism for Fe-S cluster synthesis. This approach was validated by the high agreement between native MS and traditional visible circular dichroism spectroscopic assays. Time-dependent native MS experiments revealed potential iron- and sulfur-based intermediates that decay as the [2Fe-2S] cluster signal developed. Additional experiments establish that (i) Zn(II) binding stabilizes IscU and protects the cysteine residues from oxidation, weakens the interactions between IscU and IscS, and inhibits Fe-S cluster biosynthesis; and (ii) Fe(II) ions bind to the IscU active site cysteine residues and another lower affinity binding site and promote the intermolecular sulfur transfer reaction from IscS to IscU. Overall, these results support an iron-first model for Fe-S cluster synthesis and highlight the power of native MS in defining protein-associated intermediates and elucidating mechanistic details of enzymatic processes.

Elevation of CD109 promotes metastasis and drug resistance in lung cancer via activation of EGFR-AKT-mTOR signaling.

Lung cancer is the most commonly diagnosed cancer worldwide, and metastasis in lung cancer is the leading cause of cancer-related deaths. Thus, understanding the mechanism of lung cancer metastasis will improve the diagnosis and treatment of lung cancer patients. Herein, we found that expression of cluster of differentiation 109 (CD109) was correlated with the invasive and metastatic capacities of lung adenocarcinoma cells. CD109 is upregulated in tumorous tissues, and CD109 overexpression was associated with tumor progression, distant metastasis, and a poor prognosis in patient with lung adenocarcinoma. Mechanistically, expression of CD109 regulates protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling via its association with the epidermal growth factor receptor (EGFR). Inhibition of CD109 decreases EGFR phosphorylation, diminishes EGF-elicited activation of AKT/mTOR, and sensitizes tumor cells to an EGFR inhibitor. Taken together, our results show that CD109 is a potential diagnostic and therapeutic target in lung cancer patients.