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INTRODUCTION: Reactive oxygen species modulator 1 (Romo1) is a novel protein that is critically involved in the intracellular production of reactive oxygen species. Evidence has revealed that Romo1 is associated with treatment outcomes of various human malignancies, including lung cancer. However, the clinical implications of this protein in surgically resected lung cancers harboring epidermal growth factor receptor (EGFR) mutations have not been investigated. METHODS: Data were collected from patients who underwent curative resection of EGFR-mutant lung adenocarcinoma. Romo1 protein expression levels were measured in the tumor tissue using immunohistochemical staining and evaluated semi-quantitatively using the histochemical score. Univariate and multivariate analyses were performed to identify the clinicopathological parameters that may be associated with clinical outcomes. RESULTS: A total of 98 samples were analyzed. Using the cutoff H score of 200, the population was classified into low (n = 73) and high (n = 25) Romo1 groups. Romo1 expression was significantly higher in smokers, patients with stage III disease, and patients who experienced recurrence after surgery (all p < 0.05). Multivariate analyses showed that advanced-stage and poorly differentiated cancers were associated with shorter disease-free survival (DFS). In addition, high Romo1 expression was independently associated with poor DFS (hazard ratio = 2.18, 95% confidence interval: 1.10-4.32, p = 0.0261). CONCLUSIONS: Our data showed that Romo1 overexpression was significantly associated with early recurrence in patients with resected EGFR-mutant lung adenocarcinoma. Although large-scale studies are required, Romo1 may play a prognostic role in this patient population.
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Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has become a promising analytical tool for molecular profiling in biological applications. However, its ultrahigh vacuum environment and matrix effects hamper the absolute quantitation of solution samples. Herein, we present a rapid high-throughput platform for quantitative ToF-SIMS analysis of amino acids in matrix deposits formed from freeze-dried solution drops through ice sublimation on a parylene film microarray substrate. Droplets of the amino acid solutions, which were mixed with stable isotope-labeled phenylalanine (F*) of high concentration (10 mM), were loaded on wells of the microarray, then frozen and evaporated slowly below the freezing point, forming continuous solid-phase F* matrix deposits. The amino acids (≤500 µM), adequately well dispersed throughout the F* matrix deposits on each well, were quantitatively analyzed by ToF-SIMS in a rapid and high-throughput fashion. The lower limit of quantitation reached below 10 µM.
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Aminoácidos , Espectrometria de Massa de Íon Secundário , Espectrometria de Massa de Íon Secundário/métodos , Congelamento , Fenilalanina , Análise em MicrossériesRESUMO
Technological developments and improvements in single-cell isolation and analytical platforms allow for advanced molecular profiling at the single-cell level, which reveals cell-to-cell variation within the admixture cells in complex biological or clinical systems. This helps to understand the cellular heterogeneity of normal or diseased tissues and organs. However, most studies focused on the analysis of nucleic acids (e.g., DNA and RNA) and mass spectrometry (MS)-based analysis for proteins and metabolites of a single cell lagged until recently. Undoubtedly, MS-based single-cell analysis will provide a deeper insight into cellular mechanisms related to health and disease. This review summarizes recent advances in MS-based single-cell analysis methods and their applications in biology and medicine.
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Although many efforts are undertaken to treat peripheral demyelinating neuropathies based on biochemical interventions, unfortunately, there is no approved treatment yet. Furthermore, previous studies have not shown improvement of the myelin membrane at the biomolecular level. Here, an electroceutical treatment is introduced as a biophysical intervention to treat Charcot-Marie-Tooth (CMT) disease-the most prevalent peripheral demyelinating neuropathy worldwide-using a mouse model. The specific electrical stimulation (ES) condition (50 mV mm-1 , 20 Hz, 1 h) for optimal myelination is found via an in vitro ES screening system, and its promyelinating effect is validated with ex vivo dorsal root ganglion model. Biomolecular investigation via time-of-flight secondary ion mass spectrometry shows that ES ameliorates distribution abnormalities of peripheral myelin protein 22 and cholesterol in the myelin membrane, revealing the restoration of myelin membrane integrity. ES intervention in vivo via flexible implantable electrodes shows not only gradual rehabilitation of mouse behavioral phenotypes (balance and endurance), but also restored myelin thickness, compactness, and membrane integrity. This study demonstrates, for the first time, that an electroceutical approach with the optimal ES condition has the potential to treat CMT disease and restore impaired myelin membrane integrity, shifting the paradigm toward practical interventions for peripheral demyelinating neuropathies.
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Doença de Charcot-Marie-Tooth , Doenças Desmielinizantes , Animais , Doença de Charcot-Marie-Tooth/terapia , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Doenças Desmielinizantes/terapia , Doenças Desmielinizantes/genética , Bainha de Mielina/metabolismo , Modelos Animais de Doenças , ProteínasRESUMO
New methods to analyze cells and tissues in ambient condition without any harsh chemical fixation or physical freezing and drying are summarized in this report. The first approach, an atmospheric pressure mass spectrometry imaging method, is based on laser ablation in atmospheric pressure assisted by atmospheric plasma and nanomaterials such as nanoparticles and graphene to enhance laser ablation. The second one is based on secondary ion mass spectrometry (SIMS) imaging of live cells in solution capped with single-layer graphene to preserve intact and hydrated biological samples even under ultrahigh vacuum for SIMS bio-imaging in solution.
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Espectrometria de Massa de Íon Secundário , Pressão Atmosférica , Grafite , Terapia a Laser , Imagem Molecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
As the myelin sheath is crucial for neuronal saltatory conduction, loss of myelin in the peripheral nervous system (PNS) leads to demyelinating neuropathies causing muscular atrophy, numbness, foot deformities and paralysis. Unfortunately, few interventions are available for such neuropathies, because previous pharmaceuticals have shown severe side effects and failed in clinical trials. Therefore, exploring new strategies to enhance PNS myelination is critical to provide solution for such intractable diseases. This study aimed to investigate the effectiveness of electrical stimulation (ES) to enhance myelination in the mouse dorsal root ganglion (DRG)-anex vivomodel of the PNS. Mouse embryonic DRGs were extracted at E13 and seeded onto Matrigel-coated surfaces. After sufficient growth and differentiation, screening was carried out by applying ES in the 1-100 Hz range at the beginning of the myelination process. DRG myelination was evaluated via immunostaining at the intermediate (19 daysin vitro(DIV)) and mature (30 DIV) stages. Further biochemical analyses were carried out by utilizing ribonucleic acid sequencing, quantitative polymerase chain reaction and biochemical assays at both intermediate and mature myelination stages. Imaging of DRG myelin lipids was carried out via time-of-flight secondary ion mass spectrometry (ToF-SIMS). With screening ES conditions, optimal condition was identified at 20 Hz, which enhanced the percentage of myelinated neurons and average myelin length not only at intermediate (129% and 61%) but also at mature (72% and 17%) myelination stages. Further biochemical analyses elucidated that ES promoted lipid biosynthesis in the DRG. ToF-SIMS imaging showed higher abundance of the structural lipids, cholesterol and sphingomyelin, in the myelin membrane. Therefore, promotion of lipid biosynthesis and higher abundance of myelin lipids led to ES-mediated myelination enhancement. Given that myelin lipid deficiency is culpable for most demyelinating PNS neuropathies, the results might pave a new way to treat such diseases via electroceuticals.
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Gânglios Espinais , Células de Schwann , Animais , Células Cultivadas , Lipídeos , Camundongos , Bainha de Mielina/fisiologia , Regulação para CimaRESUMO
The ε4 allele of APOE-encoding apolipoprotein (ApoE) is one of the strongest genetic risk factors for Alzheimer's disease (AD). One of the overarching questions is whether and how this astrocyte-enriched risk factor initiates AD-associated pathology in neurons such as amyloid-ß (Aß) accumulation. Here, we generate neurons and astrocytes from isogenic human induced pluripotent stem cells (hiPSCs) carrying either APOE ε3 or APOE ε4 allele and investigate the effect of astrocytic ApoE4 on neuronal Aß production. Secretory factors in conditioned media from ApoE4 astrocytes significantly increased amyloid precursor protein (APP) levels and Aß secretion in neurons. We further found that increased cholesterol secretion from ApoE4 astrocytes was necessary and sufficient to induce the formation of lipid rafts that potentially provide a physical platform for APP localization and facilitate its processing. Our study reveals the contribution of ApoE4 astrocytes to amyloidosis in neurons by expanding lipid rafts and facilitating Aß production through an oversupply of cholesterol.
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Peptídeos beta-Amiloides/biossíntese , Apolipoproteína E4/genética , Astrócitos/metabolismo , Colesterol/metabolismo , Microdomínios da Membrana/metabolismo , Neurônios/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Apolipoproteína E4/metabolismo , Biomarcadores , Comunicação Celular , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Espaço Extracelular/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/efeitos dos fármacosRESUMO
There is growing evidence that myeloid-derived suppressor cells (MDSCs) are directly involved in all stages leading to metastasis. Many mechanisms for this effect have been proposed, but mechanisms of coregulation between tumor cells and MDSCs remain poorly understood. In this study, we demonstrate that MDSCs are a source of milk fat globule-epidermal growth factor (EGF) factor 8 (MFGE8), which is known to be involved in tumor metastasis. Interestingly, TGF-ß, an abundant cytokine in the tumor microenvironment (TME), increased MFGE8 production by MDSCs. In addition, co-culturing MDSCs with B16F10 melanoma cells increased B16F10 cell migration, while MFGE8 neutralization decreased their migration. Taken together, these findings suggest that MFGE8 is an important effector molecule through which MDSCs promote tumor metastasis, and the TME positively regulates MFGE8 production by MDSCs through TGF-ß.
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We report a means by which atomic and molecular secondary ions, including cholesterol and fatty acids, can be sputtered through single-layer graphene to enable secondary ion mass spectrometry (SIMS) imaging of untreated wet cell membranes in solution at subcellular spatial resolution. We can observe the intrinsic molecular distribution of lipids, such as cholesterol, phosphoethanolamine and various fatty acids, in untreated wet cell membranes without any labeling. We show that graphene-covered cells prepared on a wet substrate with a cell culture medium reservoir are alive and that their cellular membranes do not disintegrate during SIMS imaging in an ultra-high-vacuum environment. Ab initio molecular dynamics calculations and ion dose-dependence studies suggest that sputtering through single-layer graphene occurs through a transient hole generated in the graphene layer. Cholesterol imaging shows that methyl-ß-cyclodextrin preferentially extracts cholesterol molecules from the cholesterol-enriched regions in cell membranes.
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Membrana Celular/metabolismo , Colesterol/análise , Etanolaminas/análise , Ácidos Graxos/análise , Espectrometria de Massa de Íon Secundário/métodos , Diagnóstico por Imagem , Grafite/química , Simulação de Dinâmica Molecular , Análise de Célula Única/métodos , beta-Ciclodextrinas/químicaRESUMO
The biofouling of marine organisms on a surface induces serious economic damage. One of the conventional anti-biofouling strategies is the use of toxic chemicals. In this study, a new eco-friendly oleamide-PDMS copolymer (OPC) is proposed for sustainable anti-biofouling and effective drag reduction. The anti-biofouling characteristics of the OPC are investigated using algal spores and mussels. The proposed OPC is found to inhibit the adhesion of algal spores and mussels. The slippery features of the fabricated OPC surfaces are examined by direct measurement of pressure drops in channel flows. The proposed OPC surface would be utilized in various industrial applications including marine vehicles and biomedical devices.
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Single protein imaging and understanding their interactions are of paramount importance to understand the life phenomena. Recently reported multiplex protein SIMS imaging methodology using metal-oxide nanoparticle conjugated antibodies can be extended to a single protein imaging methodology using He ion microscopy (HIM). It is proposed here that single protein can be imaged in the microscale and the nanoscale by the complementary use of SIMS and HIM.
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Hélio/química , Imageamento Tridimensional , Microscopia , Proteínas/análise , Espectrometria de Massa de Íon Secundário , Linhagem Celular Tumoral , Humanos , Íons , Microscopia de FluorescênciaRESUMO
Blocking of the interaction between Programmed cell death 1 (PD-1) and its ligand PD-L1 by monoclonal antibodies has elicited unprecedented therapeutic benefits and achieved a major breakthrough in immunotherapy of multiple types of tumors. Here, we determined the crystal structure of PD-1 in complex with the Fab fragment of tislelizumab. This monoclonal antibody was approved in December 2019 by the China National Medical Product Administration for Hodgkin's lymphoma and is under multiple clinical trials in China and the US. While the three complementarity determining regions (CDRs) in the light chain are involved in the target interaction, only CDR3 within the heavy chain interacts with PD-1. Tislelizumab binds the front ß-sheet of PD-1 in a very similar way as PD-L1 binds to PD-1, thereby blocking the PD-1/PD-L1 interaction with a higher affinity. A comparative analysis of PD-1 interactions with therapeutic antibodies targeting PD-1 provides a better understanding of the blockade mechanism of PD-1/PD-L1 interaction in addition to useful information for the improvement of therapeutic antibodies capable of diminishing checkpoint signaling for cancer immunotherapy.
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Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/uso terapêutico , Doença de Hodgkin/terapia , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia , Receptor de Morte Celular Programada 1/química , Cristalografia por Raios X , Doença de Hodgkin/imunologia , Humanos , Inibidores de Checkpoint Imunológico/química , Modelos Moleculares , Receptor de Morte Celular Programada 1/metabolismoRESUMO
In spite of recent developments in mass spectrometry imaging techniques, high-resolution multiplex protein bioimaging techniques are required to unveil the complex inter- and intracellular biomolecular interactions for accurate understanding of life phenomena and disease mechanisms. Herein, we report multiplex protein imaging with secondary ion mass spectrometry (SIMS) using metal oxide nanoparticle (MONP)-conjugated antibodies with <300 nm spatial resolution in the low ion dose without ion beam damage because of the high secondary ion yields of the MONPs, which can provide simultaneous imaging of several proteins, especially from cell membranes. We applied our new imaging technique for the study of hippocampal tissue samples from control and Alzheimer's disease (AD) model mice; the proximity of protein clusters in the hippocampus CA1 region showed intriguing dependence on aging and AD progress, suggesting that protein cluster proximity may be helpful for understanding pathological pathways in the microscopic cellular level.
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Anticorpos/imunologia , Nanopartículas Metálicas/química , Proteínas/imunologia , Espectrometria de Massa de Íon Secundário/métodos , Doença de Alzheimer/diagnóstico por imagem , Animais , Anticorpos/química , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Região CA1 Hipocampal/diagnóstico por imagem , Região CA1 Hipocampal/metabolismo , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Óxidos , Tamanho da Partícula , Proteínas/metabolismoRESUMO
Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a powerful tool to obtain both chemical information and spatial distribution of specific molecules of interest on a specimen surface. However, since the focused ion beam requires ultrahigh vacuum conditions for desorption and ionization of analytes, proper specimen preparation, such as drying, freeze-drying, and frozen dehydration, is required for ToF-SIMS analysis. In particular, biological specimens with high moisture content generally have a problem of specimen deformation during the normal drying process for a vacuum environment. In this study, the authors propose a cellular specimen preparation method to improve the ion imaging of cells by reducing the deformation of specimens in ToF-SIMS analysis. When the cells on the slide substrate are completely covered with single-layer graphene, the ToF-SIMS imaging is improved by reduced cell deformation due to slow drying. In addition, the graphene encapsulation also induces a reduction in the yield of secondary ions, thereby suppressing the background ion spectra generated by the unwanted organic residues on the substrate, resulting in the improvement of ToF-SIMS imaging. The authors also found that adding plasma treatment to this sample preparation can further improve ion imaging of cells. After cell dehydration is completed, the covered graphene layer can be peeled off by air-plasma treatment and the unwanted organic residues on the substrate can be removed due to plasma cleaning, thereby much improving ion imaging of cells.
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Grafite/química , Neurônios , Pele , Animais , Dessecação , Neurônios/química , Neurônios/citologia , Ratos , Ratos Sprague-Dawley , Pele/química , Pele/diagnóstico por imagem , Espectrometria de Massa de Íon SecundárioRESUMO
The atmospheric pressure mass spectrometric (AP-MS) imaging technology combined with an inverted optical microscopic system is a powerful tool for determining the presence and spatial distributions of specific biomolecules of interest in live tissues. Efficient desorption and ionization are essential to acquire mass spectrometric (MS) information in an ambient environment. In this study, we demonstrate a new and efficient desorption process using a graphene-coated glass substrate and a continuous wave (CW) laser for high-resolution AP-MS imaging of a live hippocampal tissue. We found that desorption of biomolecules in a live tissue slice was possible with the aid of a graphene-coated glass substrate and indirect application of a 532 nm CW laser on the graphene substrate. Interestingly, the desorption efficiency of a live tissue on the graphene-coated substrate was strongly dependent on the number of graphene layers. Single-layer graphene was found to be the most sensitive substrate for efficient desorption and reproducible high-resolution hippocampal tissue imaging applications. The subsequent ionization process using nonthermal plasma generated sufficient amounts of molecular ions to obtain high-resolution two-dimensional MS images of the cornu ammonis and the dentate gyrus regions of the hippocampus. Therefore, graphene-coated substrates could be a promising platform to induce an efficient desorption process essential for highly reproducible ambient MS imaging.
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Materiais Revestidos Biocompatíveis/química , Grafite/química , Hipocampo/ultraestrutura , Imagem Molecular , Animais , Pressão Atmosférica , Materiais Revestidos Biocompatíveis/farmacologia , Vidro/química , Grafite/farmacologia , Hipocampo/efeitos dos fármacos , Lasers , Espectrometria de Massas , CamundongosRESUMO
We report on sample preparation methods based on plasma treatment for an improvement of multiple molecular ion images of cellular membranes in the ToF-SIMS method. The air-plasma treatment of fixed cellular samples efficiently removed the organic residues of any solutions used during sample preparation and improved the quality of ToF-SIMS images due to the resulting clean surface. We also studied cell preparation methods that combine single-layer graphene covering with air-plasma treatment to achieve a synergistic effect that eliminates background spectra by organic impurities while minimizing morphological cell deformation in a vacuum environmental analysis. When the cellular sample on the glass substrate is completely covered with the single-layer graphene, the cells trapped between the graphene and the substrate can effectively reduce morphological deformation by slow-dehydration. After slow-dehydration of cells is completed inside the graphene-cover, the covered graphene layer can be peeled off by air-plasma treatment, and the unwanted organic residues on the surface of cells and substrate can also be removed by plasma cleaning, thereby much improving ion imaging of cells with the ToF-SIMS method. It is confirmed that the cell samples in which the graphene-cover was removed by air-plasma treatment maintained their morphology well in comparison with the rapid air-dried cells in atomic force microscopy (AFM) and ToF-SIMS images.
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One of the remaining challenges in material chemistry is to unveil the quantitative compositional/structural information and thermodynamic nature of inorganic materials especially in the initial nucleation and growth step. In this report, we adopted newly developed time-of-flight medium-energy-ion-scattering (TOF-MEIS) spectroscopy to address this challenge and explored heterogeneously grown nanometer-sized calcium phosphate as a model system. With TOF-MEIS, we discovered the existence of calcium-rich nanoclusters (Ca/P â¼ 3) in the presence of the non-collagenous-protein-mimicking passivating ligands. Over the reaction, these clusters progressively changed their compositional ratio toward that of a bulk phase (Ca/P â¼ 1.67) with a concurrent increase in their size to â¼2 nm. First-principles studies suggested that the calcium-rich nanoclusters can be stabilized through specific interactions between the ligands and clusters, emphasizing the important role of template on guiding the chemical and thermodynamic nature of inorganic materials at the nanoscale.
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Myeloid-derived suppressor cells (MDSCs) play roles in immune regulation during neoplastic and non-neoplastic inflammatory responses. This immune regulatory function is directed mainly toward T cells. However, MDSCs also regulate other cell populations, including B cells, during inflammatory responses. Indeed, B cells are essential for antibody-mediated immune responses. MDSCs regulate B cell immune responses directly via expression of effector molecules and indirectly by controlling other immune regulatory cells. B cell-mediated immune responses are a major component of the overall immune response; thus, MDSCs play a prominent role in their regulation. Here, we review the current knowledge about MDSC-mediated regulation of B cell responses.
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Linfócitos B/imunologia , Células Supressoras Mieloides/imunologia , Antígenos de Superfície/metabolismo , Citocinas/metabolismo , Humanos , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
BAFF, a member of the TNF superfamily, has been recognized as a good target for autoimmune diseases. Belimumab, an anti-BAFF monoclonal antibody, was approved by the FDA for use in treating systemic lupus erythematosus. However, the molecular basis of BAFF neutralization by belimumab remains unclear. Here our crystal structure of the BAFF-belimumab Fab complex shows the precise epitope and the BAFF-neutralizing mechanism of belimumab, and demonstrates that the therapeutic activity of belimumab involves not only antagonizing the BAFF-receptor interaction, but also disrupting the formation of the more active BAFF 60-mer to favor the induction of the less active BAFF trimer through interaction with the flap region of BAFF. In addition, the belimumab HCDR3 loop mimics the DxL(V/L) motif of BAFF receptors, thereby binding to BAFF in a similar manner as endogenous BAFF receptors. Our data thus provides insights for the design of new drugs targeting BAFF for the treatment of autoimmune diseases.