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There is growing interest in limiting the use of fungicides and implementing innovative, environmentally friendly strategies, such as the use of beneficial bacteria-triggered immunity, to protect grapevines from natural pathogens. Therefore, we need rapid and innovative ways to translate the knowledge of the molecular mechanisms underlying the activation of grapevine defenses against pathogens to induced resistance. Here, we have implemented an in vivo minimally invasive approach to study the interaction between plants and beneficial bacteria based on metabolic signatures. Paraburkholderia phytofirmans strain PsJN and PsJN-grapevine were used as bacterial and plant-bacterium interaction models, respectively. Using an innovative tool, SpiderMass, based on water-assisted laser desorption ionization with an IR microsampling probe, we simultaneously detect metabolic and lipidomic species. A metabolomic spectrum was thus generated, which was used to build a library and identify the most variable and discriminative peaks between the two conditions. We then showed that caftaric acid (m/z 311.04), caftaric acid dimer (m/z 623.09), derived caftaric acid (m/z 653.15), and quercetin-O-glucuronide tended to accumulate in grapevine leaves after root bacterization with PsJN. In addition, together with these phenolic messengers, we identified lipid biomarkers such as palmitic acid, linoleic acid, and α-linoleic acid as important messengers of enhanced defense mechanisms in Chardonnay plantlets. Taken together, SpiderMass is the next-generation methodology for studying plant-microorganism metabolic interactions with the prospect of in vivo real-time analysis in viticulture.
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BACKGROUND: Ballet dancers are expected to use their bodies symmetrically during training, because dance movements are performed on both sides. However, there is a general belief that ballet training encourages the use of one side of the body more than the other. Frequent repetition of a particular exercise can lead to body asymmetries and musculoskeletal injuries. The aim of this cross-sectional study was to investigate the presence of lower limbs and trunk muscle strength asymmetries in ballet dancers and secondly to assess whether there is a difference between professional dancers and ballet students. METHODS: Ballet students (n=19) and professional ballet dancers (n=23) performed maximal voluntary isometric contractions of the trunk (flexion, extension, lateral flexion), hip (flexion, extension, adduction, abduction, external and internal rotation), knee (flexion, extension) and ankle (flexion, extension) on isometric dynamometer. RESULTS: The results showed that the percentage of ballet dancers with contralateral muscle strength asymmetries >10% ranged from 22.5% (ballet students) to 31.6% (professional dancers). The percentage of ballet dancers deviating by >10% from the normative maximum torque agonist/antagonist ratio ranged from 56.5% to 100%. A statistically significant difference between ballet students and professional ballet dancers was found in the trunk flexion/extension ratio (t(40) = -3 .55; p = 0.001; d = 0.55). CONCLUSION: This study revealed strength asymmetries in the lower limbs and trunk in ballet dancers, both professionals and students. Further research is needed to develop appropriate complementary exercise to address and eliminate asymmetries in muscle strength in ballet dancers.
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Dança , Contração Isométrica , Extremidade Inferior , Força Muscular , Músculo Esquelético , Humanos , Dança/fisiologia , Força Muscular/fisiologia , Feminino , Estudos Transversais , Adulto Jovem , Masculino , Extremidade Inferior/fisiologia , Músculo Esquelético/fisiologia , Contração Isométrica/fisiologia , Tronco/fisiologia , Adulto , Amplitude de Movimento Articular/fisiologiaRESUMO
In this study, we conducted a direct comparison of water-assisted laser desorption ionization (WALDI) and matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging, with MALDI serving as the benchmark for label-free molecular tissue analysis in biomedical research. Specifically, we investigated the lipidomic profiles of several biological samples and calculated the similarity of detected peaks and Pearson's correlation of spectral profile intensities between the two techniques. We show that, overall, MALDI MS and WALDI MS present very close lipidomic analyses and that the highest similarity is obtained for the norharmane MALDI matrix. Indeed, for norharmane in negative ion mode, the lipidomic spectra revealed 100% similarity of detected peaks and over 0.90 intensity correlation between both technologies for five samples. The MALDI-MSI positive ion lipid spectra displayed more than 83% similarity of detected peaks compared to those of WALDI-MSI. However, we observed a lower percentage (77%) of detected peaks when comparing WALDI-MSI with MALDI-MSI due to the rich WALDI-MSI lipid spectra. Despite this difference, the global lipidomic spectra showed high consistency between the two technologies, indicating that they are governed by similar processes. Thanks to this similarity, we can increase datasets by including data from both modalities to either co-train classification models or obtain cross-interrogation.
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Water-assisted laser desorption/ionization mass spectrometry (WALDI-MS), also known as SpiderMass, is an emerging ambient ionization technique for in vivo and real-time analysis. It employs a remote infrared (IR) laser tuned to excite the most intense vibrational band (O-H) of water. The water molecules act as an endogenous matrix leading to the desorption/ionization of a variety of biomolecules from tissues, particularly metabolites and lipids. WALDI-MS was recently advanced into an imaging modality for ex vivo 2D sections and 3D in vivo real-time imaging. Here, we describe the methodological aspects for performing 2D and 3D imaging experiments with WALDI-MSI and the parameters for optimizing the image acquisition.
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Luz , Água , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Imageamento Tridimensional , LasersRESUMO
OBJECTIVES: The study aimed to determine the incidence and expression of body asymmetries in dancers of three different dance styles: dancesport (n = 14), hip-hop (n = 21) and ballet (n = 20) and to examine how body asymmetries (muscle strength and power, stability and range of motion) are associated with musculoskeletal injuries occurring over the past 12 months. DESIGN: Cross-sectional and retrospective study. METHODS: Maximal isometric voluntary contraction was measured for trunk, hip, knee and ankle movements. Participants performed a single leg stance, unilateral landing, weight bearing symmetry, squat and countermovement jump on force platforms. Passive range of motion was measured for hip, knee and ankle with two-arm goniometer or digital inclinometer (hip flexion, extension and rotations). A retrospective questionnaire was used to collect data on musculoskeletal injuries occurring in the last 12 months. RESULTS: Different dance styles were associated with different body asymmetries, including strength asymmetries (hip flexion and external rotation), agonist/antagonist asymmetries (trunk flexion/extension, hip abduction/adduction, ankle dorsi/plantar flexion) and hip adduction and internal rotation range of motion asymmetries. Moreover, strength asymmetries of hip flexion, adduction and abduction/adduction as well as stability asymmetries were associated with the total number of musculoskeletal injuries. CONCLUSIONS: The incidence of body asymmetries (> 10%) in dancesport, hip-hop and ballet dancers was confirmed, as well as the association of some asymmetries with self-reported injuries occurring over the last 12 months. The cause-effect relationship should be clarified by further studies.
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Molecular heterogeneity is a key feature of glioblastoma that impedes patient stratification and leads to large discrepancies in mean patient survival. Here, we analyze a cohort of 96 glioblastoma patients with survival ranging from a few months to over 4 years. 46 tumors are analyzed by mass spectrometry-based spatially-resolved proteomics guided by mass spectrometry imaging. Integration of protein expression and clinical information highlights three molecular groups associated with immune, neurogenesis, and tumorigenesis signatures with high intra-tumoral heterogeneity. Furthermore, a set of proteins originating from reference and alternative ORFs is found to be statistically significant based on patient survival times. Among these proteins, a 5-protein signature is associated with survival. The expression of these 5 proteins is validated by immunofluorescence on an additional cohort of 50 patients. Overall, our work characterizes distinct molecular regions within glioblastoma tissues based on protein expression, which may help guide glioblastoma prognosis and improve current glioblastoma classification.
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Neoplasias Encefálicas , Glioblastoma , Humanos , Glioblastoma/metabolismo , Proteoma , Neoplasias Encefálicas/metabolismo , Proteômica/métodos , Análise Espacial , Análise de SobrevidaRESUMO
In recent years, cannabis and hemp-based products have become increasingly popular for recreational use, edibles, beverages, health care products, and medicines. The rapid detection and differentiation of phytocannabinoids is, therefore, essential to assess the potency and the therapeutic and nutritional values of cannabis cultivars. Here, we implemented SpiderMass technology for in vivo detection of cannabidiolic acid (CBDA) and ∆9-tetrahydrocannabinolicacid (∆9-THCA), and other endogenous organic plant compounds, to access distribution gradients within the plants and differentiate between cultivars. The SpiderMass system is composed of an IR-laser handheld microsampling probe connected to a mass spectrometer through a transfer tube. The analysis was performed on different plant organs from freshly cultivated cannabis plants in only a few seconds. SpiderMass analysis easily discriminated the two acid phytocannabinoid isomers via MS/MS, and the built statistical models differentiated between four cannabis cultivars. Different abundancies of the two acid phytocannabinoids were found along the plant as well as between different cultivars. Overall, these results introduce direct analysis by SpiderMass as a compelling analytical alternative for rapid hemp analysis.
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Oral cavity cancers are the 15th most common cancer with more than 350,000 new cases and ~178,000 deaths each year. Among them, squamous cell carcinoma (SCC) accounts for more than 90% of tumors located in the oral cavity and on oropharynx. For the oral cavity SCC, the surgical resection remains the primary course of treatment. Generally, surgical margins are defined intraoperatively using visual and tactile elements. However, in 15-30% of cases, positive margins are found after histopathological examination several days postsurgery. Technologies based on mass spectrometry (MS) were recently developed to help guide surgical resection. The SpiderMass technology is designed for in-vivo real-time analysis under minimally invasive conditions. This instrument achieves tissue microsampling and real-time molecular analysis with the combination of a laser microprobe and a mass spectrometer. It ultimately acts as a tool to support histopathological decision-making and diagnosis. This pilot study included 14 patients treated for tongue SCC (T1 to T4) with the surgical resection as the first line of treatment. Samples were first analyzed by a pathologist to macroscopically delineate the tumor, dysplasia, and peritumoral areas. The retrospective and prospective samples were sectioned into three consecutive sections and thaw-mounted on slides for H&E staining (7 µm), SpiderMass analysis (20 µm), and matrix-assisted laser desorption ionization (MALDI) MS imaging (12 µm). The SpiderMass microprobe collected lipidometabolic profiles of the dysplasia, tumor, and peritumoral regions annotated by the pathologist. The MS spectra were then subjected to the multivariate statistical analysis. The preliminary data demonstrate that the lipidometabolic molecular profiles collected with the SpiderMass are significantly different between the tumor and peritumoral regions enabling molecular classification to be established by linear discriminant analysis (LDA). MALDI images of the different samples were submitted to segmentation for cross instrument validation and revealed additional molecular discrimination within the tumor and nontumor regions. These very promising preliminary results show the applicability of the SpiderMass to SCC of the tongue and demonstrate its interest in the surgical treatment of head and neck cancers.
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Human hair absorbs numerous biomolecules from the body during its growth. This can act as a fingerprint to determine substance intake of an individual, which can be useful in forensic studies. The cocaine concentration profile along the growth axis of hair indicates the time evolution of the metabolic incorporation of cocaine usage. It could be either assessed by chemical extraction and further analysis of hair bundels, or by direct single hair fibre analysis with mass spectroscopy imaging (MSI). Within this work, we analyzed the cocaine distribution in individual hair samples using MeV-SIMS. Unlike conventional surface analysis methods, we demonstrate high yields of nonfragmented molecular ions from the surface of biological materials, resulting in high chemical sensitivity and non-destructive characterisation. Hair samples were prepared by longitudinally cutting along the axis of growth, leaving half-cylindrical shape to access the interior structure of the hair by the probing ion beam, and attached to the silicon wafer. A focused 5.8 MeV 35Cl6+ beam was scanned across the intact, chemically pristine hair structure. A non-fragmented protonated [M+ H]+ cocaine molecular peak at m/z = 304 was detected and localized along the cross-section of the hair. Its intensity exhibits strong fluctuations along the direction of the hair's growth, with pronounced peaks as narrow as 50 micrometres, corresponding to a metabolic incorporation time of approx. three hours.
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Cocaína , Cabelo/química , Análise do Cabelo , Humanos , Espectrometria de Massas , Imagem Molecular , Detecção do Abuso de Substâncias/métodosRESUMO
Mass spectrometry imaging (MSI) has shown to bring invaluable information for biological and clinical applications. However, conventional MSI is generally performed ex vivo from tissue sections. Here, we developed a novel MS-based method for in vivo mass spectrometry imaging. By coupling the SpiderMass technology, that provides in vivo minimally invasive analysis-to a robotic arm of high accuracy, we demonstrate that images can be acquired from any surface by moving the laser probe above the surface. By equipping the robotic arm with a sensor, we are also able to both get the topography image of the sample surface and the molecular distribution, and then and plot back the molecular data, directly to the 3D topographical image without the need for image fusion. This is shown for the first time with the 3D topographic MS-based whole-body imaging of a mouse. Enabling fast in vivo MSI bridged to topography paves the way for surgical applications to excision margins.
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Robótica , Animais , Imageamento Tridimensional , Camundongos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
BACKGROUND: Formalin-fixed paraffin-embedded (FFPE) tissue has been the gold standard for routine pathology for general and cancer postoperative diagnostics. Despite robust histopathology, immunohistochemistry, and molecular methods, accurate diagnosis remains difficult for certain cases. Overall, the entire process can be time consuming, labor intensive, and does not reach over 90% diagnostic sensitivity and specificity. There is a growing need in onco-pathology for adjunct novel rapid, accurate, reliable, diagnostically sensitive, and specific methods for high-throughput biomolecular identification. Lipids have long been considered only as building blocks of cell membranes or signaling molecules, but have recently been introduced as central players in cancer. Due to sample processing, which limits their detection, lipid analysis directly from unprocessed FFPE tissues has never been reported. METHODS: We present a proof-of-concept with direct analysis of tissue-lipidomic signatures from FFPE tissues without dewaxing and minimal sample preparation using water-assisted laser desorption ionization mass spectrometry and deep-learning. RESULTS: On a cohort of difficult canine and human sarcoma cases, classification for canine sarcoma subtyping was possible with 99.1% accuracy using "5-fold" and 98.5% using "leave-one-patient out," and 91.2% accuracy for human sarcoma using 5-fold and 73.8% using leave-one-patient out. The developed classification model enabled stratification of blind samples in <5 min and showed >95% probability for discriminating 2 human sarcoma blind samples. CONCLUSION: It is possible to create a rapid diagnostic platform to screen clinical FFPE tissues with minimal sample preparation for molecular pathology.
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Lipidômica , Sarcoma , Animais , Cães , Formaldeído/química , Humanos , Lasers , Inclusão em Parafina , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Fixação de Tecidos/métodos , ÁguaRESUMO
In vivo cancer margin delineation during surgery remains a major challenge. Despite the availability of several image guidance techniques and intraoperative assessment, clear surgical margins and debulking efficiency remain scarce. For this reason, there is particular interest in developing rapid intraoperative tools with high sensitivity and specificity to help guide cancer surgery in vivo. Recently, several emerging technologies including intraoperative mass spectrometry have paved the way for molecular guidance in a clinical setting. We evaluate these techniques and assess their relevance for intraoperative surgical guidance and how they can transform the future of molecular cancer surgery, diagnostics, patient management and care.
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Diagnóstico por Imagem/métodos , Cuidados Intraoperatórios , Margens de Excisão , Neoplasias/cirurgia , Cirurgia Assistida por Computador/métodos , Animais , Humanos , Neoplasias/diagnóstico por imagem , Neoplasias/patologiaRESUMO
Rapid and accurate clinical diagnosis remains challenging. A component of diagnosis tool development is the design of effective classification models with Mass spectrometry (MS) data. Some Machine Learning approaches have been investigated but these models require time-consuming preprocessing steps to remove artifacts, making them unsuitable for rapid analysis. Convolutional Neural Networks (CNNs) have been found to perform well under such circumstances since they can learn representations from raw data. However, their effectiveness decreases when the number of available training samples is small, which is a common situation in medicine. In this work, we investigate transfer learning on 1D-CNNs, then we develop a cumulative learning method when transfer learning is not powerful enough. We propose to train the same model through several classification tasks over various small datasets to accumulate knowledge in the resulting representation. By using rat brain as the initial training dataset, a cumulative learning approach can have a classification accuracy exceeding 98% for 1D clinical MS-data. We show the use of cumulative learning using datasets generated in different biological contexts, on different organisms, and acquired by different instruments. Here we show a promising strategy for improving MS data classification accuracy when only small numbers of samples are available.
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Aprendizado Profundo , Espectrometria de Massas/métodos , Redes Neurais de Computação , Animais , Bases de Dados Factuais , Diagnóstico por Computador , Humanos , Aprendizado de Máquina , Espectrometria de Massas/estatística & dados numéricosRESUMO
Rapid, sensitive, precise and accurate analysis of samples in their native in vivo environment is critical to better decipher physiological and physiopathological mechanisms. SpiderMass is an ambient mass spectrometry (MS) system designed for mobile in vivo and real-time surface analyses of biological tissues. The system uses a fibered laser, which is tuned to excite the most intense vibrational band of water, resulting in a process termed water-assisted laser desorption/ionization (WALDI). The water molecules act as an endogenous matrix in a matrix-assisted laser desorption ionization (MALDI)-like scenario, leading to the desorption/ionization of biomolecules (lipids, metabolites and proteins). The ejected material is transferred to the mass spectrometer through an atmospheric interface and a transfer line that is several meters long. Here, we formulate a three-stage procedure that includes (i) a laser system setup coupled to a Waters Q-TOF or Thermo Fisher Q Exactive mass analyzer, (ii) analysis of specimens and (iii) data processing. We also describe the optimal setup for the analysis of cell cultures, fresh-frozen tissue sections and in vivo experiments on skin. With proper optimization, the system can be used for a variety of different targets and applications. The entire procedure takes 1-2 d for complex samples.
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Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Linhagem Celular , Cães , Desenho de Equipamento , Secções Congeladas , Humanos , Neoplasias/química , Ratos , Pele/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentação , Água/químicaRESUMO
Inhibition of the initial stages of amyloid-ß peptide self-assembly is a key approach in drug development for Alzheimer's disease, in which soluble and highly neurotoxic low molecular weight oligomers are produced and aggregate in the brain over time. Here we report a high-throughput method based on ion mobility mass spectrometry and multivariate statistical analysis to rapidly select statistically significant early-stage species of amyloid-ß1-40 whose formation is inhibited by a candidate theranostic agent. Using this method, we have confirmed the inhibition of a Zn-porphyrin-peptide conjugate in the early self-assembly of Aß40 peptide. The MS/MS fragmentation patterns of the species detected in the samples containing the Zn-porphyrin-peptide conjugate suggested a porphyrin-catalyzed oxidation at Met-35(O) of Aß40. We introduce ion mobility MS combined with multivariate statistics as a systematic approach to perform data analytics in drug discovery/amyloid research that aims at the evaluation of the inhibitory effect on the Aß early assembly in vitro models at very low concentration levels of Aß peptides.
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Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Espectrometria de Mobilidade Iônica/métodos , Fragmentos de Peptídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Doença de Alzheimer/tratamento farmacológico , Humanos , Análise MultivariadaRESUMO
Gold nanoparticles (GNPs) are claimed as outstanding biomedical tools for cancer diagnostics and photo-thermal therapy, but without enough evidence on their potentially adverse immunological effects. Using a model of human dendritic cells (DCs), we showed that 10 nm- and 50 nm-sized GNPs (GNP10 and GNP50, respectively) were internalized predominantly via dynamin-dependent mechanisms, and they both impaired LPS-induced maturation and allostimulatory capacity of DCs, although the effect of GNP10 was more prominent. However, GNP10 inhibited LPS-induced production of IL-12p70 by DCs, and potentiated their Th2 polarization capacity, while GNP50 promoted Th17 polarization. Such effects of GNP10 correlated with a stronger inhibition of LPS-induced changes in Ca2+ oscillations, their higher number per DC, and more frequent extra-endosomal localization, as judged by live-cell imaging, proton, and electron microscopy, respectively. Even when released from heat-killed necrotic HEp-2 cells, GNP10 inhibited the necrotic tumor cell-induced maturation and functions of DCs, potentiated their Th2/Th17 polarization capacity, and thus, impaired the DCs' capacity to induce T cell-mediated anti-tumor cytotoxicity in vitro. Therefore, GNP10 could potentially induce more adverse DC-mediated immunological effects, compared to GNP50.
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Antineoplásicos/imunologia , Células Dendríticas/imunologia , Ouro/imunologia , Nanopartículas/administração & dosagem , Tamanho da Partícula , Antineoplásicos/administração & dosagem , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/imunologia , Polaridade Celular/efeitos dos fármacos , Polaridade Celular/imunologia , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Ouro/administração & dosagem , Humanos , Interleucina-12/imunologia , Lipopolissacarídeos/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Células Th2/efeitos dos fármacos , Células Th2/imunologiaRESUMO
With a model invertebrate animal, we have assessed the fate of magnetic nanoparticles in biologically relevant media, i.e., digestive juices. The toxic potential and the internalization of such nanoparticles by nontarget cells were also examined. The aim of this study was to provide experimental evidence on the formation of Co(2+), Fe(2+), and Fe(3+) ions from CoFe2O4 nanoparticles in the digestive juices of a model organism. Standard toxicological parameters were assessed. Cell membrane stability was tested with a modified method for measurement of its quality. Proton-induced X-ray emission and low energy synchrotron radiation X-ray fluorescence were used to study internalization and distribution of Co and Fe. Co(2+) ions were found to be more toxic than nanoparticles. We confirmed that Co(2+) ions accumulate in the hepatopancreas, but Fe(n+) ions or CoFe2O4 nanoparticles are not retained in vivo. A model biological system with a terrestrial isopod is suited to studies of the potential dissolution of ions and other products from metal-containing nanoparticles in biologically complex media.