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Chronic hepatitis B virus (HBV) infection affects 300 million patients worldwide1,2, in whom virus-specific CD8 T cells by still ill-defined mechanisms lose their function and cannot eliminate HBV-infected hepatocytes3-7. Here we demonstrate that a liver immune rheostat renders virus-specific CD8 T cells refractory to activation and leads to their loss of effector functions. In preclinical models of persistent infection with hepatotropic viruses such as HBV, dysfunctional virus-specific CXCR6+ CD8 T cells accumulated in the liver and, as a characteristic hallmark, showed enhanced transcriptional activity of cAMP-responsive element modulator (CREM) distinct from T cell exhaustion. In patients with chronic hepatitis B, circulating and intrahepatic HBV-specific CXCR6+ CD8 T cells with enhanced CREM expression and transcriptional activity were detected at a frequency of 12-22% of HBV-specific CD8 T cells. Knocking out the inhibitory CREM/ICER isoform in T cells, however, failed to rescue T cell immunity. This indicates that CREM activity was a consequence, rather than the cause, of loss in T cell function, further supported by the observation of enhanced phosphorylation of protein kinase A (PKA) which is upstream of CREM. Indeed, we found that enhanced cAMP-PKA-signalling from increased T cell adenylyl cyclase activity augmented CREM activity and curbed T cell activation and effector function in persistent hepatic infection. Mechanistically, CD8 T cells recognizing their antigen on hepatocytes established close and extensive contact with liver sinusoidal endothelial cells, thereby enhancing adenylyl cyclase-cAMP-PKA signalling in T cells. In these hepatic CD8 T cells, which recognize their antigen on hepatocytes, phosphorylation of key signalling kinases of the T cell receptor signalling pathway was impaired, which rendered them refractory to activation. Thus, close contact with liver sinusoidal endothelial cells curbs the activation and effector function of HBV-specific CD8 T cells that target hepatocytes expressing viral antigens by means of the adenylyl cyclase-cAMP-PKA axis in an immune rheostat-like fashion.
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Linfocitos T CD8-positivos , Hepatitis B Crónica , Hígado , Animales , Humanos , Masculino , Ratones , Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Modulador del Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/virología , Hepatocitos/inmunología , Hepatocitos/virología , Hígado/inmunología , Hígado/virología , Fosforilación , Transducción de Señal , Activación de LinfocitosRESUMEN
Bone fracture healing is a complex process in which specific molecular knowledge is still lacking. The citrulline-arginine-nitric oxide metabolism is one of the involved pathways, and its enrichment via citrulline supplementation can enhance fracture healing. This study investigated the molecular effects of citrulline supplementation during the different fracture healing phases in a rat model. Microcomputed tomography (µCT) was applied for the analysis of the fracture callus formation. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and liquid-chromatography tandem mass spectrometry (LC-MS/MS) were used for lipid and protein analyses, respectively. µCT analysis showed no significant differences in the fracture callus volume and volume fraction between the citrulline supplementation and control group. The observed lipid profiles for the citrulline supplementation and control group were distinct for the different fracture healing stages. The main contributing lipid classes were phosphatidylcholines (PCs) and lysophosphatidylcholines (LPCs). The changing effect of citrulline supplementation throughout fracture healing was indicated by changes in the differentially expressed proteins between the groups. Pathway analysis showed an enhancement of fracture healing in the citrulline supplementation group in comparison to the control group via improved angiogenesis and earlier formation of the soft and hard callus. This study showed the molecular effects on lipids, proteins, and pathways associated with citrulline supplementation during bone fracture healing, even though no effect was visible with µCT.
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Citrulina , Curación de Fractura , Ratas Sprague-Dawley , Espectrometría de Masas en Tándem , Microtomografía por Rayos X , Animales , Curación de Fractura/efectos de los fármacos , Ratas , Citrulina/análisis , Citrulina/metabolismo , Citrulina/farmacología , Espectrometría de Masas en Tándem/métodos , Microtomografía por Rayos X/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Suplementos Dietéticos/análisis , Modelos Animales de Enfermedad , Masculino , Callo Óseo/efectos de los fármacos , Callo Óseo/diagnóstico por imagen , Callo Óseo/metabolismo , Cromatografía Liquida/métodos , Lisofosfatidilcolinas/metabolismo , Lisofosfatidilcolinas/análisis , Fosfatidilcolinas/metabolismo , Fosfatidilcolinas/análisis , Fosfatidilcolinas/farmacologíaRESUMEN
The spatial distribution of molecules and compounds responsible for the flavor profile of edible button mushrooms (Agaricus bisporous) has never been determined. The food industry is interested in knowing the localization of these compounds. Such knowledge would enable extraction of flavor compounds from a particular regions of the mushroom, which is safer for consumption compared to alternatives such as synthetic flavoring agents. The present study utilizes matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI), to determine the spatial distribution of flavor compounds in a mushroom. As MALDI-MSI requires very thin sections, a sample preparation protocol was optimized and sectioning fresh frozen mushrooms at 35 µm thickness was considered the best method to evaluate the distribution of flavor compounds. Further, the effect of heat on the spatial distribution of flavor compounds was investigated by heating whole mushrooms to 140 â prior to sectioning. Heating reduced the water content of the mushroom and thus enabled the generation of even-thinner 17 µm thick sections. MALDI-MSI measurements performed on underivatized and on-tissue derivatized fresh frozen and heat-treated mushroom sections elucidated the spatial distribution of several flavor-related compounds. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05883-0.
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We introduce a novel approach for comprehensive molecular profiling in biological samples. Our single-section methodology combines quantitative mass spectrometry imaging (Q-MSI) and a single step extraction protocol enabling lipidomic and proteomic liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis on the same tissue area. The integration of spatially correlated lipidomic and proteomic data on a single tissue section allows for a comprehensive interpretation of the molecular landscape. Comparing Q-MSI and Q-LC-MS/MS quantification results sheds new light on the effect of MSI and related sample preparation. Performing MSI before Q-LC-MS on the same tissue section led to fewer protein identifications and a lower correlation between lipid quantification results. Also, the critical role and influence of internal standards in Q-MSI for accurate quantification is highlighted. Testing various slide types and the evaluation of different workflows for single-section spatial multiomics analysis emphasized the need for critical evaluation of Q-MSI data. These findings highlight the necessity for robust quantification methods comparable to current gold-standard LC-MS/MS techniques. The spatial information from MSI allowed region-specific insights within heterogeneous tissues, as demonstrated for glioblastoma multiforme. Additionally, our workflow demonstrated the efficiency of a single step extraction for lipidomic and proteomic analyses on the same tissue area, enabling the examination of significantly altered proteins and lipids within distinct regions of a single section. The integration of these insights into a lipid-protein interaction network expands the biological information attainable from a tissue section, highlighting the potential of this comprehensive approach for advancing spatial multiomics research.
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Lipidómica , Espectrometría de Masas en Tándem , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Cromatografía Liquida , Flujo de Trabajo , Cromatografía Líquida con Espectrometría de Masas , Proteómica/métodos , Lípidos/análisisRESUMEN
There is a risk of crimes remaining unsolved when no matching DNA profiles or fingermarks are found. If this is the case, forensic investigations are faced with a significant shortage of evidence and information regarding the unknown perpetrator and/or victim as well as any missing persons. However, a rather commonly found biological trace encountered at crime scenes is human hair. As hair acts as a biochemical reservoir, it may contain valuable information regarding one's characteristics and habits. This study aimed to build an analytical method capable of determining a marker set of relevant metabolites in hair, eventually building up a profile of its donor. To find potential markers, an untargeted metabolomics approach was developed to select and identify statistically significant features. For that purpose, a total of 68 hair samples were collected at several hairdresser shops in varying neighbourhoods. Compound extraction was achieved via methanolic incubation overnight and analysis performed using a high-resolution mass spectrometry (HRMS) Orbitrap Q Exactive Focus. The acquired data was uploaded and statistically evaluated using two free online software/libraries, where a total of eight compounds have given a match on both tools. Their presumptive identity was confirmed using reference standards and consequently added to a dynamic target donor profiling list. These results show the potential of using untargeted metabolomics for the search for lifestyle biomarkers capable of differentiating individuals. Such tools are of paramount importance in a forensic setting with little or no evidence available and no clear tactical leads.
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Metabolómica , Programas Informáticos , Humanos , Metabolómica/métodos , Espectrometría de Masas/métodos , Biomarcadores/análisis , Cabello/químicaRESUMEN
Synthetic polymers are ubiquitous in daily life, and their properties offer diverse benefits in numerous applications. However, synthetic polymers also present an increasing environmental burden through their improper disposal and subsequent degradation into secondary micro- and nanoparticles (MNPs). These MNPs accumulate in soil and water environments and can ultimately end up in the food chain, resulting in potential health risks. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) has the potential to study localized biological or toxicological changes in organisms exposed to MNPs. Here, we investigate whether MALDI-2 postionization can provide a sensitivity enhancement in polymer analysis that could contribute to the study of MNPs. We evaluated the effect of MALDI-2 by comparing MALDI and MALDI-2 ion yields from polyethyleneglycol (PEG), polypropylene glycol (PPG), polytetrahydrofuran (PTHF), nylon-6, and polystyrene (PS). MALDI-2 caused a signal enhancement of the protonated species for PEG, PPG, PTHF, and nylon-6. PS, by contrast, preferentially formed radical ions, which we attribute to direct resonance-enhanced multiphoton ionization (REMPI). REMPI of PS led to an improvement in sensitivity by several orders of magnitude, even without cationizing salts. The improved sensitivity demonstrated by MALDI-2 for all polymers tested highlights its potential for studying the distribution of certain classes of polymers in biological systems.
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Three-dimensional (3D) cell cultures, including organ-on-a-chip (OOC) devices, offer the possibility to mimic human physiology conditions better than 2D models. The organ-on-a-chip devices have a wide range of applications, including mechanical studies, functional validation, and toxicology investigations. Despite many advances in this field, the major challenge with the use of organ-on-a-chips relies on the lack of online analysis methods preventing the real-time observation of cultured cells. Mass spectrometry is a promising analytical technique for real-time analysis of cell excretes from organ-on-a-chip models. This is due to its high sensitivity, selectivity, and ability to tentatively identify a large variety of unknown compounds, ranging from metabolites, lipids, and peptides to proteins. However, the hyphenation of organ-on-a-chip with MS is largely hampered by the nature of the media used, and the presence of nonvolatile buffers. This in turn stalls the straightforward and online connection of organ-on-a-chip outlet to MS. To overcome this challenge, multiple advances have been made to pre-treat samples right after organ-on-a-chip and just before MS. In this review, we summarised these technological advances and exhaustively evaluated their benefits and shortcomings for successful hyphenation of organ-on-a-chip with MS.
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Brain tumour identification and delineation in a timeframe of seconds would significantly guide and support surgical decisions. Here, treatment is often complicated by the infiltration of gliomas in the surrounding brain parenchyma. Accurate delineation of the invasive margins is essential to increase the extent of resection and to avoid postoperative neurological deficits. Currently, histopathological annotation of brain biopsies and genetic phenotyping still define the first line treatment, where results become only available after surgery. Furthermore, adjuvant techniques to improve intraoperative visualisation of the tumour tissue have been developed and validated. In this review, we focused on the sensitivity and specificity of conventional techniques to characterise the tumour type and margin, specifically fluorescent-guided surgery, neuronavigation and intraoperative imaging as well as on more experimental techniques such as mass spectrometry-based diagnostics, Raman spectrometry and hyperspectral imaging. Based on our findings, all investigated methods had their advantages and limitations, guiding researchers towards the combined use of intraoperative imaging techniques. This can lead to an improved outcome in terms of extent of tumour resection and progression free survival while preserving neurological outcome of the patients.
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Cells often adopt different phenotypes, dictated by tissue-specific or local signals such as cell-cell and cell-matrix contacts or molecular micro-environment. This holds in extremis for macrophages with their high phenotypic plasticity. Their broad range of functions, some even opposing, reflects their heterogeneity, and a multitude of subsets has been described in different tissues and diseases. Such micro-environmental imprint cannot be adequately studied by single-cell applications, as cells are detached from their context, while histology-based assessment lacks the phenotypic depth due to limitations in marker combination. Here, we present a novel, integrative approach in which 15-color multispectral imaging allows comprehensive cell classification based on multi-marker expression patterns, followed by downstream analysis pipelines to link their phenotypes to contextual, micro-environmental cues, such as their cellular ("community") and metabolic ("local lipidome") niches in complex tissue. The power of this approach is illustrated for myeloid subsets and associated lipid signatures in murine atherosclerotic plaque.
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Aterosclerosis , Placa Aterosclerótica , Animales , Aterosclerosis/metabolismo , Biomarcadores/metabolismo , Macrófagos/metabolismo , Espectrometría de Masas , Ratones , FenotipoRESUMEN
Introduction: The main goal of brain tumour surgery is to maximize tumour resection while avoiding neurological deficits. Accurate characterization of tissue and delineation of resection margins are, therefore, essential to achieve optimal surgical results. Objectives: The primary objective of this study was to develop and validate a mass spectrometry- based technique for the molecular characterization of high- and low-grade glioma tissue during surgery. Methods: An electrosurgical knife is connected to a mass spectrometer (iKnife). Using this system, an aerosol created during electrosurgical resection is aspirated to a mass spectrometer to determine the molecular profile of the tissue within seconds. This rapid evaporative ionization mass spectrometry (REIMS) technique is used to create a chemical profile database and develop a real-time tissue recognition system based on machine learning. Results: Classification models were built by analysing biopsies from 36 patients who underwent brain tumour resection. Our multivariate statistical model could differentiate between astrocytoma grade II and III, glioblastoma, oligodendroglioma grade II and III, and normal brain tissue with an 88% overall accuracy. Astrocytoma and oligodendroglioma grade II were separated from normal brain with a 96% correct classification rate. REIMS could differentiate between different percentages of GBM with 99.2% sensitivity and different percentages of astrocytoma grade II with 97.5% sensitivity. Conclusion: Real-time information during electrosurgical dissection can improve intra-operative decision-making, leading to a more accurate tumour removal for different glioma subtypes.
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The molecular pathology of breast cancer is challenging due to the complex heterogeneity of cellular subtypes. The ability to directly identify and visualize cell subtype distribution at the single-cell level within a tissue section enables precise and rapid diagnosis and prognosis. Here, we applied mass spectrometry imaging (MSI) to acquire and visualize the molecular profiles at the single-cell and subcellular levels of 14 different breast cancer cell lines. We built a molecular library of genetically well-characterized cell lines. Multistep processing, including deep learning, resulted in a breast cancer subtype, the cancer's hormone status, and a genotypic recognition model based on metabolic phenotypes with cross-validation rates of up to 97%. Moreover, we applied our single-cell-based recognition models to complex tissue samples, identifying cell subtypes in tissue context within seconds during measurement. These data demonstrate "on the spot" digital pathology at the single-cell level using MSI, and they provide a framework for fast and accurate high spatial resolution diagnostics and prognostics.
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Neoplasias de la Mama , Neoplasias de la Mama/diagnóstico por imagen , Diagnóstico por Imagen , Femenino , Humanos , Espectrometría de Masas , Análisis EspectralRESUMEN
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 , Cabello/química , Análisis de Cabello , Humanos , Espectrometría de Masas , Imagen Molecular , Detección de Abuso de Sustancias/métodosRESUMEN
Every year, thousands of suspicious deaths are accounted for by an overdose of opioids. Occasionally all traditional matrices are unavailable due to decomposition. Skeletal tissue may pose a valid alternative. However, reference data on postmortem concentrations in bone tissue and bone marrow (BM) is sparse. Therefore, a liquid chromatography--tandem mass spectrometry method was developed and fully validated for the analysis of four opioids and two metabolites (tramadol, O-desmethyltramadol, morphine, fentanyl, norfentanyl, codeine) in bone tissue and BM. Sample preparation was performed using solid phase extraction (BM), methanolic extraction (bone) and a protein precipitation (whole blood). All validation parameters were successfully fulfilled. This method was applied to analyze 22 forensic cases involving opioids. All six opioids were proven to be detectable and quantifiable in all specimens sampled. When tramadol blood concentrations were correlated with bone concentrations, a linear trend could be detected. The same was seen between tramadol blood and BM concentration. A similar linear trend was seen when correlating codeine blood concentration with bone and BM concentration. Although some variability was detected, the same linear trend was seen for morphine. For fentanyl and norfentanyl, the sample size was too small to draw conclusions, regarding correlation. As far as the authors know this is the first-time fentanyl and norfentanyl are quantified in skeletal tissue. In conclusion, due to the absence of reference data for drugs in skeletal tissue, these findings are a step forward toward a more thorough understanding of drug concentration found in postmortem skeletal tissue.
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Analgésicos Opioides , Tramadol , Cromatografía Liquida/métodos , Codeína , Fentanilo , MorfinaRESUMEN
Fast, accurate and sensitive detection of drugs in human tissue is of crucial importance in an investigation of a suspicious death. Here, we aimed to screen cocaine, diazepam, methadone and morphine in post-mortem muscle samples without sample preparation and in quasi-real time using rapid evaporative ionisation mass spectrometry (REIMS). REIMS enables the online MS analysis of vapours generated from tissue dissection by a diathermic knife. Human muscle samples were soaked in solutions of 4 drugs at different concentrations and multiple incubation times to check the feasibility of REIMS for this innovative application. Muscle samples soaked in blank saline were used as a control. The classification model was able to distinguish between 30 µg g-1 cocaine (m/z 304.2), 200 µg g-1 morphine (m/z 286.2), 10 µg g-1 methadone (m/z 310.2) and 10 µg g-1 muscle of diazepam (m/z 285.1). REIMS tandem MS confirmed that the mass peaks that contributed to the class separation, originated from the drugs of interest. As a proof-of-concept, a forensic case muscle sample from a methadone overdose was investigated using REIMS. Here, using our classification model, the recognition software was able to detect methadone, demonstrating that the REIMS method opens new possibilities in forensic toxicology and during autopsy, leading to faster crime solving and decreased costs.
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Preparaciones Farmacéuticas , Detección de Abuso de Sustancias , Toxicología Forense , Humanos , Espectrometría de Masas , Manejo de EspecímenesRESUMEN
In the past decades, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been applied to a broad range of biological samples, e.g., forensics and preclinical samples. The use of MALDI-MSI for the analysis of bone tissue has been limited due to the insulating properties of the material but more importantly the absence of a proper sample preparation protocol for undecalcified bone tissue. Undecalcified sections are preferred to retain sample integrity as much as possible or to study the tissue-bone bio interface in particular. Here, we optimized the sample preparation protocol of undecalcified bone samples, aimed at both targeted and untargeted applications for forensic and preclinical applications, respectively. Different concentrations of gelatin and carboxymethyl cellulose (CMC) were tested as embedding materials. The composition of 20% gelatin and 7.5% CMC showed to support the tissue best while sectioning. Bone tissue has to be sectioned with a tungsten carbide knife in a longitudinal fashion, while the sections need to be supported with double-sided tapes to maintain the morphology of the tissue. The developed sectioning method was shown to be applicable on rat and mouse as well as human bone samples. Targeted (methadone and EDDP) as well as untargeted (unknown lipids) detection was demonstrated. DHB proved to be the most suitable matrix for the detection of methadone and EDDP in positive ion mode. The limit of detection (LOD) is estimated to approximately 50 pg/spot on bone tissue. The protocol was successfully applied to detect the presence of methadone and EDDP in a dosed rat femur and a dosed human clavicle. The best matrices for the untargeted detection of unknown lipids in mouse hind legs in positive ion mode were CHCA and DHB based on the number of tissue-specific peaks and signal-to-noise ratios. The developed and optimized sample preparation method, applicable on animal and human bones, opens the door for future forensic and (pre)clinical investigations.
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Huesos/química , Lípidos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Adhesión del Tejido/métodos , Animales , Carboximetilcelulosa de Sodio/química , Medicina Legal/métodos , Gelatina/química , Masculino , Microtomía/métodos , Ratas WistarRESUMEN
Blood analysis is the golden standard in the field of forensic toxicology. However, when extended decomposition of the remains has occurred, alternative matrices are required. Skeletal tissue may provide an appropriate sample of choice since it is very resistant to putrefaction. However, today, the absence of reference data of drug concentrations in skeletal tissue poses a problem to meaningfully and reliably conduct toxicological testing on human skeletal material. The present study investigates the viability of skeletal tissue as an alternative matrix to evaluate xenobiotic consumption in legal cases. Blood, bone tissue and bone marrow of different forensic cases were screened for 415 compounds of forensic interest. Afterwards, methadone, clomipramine, citalopram and their respectively metabolites positive samples were quantified using fully validated methods. Sample preparation was carried out by SPE (whole blood and bone marrow), methanol extraction (bone sections) or protein precipitation (whole blood). All samples were analyzed using liquid chromatography coupled to a triple quad mass spectrometer. Multiple drugs were successfully identified in all sampled matrices. In bone (marrow) not as many substances were detected as in blood but it poses a valid alternative when blood is not available. Especially bone marrow showed big potential with a concordance of 80.5% with blood. Clomipramine, citalopram and their metabolites were proven to be detectable and quantifiable in all specimens sampled. Bone marrow showed the highest concentrations followed by blood and bone tissue. When citalopram blood concentrations were correlated with the bone concentrations, a linear trend could be detected. The same was seen between blood and bone marrow for citalopram concentrations. Methadone was also proven to be detectable in all specimens sampled. However, its metabolites EMDP and EDPP were absent or below the LOD in some samples. Overall, methadone concentrations were higher in bone marrow than in bone. With exception of one case, blood concentrations were higher than bone concentrations. For methadone, a linear trend could be found between blood and bone concentration. Comparing methadone concentrations in blood and bone marrow an exponential trend could be seen. In conclusion, these findings show the potential forensic value of bone and bone marrow as an alternative matrix. Aside to that, a standard protocol for the sample collection and processing is proposed.
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Analgésicos Opioides/química , Médula Ósea/química , Huesos/química , Metadona/química , Cambios Post Mortem , Cromatografía Liquida , Toxicología Forense , Humanos , Espectrometría de Masas en TándemRESUMEN
Phlotoxin-1 (PhlTx1) is a peptide previously identified in tarantula venom (Phlogius species) that belongs to the inhibitory cysteine-knot (ICK) toxin family. Like many ICK-based spider toxins, the synthesis of PhlTx1 appears particularly challenging, mostly for obtaining appropriate folding and concomitant suitable disulfide bridge formation. Herein, we describe a procedure for the chemical synthesis and the directed sequential disulfide bridge formation of PhlTx1 that allows for a straightforward production of this challenging peptide. We also performed extensive functional testing of PhlTx1 on 31 ion channel types and identified the voltage-gated sodium (Nav) channel Nav1.7 as the main target of this toxin. Moreover, we compared PhlTx1 activity to 10 other spider toxin activities on an automated patch-clamp system with Chinese Hamster Ovary (CHO) cells expressing human Nav1.7. Performing these analyses in reproducible conditions allowed for classification according to the potency of the best natural Nav1.7 peptide blockers. Finally, subsequent in vivo testing revealed that intrathecal injection of PhlTx1 reduces the response of mice to formalin in both the acute pain and inflammation phase without signs of neurotoxicity. PhlTx1 is thus an interesting toxin to investigate Nav1.7 involvement in cellular excitability and pain.
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Analgésicos/aislamiento & purificación , Péptidos/aislamiento & purificación , Venenos de Araña/química , Bloqueadores del Canal de Sodio Activado por Voltaje/aislamiento & purificación , Secuencia de Aminoácidos , Analgésicos/química , Analgésicos/farmacología , Analgésicos/uso terapéutico , Animales , Células CHO , Cricetulus , Femenino , Formaldehído , Ratones Endogámicos C57BL , Canal de Sodio Activado por Voltaje NAV1.7/fisiología , Oocitos , Dolor/inducido químicamente , Dolor/tratamiento farmacológico , Péptidos/química , Péptidos/farmacología , Péptidos/uso terapéutico , Pliegue de Proteína , Arañas , Bloqueadores del Canal de Sodio Activado por Voltaje/química , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Bloqueadores del Canal de Sodio Activado por Voltaje/uso terapéutico , Xenopus laevisRESUMEN
In recent years, the use of skeletal tissue as an alternative matrix in forensic toxicology has received new interest. In cases where extreme decomposition has taken place, analysis of skeletal tissue is often the only option left. In this article, a fully validated method is presented and the distribution of clomipramine, citalopram, midazolam, and metabolites after chronically administration is examined within skeletal tissue. Rats were chronically dosed with respectively clomipramine, citalopram, or midazolam. Extracts were quantitatively analyzed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Clomipramine, citalopram, and metabolites, respectively desmethylclomipramine and desmethylcitalopram are shown to be detectable in all bone types sampled. Midazolam and its metabolite α-OH-midazolam could not be detected. The absence of midazolam in extracts gives an indication that drugs with pKa values under physiological pH are badly or not incorporated in bone tissue. Bone and post-mortem blood concentrations were compared. A range of different bone types was compared and showed that the concentration is strongly dependent on the bone type. In concordance with previous publications, the humerus shows the highest drug levels. Skeletal tissue concentrations found ranged from 1.1 to 587.8 ng/g. Comparison of the same bone type between the different rats showed high variances. However, the drugs-metabolite ratio proved to have lower variances (<20%). Moreover, the drugs-metabolite ratio in the sampled bones is in close concordance to the ratios seen in blood within a rat. From this, we can assume that the drugs-metabolite ratio in skeletal tissue may prove to be more useful than absolute found concentration.
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Ansiolíticos/farmacocinética , Antidepresivos/farmacocinética , Huesos/metabolismo , Citalopram/farmacocinética , Clomipramina/farmacocinética , Midazolam/farmacocinética , Animales , Ansiolíticos/administración & dosificación , Ansiolíticos/metabolismo , Antidepresivos/administración & dosificación , Antidepresivos/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Citalopram/administración & dosificación , Citalopram/metabolismo , Clomipramina/administración & dosificación , Clomipramina/metabolismo , Límite de Detección , Masculino , Midazolam/administración & dosificación , Midazolam/metabolismo , Ratas , Ratas Wistar , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
The analysis of biological samples, such as whole blood, comes with several sample preparation challenges. Biological matrices often contain a variety of endogenous components that can interfere with the determination of xenobiotics. Especially blood plasma proteins (e.g. serum albumin) are known to interfere with electrospray ionization and result in analyte ion suppression. Sample preparation techniques should guarantee adequate removal of these biomolecules. The current study aims to determine to which extent proteins are removed from whole blood samples, using ionic liquid-based dispersive liquid-liquid microextraction (IL-DLLME). A qualitative comparison of the protein presence in extracts of IL-DLLME, solid-phase extraction (SPE) and protein precipitation (PP) was performed, using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, UV/VIS spectrophotometry was used to determine the protein content of a whole blood sample and IL-DLLME, SPE and PP extracts of the same sample. Finally, a quantitative comparison of matrix effects of benzodiazepines present in both whole blood and water samples. SDS-PAGE results showed that IL-DLLME extracts still contained proteins (i.e. albumin, hemoglobin); however, band intensities were comparable to SPE extracts. Spectrophotometric tests showed a total protein content of approximately 2 mg/mL in the final extracts. PP showed the highest protein extraction rate (19 mg/mL). Quantitative ME results showed no significant differences (α = 0.05) between blood and water IL-DLLME extracts. Overall, this is the first study to conclude that IL-DLLME is able to sufficiently remove blood proteins from whole blood samples, in order to avoid significant ion suppression.
Asunto(s)
Proteínas Sanguíneas/análisis , Líquidos Iónicos/química , Microextracción en Fase Líquida/métodos , Xenobióticos/sangre , Benzodiazepinas/sangre , Benzodiazepinas/química , Proteínas Sanguíneas/química , Cromatografía Líquida de Alta Presión/instrumentación , Cromatografía Líquida de Alta Presión/métodos , Humanos , Límite de Detección , Microextracción en Fase Líquida/instrumentación , Extracción en Fase Sólida/instrumentación , Extracción en Fase Sólida/métodosRESUMEN
OBJECTIVE: To investigate the comparative analgesic efficacy of systemic lidocaine and quadratus lumborum (QL) block in laparoscopic colorectal surgery. BACKGROUND: Although epidural analgesia is the standard to control pain in patients undergoing open colorectal surgery, optimal analgesic management in laparoscopic surgery is less well-defined. There is need for effective and efficient alternatives to epidural analgesia for pain management in patients undergoing laparoscopic colorectal surgery. METHODS: A total of 125 patients undergoing laparoscopic colorectal surgery were included in this randomized, double-blind controlled clinical trial. Patients randomly received an intravenous infusion with placebo plus a QL-block with placebo, a QL-block with ropivacaine 0.25% plus intravenous placebo, or intravenous lidocaine plus a QL-block with placebo. Postoperatively, all patients received patient-controlled intravenous anesthesia (PCIA) with morphine. Primary outcome parameter was the opioid consumption during the first 24 hours postoperatively. Secondary endpoints included severity of postoperative pain, time to return of intestinal function, incidence of postoperative nausea and vomiting, and length of hospital stay. RESULTS: The QL-block was not superior to systemic lidocaine for the reduction of morphine requirements in the first 24 hours postoperatively {QL-group: 37.5 (28.4) mg [mean (standard deviation)] vs lidocaine group: 40.2 (25) mg, P = 0.15}. For the majority of secondary outcome parameters, no significant differences were found between the groups. Morphine consumption in the postanesthesia care unit, the number of PCIA-boli demanded by the patient, and the number of PCIA-boli delivered by the PCIA-pump during the first 24 hours postoperatively were lower in the placebo group. CONCLUSIONS: In our trial, the QL-block did not provide superior postoperative analgesia when compared to systemic lidocaine in laparoscopic colorectal surgery. TRIAL REGISTRATION: Eudra CT: 2014-001499-73; 31/7/2014.