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Hypoxia and low glucose abundance often occur simultaneously at sites of inflammation. In monocytes and macrophages, glucose-oxygen deprivation stimulates the assembly of the NLRP3 inflammasome to generate the proinflammatory cytokine IL-1ß. We found that concomitant glucose deprivation and hypoxia activated the NLRP3 inflammasome by constraining the function of HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate kinase pathway. HMGCR is involved in the synthesis of geranylgeranyl pyrophosphate (GGPP), which is required for the prenylation and lipid membrane integration of proteins. Under glucose-oxygen deprivation, GGPP synthesis was decreased, leading to reduced prenylation of the small GTPase Rac1, increased binding of nonprenylated Rac1 to the scaffolding protein IQGAP1, and enhanced activation of the NLRP3 inflammasome. In response to restricted oxygen and glucose supply, patient monocytes with a compromised mevalonate pathway due to mevalonate kinase deficiency or Muckle-Wells syndrome released more IL-1ß than did control monocytes. Thus, reduced GGPP synthesis due to inhibition of HMGCR under glucose-oxygen deprivation results in proinflammatory innate responses, which are normally kept in check by the prenylation of Rac1. We suggest that this mechanism is also active in inflammatory autoimmune conditions.
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Glucosa , Hidroximetilglutaril-CoA Reductasas , Inflamasomas , Monocitos , Proteína con Dominio Pirina 3 de la Familia NLR , Proteína de Unión al GTP rac1 , Humanos , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética , Monocitos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Hidroximetilglutaril-CoA Reductasas/metabolismo , Hidroximetilglutaril-CoA Reductasas/genética , Inflamasomas/metabolismo , Glucosa/metabolismo , Fosfatos de Poliisoprenilo/metabolismo , Interleucina-1beta/metabolismo , Oxígeno/metabolismo , Prenilación de Proteína , Deficiencia de Mevalonato Quinasa/metabolismo , Deficiencia de Mevalonato Quinasa/genética , Ácido Mevalónico/metabolismoRESUMEN
The peptide/histidine transporter PHT1 (SLC15A4) is expressed in the lysosomal membranes of immune cells where it plays an important role in metabolic and inflammatory signaling. PHT1 is an H+-coupled/histidine symporter that can transport a wide range of oligopeptides, including a variety of bacterial-derived peptides. Moreover, it enables the scaffolding of various metabolic signaling molecules and interacts with key regulatory elements of the immune response. Not surprisingly, PHT1 has been implicated in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). Unfortunately, the pharmacological development of PHT1 modulators has been hampered by the lack of suitable transport assays. To address this shortcoming, a novel transport assay based on solid-supported membrane-based electrophysiology (SSME) is presented. Key findings of the present SSME studies include the first recordings of electrophysiological properties, a pH dependence analysis, an assessment of PHT1 substrate selectivity, as well as the transport kinetics of the identified substrates. In contrast to previous work, PHT1 is studied in its native lysosomal environment. Moreover, observed substrate selectivity is validated by molecular docking. Overall, this new SSME-based assay is expected to contribute to unlocking the pharmacological potential of PHT1 and to deepen the understanding of its functional properties.
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Lisosomas , Humanos , Lisosomas/metabolismo , Concentración de Iones de Hidrógeno , Simulación del Acoplamiento Molecular , Electrofisiología/métodos , Fenómenos Electrofisiológicos , Histidina/metabolismo , Histidina/química , CinéticaRESUMEN
Introduction: Δ9-tetrahydrocannabinolic acid A (THCA-A) is one of the main ingredients of cannabis plants and is converted to the psychoactive substance Δ9-tetrahydrocannabinol (THC) by decarboxylation during heating above â¼90°C. During the consumption of cannabis, a varying proportion of THCA-A is absorbed into the body. Therefore, the quantification of THCA-A in serum/plasma might provide additional information on consumption behavior in driving under the influence of cannabis cases. Materials and Methods: In this study, an already established gas-chromatography mass-spectrometry (GC-MS) method for the quantification of THC, 11-OH-THC, and THC-COOH in serum and plasma samples was extended to include THCA-A. This validated method was then applied to 1228 routinely achieved serum/plasma samples from drivers suspected of cannabis consumption in Western Saxony. Two different grouping systems for chronic/occasional consumption, one system for acute/subacute consumption, Huestis formulas, and the cannabis influence factor (CIF) were used for evaluation. Results: Method validation showed appropriate results for forensic toxicological routine analysis. Limit of detection and lower limit of quantification (LLOQ) for THCA-A were 0.3 and 1.0 ng/mL, respectively. Reproducibility was <11% and accuracy ranged between 104% and 107%. THCA-A was stable in native samples at least for 2 weeks at room temperature or 4°C as well as 1 month at -20°C. Freeze-thaw stability for three cycles and processed sample stability over 3 days was proven. A total of 865 cases with a THC concentration above the German analytical cutoff of 1 ng/mL as well as the analytical LLOQs of 0.9 and 2.5 ng/mL for 11-OH-THC and THC-COOH, respectively, were included in further statistical analysis. In 407 (47.1%) of these samples, THCA-A was quantifiable. Different statistical analyses indicated a correlation between THCA-A and THC concentrations in cases of chronic and acute consumption. In addition, an increase of chronic and acute cases with increasing THCA-A concentrations was observed. However, no correlation between THCA-A and CIF was found. Discussion: These data show that THCA-A might be an additional indicative marker to provide information about consumption frequency and acuteness. Additional studies with known consumption frequencies and times are required to verify these findings.
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BACKGROUND: We describe the spatial distribution of Echinococcus multilocularis in its main definitive host, the red fox, and the distribution of human cases of alveolar echinococcosis (AE) within a highly endemic focus in southern Germany (13.7-19.9/100,000 in 1992-2018). Human cases were unequally distributed within the endemicity focus. The purpose of the study was to test whether this is reflected in the small-scale distribution of E. multilocularis in foxes. METHODS: Three areas with contrasting numbers of human cases were selected within the counties of Ravensburg and Alb-Donau, Baden-Württemberg, Germany. From 2018 to 2020, a total of 240 fox carcasses were obtained from traditional hunters in these areas. Carcasses were necropsied and examined for the presence of intestinal helminths. The statistical analysis was performed with SAS version 9.4, and the geo-mapping with QGIS version 3.16.0 Hannover. RESULTS: The prevalence of E. multilocularis in foxes was 44/106 (41.5%) in area I (commune Leutkirch and environs), 30/59 (50.8%) in area II (commune Isny and environs), and 31/75 (41.3%) in area III (commune Ehingen and environs). From 1992 to 2018, a total of nine human cases of alveolar echinococcosis were recorded in area I, five cases were recorded in study area III, and no cases were recorded in area II. No statistically significant differences between the areas were observed (P > 0.05) for intestinal infections with E. multilocularis, and no apparent spatial correlation with the small-scale distribution of human cases was found. Concerning other zoonotic helminths, Toxocara spp. were equally common, with prevalence of 38.7%, 47.4% and 48.0%, respectively, while the frequency of Alaria alata varied among the study areas (0.0-9.4%), probably reflecting the specific habitat requirements for the establishment of its complex life cycle. CONCLUSIONS: Echinococcus multilocularis is highly prevalent in foxes in all the studied areas. The varying number of human AE cases within these areas should therefore be caused by factors other than the intensity of parasite transmission in foxes.
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Equinococosis , Echinococcus multilocularis , Parasitosis Intestinales , Animales , Humanos , Zorros/parasitología , Equinococosis/epidemiología , Equinococosis/veterinaria , Equinococosis/parasitología , PrevalenciaRESUMEN
The semisynthetic cannabinoid hexahydrocannabinol (HHC) is currently getting a lot of media attention because the legal status in many countries is not clearly specified. In this study, a GC-MS method for the quantification of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) was extended to (9R)- and (9S)-HHC. The applicability was proven by serum/plasma samples from drivers suspected of cannabis consumption. Limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.15 and 0.25 ng/mL, respectively. Within-run imprecision was <6.5% and between-run imprecision was <10.0%. Inter-injection stability, processed sample stability (3 days), freeze-thaw stability (three cycles), and storage stability (1 week room temperature; 1 month 4°C, -20°C) could be proven. Both HHC diastereomers could be detected in 17 (5.3%) out of 321 analyzed samples from traffic controls in Western Saxony. The mean ratio between (9R)- and (9S)-HHC was 1.99 (CV = 14.6%). Quantification resulted in concentrations between
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BACKGROUND: Human life without sperm is not possible. Therefore, it is alarming that the fertilizing ability of human spermatozoa is continuously decreasing. The reasons for that are widely unknown, but there is hope that metabolomics-based investigations may be able to contribute to overcoming this problem. This review summarizes the attempts made so far. METHODS: We will discuss liquid chromatography-mass spectrometry (LC-MS), gas chromatography (GC), infrared (IR) and Raman as well as nuclear magnetic resonance (NMR) spectroscopy. Almost all available studies apply one of these methods. RESULTS: Depending on the methodology used, different compounds can be detected, which is (in combination with sophisticated methods of bioinformatics) helpful to estimate the state of the sperm. Often, but not in all cases, there is a correlation with clinical parameters such as the sperm mobility. CONCLUSIONS: LC-MS detects the highest number of metabolites and can be considered as the method of choice. Unfortunately, the reproducibility of some studies is poor, and, thus, further improvements of the study designs are needed to overcome this problem. Additionally, a stronger focus on the biochemical consequences of the altered metabolite concentrations is also required.
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Metaboloma , Semen , Biomarcadores/metabolismo , Fertilidad , Cromatografía de Gases y Espectrometría de Masas , Humanos , Masculino , Metabolómica/métodos , Reproducibilidad de los Resultados , Semen/metabolismoRESUMEN
Currently, spermiogram analysis is the most relevant method used to clarify the potential infertility of a couple. However, in some cases, the reasons for infertility remain obscure. Smoking is among the factors that have been described to adversely affect male fertility. Smoking increases oxidative stress and thus promotes various pathological processes. Comparative studies, particularly those on metabolomic changes in sperm and seminal plasma caused by smoking, have not yet been published. Thus, the present pilot study aimed at the mass spectrometric characterization of the metabolomes of specimens from both smoking and nonsmoking subjects and the comparison of the evaluated data in terms of sperm apoptosis and spermiogram parameters. The results provided evidence that the conventional spermiogram is not altered in smokers compared to nonsmokers. However, a more careful investigation of sperm cells by metabolomic profiling reveals profound effects of smoking on sperm: first, nitrogen oxide synthase, a marker of oxidative stress, is activated. Second, the uptake of fatty acids into sperm mitochondria is reduced, leading to an impaired energy supply. Third, phenylalanine hydroxylation and tryptophan degradation, which are both indications of altered tetrahydrobiopterin biosynthesis, are reduced. Moreover, flow cytometry approaches indicated increased sperm caspase-3 activity, a sign of apoptosis. The present study clearly shows the negative effects of smoking on semen quality. Especially for idiopathic cases, metabolomic profiling can help to shed light on male subfertility or infertility.
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Apoptosis/efectos de los fármacos , Metaboloma/efectos de los fármacos , Fumar/efectos adversos , Espermatozoides/efectos de los fármacos , Adulto , Biopterinas/análogos & derivados , Biopterinas/biosíntesis , Ácidos Grasos/metabolismo , Alemania , Humanos , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Estrés Oxidativo/efectos de los fármacos , Proyectos Piloto , Análisis de Semen , Espermatozoides/metabolismo , Adulto JovenRESUMEN
OBJECTIVE: Alveolar echinococcosis (AE) is a rare disease in Austria, Switzerland and Germany (DACh) caused by an infection with the parasite Echinococcus multilocularis. The aim of the study was to describe differences in the detection and reporting systems of alveolar echinococcosis in Austria, Switzerland and Germany and to describe epidemiological trends. METHODOLOGY: As part of an epidemiological update on 6th September 2019 in Ulm, Germany, experts and representatives discussed differences in the reporting and recording systems as well as the current epidemiological situation. RESULTS: Since 2004, Austria has had an obligation to report suspected cases, diseases and deaths of alveolar echinococcosis by name in accordance with §1 Para. 1 of the Epidemiegesetz 1950 (EpidemieG) and the Ordinance on Notifiable Communicable Diseases. According to §7 Para. 3 of the German Infection Protection Act (IfSG), Germany has also been subject to a reporting obligation since 2001, but not by name. In addition, national registers are available in both countries, which can be used to answer scientific questions. In Switzerland, there is no obligation to report human alveolar echinococcosis since 1997. Efforts are currently being made to implement a national register for alveolar echinococcosis in Switzerland. Despite different reporting and recording systems, a similar epidemiological trend can be observed for DACh. CONCLUSIONS: In Austria, Switzerland and Germany there is a slightly increasing trend of human cases with alveolar echinococcosis. The direct comparability is limited due to different reporting obligations. The structures often do not allow a joint answering of scientific questions concerning diagnostics, treatment and care.
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Equinococosis , Austria/epidemiología , Equinococosis/epidemiología , Alemania/epidemiología , Humanos , Suiza/epidemiologíaRESUMEN
Background Despite the increasing role of ultrasound, structured ultrasound teaching is only slowly being integrated into the curricula of medical schools and universities all over Europe. Aim To survey the current situation at European universities regarding the integration of ultrasound in student medical education and to report on models of student ultrasound training from selected European universities. Methods A questionnaire survey focusing on the implementation of curricular ultrasound education was sent out to the 28 presidents of the national ultrasound societies of the European Federation of Societies for Ultrasound in Medicine and Biology (EFSUMB), who were asked to distribute the questionnaires to the medical universities of their countries. Results Overall, 53 questionnaires were returned from 46 universities in 17 European countries. In most of the universities (40/46 universities, 87%), the theoretical background of ultrasound is taught. However, in only a minority of universities is ultrasound integrated in anatomy courses (8/46 universities, 17%) or basic science courses (16/46 universities, 35%). Practical skills in ultrasound are taught in 56% of the universities (26/46 universities) and tested in a practical exam in seven of the responding universities (15%). The number of hours in which ultrasound was taught ranged from one to 58 (mean, seven). The respondents reported that lack of time and limited faculty funding were major hurdles. Conclusion According to our survey, only a minority of European universities has integrated ultrasound into the preclinical curriculum thus far. Future EFSUMB initiatives will continue to promote the introduction of ultrasound as an integrative part of the core curriculum of student medical education, and the preparation of proper teaching material.
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PURPOSE: Human alveolar echinococcosis (AE) is a potentially lethal zoonosis caused by the cestode Echinococcus multilocularis. The aim of this systematic review is to establish a comprehensive global AE literature overview taking into account the epidemiologically relevant AE research of the twenty-first century. METHODS: We systematically searched the global literature published from 2001 through 2018 via MEDLINE, EMBASE, the Russian databases eLIBRARY.RU, CyberLeninka, the Chinese databases CNKI, VIP, Journals. RESEARCH: ac.ir (Farsi language-based), Jordan E-Library (Arab language-based) and supplementary Google Scholar, in accordance with the PRISMA guidelines. QGIS software was used for the mapping of the affected countries. RESULTS: We have listed 154 relevant publications in the final literature synopsis in consideration of our quality assessment. Including non-autochthonous cases, human AE was reported in 36 countries within the northern hemisphere from 2001 to 2018. The first publication of AE in Tajikistan, Pakistan, South Korea, Belgium, the Netherlands, Slovakia, Hungary, Lithuania, Latvia, Slovenia and Morocco occurred in this century; further first cases in Taiwan, Thailand, and Denmark were considered to be non-autochthonous by the authors. The highest total case numbers (n ≥ 100 in a single article) were reported in France, Germany, Switzerland, Poland, and Lithuania, including China and Kyrgyzstan with by far the highest prevalence figures. CONCLUSIONS: Our paper emphasises the increasing spread of reported cases and the rise in its numbers in the literature of the twenty-first century, especially in western, northern and eastern Europe, as well as in central Asia. Epidemiological studies on human infections are lacking in many parts of the world.
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Equinococosis/epidemiología , Salud Global , Animales , China/epidemiología , Echinococcus multilocularis , Europa (Continente)/epidemiología , Geografía , Humanos , Prevalencia , República de Corea/epidemiologíaRESUMEN
Ultrasound is the most commonly used diagnostic method in everyday clinical practice aside from conventional radiography. For prospective physicians, there is a gap between the increased requirements in everyday clinical practice and the skills acquired thus far, especially when dealing with sonography. In this work, we would like to present the structure of our sonography course and its implementation in the Ulm core curriculum of medical studies. The sonography course is based on two pillars. The Sonography Seminar (Pillar 1) has a lecturer who regularly conveys interdisciplinary theoretical content, fundamentals and possible fields of application for sonography during 13 appointments of 45 minutes. The internship (Pillar 2) consists of four appointments of two hours each. The groups of four students are supervised by a student tutor. In addition to the technical basics and an introduction to the device customer, the organ systems liver, gallbladder and biliary tract, kidneys and adrenal glands, spleen, pancreas, abdominal vessels and lymph nodes are mediated. The focus is on the realistic exercises with high-end ultrasound equipment used in small groups with a final practical test. The structure and procedure of the course was evaluated as being very positive by the students.
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Educación de Postgrado en Medicina , Ultrasonografía , Competencia Clínica , Curriculum , Humanos , MédicosRESUMEN
In 50% of all infertility cases, the male is subfertile or infertile, however, the underlying mechanisms are often unknown. Even when assisted reproductive procedures such as in vitro fertilization and intracytoplasmic sperm injection are performed, the causes of male factor infertility frequently remain elusive. Since the overall activity of cells is closely linked to their metabolic capacity, we analyzed a panel of 180 metabolites in human sperm and seminal plasma and elucidated their associations with spermiogram parameters. Therefore, metabolites from a group of 20 healthy donors were investigated using a targeted LC-MS/MS approach. The correlation analyses of the amino acids, biogenic amines, acylcarnitines, lysophosphatidylcholines, phosphatidylcholines, sphingomyelins and sugars from sperm and seminal plasma with standard spermiogram parameters revealed that metabolites in sperm are closely related to sperm motility, whereas those in seminal plasma are closely related to sperm concentration and morphology. This study provides essential insights into the metabolome of human sperm and seminal plasma and its associations with sperm functions. This metabolomics technique could be a promising screening tool to detect the factors of male infertility in cases where the cause of infertility is unclear.
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Metaboloma , Semen/química , Espermatozoides/química , Adulto , Aminoácidos/análisis , Aminas Biogénicas/análisis , Carnitina/análogos & derivados , Carnitina/análisis , Centrifugación por Gradiente de Densidad , Cromatografía de Gases y Espectrometría de Masas , Humanos , Lisofosfatidilcolinas/análisis , Masculino , Fosfatidilcolinas/análisis , Semen/metabolismo , Análisis de Semen , Espermatozoides/metabolismo , Esfingomielinas/análisis , Azúcares/análisisRESUMEN
Monensin (Mon) is an anticoccidial polyether ionophore widely used to control coccidiosis. The extensive use of polyether ionophores on poultry farms resulted in widespread resistance, but the underlying resistance mechanisms are unknown in detail. For analysing the mode of action by which resistance against polyether ionophores is obtained, we induced in vitro Mon resistance in Toxoplasma gondii-RH strain (MonR-RH) and compared it with the sensitive parental strain (Sen-RH). The proteome assessment of MonR-RH and Sen-RH strains was obtained after isotopic labelling using stable isotope labelling by amino acid in cell culture. Relative proteomic quantification between resistant and sensitive strains was performed using liquid chromatography-mass spectrometry/mass spectrometry. Overall, 1024 proteins were quantified and 52 proteins of them were regulated. The bioinformatic analysis revealed regulation of cytoskeletal and transmembrane proteins being involved in transport mechanisms, metal ion-binding and invasion. During invasion, actin and microneme protein 8 (MIC8) are seem to be important for conoid extrusion and forming moving junction with host cells, respectively. Actin was significantly upregulated, while MIC8 was downregulated, which indicate an invasion reduction in the resistant strain. Resistance against Mon is not a simple process but it involves reduced invasion and egress activity of T. gondii tachyzoites while intracellular replication is enhanced.
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Coccidiostáticos/farmacología , Citoplasma/parasitología , Resistencia a Medicamentos , Monensina/farmacología , Proteínas Protozoarias/genética , Toxoplasma/efectos de los fármacos , Actinas/genética , Cromatografía Liquida , Biología Computacional , Fibroblastos/parasitología , Prepucio/citología , Prepucio/parasitología , Interacciones Huésped-Parásitos , Humanos , Masculino , Proteoma , Proteómica , Proteínas Protozoarias/análisis , Proteínas Protozoarias/aislamiento & purificación , Proteínas Protozoarias/metabolismo , Espectrometría de Masas en Tándem , Toxoplasma/fisiologíaRESUMEN
Monocytes enter sites of microbial or sterile inflammation as the first line of defense of the immune system and initiate pro-inflammatory effector mechanisms. We show that activation with bacterial lipopolysaccharide (LPS) induces them to undergo a metabolic shift toward aerobic glycolysis, similar to the Warburg effect observed in cancer cells. At sites of inflammation, however, glucose concentrations are often drastically decreased, which prompted us to study monocyte function under conditions of glucose deprivation and abrogated Warburg effect. Experiments using the Seahorse Extracellular Flux Analyzer revealed that limited glucose supply shifts monocyte metabolism toward oxidative phosphorylation, fueled largely by fatty acid oxidation at the expense of lipid droplets. While this metabolic state appears to provide sufficient energy to sustain functional properties like cytokine secretion, migration, and phagocytosis, it cannot prevent a rise in the AMP/ATP ratio and a decreased respiratory burst. The molecular trigger mediating the metabolic shift and the functional consequences is activation of AMP-activated protein kinase (AMPK). Taken together, our results indicate that monocytes are sufficiently metabolically flexible to perform pro-inflammatory functions at sites of inflammation despite glucose deprivation and inhibition of the LPS-induced Warburg effect. AMPK seems to play a pivotal role in orchestrating these processes during glucose deprivation in monocytes.
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The disruptive potential of xenoestrogens like bisphenol A (BPA) lies in their 17ß-estradiol (E2)-like binding to estrogen receptors (ERs) followed by concomitant modulation of ER target gene expression. Unsurprisingly, most endocrine testing systems focus on the quantification of canonical transcripts or ER-sensitive reporters. However, only little information is available about the corresponding metabolomic changes in vitro. This knowledge gap becomes particularly relevant in the context of potential mixture effects, for example, as a consequence of coexposure to potentially estrogenically active pollutants (e.g., Cd2+). Such effects are often difficult to dissect with molecular tools, especially with regard to potential physiological relevance. Metabolomic biomarkers are well-suited to address this latter aspect as they provide a comprehensive readout of whole-cell physiology. Applying a targeted metabolomics approach (FIA-MS/MS), this study looked for biomarkers indicative of xenoestrogenic exposure in MCF-7 cells. Cells were treated with E2 and BPA in the presence or absence of Cd2+. Statistical analysis revealed a total of 11 amino acids and phospholipids to be related to the compound's estrogenic potency. Co-exposure to Cd2+ modulated the estrogenic profile. However, the corresponding changes were found to be moderate with cellular assays such as the E-screen failing to record any Cd2+-specific estrogenic effects. Overall, metabolomics analysis identified proline as the most prominent estrogenic biomarker. Its increase could clearly be related to estrogenic exposure and concomitant ERα-mediated induction of proliferation. Involvement of the latter was confirmed by siRNA-mediated knockdown studies as well as by receptor inhibition. Further, the underlying signaling was also found to involve the oncoprotein MYC. Taken together, this study provides insights into the underlying mechanisms of xenoestrogenic effects and exemplify the strength of the complementary use of metabolomics and cellular and molecular assays.
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Biomarcadores/metabolismo , Proliferación Celular/efectos de los fármacos , Disruptores Endocrinos/toxicidad , Metabolómica , Compuestos de Bencidrilo/química , Compuestos de Bencidrilo/toxicidad , Cadmio/química , Colorimetría , Análisis Discriminante , Disruptores Endocrinos/química , Estradiol/química , Estradiol/toxicidad , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Genes Reporteros , Humanos , Células MCF-7 , Metaboloma/efectos de los fármacos , Fenoles/química , Fenoles/toxicidad , Prolina/metabolismo , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Espectrometría de Masas en TándemRESUMEN
Bone healing involves a variety of different cell types and biological processes. Although certain key molecules have been identified, the molecular interactions of the healing progress are not completely understood. Moreover, a clinical routine for predicting the quality of bone healing after a fracture in an early phase is missing. This is mainly due to a lack of techniques to comprehensively screen for cytokines, growth factors and metabolites at their local site of action. Since all soluble molecules of interest are present in the fracture hematoma, its in-depth assessment could reveal potential markers for the monitoring of bone healing. Here, we describe an approach for sampling and quantification of cytokines and metabolites by using microdialysis, combined with solid phase extractions of proteins from wound fluids. By using a control group with an isolated soft tissue wound, we could reveal several bone defect-specific molecular features. In bone defect dialysates the neutrophil chemoattractants CXCL1, CXCL2 and CXCL3 were quantified with either a higher or earlier response compared to dialysate from soft tissue wound. Moreover, by analyzing downstream adaptions of the cells on protein level and focusing on early immune response, several proteins involved in the immune cell migration and activity could be identified to be specific for the bone defect group, e.g. immune modulators, proteases and their corresponding inhibitors. Additionally, the metabolite screening revealed different profiles between the bone defect group and the control group. In summary, we identified potential biomarkers to indicate imbalanced healing progress on all levels of analysis.
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Líquidos Corporales/metabolismo , Huesos/patología , Citocinas/metabolismo , Metaboloma , Microdiálisis , Heridas y Lesiones/metabolismo , Heridas y Lesiones/patología , Adsorción , Animales , Biomarcadores/metabolismo , Huesos/efectos de los fármacos , Quimiocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Proteínas de la Matriz Extracelular/metabolismo , Curación de Fractura/efectos de los fármacos , Fracturas Óseas/metabolismo , Fracturas Óseas/patología , Hematoma/metabolismo , Hematoma/patología , Inmunomodulación/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Metaboloma/efectos de los fármacos , Metabolómica , Análisis de Componente Principal , Inhibidores de Proteasas/farmacología , Proteómica , Ratas Wistar , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacos , Traumatismos de los Tejidos Blandos/metabolismo , Traumatismos de los Tejidos Blandos/patologíaRESUMEN
The challenge of performing a time-resolved comprehensive analysis of molecular systems has led to the quest to optimize extraction methods. When the size of a biological sample is limited, there is demand for the simultaneous extraction of molecules representing the four areas of "omics": genomics, transcriptomics, proteomics, and metabolomics. Here we optimized a protocol for the simultaneous extraction of DNA, RNA, proteins, and metabolites and compared it with two existing protocols. Our optimization comprised the addition of a methanol/chloroform metabolite purification before the separation of DNA/RNA and proteins. Extracted DNA, RNA, proteins, and metabolites were quantitatively and/or qualitatively analyzed. Of the three methods, only the newly developed protocol yielded all biomolecule classes of adequate quantity and quality.
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Técnicas de Química Analítica/métodos , Biología Computacional/métodos , ADN/aislamiento & purificación , Proteínas/aislamiento & purificación , ARN/aislamiento & purificación , ADN/química , Proteínas/química , Proteínas/metabolismo , ARN/químicaRESUMEN
In human and mice ATP-binding cassette efflux transporter ABCG2 represents the main route for active drug transport into milk. However, there is no detailed information on the role of ABCG2 in drug secretion and accumulation in milk of dairy animals. We therefore examined ABCG2-mediated drug transport in the bovine mammary gland by parallel pharmacokinetic studies in lactating Jersey cows and in vitro flux studies using the anthelmintic drug monepantel (MNP) as representative bovine ABCG2 (bABCG2) drug substrate. Animals received MNP (Zolvix, Novartis Animal Health Inc.) once (2.5 mg/kg per os) and the concentrations of MNP and the active MNP metabolite MNPSO2 were assessed by high-performance liquid chromatography. Compared with the parent drug MNP, we detected higher MNPSO2 plasma concentrations (expressed as area under the concentration-versus-time curve). Moreover, we observed MNPSO2 excretion into milk of dairy cows with a high milk-to-plasma ratio of 6.75. In mechanistic flux assays, we determined a preferential time-dependent basolateral-to-apical (B > A) MNPSO2 transport across polarized Madin-Darby canine kidney II cells-bABCG2 monolayers using liquid chromatography coupled with tandem mass spectrometry analysis. The B > A MNPSO2 transport was significantly inhibited by the ABCG2 inhibitor fumitremorgin C in bABCG2- but not in mock-transduced MDCKII cells. Additionally, the antibiotic drug enrofloxacin, the benzimidazole anthelmintic oxfendazole and the macrocyclic lactone anthelmintic moxidectin caused a reduction in the MNPSO2(B > A) net efflux. Altogether, this study indicated that therapeutically relevant drugs like the anthelmintic MNP represent substrates of the bovine mammary ABCG2 transporter and may thereby be actively concentrated in dairy milk.