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Mass spectrometry-based omics technologies are increasingly used in perturbation studies to map drug effects to biological pathways by identifying significant molecular events. Significance is influenced by fold change and variation of each molecular parameter, but also by multiple testing corrections. While the fold change is largely determined by the biological system, the variation is determined by experimental workflows. Here, it is shown that memory effects of prior subculture can influence the variation of perturbation profiles using the two colon carcinoma cell lines SW480 and HCT116. These memory effects are largely driven by differences in growth states that persist into the perturbation experiment. In SW480 cells, memory effects combined with moderate treatment effects amplify the variation in multiple omics levels, including eicosadomics, proteomics, and phosphoproteomics. With stronger treatment effects, the memory effect was less pronounced, as demonstrated in HCT116 cells. Subculture homogeneity was controlled by real-time monitoring of cell growth. Controlled homogeneous subculture resulted in a perturbation network of 321 causal conjectures based on combined proteomic and phosphoproteomic data, compared to only 58 causal conjectures without controlling subculture homogeneity in SW480 cells. Some cellular responses and regulatory events were identified that extend the mode of action of arsenic trioxide (ATO) only when accounting for these memory effects. Controlled prior subculture led to the finding of a synergistic combination treatment of ATO with the thioredoxin reductase 1 inhibitor auranofin, which may prove useful in the management of NRF2-mediated resistance mechanisms.
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Proteómica , Humanos , Proteómica/métodos , Línea Celular Tumoral , Células HCT116 , Técnicas de Cultivo de Célula/métodos , Neoplasias del Colon/metabolismo , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Trióxido de Arsénico/farmacología , Auranofina/farmacología , Proliferación Celular/efectos de los fármacos , Espectrometría de Masas/métodosRESUMEN
The aim of the present study was to investigate retinal microcirculatory and functional metabolic changes in patients after they had recovered from a moderate to severe acute COVID-19 infection. Retinal perfusion was quantified using laser speckle flowgraphy. Oxygen saturation and retinal calibers were assessed with a dynamic vessel analyzer. Arterio-venous ratio (AVR) was calculated based on retinal vessel diameter data. Blood plasma samples underwent mass spectrometry-based multi-omics profiling, including proteomics, metabolomics and eicosadomics. A total of 40 subjects were included in the present study, of which 29 had recovered from moderate to severe COVID-19 within 2 to 23 weeks before inclusion and 11 had never had COVID-19, as confirmed by antibody testing. Perfusion in retinal vessels was significantly lower in patients (60.6 ± 16.0 a.u.) than in control subjects (76.2 ± 12.1 a.u., p = 0.006). Arterio-venous (AV) difference in oxygen saturation and AVR was significantly lower in patients compared to healthy controls (p = 0.021 for AVR and p = 0.023 for AV difference in oxygen saturation). Molecular profiles demonstrated down-regulation of cell adhesion molecules, NOTCH3 and fatty acids, and suggested a bisphasic dysregulation of nitric oxide synthesis after COVID-19 infection. The results of this study imply that retinal perfusion and oxygen metabolism is still significantly altered in patients well beyond the acute phase of COVID-19. This is also reflected in the molecular profiling analysis of blood plasma, indicating a down-regulation of nitric oxide-related endothelial and immunological cell functions.Trial Registration: ClinicalTrials.gov ( https://clinicaltrials.gov ) NCT05650905.
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COVID-19 , Oxígeno , Humanos , Oxígeno/metabolismo , Microcirculación , Óxido Nítrico , Oximetría/métodos , Vasos Retinianos , Perfusión , Proteínas Sanguíneas , LípidosRESUMEN
Metabolomics is an emerging and powerful bioanalytical method supporting clinical investigations. Serum and plasma are commonly used without rational prioritization. Serum is collected after blood coagulation, a complex biochemical process involving active platelet metabolism. This may affect the metabolome and increase the variance, as platelet counts and function may vary substantially in individuals. A multiomics approach systematically investigating the suitability of serum and plasma for clinical studies demonstrated that metabolites correlated well (n = 461, R2 = 0.991), whereas lipid mediators (n = 83, R2 = 0.906) and proteins (n = 322, R2 = 0.860) differed substantially between specimen. Independently, analysis of platelet releasates identified most biomolecules significantly enriched in serum compared to plasma. A prospective, randomized, controlled parallel group metabolomics trial with acetylsalicylic acid administered for 7 days demonstrated that the apparent drug effects significantly differ depending on the analyzed specimen. Only serum analyses of healthy individuals suggested a significant downregulation of TXB2 and 12-HETE, which were specifically formed during coagulation in vitro. Plasma analyses reliably identified acetylsalicylic acid effects on metabolites and lipids occurring in vivo such as an increase in serotonin, 15-deoxy-PGJ2 and sphingosine-1-phosphate and a decrease in polyunsaturated fatty acids. The present data suggest that plasma should be preferred above serum for clinical metabolomics studies as the serum metabolome may be substantially confounded by platelets.
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Aspirina , Plaquetas , Metabolómica , Plasma , Humanos , Plaquetas/metabolismo , Plaquetas/efectos de los fármacos , Metabolómica/métodos , Aspirina/farmacología , Plasma/metabolismo , Plasma/química , Suero/metabolismo , Suero/química , Lisofosfolípidos/sangre , Esfingosina/análogos & derivados , Esfingosina/sangre , Metaboloma/efectos de los fármacos , Tromboxano B2/sangre , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/sangre , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Masculino , Femenino , Estudios Prospectivos , AdultoRESUMEN
PURPOSE: Despite high clinical need, there are no biomarkers that accurately predict the response of patients with metastatic melanoma to anti-PD-1 therapy. EXPERIMENTAL DESIGN: In this multicenter study, we applied protein depletion and enrichment methods prior to various proteomic techniques to analyze a serum discovery cohort (n = 56) and three independent serum validation cohorts (n = 80, n = 12, n = 17). Further validation analyses by literature and survival analysis followed. RESULTS: We identified several significantly regulated proteins as well as biological processes such as neutrophil degranulation, cell-substrate adhesion, and extracellular matrix organization. Analysis of the three independent serum validation cohorts confirmed the significant differences between responders (R) and nonresponders (NR) observed in the initial discovery cohort. In addition, literature-based validation highlighted 30 markers overlapping with previously published signatures. Survival analysis using the TCGA database showed that overexpression of 17 of the markers we identified correlated with lower overall survival in patients with melanoma. CONCLUSIONS: Ultimately, this multilayered serum analysis led to a potential marker signature with 10 key markers significantly altered in at least two independent serum cohorts: CRP, LYVE1, SAA2, C1RL, CFHR3, LBP, LDHB, S100A8, S100A9, and SAA1, which will serve as the basis for further investigation. In addition to patient serum, we analyzed primary melanoma tumor cells from NR and found a potential marker signature with four key markers: LAMC1, PXDN, SERPINE1, and VCAN.
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Melanoma , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/metabolismo , Proteómica , Biomarcadores de Tumor/metabolismo , Análisis de SupervivenciaRESUMEN
During megakaryopoiesis, megakaryocytes (MK) undergo cellular morphological changes with strong modification of membrane composition and lipid signaling. Here we adopt a lipid-centric multiomics approach to create a quantitative map of the MK lipidome during maturation and proplatelet formation. Data reveal that MK differentiation is driven by an increased fatty acyl import and de novo lipid synthesis, resulting in an anionic membrane phenotype. Pharmacological perturbation of fatty acid import and phospholipid synthesis blocked membrane remodeling and directly reduced MK polyploidization and proplatelet formation resulting in thrombocytopenia. The anionic lipid shift during megakaryopoiesis was paralleled by lipid-dependent relocalization of the scaffold protein CKIP-1 and recruitment of the kinase CK2α to the plasma membrane, which seems to be essential for sufficient platelet biogenesis. Overall, this study provides a framework to understand how the MK lipidome is altered during maturation and the impact of MK membrane lipid remodeling on MK kinase signaling involved in thrombopoiesis.
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Given the absence of written records, the main source of information available to analyze gender inequalities in early complex societies is the human body itself. And yet, for decades, archaeologists have struggled with the sex estimation of poorly preserved human remains. Here we present an exceptional case study that shows how ground-breaking new scientific methods may address this problem. Through the analysis of sexually dimorphic amelogenin peptides in tooth enamel, we establish that the most socially prominent person of the Iberian Copper Age (c. 3200-2200 BC) was not male, as previously thought, but female. The analysis of this woman, discovered in 2008 at Valencina, Spain, reveals that she was a leading social figure at a time where no male attained a remotely comparable social position. Only other women buried a short time after in the Montelirio tholos, part of the same burial area, appear to have enjoyed a similarly high social position. Our results invite to reconsider established interpretations about the political role of women at the onset of early social complexity, and question traditionally held views of the past. Furthermore, this study anticipates the changes that newly developed scientific methods may bring to prehistoric archaeology and the study of human social evolution.
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Liderazgo , Péptidos , Humanos , Femenino , Amelogenina , España , ArqueologíaRESUMEN
Metastasis is the major cause of cancer-related deaths. Neuroblastoma (NB), a childhood tumor has been molecularly defined at the primary cancer site, however, the bone marrow (BM) as the metastatic niche of NB is poorly characterized. Here we perform single-cell transcriptomic and epigenomic profiling of BM aspirates from 11 subjects spanning three major NB subtypes and compare these to five age-matched and metastasis-free BM, followed by in-depth single cell analyses of tissue diversity and cell-cell interactions, as well as functional validation. We show that cellular plasticity of NB tumor cells is conserved upon metastasis and tumor cell type composition is NB subtype-dependent. NB cells signal to the BM microenvironment, rewiring via macrophage mgration inhibitory factor and midkine signaling specifically monocytes, which exhibit M1 and M2 features, are marked by activation of pro- and anti-inflammatory programs, and express tumor-promoting factors, reminiscent of tumor-associated macrophages. The interactions and pathways characterized in our study provide the basis for therapeutic approaches that target tumor-to-microenvironment interactions.
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Neoplasias de la Médula Ósea , Neuroblastoma , Humanos , Niño , Médula Ósea/patología , Monocitos/metabolismo , Transcriptoma , Epigenómica , Neoplasias de la Médula Ósea/genética , Neoplasias de la Médula Ósea/metabolismo , Neoplasias de la Médula Ósea/patología , Neuroblastoma/metabolismo , Microambiente Tumoral/genéticaRESUMEN
The ABC transporter ABCA7 has been found to be aberrantly expressed in a variety of cancer types, including breast cancer. We searched for specific epigenetic and genetic alterations and alternative splicing variants of ABCA7 in breast cancer and investigated whether these alterations are associated with ABCA7 expression. By analyzing tumor tissues from breast cancer patients, we found CpGs at the exon 5-intron 5 boundary aberrantly methylated in a molecular subtype-specific manner. The detection of altered DNA methylation in tumor-adjacent tissues suggests epigenetic field cancerization. In breast cancer cell lines, DNA methylation levels of CpGs in promoter-exon 1, intron 1, and at the exon 5-intron 5 boundary were not correlated with ABCA7 mRNA levels. By qPCR involving intron-specific and intron-flanking primers, we identified intron-containing ABCA7 mRNA transcripts. The occurrence of intron-containing transcripts was neither molecular subtype-specific nor directly correlated with DNA methylation at the respective exon-intron boundaries. Treatment of breast cancer cell lines MCF-7, BT-474, SK-BR3, and MDA-MB-231 with doxorubicin or paclitaxel for 72 h resulted in altered ABCA7 intron levels. Shotgun proteomics revealed that an increase in intron-containing transcripts was associated with significant dysregulation of splicing factors linked to alternative splicing.
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Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/patología , Metilación de ADN/genética , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Empalme Alternativo/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Intestinal cells are continuously exposed to food constituents while adapting to peristaltic movement and fluid shear stress. Oleic acid (OA) and palmitic acid (PA) are among the most prevalent fatty acids with respect to dietary lipids. Despite the central importance of dietary lipids for a balanced diet, awareness about potential detrimental effects related to excessive consumption is increasing; this includes toxicity, metabolic deregulation, and, particularly for cancer cells, a benefit from the uptake of fatty acids related to promotion of metastasis. Expanding on this, we started elucidating the effects of OA and PA (25-500 µM) on non-transformed human intestinal epithelial cells (HCEC-1CT) in comparison to colon carcinoma cells (HCT116), with regard to the mechanosensory apparatus. Hence, intestinal cells' motility is on the one side essential to ensure adaption to peristaltic movement and barrier function, but also to enable metastatic progression. Incubation with both OA and PA (≥ 25 µM) significantly decreased membrane fluidity of HCT116 cells, whereas the effect on HCEC-1CT was more limited. Application of rhodamine-labelled PA demonstrated that the fatty acid is incorporated into the plasma membrane of HCT116, which could not be observed in the non-tumorigenic cell line. Down-streaming into the intracellular compartment, a pronounced rearrangement of actin cytoskeleton was evident in both cell lines (OA and PA; 25 and 100 µM). This was accompanied by a variation of translocation efficiency of the mechanosensitive co-transcription factor YAP1, albeit with a stronger effect seen for PA and the cancer cells. Untargeted proteomic analysis confirmed that exposure to OA and PA could alter the response capacity of HCT116 cells to fluid shear stress. Taken together, OA and PA were able to functionally modulate the mechanosensory apparatus of intestinal cells, implying a novel role for dietary fatty acids in the regulation of intestinal pathophysiology.
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Mecanotransducción Celular , Ácido Palmítico , Humanos , Ácido Palmítico/toxicidad , Ácido Palmítico/metabolismo , Proteómica , Ácidos Grasos , Ácido Oléico/metabolismoRESUMEN
Proteomics is an indispensable analytical technique to study the dynamic functioning of biological systems via different proteins and their proteoforms. In recent years, bottom-up shotgun has become more popular than gel-based top-down proteomics. The current study examined the qualitative and quantitative performance of these two fundamentally different methodologies by the parallel measurement of six technical and three biological replicates of the human prostate carcinoma cell line DU145 using its two most common standard techniques, label-free shotgun and two-dimensional differential gel electrophoresis (2D-DIGE). The analytical strengths and limitations were explored, finally focusing on the unbiased detection of proteoforms, exemplified by discovering a prostate cancer-related cleavage product of pyruvate kinase M2. Label-free shotgun proteomics quickly yields an annotated proteome but with reduced robustness, as determined by three times higher technical variation compared to 2D-DIGE. At a glance, only 2D-DIGE top-down analysis provided valuable, direct stoichiometric qualitative and quantitative information from proteins to their proteoforms, even with unexpected post-translational modifications, such as proteolytic cleavage and phosphorylation. However, the 2D-DIGE technology required almost 20 times as much time per protein/proteoform characterization with more manual work. Ultimately, this work should expose both techniques' orthogonality with their different contents of data output to elucidate biological questions.
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Proteoma , Proteómica , Masculino , Humanos , Proteómica/métodos , Proteoma/análisis , Procesamiento Proteico-Postraduccional , Electroforesis en Gel Bidimensional , FosforilaciónRESUMEN
Clinical management of melanomas with NRAS mutations is challenging. Targeting MAPK signaling is only beneficial to a small subset of patients due to resistance that arises through genetic, transcriptional, and metabolic adaptation. Identification of targetable vulnerabilities in NRAS-mutated melanoma could help improve patient treatment. Here, we used multiomics analyses to reveal that NRAS-mutated melanoma cells adopt a mesenchymal phenotype with a quiescent metabolic program to resist cellular stress induced by MEK inhibition. The metabolic alterations elevated baseline reactive oxygen species (ROS) levels, leading these cells to become highly sensitive to ROS induction. In vivo xenograft experiments and single-cell RNA sequencing demonstrated that intratumor heterogeneity necessitates the combination of a ROS inducer and a MEK inhibitor to inhibit both tumor growth and metastasis. Ex vivo pharmacoscopy of 62 human metastatic melanomas confirmed that MEK inhibitor-resistant tumors significantly benefited from the combination therapy. Finally, oxidative stress response and translational suppression corresponded with ROS-inducer sensitivity in 486 cancer cell lines, independent of cancer type. These findings link transcriptional plasticity to a metabolic phenotype that can be inhibited by ROS inducers in melanoma and other cancers. SIGNIFICANCE: Metabolic reprogramming in drug-resistant NRAS-mutated melanoma cells confers sensitivity to ROS induction, which suppresses tumor growth and metastasis in combination with MAPK pathway inhibitors.
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Melanoma , Neoplasias Cutáneas , Humanos , Especies Reactivas de Oxígeno , Proteínas Proto-Oncogénicas B-raf/genética , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Neoplasias Cutáneas/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Línea Celular Tumoral , Mutación , Proteínas de la Membrana/genética , GTP Fosfohidrolasas/genéticaRESUMEN
INTRODUCTION: Ulcerative colitis [UC] is a chronic disease with rising incidence and unclear aetiology. Deep molecular phenotyping by multiomics analyses may provide novel insights into disease processes and characteristic features of remission states. METHODS: UC pathomechanisms were assessed by proteome profiling of human tissue specimens, obtained from five distinct colon locations for each of the 12 patients included in the study. Systemic disease-associated alterations were evaluated thanks to a cross-sectional setting of mass spectrometry-based multiomics analyses comprising proteins, metabolites, and eicosanoids of plasma obtained from UC patients during acute episodes and upon remission, in comparison with healthy controls. RESULTS: Tissue proteome profiling indicated colitis-associated activation of neutrophils, macrophages, B and T cells, fibroblasts, endothelial cells and platelets, and hypoxic stress, and suggested a general downregulation of mitochondrial proteins accompanying the establishment of apparent wound healing-promoting activities including scar formation. Whereas pro-inflammatory proteins were apparently upregulated by immune cells, the colitis-associated epithelial cells, fibroblasts, endothelial cells, and platelets seemed to predominantly contribute anti-inflammatory and wound healing-promoting proteins. Blood plasma proteomics indicated chronic inflammation and platelet activation, whereas plasma metabolomics identified disease-associated deregulations of gut and gut microbiome-derived metabolites. Upon remission several, but not all, molecular candidate biomarker levels recovered back to normal. CONCLUSION: The findings may indicate that microvascular damage and platelet deregulation hardly resolve upon remission, but apparently persist as disease-associated molecular signatures. This study presents local and systemic molecular alterations integrated in a model for UC pathomechanisms, potentially supporting the assessment of disease and remission states in UC patients.
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Obesity causes genetic instability, which plays a key-role in the etiology of cancer and aging. We investigated the impact of bariatric surgery (BS) on DNA repair, oxidative DNA damage, telomere lengths, alterations of antioxidant enzymes and, selected proteins which reflect inflammation. The study was realized with BS patients (n = 35). DNA damage, base oxidation, BER, and NER were measured before and 1 month and 6 months after surgery with the single-cell gel electrophoresis technique. SOD and GPx were quantified spectrophotometrically, malondealdehyde (MDA) was quantified by HPLC. Telomere lengths were determined with qPCR, and plasma proteome profiling was performed with high-resolution mass spectrophotometry. Six months after the operations, reduction of body weight by 27.5% was observed. DNA damage decreased after this period, this effect was paralleled by reduced formation of oxidized DNA bases, a decline in the MDA levels and of BER and NER, and an increase in the telomere lengths. The activities of antioxidant enzymes were not altered. Clear downregulation of certain proteins (CRP, SAA1) which reflect inflammation and cancer risks was observed. Our findings show that BS causes reduced oxidative damage of DNA bases, possibly as a consequence of reduction of inflammation and lipid peroxidation, and indicate that the surgery has beneficial long-term health effects.
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To investigate long COVID-19 syndrome (LCS) pathophysiology, we performed an exploratory study with blood plasma derived from three groups: 1) healthy vaccinated individuals without SARS-CoV-2 exposure; 2) asymptomatic recovered patients at least three months after SARS-CoV-2 infection and; 3) symptomatic patients at least 3 months after SARS-CoV-2 infection with chronic fatigue syndrome or similar symptoms, here designated as patients with long COVID-19 syndrome (LCS). Multiplex cytokine profiling indicated slightly elevated pro-inflammatory cytokine levels in recovered individuals in contrast to patients with LCS. Plasma proteomics demonstrated low levels of acute phase proteins and macrophage-derived secreted proteins in LCS. High levels of anti-inflammatory oxylipins including omega-3 fatty acids in LCS were detected by eicosadomics, whereas targeted metabolic profiling indicated high levels of anti-inflammatory osmolytes taurine and hypaphorine, but low amino acid and triglyceride levels and deregulated acylcarnitines. A model considering alternatively polarized macrophages as a major contributor to these molecular alterations is presented.
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A series of six highly lipophilic Cp-substituted molybdenocenes bearing different bioactive chelating ligands was synthesized and characterized by NMR spectroscopy, mass spectrometry and X-ray crystallography. In vitro experiments showed a greatly increased cytotoxic potency when compared to the non-Cp-substituted counterparts. In vivo experiments performed with the dichlorido precursor, (Ph2 C-Cp)2 MoCl2 and the inâ vitro most active complex, containing the thioflavone ligand, showed an inhibition of tumour growth. Proteomic studies on the same two compounds demonstrated a significant regulation of tubulin-associated and mitochondrial inner membrane proteins for both compounds and a strong metabolic effect of the thioflavone containing complex.
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Antineoplásicos , Neoplasias , Animales , Ratones , Estructura Molecular , Proteómica , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Antineoplásicos/química , Quelantes/química , Cristalografía por Rayos X , Ligandos , Línea Celular TumoralRESUMEN
Pathogen inactivation techniques for blood products have been implemented to optimize clinically safe blood components supply. The INTERCEPT system uses amotosalen together with ultraviolet light wavelength A (UVA) irradiation. Irradiation-induced inactivation of nucleic acids may actually be accompanied by modifications of chemically reactive polyunsaturated fatty acids known to be important mediators of platelet functions. Thus, here, we investigated eicosanoids and the related fatty acids released upon treatment and during storage of platelet concentrates for 7 days, complemented by the analysis of functional and metabolic consequences of these treatments. Metabolic and functional issues like glucose consumption, lactate formation, platelet aggregation, and clot firmness hardly differed between the two treatment groups. In contrast to gamma irradiation, here, we demonstrated that INTERCEPT treatment immediately caused new formation of trans-arachidonic acid isoforms, while 11-hydroxyeicosatetraenoic acid (11-HETE) and 15-HETE were increased and two hydroperoxyoctadecadienoic acid (HpODE) isoforms decreased. During further storage, these alterations remained stable, while the release of 12-lipoxygenase (12-LOX) products such as 12-HETE and 12-hydroxyeicosapentaenoic acid (12-HEPE) was further attenuated. In vitro synthesis of trans-arachidonic acid isoforms suggested that thiol radicals formed by UVA treatment may be responsible for the INTERCEPT-specific effects observed in platelet concentrates. It is reasonable to assume that UVA-induced molecules may have specific biological effects which need to be further investigated.
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Ácidos Araquidónicos , Ácidos Nucleicos , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacología , Araquidonato 12-Lipooxigenasa/metabolismo , Ácido Araquidónico/metabolismo , Ácido Araquidónico/farmacología , Ácidos Araquidónicos/metabolismo , Plaquetas , Glucosa/metabolismo , Ácidos Hidroxieicosatetraenoicos/metabolismo , Ácidos Hidroxieicosatetraenoicos/farmacología , Lactatos/metabolismo , Ácidos Nucleicos/metabolismo , Compuestos de Sulfhidrilo/metabolismoRESUMEN
Background: The age-related loss of muscle mass significantly contributes to the development of chronic diseases, loss of mobility and dependency on others, yet could be improved by an optimized lifestyle. Objective: The goal of this randomized controlled trial was to compare the influence of a habitual diet (CON) with either a diet containing the recommended protein intake (RP) or a high protein intake (HP), both with and without strength training, on the plasma proteome in older adults. Methods: One hundred and thirty-six women and men (65-85 years) were randomly assigned to three intervention groups. CON continued their habitual diet; participants of the HP and RP group consumed either high protein or standard foods. After 6 weeks of dietary intervention, HP and RP groups additionally started a strength training intervention twice per week for 8 weeks. Twenty-four hours dietary recalls were performed every 7-10 days. Body composition was assessed and blood taken. Plasma proteomics were assessed with LC-MS. Results: Participants of the HP group doubled their baseline protein intake from 0.80 ± 0.31 to 1.63 ± 0.36 g/kg BW/d; RP increased protein intake from 0.89 ± 0.28 to 1.06 ± 0.26 g/kg BW/d. The CON group kept the protein intake stable throughout the study. Combined exercise and HP initiated notable changes, resulting in a reduction in bodyfat and increased muscle mass. Proteomics analyses revealed 14 significantly affected proteins by HP diet, regulating innate immune system, lipid transport and blood coagulation, yet the additional strength training did not elicit further changes. Conclusions: Combined HP and resistance exercise in healthy older adults seem to induce favorable changes in the body composition. Changes in the plasma proteome due to the high protein diet point to a beneficial impact for the innate immune system, lipid transport and blood coagulation system, all of which are involved in chronic disease development. Clinical trial registration: The study was registered at ClinicalTrials.gov (NCT04023513).
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Target identification remains a critical challenge in inorganic drug discovery to deconvolute potential polypharmacology. Herein, we describe an improved approach to prioritize candidate protein targets based on a combination of dose-dependent chemoproteomics and treatment effects in living cancer cells for the rhenium tricarbonyl compound TRIP. Chemoproteomics revealed 89 distinct dose-dependent targets with concentrations of competitive saturation between 0.1 and 32â µM despite the broad proteotoxic effects of TRIP. Target-response networks revealed two highly probable targets of which the Fe-S cluster biogenesis factor NUBP2 was competitively saturated by free TRIP at nanomolar concentrations. Importantly, TRIP treatment led to a down-regulation of Fe-S cluster containing proteins and upregulated ferritin. Fe-S cluster depletion was further verified by assessing mitochondrial bioenergetics. Consequently, TRIP emerges as a first-in-class modulator of the scaffold protein NUBP2, which disturbs Fe-S cluster biogenesis at sub-cytotoxic concentrations in ovarian cancer cells.
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Proteínas Hierro-Azufre , Neoplasias Ováricas , Renio , Humanos , Femenino , Proteínas Hierro-Azufre/metabolismo , Mitocondrias/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Ferritinas/metabolismoRESUMEN
Background/aims: Concerning healthcare approaches, a paradigm change from reactive medicine to predictive approaches, targeted prevention, and personalisation of medical services is highly desirable. This raises demand for biomarker signatures that support the prediction and diagnosis of diseases, as well as monitoring strategies regarding therapeutic efficacy and supporting individualised treatments. New methodological developments should preferably rely on non-invasively sampled biofluids like sweat and tears in order to provide optimal compliance, reduce costs, and ensure availability of the biomaterial. Here, we have thus investigated the metabolic composition of human tears in comparison to finger sweat in order to find biofluid-specific marker molecules derived from distinct secretory glands. The comprehensive investigation of numerous biofluids may lead to the identification of novel biomarker signatures. Moreover, tear fluid analysis may not only provide insight into eye pathologies but may also be relevant for the prediction and monitoring of disease progression and/ or treatment of systemic disorders such as type 2 diabetes mellitus. Methods: Sweat and tear fluid were sampled from 20 healthy volunteers using filter paper and commercially available Schirmer strips, respectively. Finger sweat analysis has already been successfully established in our laboratory. In this study, we set up and evaluated methods for tear fluid extraction and analysis using high-resolution mass spectrometry hyphenated with liquid chromatography, using optimised gradients each for metabolites and eicosanoids. Sweat and tears were systematically compared using statistical analysis. As second approach, we performed a clinical pilot study with 8 diabetic patients and compared them to 19 healthy subjects. Results: Tear fluid was found to be a rich source for metabolic phenotyping. Remarkably, several molecules previously identified by us in sweat were found significantly enriched in tear fluid, including creatine or taurine. Furthermore, other metabolites such as kahweol and various eicosanoids were exclusively detectable in tears, demonstrating the orthogonal power for biofluid analysis in order to gain information on individual health states. The clinical pilot study revealed that many endogenous metabolites that have previously been linked to type 2 diabetes such as carnitine, tyrosine, uric acid, and valine were indeed found significantly up-regulated in tears of diabetic patients. Nicotinic acid and taurine were elevated in the diabetic cohort as well and may represent new biomarkers for diabetes specifically identified in tear fluid. Additionally, systemic medications, like metformin, bisoprolol, and gabapentin, were readily detectable in tears of patients. Conclusions: The high number of identified marker molecules found in tear fluid apparently supports disease development prediction, developing preventive approaches as well as tailoring individual patients' treatments and monitoring treatment efficacy. Tear fluid analysis may also support pharmacokinetic studies and patient compliance control. Supplementary Information: The online version contains supplementary material available at 10.1007/s13167-022-00272-7.
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Von Willebrand factor (VWF) and factor VIII (FVIII) circulate in a noncovalent complex in blood and promote primary hemostasis and clotting, respectively. A new VWF A1-domain binding aptamer, BT200, demonstrated good subcutaneous bioavailability and a long half-life in non-human primates. This first-in-human, randomized, placebo-controlled, doubleblind trial tested the hypothesis that BT200 is well tolerated and has favorable pharmacokinetic and pharmacodynamic effects in 112 volunteers. Participants received one of the following: a single ascending dose of BT200 (0.18-48 mg) subcutaneously, an intravenous dose, BT200 with concomitant desmopressin or multiple doses. Pharmacokinetics were characterized, and the pharmacodynamic effects were measured by VWF levels, FVIII clotting activity, ristocetin-induced aggregation, platelet function under high shear rates, and thrombin generation. The mean half-lives ranged from 7-12 days and subcutaneous bioavailability increased dose-dependently exceeding 55% for doses of 6-48 mg. By blocking free A1 domains, BT200 dose-dependently decreased ristocetin-induced aggregation, and prolonged collagen-adenosine diphosphate and shear-induced platelet plug formation times. However, BT200 also increased VWF antigen and FVIII levels 4-fold (P<0.001), without increasing VWF propeptide levels, indicating decreased VWF/FVIII clearance. This, in turn, increased thrombin generation and accelerated clotting. Desmopressin-induced VWF/FVIII release had additive effects on a background of BT200. Tolerability and safety were generally good, but exaggerated pharmacology was seen at saturating doses. This trial identified a novel mechanism of action for BT200: BT200 dose-dependently increases VWF/FVIII by prolonging half-life at doses well below those which inhibit VWF-mediated platelet function. This novel property can be exploited therapeutically to enhance hemostasis in congenital bleeding disorders.