Memory effects of prior subculture may impact the quality of multiomic perturbation profiles.

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.

Systematic Optimization of Automated Phosphopeptide Enrichment for High-Sensitivity Phosphoproteomics.

Improving coverage, robustness, and sensitivity is crucial for routine phosphoproteomics analysis by single-shot liquid chromatography-tandem mass spectrometry (LC-MS/MS) from minimal peptide inputs. Here, we systematically optimized key experimental parameters for automated on-bead phosphoproteomics sample preparation with a focus on low-input samples. Assessing the number of identified phosphopeptides, enrichment efficiency, site localization scores, and relative enrichment of multiply-phosphorylated peptides pinpointed critical variables influencing the resulting phosphoproteome. Optimizing glycolic acid concentration in the loading buffer, percentage of ammonium hydroxide in the elution buffer, peptide-to-beads ratio, binding time, sample, and loading buffer volumes allowed us to confidently identify >16,000 phosphopeptides in half-an-hour LC-MS/MS on an Orbitrap Exploris 480 using 30 µg of peptides as starting material. Furthermore, we evaluated how sequential enrichment can boost phosphoproteome coverage and showed that pooling fractions into a single LC-MS/MS analysis increased the depth. We also present an alternative phosphopeptide enrichment strategy based on stepwise addition of beads thereby boosting phosphoproteome coverage by 20%. Finally, we applied our optimized strategy to evaluate phosphoproteome depth with the Orbitrap Astral MS using a cell dilution series and were able to identify >32,000 phosphopeptides from 0.5 million HeLa cells in half-an-hour LC-MS/MS using narrow-window data-independent acquisition (nDIA).

Plasma Instead of Serum Avoids Critical Confounding of Clinical Metabolomics Studies by Platelets.

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.

Dichotomous Responses to Chronic Fetal Hypoxia Lead to a Predetermined Aging Phenotype.

Hypoxia-induced intrauterine growth restriction increases the risk for cardiovascular, renal, and other chronic diseases in adults, representing thus a major public health problem. Still, not much is known about the fetal mechanisms that predispose these individuals to disease. Using a previously validated mouse model of fetal hypoxia and bottom-up proteomics, we characterize the response of the fetal kidney to chronic hypoxic stress. Fetal kidneys exhibit a dichotomous response to chronic hypoxia, comprising on the one hand cellular adaptations that promote survival (glycolysis, autophagy, and reduced DNA and protein synthesis), but on the other processes that induce a senescence-like phenotype (infiltration of inflammatory cells, DNA damage, and reduced proliferation). Importantly, chronic hypoxia also reduces the expression of the antiaging proteins klotho and Sirt6, a mechanism that is evolutionary conserved between mice and humans. Taken together, we uncover that predetermined aging during fetal development is a key event in chronic hypoxia, establishing a solid foundation for Barker's hypothesis of fetal programming of adult diseases. This phenotype is associated with a characteristic biomarker profile in tissue and serum samples, exploitable for detecting and targeting accelerated aging in chronic hypoxic human diseases.

Proteomic identification of a marker signature for MAPKi resistance in melanoma.

MAPK inhibitors (MAPKi) show outstanding clinical response rates in melanoma patients harbouring BRAF mutations, but resistance is common. The ability of melanoma cells to switch from melanocytic to mesenchymal phenotypes appears to be associated with therapeutic resistance. High-throughput, subcellular proteome analyses and RNAseq on two panels of primary melanoma cells that were either sensitive or resistant to MAPKi revealed that only 15 proteins were sufficient to distinguish between these phenotypes. The two proteins with the highest discriminatory power were PTRF and IGFBP7, which were both highly upregulated in the mesenchymal-resistant cells. Proteomic analysis of CRISPR/Cas-derived PTRF knockouts revealed targets involved in lysosomal activation, endocytosis, pH regulation, EMT, TGFß signalling and cell migration and adhesion, as well as a significantly reduced invasive index and ability to form spheres in 3D culture. Overexpression of PTRF led to MAPKi resistance, increased cell adhesion and sphere formation. In addition, immunohistochemistry of patient samples showed that PTRF expression levels were a significant biomarker of poor progression-free survival, and IGFBP7 levels in patient sera were shown to be higher after relapse.

Exploring the Potential of Metal-Based Candidate Drugs as Modulators of the Cytoskeleton.

During recent years, accumulating evidence suggested that metal-based candidate drugs are promising modulators of cytoskeletal and cytoskeleton-associated proteins. This was substantiated by the identification and validation of actin, vimentin and plectin as targets of distinct ruthenium(II)- and platinum(II)-based modulators. Despite this, structural information about molecular interaction is scarcely available. Here, we compile the scattered reports about metal-based candidate molecules that influence the cytoskeleton, its associated proteins and explore their potential to interfere in cancer-related processes, including proliferation, invasion and the epithelial-to-mesenchymal transition. Advances in this field depend crucially on determining binding sites and on gaining comprehensive insight into molecular drug-target interactions. These are key steps towards establishing yet elusive structure-activity relationships.

Highly Cytotoxic Molybdenocenes with Strong Metabolic Effects Inhibit Tumour Growth in Mice.

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 in 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.

RNase P without RNA: identification and functional reconstitution of the human mitochondrial tRNA processing enzyme.

tRNAs are synthesized as immature precursors, and on their way to functional maturity, extra nucleotides at their 5' ends are removed by an endonuclease called RNase P. All RNase P enzymes characterized so far are composed of an RNA plus one or more proteins, and tRNA 5' end maturation is considered a universal ribozyme-catalyzed process. Using a combinatorial purification/proteomics approach, we identified the components of human mitochondrial RNase P and reconstituted the enzymatic activity from three recombinant proteins. We thereby demonstrate that human mitochondrial RNase P is a protein enzyme that does not require a trans-acting RNA component for catalysis. Moreover, the mitochondrial enzyme turns out to be an unexpected type of patchwork enzyme, composed of a tRNA methyltransferase, a short-chain dehydrogenase/reductase-family member, and a protein of hitherto unknown functional and evolutionary origin, possibly representing the enzyme's metallonuclease moiety. Apparently, animal mitochondria lost the seemingly ubiquitous RNA world remnant after reinventing RNase P from preexisting components.

Activation of autophagy triggers mitochondrial loss and changes acetylation profile relevant for mechanotransduction in bladder cancer cells.

Bladder cells are constantly exposed to multiple xenobiotics and bioactive metabolites. In addition to this challenging chemical environment, they are also exposed to shear stress originating from urine and interstitial fluids. Hence, physiological function of bladder cells relies on a high biochemical and biomechanical adaptive competence, which, in turn, is largely supported via autophagy-related mechanisms. As a negative side of this plasticity, bladder cancer cells are known to adapt readily to chemotherapeutic programs. At the molecular level, autophagy was described to support resistance against pharmacological treatments and to contribute to the maintenance of cell structure and metabolic competence. In this study, we enhanced autophagy with rapamycin (1-100 nM) and assessed its effects on the motility of bladder cells, as well as the capability to respond to shear stress. We observed that rapamycin reduced cell migration and the mechanical-induced translocation potential of Krüppel-like transcription factor 2 (KLF2). These effects were accompanied by a rearrangement of cytoskeletal elements and mitochondrial loss. In parallel, intracellular acetylation levels were decreased. Mechanistically, inhibition of the NAD + -dependent deacetylase sirtuin-1 (SIRT1) with nicotinamide (NAM; 0.1-5 mM) restored acetylation levels hampered by rapamycin and cell motility. Taken together, we described the effects of rapamycin on cytoskeletal elements crucial for mechanotransduction and the dependency of these changes on the mitochondrial turnover caused by autophagy activation. Additionally, we could show that targeted metabolic intervention could revert the outcome of autophagy activation, reinforcing the idea that bladder cells can easily adapt to multiple xenobiotics and circumvent in this way the effects of single chemicals.

Exposure to dietary fatty acids oleic and palmitic acid alters structure and mechanotransduction of intestinal cells in vitro.

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.

Corazonin signaling integrates energy homeostasis and lunar phase to regulate aspects of growth and sexual maturation in Platynereis.

The molecular mechanisms by which animals integrate external stimuli with internal energy balance to regulate major developmental and reproductive events still remain enigmatic. We investigated this aspect in the marine bristleworm, Platynereis dumerilii, a species where sexual maturation is tightly regulated by both metabolic state and lunar cycle. Our specific focus was on ligands and receptors of the gonadotropin-releasing hormone (GnRH) superfamily. Members of this superfamily are key in triggering sexual maturation in vertebrates but also regulate reproductive processes and energy homeostasis in invertebrates. Here we show that 3 of the 4 gnrh-like (gnrhl) preprohormone genes are expressed in specific and distinct neuronal clusters in the Platynereis brain. Moreover, ligand-receptor interaction analyses reveal a single Platynereis corazonin receptor (CrzR) to be activated by CRZ1/GnRHL1, CRZ2/GnRHL2, and GnRHL3 (previously classified as AKH1), whereas 2 AKH-type hormone receptors (GnRHR1/AKHR1 and GnRHR2/AKHR2) respond only to a single ligand (GnRH2/GnRHL4). Crz1/gnrhl1 exhibits a particularly strong up-regulation in sexually mature animals, after feeding, and in specific lunar phases. Homozygous crz1/gnrhl1 knockout animals exhibit a significant delay in maturation, reduced growth, and attenuated regeneration. Through a combination of proteomics and gene expression analysis, we identify enzymes involved in carbohydrate metabolism as transcriptional targets of CRZ1/GnRHL1 signaling. Our data suggest that Platynereis CRZ1/GnRHL1 coordinates glycoprotein turnover and energy homeostasis with growth and sexual maturation, integrating both metabolic and developmental demands with the worm's monthly cycle.

Probabilistic quotient's work and pharmacokinetics' contribution: countering size effect in metabolic time series measurements.

Metabolomic time course analyses of biofluids are highly relevant for clinical diagnostics. However, many sampling methods suffer from unknown sample sizes, commonly known as size effects. This prevents absolute quantification of biomarkers. Recently, several mathematical post acquisition normalization methods have been developed to overcome these problems either by exploiting already known pharmacokinetic information or by statistical means. Here we present an improved normalization method, MIX, that combines the advantages of both approaches. It couples two normalization terms, one based on a pharmacokinetic model (PKM) and the other representing a popular statistical approach, probabilistic quotient normalization (PQN), in a single model. To test the performance of MIX, we generated synthetic data closely resembling real finger sweat metabolome measurements. We show that MIX normalization successfully tackles key weaknesses of the individual strategies: it (i) reduces the risk of overfitting with PKM, and (ii), contrary to PQN, it allows to compute sample volumes. Finally, we validate MIX by using real finger sweat as well as blood plasma metabolome data and demonstrate that MIX allows to better and more robustly correct for size effects. In conclusion, the MIX method improves the reliability and robustness of quantitative biomarker detection in finger sweat and other biofluids, paving the way for biomarker discovery and hypothesis generation from metabolomic time course data.

Packed red blood cells inhibit T-cell activation via ROS-dependent signaling pathways.

Numerous observations indicate that red blood cells (RBCs) affect T-cell activation and proliferation. We have studied effects of packed RBCs (PRBCs) on T-cell receptor (TCR) signaling and the molecular mechanisms whereby (P)RBCs modulate T-cell activation. In line with previous reports, PRBCs attenuated the expression of T-cell activation markers CD25 and CD69 upon costimulation via CD3/CD28. In addition, T-cell proliferation and cytokine expression were markedly reduced when T-cells were stimulated in the presence of PRBCs. Inhibitory activity of PRBCs required direct cell-cell contact and intact PRBCs. The production of activation-induced cellular reactive oxygen species, which act as second messengers in T-cells, was completely abrogated to levels of unstimulated T-cells in the presence of PRBCs. Phosphorylation of the TCR-related zeta chain and thus proximal TCR signal transduction was unaffected by PRBCs, ruling out mechanisms based on secreted factors and steric interaction restrictions. In large part, downstream signaling events requiring reactive oxygen species for full functionality were affected, as confirmed by an untargeted MS-based phosphoproteomics approach. PRBCs inhibited T-cell activation more efficiently than treatment with 1 mM of the antioxidant N-acetyl cysteine. Taken together, our data imply that inflammation-related radical reactions are modulated by PRBCs. These immunomodulating effects may be responsible for clinical observations associated with transfusion of PRBCs.

A novel nanobody as therapeutics target for EGFR-positive colorectal cancer therapy: exploring the effects of the nanobody on SW480 cells using proteomics approach.

BACKGROUND: The epidermal growth factor receptor (EGFR) overexpression is found in metastatic colorectal cancer (mCRC). Targeted molecular therapies such as monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKI) are becoming more precise, targeting specifically for cancer therapeutics. However, there are adverse effects of currently available anti-EGFR drugs, including drug-resistant and side effects. Nanobodies can overcome these limitations. Our previous study has found that cell-penetrable nanobodies targeted at EGFR-tyrosine kinase were significantly reduced EGFR-positive lung cancer cells viability and proliferation. The aim of the present study was to determine the effect of cell-penetrable nanobody (R9VH36) on cell viability and proteomic profile in EGFR-positive human colorectal cancer cell lines. METHODS: The human colorectal carcinoma cell line (SW480) was treated with R9VH36, compared with gefitinib. Cell viability was monitored using the MTT cell viability assay. The proteomic profiling was analyzed by LC-MS/MS . RESULTS: The half-maximal inhibitory concentration (IC50) values determined for R9VH36 and gefitinib against SW480 were 527 ± 0.03 nM and 13.31 ± 0.02 µM, respectively. Moreover, both the gefitinib-treated group and nanobody-treated group had completely different proteome profiles. A total 6626 differentially expressed proteins were identified. PCA analysis revealed different proteome profiling in R9VH36 experiment. There were 8 proteins in R9VH36 that significantly exhibited opposite expression directions when compared to gefitinib. These proteins are involved in DNA-damage checkpoint processes. CONCLUSION: The proteomics explored those 6,626 proteins had different expressions between R9VH36 and gefitinib. There were 8 proteins in R9VH36 exhibited opposite expression direction when comparing to gefitinib. Our findings suggest that R9VH36 has the potential to be an alternative remedy for treating EGFR-positive colon cancer.

Daily Caffeine Intake Induces Concentration-Dependent Medial Temporal Plasticity in Humans: A Multimodal Double-Blind Randomized Controlled Trial.

Caffeine is commonly used to combat high sleep pressure on a daily basis. However, interference with sleep-wake regulation could disturb neural homeostasis and insufficient sleep could lead to alterations in human gray matter. Hence, in this double-blind, randomized, cross-over study, we examined the impact of 10-day caffeine (3 × 150 mg/day) on human gray matter volumes (GMVs) and cerebral blood flow (CBF) by fMRI MP-RAGE and arterial spin-labeling sequences in 20 habitual caffeine consumers, compared with 10-day placebo (3 × 150 mg/day). Sleep pressure was quantified by electroencephalographic slow-wave activity (SWA) in the previous nighttime sleep. Nonparametric voxel-based analyses revealed a significant reduction in GMV in the medial temporal lobe (mTL) after 10 days of caffeine intake compared with 10 days of placebo, voxel-wisely adjusted for CBF considering the decreased perfusion after caffeine intake compared with placebo. Larger GMV reductions were associated with higher individual concentrations of caffeine and paraxanthine. Sleep SWA was, however, neither different between conditions nor associated with caffeine-induced GMV reductions. Therefore, the data do not suggest a link between sleep depth during daily caffeine intake and changes in brain morphology. In conclusion, daily caffeine intake might induce neural plasticity in the mTL depending on individual metabolic processes.

The Challenge of Classifying Metastatic Cell Properties by Molecular Profiling Exemplified with Cutaneous Melanoma Cells and Their Cerebral Metastasis from Patient Derived Mouse Xenografts.

The prediction of metastatic properties from molecular analyses still poses a major challenge. Here we aimed at the classification of metastasis-related cell properties by proteome profiling making use of cutaneous and brain-metastasizing variants from single melanomas sharing the same genetic ancestry. Previous experiments demonstrated that cultured cells derived from these xenografted variants maintain a stable phenotype associated with a differential metastatic behavior: The brain metastasizing variants produce more spontaneous micro-metastases than the corresponding cutaneous variants. Four corresponding pairs of cutaneous and metastatic cells were obtained from four individual patients, resulting in eight cell-lines presently investigated. Label free proteome profiling revealed significant differences between corresponding pairs of cutaneous and cerebellar metastases from the same patient. Indeed, each brain metastasizing variant expressed several apparently metastasis-associated proteomic alterations as compared with the corresponding cutaneous variant. Among the differentially expressed proteins we identified cell adhesion molecules, immune regulators, epithelial to mesenchymal transition markers, stem cell markers, redox regulators and cytokines. Similar results were observed regarding eicosanoids, considered relevant for metastasis, such as PGE2 and 12-HETE. Multiparametric morphological analysis of cells also revealed no characteristic alterations associated with the cutaneous and brain metastasis variants. However, no correct classification regarding metastatic potential was yet possible with the present data. We thus concluded that molecular profiling is able to classify cells according to known functional categories but is not yet able to predict relevant cell properties emerging from networks consisting of many interconnected molecules. The presently observed broad diversity of molecular patterns, irrespective of restricting to one tumor type and two main classes of metastasis, highlights the important need to develop meta-analysis strategies to predict cell properties from molecular profiling data. Such base knowledge will greatly support future individualized precision medicine approaches.

Prediction of Neurological Recovery After Cardiac Arrest Using Neurofilament Light Chain is Improved by a Proteomics-Based Multimarker Panel.

BACKGROUND: Continuous advances in resuscitation care have increased survival, but the rate of favorable neurological outcome remains low. We have shown the usefulness of proteomics in identifying novel biomarkers to predict neurological outcome. Neurofilament light chain (NfL), a marker of axonal damage, has since emerged as a promising single marker. The aim of this study was to assess the predictive value of NfL in comparison with and in addition to our established model. METHODS: NfL was measured in plasma samples drawn at 48 h after cardiac arrest using single-molecule assays. Neurological function was recorded on the cerebral performance category (CPC) scale at discharge from the intensive care unit and after 6 months. The ability to predict a dichotomized outcome (CPC 1-2 vs. 3-5) was assessed with receiver operating characteristic (ROC) curves. RESULTS: Seventy patients were included in this analysis, of whom 21 (30%) showed a favorable outcome (CPC 1-2), compared with 49 (70%) with an unfavorable outcome (CPC 3-5) at discharge. NfL increased from CPC 1 to 5 (16.5 pg/ml to 641 pg/ml, p < 0.001). The addition of NfL to the existing model improved it significantly (Wald test, p < 0.001), and the combination of NfL with a multimarker model showed high areas under the ROC curve (89.7% [95% confidence interval 81.7-97.7] at discharge and 93.7% [88.2-99.2] at 6 months) that were significantly greater than each model alone. CONCLUSIONS: The combination of NfL with other plasma and clinical markers is superior to that of either model alone and achieves high areas under the ROC curve in this relatively small sample.

An Anticancer Rhenium Tricarbonyl Targets Fe-S Cluster Biogenesis in Ovarian Cancer Cells.

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 32 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.

MULTIOMIC PATTERNS IN BODY FLUIDS: TECHNOLOGICAL CHALLENGE WITH A GREAT POTENTIAL TO IMPLEMENT THE ADVANCED PARADIGM OF 3P MEDICINE.

Liquid biopsy (LB) is defined as a sample of any of body fluids (blood, saliva, tear fluid, urine, sweat, amniotic, cerebrospinal and pleural fluids, cervicovaginal secretion, and wound efflux, amongst others), which can be ex vivo analysed to detect and quantity the target(s) of interest. LB represents diagnostic approach relevant for organ-specific changes and systemic health conditions including both manifested diseases and their prestages such as suboptimal health. Further, experts emphasise that DNA-based analysis alone does not provide sufficient information for optimal diagnostics and effective treatments. Consequently, of great scientific and clinical utility are molecular patterns detected by hybrid technologies such as metabolomic tools and molecular imaging. Future proposed strategies utilise multiomic pillars (generally genome, tanscriptome, proteome, metabolome, epigenome, radiome, and microbiome), system-biological approach, and multivariable algorithms for diagnostic, prognostic, and therapeutic purposes. Current article analyses pros and cons of the mass spectrometry-based technologies, provides eminent examples of a success story "from discovery to clinical application," and demonstrates a "road-map" for the technology-driven paradigm change from reactive to predictive, preventive and personalised medical services as the medicine of the future benefiting the patient and healthcare at large. © 2019 The Authors. Mass Spectrometry Reviews published by John Wiley & Sons Ltd. Mass Spec Rev.

Exploring the dermotoxicity of the mycotoxin deoxynivalenol: combined morphologic and proteomic profiling of human epidermal cells reveals alteration of lipid biosynthesis machinery and membrane structural integrity relevant for skin barrier function.

Deoxynivalenol (vomitoxin, DON) is a secondary metabolite produced by Fusarium spp. fungi and it is one of the most prevalent mycotoxins worldwide. Crop infestation results not only in food and feed contamination, but also in direct dermal exposure, especially during harvest and food processing. To investigate the potential dermotoxicity of DON, epidermoid squamous cell carcinoma cells A431 were compared to primary human neonatal keratinocytes (HEKn) cells via proteome/phosphoproteome profiling. In A431 cells, 10 µM DON significantly down-regulated ribosomal proteins, as well as mitochondrial respiratory chain elements (OXPHOS regulation) and transport proteins (TOMM22; TOMM40; TOMM70A). Mitochondrial impairment was reflected in altered metabolic competence, apparently combined with interference of the lipid biosynthesis machinery. Functional effects on the cell membrane were confirmed by live cell imaging and membrane fluidity assays (0.1-10 µM DON). Moreover, a common denominator for both A431 and HEKn cells was a significant downregulation of the squalene synthase (FDFT1). In sum, proteome alterations could be traced back to the transcription factor Klf4, a crucial regulator of skin barrier function. Overall, these results describe decisive molecular events sustaining the capability of DON to impair skin barrier function. Proteome data generated in the study are fully accessible via ProteomeXchange with the accession numbers PXD011474 and PXD013613.