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

iTAP, a novel iRhom interactor, controls TNF secretion by policing the stability of iRhom/TACE

The apical inflammatory cytokine TNF regulates numerous important biological processes including inflammation and cell death, and drives inflammatory diseases. TNF secretion requires TACE (also called ADAM17), which cleaves TNF from its transmembrane tether. The trafficking of TACE to the cell surface, and stimulation of its proteolytic activity, depends on membrane proteins, called iRhoms. To delineate how the TNF/TACE/iRhom axis is regulated, we performed an immunoprecipitation/mass spectrometry screen to identify iRhom-binding proteins. This identified a novel protein, that we name iTAP (iRhom Tail-Associated Protein) that binds to iRhoms, enhancing the cell surface stability of iRhoms and TACE, preventing their degradation in lysosomes. Depleting iTAP in primary human macrophages profoundly impaired TNF production and tissues from iTAP KO mice exhibit a pronounced depletion in active TACE levels. Our work identifies iTAP as a physiological regulator of TNF signalling and a novel target for the control of inflammation.info:eu-repo/semantics/publishedVersio

How high-resolution techniques enable reliable steroid identification and quantification

Due to possible matrix interferences and artefact generation during sample preparation, careful method validation is required for quantitative bioanalytical methods, especially for analytes that are only present in low concentrations. Using the identification and quantification of progesterone metabolite in the urine of newborns as an example, we show how modern high-resolution instruments can be used to verify analyte assignment and avoid pitfalls commonly encountered by the use of low-resolution instruments

A comprehensive urinary steroid analysis strategy using two-dimensional gas chromatography - time of flight mass spectrometry.

Steroids are key players in a high variety of physiological processes and are typically analyzed for the diagnosis of hormonal disorders. Due to their chemical and structural similarity many of these metabolites cannot be separated by conventional techniques such as liquid chromatography. Herein, we present an analysis strategy based on two dimensional gas chromatography (GC×GC) coupled to time-of-flight mass spectrometry (TOF MS) which demonstrates superior separation power and enables comprehensive screening of steroids. We show absolute quantitation of 40 steroids in human urine over three orders of magnitude with limits of detection ≤50 nM and the tentative identification of additional 30 steroids based on accurate mass, isotopic pattern analysis and spectral similarity matching to known steroids. The method displays excellent inter- and intra-day stability, repeatability and recovery and was validated for clinical routine analysis. Additionally, we demonstrate the potential of the approach for untargeted analysis of urinary steroids in mouse and rat

Quantification of Cytokines secreted by primary human cells using multiple reaction monitoring: evaluation of analytical parameters

Determination of secreted proteins provides highly valuable information about cell functions. While the typical methods for the determination of biologically relevant but low-abundant molecular species still relies on the use of specific antibodies, mass spectrometry-based methods are now gaining sufficient sensitivity to cope with such challenges as well. In the current study we have identified several cytokines and chemokines which were induced in primary human umbilical vein endothelial cells upon inflammatory activation. Based on the high-resolution mass spectrometry data obtained with a Q Exactive orbitrap, we built an MRM method to quantify the most relevant molecules selected from the screening experiment. All experimental data are available via ProteomeXchange, PXD002211/12, and Panorama, www.panoramaweb.org. Using nano-flow Chip-HPLC coupled to a 6490 triple-quadrupole MS for MRM analyses we achieved calibration curves covering a linear range of four orders of magnitude and detection limits in the low attomol per microliter concentration range. Carryover was consistently less than 0.005%, the accuracy was between 80% and 120%, and the median coefficient of variation for LC/MS was only 2.2%. When including the variance of quantification introduced by cell culture and digestion, the coefficient of variation was less than 20% for most peptides. With appropriate marker molecules we monitored typical variations introduced by cell culture caused by differences in cell numbers, proliferative states and cell death. As a result, here, we present a robust and efficient MRM-based assay for the accurate and sensitive determination of cytokines and chemokines representative for functional cell states and including comprehensive quality controls

Fetal Immunomodulatory Environment Following Cartilage Injury—The Key to CARTILAGE Regeneration?

Fetal cartilage fully regenerates following injury, while in adult mammals cartilage injury leads to osteoarthritis (OA). Thus, in this study, we compared the in vivo injury response of fetal and adult ovine articular cartilage histologically and proteomically to identify key factors of fetal regeneration. In addition, we compared the secretome of fetal ovine mesenchymal stem cells (MSCs) in vitro with injured fetal cartilage to identify potential MSC-derived therapeutic factors. Cartilage injury caused massive cellular changes in the synovial membrane, with macrophages dominating the fetal, and neutrophils the adult, synovial cellular infiltrate. Correspondingly, proteomics revealed differential regulation of pro- and anti-inflammatory mediators and growth-factors between adult and fetal joints. Neutrophil-related proteins and acute phase proteins were the two major upregulated protein groups in adult compared to fetal cartilage following injury. In contrast, several immunomodulating proteins and growth factors were expressed significantly higher in the fetus than the adult. Comparison of the in vitro MSCs proteome with the in vivo fetal regenerative signature revealed shared upregulation of 17 proteins, suggesting their therapeutic potential. Biomimicry of the fetal paracrine signature to reprogram macrophages and modulate inflammation could be an important future research direction for developing novel therapeutics

Comprehensive Assessment of Proteins Regulated by Dexamethasone Reveals Novel Effects in Primary Human Peripheral Blood Mononuclear Cells

Inflammation is a physiological process involved in many diseases. Monitoring proteins involved in regulatory effects may help to improve our understanding of inflammation. We have analyzed proteome alterations induced in peripheral blood mononuclear cells (PBMCs) upon inflammatory activation in great detail using high-resolution mass spectrometry. Moreover, the activated cells were treated with dexamethasone to investigate their response to this antiphlogistic drug. From a total of 6886 identified proteins, 469 proteins were significantly regulated upon inflammatory activation. Data are available via ProteomeXchange with identifiers PXD001415–23. Most of these proteins were counter-regulated by dexamethasone, with some exceptions concerning members of the interferon-induced protein family. To confirm some of these results, we performed targeted MRM analyses of selected peptides. The inflammation-induced upregulation of proteins such as IL-1β, IL-6, CXCL2, and GROα was confirmed, however, with strong quantitative interindividual differences. Furthermore, the inability of dexamethasone to downregulate inflammation-induced proteins such as PTX3 and TSG6 was clearly demonstrated. In conclusion, the relation of cell function as well as drug-induced modulation thereof was successfully mapped to proteomes, suggesting targeted analysis as a novel and powerful drug evaluation method. Although most consequences of dexamethasone were found to be compatible with the expected mode of action, some unexpected but significant observations may be related to adverse effects

MOESM4 of Proteomic profiling identifies markers for inflammation-related tumor–fibroblast interaction

Additional file 4. Table S3. GO-term enrichment analysis based on biological process

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.status: publishe

Amelogenin peptide analyses reveal female leadership in Copper Age Iberia (c. 2900–2650 BC)

Abstract 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