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Background The function of skin mast cells has been well documented in IgE-mediated allergic reactions, whereas other mast cell functions are poorly defined. This study aimed at identifying novel mast cell proteins by proteome analysis of primary human skin mast cells. Methods The proteome of skin mast cells was compared to other cell types and analyzed using bioinformatics. The expression and function of two proteins hitherto not described in skin mast cells was investigated in isolated mast cells as well as in mast cells in situ. Results Within the mast cell proteome, we identified 49 highly expressed proteins previously not described in mast cells; 21 of these proteins were found to be selectively expressed in mast cells. Two proteins, the neural cell adhesion molecule L1 and dipeptidyl peptidase 4, were further studied. L1 was found to be highly expressed in mast cells in normal, psoriasis, and mastocytosis skin. Dipeptidyl peptidase 4 was found to be expressed in mast cells in normal, psoriasis, and mastocytosis skin as well as in bone marrow mast cells in patients with systemic mastocytosis. In normal skin, mast cells were identified as a major source of dipeptidyl peptidase 4 and we also found that skin mast cells and fibroblasts secrete an active form of this enzyme. Conclusions In a systematic proteomics approach we identified two novel mast cell proteins potentially relevant to skin homeostasis: neural cell adhesion molecule L1 and dipeptidyl peptidase 4. [ABSTRACT FROM AUTHOR]

The pro-peptide of transforming growth factor alpha (proTGFalpha) was recently found in hepatocyte nuclei preparing for DNA replication, which suggests a role of nuclear proTGFalpha for mitogenic signalling. This study investigates whether the nuclear occurrence of the pro-peptide is involved in the altered growth regulation of (pre)malignant hepatocytes. In human hepatocarcinogenesis, the incidence of proTGFalpha-positive and replicating nuclei gradually increased from normal liver, to dysplastic nodules, to hepatocellular carcinoma. ProTGFalpha-positive nuclei almost always were in DNA synthesis. Also, in rat hepatocarcinogenesis, proTGFalpha-positive nuclei occurred in (pre)malignant hepatocytes at significantly higher incidences than in unaltered hepatocytes. For functional studies unaltered (GSTp(-)) and premalignant (GSTp(+)) rat hepatocytes were isolated by collagenase perfusion and cultivated. Again, DNA synthesis occurred almost exclusively in proTGFalpha-positive nuclei. GSTp(+) hepatocytes showed an approximately 3-fold higher frequency of proTGFalpha-positive nuclei and DNA replication than GSTp(-) cells. Treatment of cultures with the mitogen cyproterone acetate (CPA) elevated the incidence of proTGFalpha-positive nuclei and DNA synthesis in parallel. Conversely, transforming growth factor beta1 (TGFbeta1) lowered both. These effects of CPA and TGFbeta1 were significantly more pronounced in GSTp(+) than in GSTp(-) hepatocytes. In conclusion, nuclear translocation of proTGFalpha increases in the course of hepatocarcinogenesis and appears to be involved in the inherent growth advantage of (pre)malignant hepatocytes. [ABSTRACT FROM AUTHOR]

Lipid-containing vacuoles in microglia were discovered more than one hundred years ago in the brain of patients showing neurodegenerative processes. Recently, molecular-biological studies demonstrated specific changes in lipid-metabolism related to neurodegeneration. Despite that already Alzheimer described a distinct glia phenotype having large, lipid-containing vacuoles (Gitterzellen), little is known about how microglia convert lipid metabolites into a vacuolated phenotype. We studied the impact of liver-derived, insoluble, lipid-enriched nanoparticles (Lef-NP) ( ~ 20 nm) and of ceramide-coated Percoll-nanoparticles (Cer-NP) ( ~ 20 nm) on vacuolization in microglia. Lipidomic analysis of Lef-NP revealed numerous distinct lipids, including pro-inflammatory ceramides, which are enriched in the brain of Alzheimer patients. Video microscopy revealed that hepatocyte-derived Lef-NP and Cer-NP enhanced macropinocytosis, followed by macropinosome swelling and formation of the Gitterzellen phenotype. Neither ceramide nor Percoll-nanoparticles induced Gitterzellen-formation. Electron-tomography visualized membrane contact-sites between nanoparticle-loaded endosomes, endoplasmic reticulum cisternae and mitochondria. Suppression of lipid-nanoparticle-induced Gitterzellen-formation by amiloride, which supresses macropinocytosis, and bafilomycin A, an endosomal acidification inhibitor, further confirmed a pinocytotic pathway in Gitterzellen-formation. Bafilomycin A also reversed Gitterzellen to a ramified microglia phenotype. Our experimental findings suggest that lipid-nanoparticles but not emulsified lipids provoke vacuolization in microglia, and provide a simple in-vitro model for a pathogenic process taking years in the human brain., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Biological sex is key information for archeological and forensic studies, which can be determined by proteomics. However, the lack of a standardized approach for fast and accurate sex identification currently limits the reach of proteomics applications. Here, we introduce a streamlined mass spectrometry (MS)-based workflow for the determination of biological sex using human dental enamel. Our approach builds on a minimally invasive sampling strategy by acid etching, a rapid online liquid chromatography (LC) gradient coupled to a high-resolution parallel reaction monitoring (PRM) assay allowing for a throughput of 200 samples per day (SPD) with high quantitative performance enabling confident identification of both males and females. Additionally, we developed a streamlined data analysis pipeline and integrated it into a Shiny interface for ease of use. The method was first developed and optimized using modern teeth and then validated in an independent set of deciduous teeth of known sex. Finally, the assay was successfully applied to archeological material, enabling the analysis of over 300 individuals. We demonstrate unprecedented performance and scalability, speeding up MS analysis by 10-fold compared to conventional proteomics-based sex identification methods. This work paves the way for large-scale archeological or forensic studies enabling the investigation of entire populations rather than focusing on individual high-profile specimens. Data are available via ProteomeXchange with the identifier PXD049326.

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, R 2 = 0.991), whereas lipid mediators ( n = 83, R 2 = 0.906) and proteins ( n = 322, R 2 = 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.