Sources of Variance in Human Tear Proteomic Samples: Statistical Evaluation, Quality Control, Normalization, and Biological Insight.

Human tear fluid contains numerous compounds, which are present in highly variable amounts owing to the dynamic and multipurpose functions of tears. A better understanding of the level and sources of variance is essential for determining the functions of the different tear components and the limitations of tear samples as a potential biomarker source. In this study, a quantitative proteomic method was used to analyze variations in the tear protein profiles of healthy volunteers. High day-to-day and inter-eye personal variances were observed in the tear volumes, protein content, and composition of the tear samples. Several normalization and outlier exclusion approaches were evaluated to decrease variances. Despite the intrapersonal variances, statistically significant differences and cluster analysis revealed that proteome profile and immunoglobulin composition of tear fluid present personal characteristics. Using correlation analysis, we could identify several correlating protein clusters, mainly related to the source of the proteins. Our study is the first attempt to achieve more insight into the biochemical background of human tears by statistical evaluation of the experimentally observed dynamic behavior of the tear proteome. As a pilot study for determination of personal protein profiles of the tear fluids of individual patients, it contributes to the application of this noninvasively collectible body fluid in personal medicine.

Synthesis and Antiproliferative Activity of Steroidal Diaryl Ethers.

Novel 13α-estrone derivatives have been synthesized via direct arylation of the phenolic hydroxy function. Chan-Lam couplings of arylboronic acids with 13α-estrone as a nucleophilic partner were carried out under copper catalysis. The antiproliferative activities of the newly synthesized diaryl ethers against a panel of human cancer cell lines (A2780, MCF-7, MDA-MB 231, HeLa, SiHa) were investigated by means of MTT assays. The quinoline derivative displayed substantial antiproliferative activity against MCF-7 and HeLa cell lines with low micromolar IC50 values. Disturbance of tubulin polymerization has been confirmed by microplate-based photometric assay. Computational calculations reveal significant interactions of the quinoline derivative with the taxoid binding site of tubulin.

Diet-Induced Hypercholesterolemia Leads to Cardiac Dysfunction and Alterations in the Myocardial Proteome.

Elevated blood cholesterol is a major risk factor for coronary heart disease. Moreover, direct effects on the myocardium also contribute to the adverse effects of hypercholesterolemia. Here, we investigated the effect of hypercholesterolemia on the cardiac proteome. Male Wistar rats were fed with a laboratory rodent chow supplemented with 2% cholesterol for 8 weeks to induce hypercholesterolemia. The protein expression data obtained from the proteomic characterization of left ventricular samples from normo- and hypercholesterolemic animals were subjected to gene ontology (GO) and protein interaction analyses. Elevated circulating cholesterol levels were accompanied by diastolic dysfunction in cholesterol-fed rats. The proteomic characterization of left ventricular samples revealed altered expression of 45 proteins due to hypercholesterolemia. Based on the Gene Ontology analysis, hypercholesterolemia was associated with disturbed expression of cytoskeletal and contractile proteins. Beta-actin was downregulated in the hypercholesterolemic myocardium, and established a prominent hub of the protein interaction network. Analysis of the unfiltered dataset revealed concordant downregulated expression patterns in proteins associated with the arrangement of the contractile system (e.g., cardiac-specific troponins and myosin complex), and in subunits of the mitochondrial respiratory chain. We conclude that the observed changes in the cardiac proteome may contribute to the development of diastolic dysfunction in hypercholesterolemia.

Indoleamine 2,3-Dioxygenase Cannot Inhibit Chlamydia trachomatis Growth in HL-60 Human Neutrophil Granulocytes.

Aims: Neutrophil granulocytes are the major cells involved in Chlamydia trachomatis (C. trachomatis)-mediated inflammation and histopathology. A key protein in human intracellular antichlamydial defense is the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) which limits the growth of the tryptophan auxotroph Chlamydia. Despite its importance, the role of IDO in the intracellular defense against Chlamydia in neutrophils is not well characterized. Methods: Global gene expression screen was used to evaluate the effect of C. trachomatis serovar D infection on the transcriptome of human neutrophil granulocytes. Tryptophan metabolite concentrations in the Chlamydia-infected and/or interferon-gamma (IFNG)-treated neutrophils were measured by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Results: Our results indicate that the C. trachomatis infection had a major impact on neutrophil gene expression, inducing 1,295 genes and repressing 1,510 genes. A bioinformatics analysis revealed that important factors involved in the induction of neutrophil gene expression were the interferon-related transcription factors such as IRF1-5, IRF7-9, STAT2, ICSB, and ISGF3. One of the upregulated genes was ido1, a known infection- and interferon-induced host gene. The tryptophan-degrading activity of IDO1 was not induced significantly by Chlamydia infection alone, but the addition of IFNG greatly increased its activity. Despite the significant IDO activity in IFNG-treated cells, C. trachomatis growth was not affected by IFNG. This result was in contrast to what we observed in HeLa human cervical epithelial cells, where the IFNG-mediated inhibition of C. trachomatis growth was significant and the IFNG-induced IDO activity correlated with growth inhibition. Conclusions: IDO activity was not able to inhibit chlamydial growth in human neutrophils. Whether the IDO activity was not high enough for inhibition or other chlamydial growth-promoting host mechanisms were induced in the infected and interferon-treated neutrophils needs to be further investigated.

Microwave-assisted Phospha-Michael addition reactions in the 13α-oestrone series and in vitro antiproliferative properties.

Microwave-assisted phospha-Michael addition reactions were carried out in the 13α-oestrone series. The exocyclic 16-methylene-17-ketones as α,ß-unsaturated ketones were reacted with secondary phosphine oxides as nucleophilic partners. The addition reactions furnished the two tertiary phosphine oxide diastereomers in high yields. The main product was the 16α-isomer. The antiproliferative activities of the newly synthesised organophosphorus compounds against a panel of nine human cancer cell lines were investigated by means of MTT assays. The most potent compound, the diphenylphosphine oxide derivative in the 3-O-methyl-13α-oestrone series (9), exerted selective cell growth-inhibitory activity against UPCI-SCC-131 and T47D cell lines with low micromolar IC50 values. Moreover, it displayed good tumour selectivity property determined against non-cancerous mouse fibroblast cells.

Small Extracellular Vesicles Isolated from Serum May Serve as Signal-Enhancers for the Monitoring of CNS Tumors.

Liquid biopsy-based methods to test biomarkers (e.g., serum proteins and extracellular vesicles) may help to monitor brain tumors. In this proteomics-based study, we aimed to identify a characteristic protein fingerprint associated with central nervous system (CNS) tumors. Overall, 96 human serum samples were obtained from four patient groups, namely glioblastoma multiforme (GBM), non-small-cell lung cancer brain metastasis (BM), meningioma (M) and lumbar disc hernia patients (CTRL). After the isolation and characterization of small extracellular vesicles (sEVs) by nanoparticle tracking analysis (NTA) and atomic force microscopy (AFM), liquid chromatography -mass spectrometry (LC-MS) was performed on two different sample types (whole serum and serum sEVs). Statistical analyses (ratio, Cohen's d, receiver operating characteristic; ROC) were carried out to compare patient groups. To recognize differences between the two sample types, pairwise comparisons (Welch's test) and ingenuity pathway analysis (IPA) were performed. According to our knowledge, this is the first study that compares the proteome of whole serum and serum-derived sEVs. From the 311 proteins identified, 10 whole serum proteins and 17 sEV proteins showed the highest intergroup differences. Sixty-five proteins were significantly enriched in sEV samples, while 129 proteins were significantly depleted compared to whole serum. Based on principal component analysis (PCA) analyses, sEVs are more suitable to discriminate between the patient groups. Our results support that sEVs have greater potential to monitor CNS tumors, than whole serum.

Prediabetes Induced by Fructose-Enriched Diet Influences Cardiac Lipidome and Proteome and Leads to Deterioration of Cardiac Function prior to the Development of Excessive Oxidative Stress and Cell Damage.

Prediabetes is a condition affecting more than 35% of the population. In some forms, excessive carbohydrate intake (primarily refined sugar) plays a prominent role. Prediabetes is a symptomless, mostly unrecognized disease which increases cardiovascular risk. In our work, we examined the effect of a fructose-enriched diet on cardiac function and lipidome as well as proteome of cardiac muscle. Male Wistar rats were divided into two groups. The control group received a normal diet while the fructose-fed group received 60% fructose-supplemented chow for 24 weeks. Fasting blood glucose measurement and oral glucose tolerance test (OGTT) showed slightly but significantly elevated values due to fructose feeding indicating development of a prediabetic condition. Both echocardiography and isolated working heart perfusion performed at the end of the feeding protocol demonstrated diastolic cardiac dysfunction in the fructose-fed group. Mass spectrometry-based, high-performance lipidomic and proteomic analyses were executed from cardiac tissue. The lipidomic analysis revealed complex rearrangement of the whole lipidome with special emphasis on defects in cardiolipin remodeling. The proteomic analysis showed significant changes in 75 cardiac proteins due to fructose feeding including mitochondria-, apoptosis-, and oxidative stress-related proteins. Nevertheless, just very weak or no signs of apoptosis induction and oxidative stress were detected in the hearts of fructose-fed rats. Our results suggest that fructose feeding induces marked alterations in the cardiac lipidome, especially in cardiolipin remodeling, which leads to mitochondrial dysfunction and impaired cardiac function. However, at the same time, several adaptive responses are induced at the proteome level in order to maintain a homeostatic balance. These findings demonstrate that even very early stages of prediabetes can impair cardiac function and can result in significant changes in the lipidome and proteome of the heart prior to the development of excessive oxidative stress and cell damage.