Browsing by Subject "proteomics"
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Item Activated human astrocyte-derived extracellular vesicles modulate neuronal uptake, differentiation and firing(Informa UK Limited, trading as Taylor & Francis Group, 2019-12-26) You, Yang; Borgmann, Kathleen; Edara, Venkata Viswanadh; Stacy, Satomi; Ghorpade, Anuja; Ikezu, TsuneyaAstrocytes in the central nervous system (CNS) provide supportive neural functions and mediate inflammatory responses from microglia. Increasing evidence supports their critical roles in regulating brain homoeostasis in response to pro-inflammatory factors such as cytokines and pathogen/damage-associated molecular pattern molecules in infectious and neurodegenerative diseases. However, the underlying mechanisms of the trans-cellular communication are still unclear. Extracellular vesicles (EVs) can transfer a large diversity of molecules such as lipids, nucleic acids and proteins for cellular communications. The purpose of this study is to characterize the EVs cargo proteins derived from human primary astrocytes (ADEVs) under both physiological and pathophysiological conditions. ADEVs were isolated from human primary astrocytes after vehicle (CTL) or interleukin-1beta (IL-1beta) pre-treatment. Label-free quantitative proteomic profiling revealed a notable up-regulation of proteins including actin-associated molecules, integrins and major histocompatibility complex in IL-1beta-ADEVs compared to CTL-ADEVs, which were involved in cellular metabolism and organization, cellular communication and inflammatory response. When fluorescently labelled ADEVs were added into primary cultured mouse cortical neurons, we found a significantly increased neuronal uptake of IL-1beta-ADEVs compared to CTL-ADEVs. We further confirmed it is likely due to the enrichment of surface proteins in IL-1beta-ADEVs, as IL-1beta-ADEVs uptake by neurons was partially suppressed by a specific integrin inhibitor. Additionally, treatment of neurons with IL-1beta-ADEVs also reduced neurite outgrowth, branching and neuronal firing. These findings provide insight for the molecular mechanism of the ADEVs' effects on neural uptake, neural differentiation and maturation, and its alteration in inflammatory conditions.Item In Vitro Culture Expansion Shifts the Immune Phenotype of Human Adipose-Derived Mesenchymal Stem Cells(Frontiers Media S.A., 2021-03-10) Jeske, Richard; Yuan, Xuegang; Fu, Qin; Bunnell, Bruce A.; Logan, Timothy M.; Li, YanHuman mesenchymal stem or stromal cells (hMSCs) are known for their potential in regenerative medicine due to their differentiation abilities, secretion of trophic factors, and regulation of immune responses in damaged tissues. Due to the limited quantity of hMSCs typically isolated from bone marrow, other tissue sources, such as adipose tissue-derived mesenchymal stem cells (hASCs), are considered a promising alternative. However, differences have been observed for hASCs in the context of metabolic characteristics and response to in vitro culture stress compared to bone marrow derived hMSCs (BM-hMSCs). In particular, the relationship between metabolic homeostasis and stem cell functions, especially the immune phenotype and immunomodulation of hASCs, remains unknown. This study thoroughly assessed the changes in metabolism, redox cycles, and immune phenotype of hASCs during in vitro expansion. In contrast to BM-hMSCs, hASCs did not respond to culture stress significantly during expansion as limited cellular senescence was observed. Notably, hASCs exhibited the increased secretion of pro-inflammatory cytokines and the decreased secretion of anti-inflammatory cytokines after extended culture expansion. The NAD+/NADH redox cycle and other metabolic characteristics associated with aging were relatively stable, indicating that hASC functional decline may be regulated through an alternative mechanism rather than NAD+/Sirtuin aging pathways as observed in BM-hMSCs. Furthermore, transcriptome analysis by mRNA-sequencing revealed the upregulation of genes for pro-inflammatory cytokines/chemokines and the downregulation of genes for anti-inflammatory cytokines for hASCs at high passage. Proteomics analysis indicated key pathways (e.g., tRNA charging, EIF2 signaling, protein ubiquitination pathway) that may be associated with the immune phenotype shift of hASCs. Together, this study advances our understanding of the metabolism and senescence of hASCs and may offer vital insights for the biomanufacturing of hASCs for clinical use.Item Mass Spectrometry-Based Characterization of Posttranslational Modifications by 4-Hydroxy-2-Nonenal(2010-05-01) Rauniyar, Navin; Prokai, LaszloItem Protein Biomakers of Estrogenic Impact on Mouse and Rat Uterus(2021-05) Thai, Stephen Duy-An; Prokai, Laszlo; Prokai-Tatrai, Katalin; Lacko, Andras G.Understanding the structural and morphological impact that occur in the uterus upon exposure to estrogens can reveal how these hormones elicit changes in the organ. This study seeks to revalidate the data obtained in studies of ovariectomized mice and rats treated with 17β-estradiol, followed by comparative analysis of the protein expression between the two animal models (the mice model studied by Prokai et al. [1] and the rat model presented in a dissertation by Rahlouni [2]). During these studies, female Swiss-Webster mice and Sprague-Dawley rats were ovariectomized (OVX) and divided respectively into two groups: 1) The control was treated with a corn oil vehicle while 2) The treatment group received 17β-estradiol injection for 5 days. Raw data gathered from the earlier proteomics studies were reanalyzed using Maxquant version 1.6.17, which utilized extracted ion chromatography technique (XIC). LFQ analyst and Ingenuity Pathway Analysis (IPA) version 5.0 were used to identify proteins that were differentially regulated by 17β-estradiol and perform comparative analyses of the protein expression between OVX mice and rats. Reanalysis of OVX mice identified 59 proteins of interest at 95% confidence with 29 upregulated and 30 downregulated significantly. Reanalysis of the OVX rats identified 126 differentially expressed proteins at 95% confidence with 98 upregulated and 27 downregulated significantly. Comparative analysis using IPA® found 27 proteins unique to mice and 85 proteins unique to rats. Conversely, the OVX mice and rats shared 19 proteins regulated by 17β-estradiol in the uterus. Ingenuity Pathway Analysis® also created networks in OVX mouse and rat models showing a relationship with estrogen receptors in the nucleus, which can bind 17β-estradiol and then initiate gene transcription. Although there is overlap between OVX mice and rat protein expression, the proteins that were found to be unique to each animal demonstrate that a complementary model using both animals provides a much broader view of uterine protein expression in OVX animals treated with 17?-estradiol. Additionally, this study illustrates the merit of reanalyzing older data with improved computational and bioinformatic tools to pinpoint proteins of interest for future analysis as potential markers of estrogenic effects in the uterus.Item Proteomics-Based Retinal Target Engagement Analysis and Retina-Targeted Delivery of 17beta-Estradiol by the DHED Prodrug for Ocular Neurotherapy in Males(MDPI, 2021-09-02) Prokai-Tatrai, Katalin; Zaman, Khadiza; Nguyen, Vien; De La Cruz, Daniel L.; Prokai, LaszloWe examined the impact of 17beta-estradiol (E2) eye drops on the modulation of the proteome profile in the male rat retina. With discovery-driven proteomics, we have identified proteins that were regulated by our treatment. These proteins were assembled to several bioinformatics-based networks implicating E2's beneficial effects on the male rat retina in a broad context of ocular neuroprotection including the maintenance of retinal homeostasis, facilitation of efficient disposal of damaged proteins, and mitochondrial respiratory chain biogenesis. We have also shown for the first time that the hormone's beneficial effects on the male retina can be constrained to this target site by treatment with the bioprecursor prodrug, DHED. A large concentration of E2 was produced after DHED eye drops not only in male rat retinae but also in those of rabbits. However, DHED treatment did not increase circulating E2 levels, thereby ensuring therapeutic safety in males. Targeted proteomics focusing on selected biomarkers of E2's target engagement further confirmed the prodrug's metabolism to E2 in the male retina and indicated that the retinal impact of DHED treatment was identical to that of the direct E2 treatment. Altogether, our study shows the potential of topical DHED therapy for an efficacious and safe protection of the male retina without the unwanted hormonal side-effects associated with current estrogen therapies.Item SASD: THE SYNTHETIC ALTERNATIVE SPLICING DATABASE FOR IDENTIFYING NOVEL ISOFORM FROM PROTEOMICS(2013-04-12) Zhang, FanPurpose: Alternative splicing is an important widespread mechanism for generating protein diversity and regulating protein expression. In human cells, about 40-60% of the genes are known to exhibit alternative splicing. Recent methodological advances, including EST sequencing, exon array, exon-exon junction array, and next-generation sequencing of all mRNA transcripts, have made it possible to perform high-throughput alternative splicing analysis. However, high-throughput identification and analysis of alternative splicing in the protein level has several advantages. For example, mRNA abundance in a cell often correlates poorly with the amount of protein synthesized, and proteins rather than mRNA transcripts are the major effector molecules in the cell. The combination of alternative splicing database and tandem mass spectrometry provides a powerful technique for identification, analysis and characterization of potential novel alternative splicing protein isoforms from proteomics. Therefore, we used a three steps pipeline to create an synthetic alternative splicing database(SASD) for tandem mass spectrometry data analysis. Methods: First we derived exons and introns from UCSC Genome Database, then we analyzed six types of combinations of exons and introns for the transcription of artificial splicing gene (exon_exon_normal, exon_exon_skipping, intron_exon, exon_intron, single exon, and single intron), and lastly we performed the translation of the artificial transcripts. Results: In addition, we built a web interface for users to browse 1) by genes/proteins, 2) by biological process, 3) by signaling and metabolic pathway, 4) by disease, 5) by drug, and 6) organ. Lastly, we presented two case studies: 1)in breast cancer and 2) in liver cancer, to demonstrate that the SASD can enable users to analyze, characterize, and understand the impact of alternative splicing on genes involved in drug, disease, pathway, function, and organ-specificity. Conclusions: The SASD provides the scientific community with an efficient means to identify and characterize novel Exon Skipping, Intron Retention, and alternative 3' splice site and 5' splice site protein isoforms from mass spectrometry data. We believe that it will be useful in annotating genome structures using rapidly accumulating proteomics data and assist scientific research on signal transduction pathways regulating pre-mRNA, clinical therapy, disease prevention, and drug development.