scholarly journals TMT-based proteomic and bioinformatic analyses of human granulosa cells from obese and normal-weight female subjects

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Chenchen Si ◽  
Nan Wang ◽  
Mingjie Wang ◽  
Yue Liu ◽  
Zhihong Niu ◽  
...  
Keyword(s):  
Protein Expression ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Expression Profiles ◽  
Normal Weight ◽  
Differentially Expressed ◽  
Tandem Mass ◽  
Differentially Expressed Proteins ◽  
Obese Subjects ◽  
Female Subjects

Abstract Background Increasing evidence supports a relationship between obesity and either infertility or subfertility in women. Most previous omics studies were focused on determining if the serum and follicular fluid expression profiles of subjects afflicted with both obesity-related infertility and polycystic ovary syndrome (PCOS) are different than those in normal healthy controls. As granulosa cells (GCs) are essential for oocyte development and fertility, we determined here if the protein expression profiles in the GCs from obese subjects are different than those in their normal-weight counterpart. Methods GC samples were collected from obese female subjects (n = 14) and normal-weight female subjects (n = 12) who were infertile and underwent in vitro fertilization (IVF) treatment due to tubal pathology. A quantitative approach including tandem mass tag labeling and liquid chromatography tandem mass spectrometry (TMT) was employed to identify differentially expressed proteins. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were then conducted to interrogate the functions and pathways of identified proteins. Clinical, hormonal, and biochemical parameters were also analyzed in both groups. Results A total of 228 differentially expressed proteins were noted, including 138 that were upregulated whereas 90 others were downregulated. Significant pathways and GO terms associated with protein expression changes were also identified, especially within the mitochondrial electron transport chain. The levels of free fatty acids in both the serum and follicular fluid of obese subjects were significantly higher than those in matched normal-weight subjects. Conclusions In GCs obtained from obese subjects, their mitochondria were damaged and the endoplasmic reticulum stress response was accompanied by dysregulated hormonal synthesis whereas none of these changes occurred in normal-weight subjects. These alterations may be related to the high FFA and TG levels detected in human follicular fluid.

scholarly journals Differential Protein Abundance in Dark-Cutting and Normal-pH Beef

2019 ◽  
Vol 3 (2) ◽  
Author(s):  
F. Kiyimba ◽  
S. Hartson ◽  
J. Rogers ◽  
G. Mafi ◽  
D. VanOverbeke ◽  
...  
Keyword(s):  
Protein Expression ◽  
Pathway Analysis ◽  
Solid Phase ◽  
Muscle Protein ◽  
Expression Profiles ◽  
Protein Profile ◽  
Differentially Expressed ◽  
Cutting Condition ◽  
Differentially Expressed Proteins ◽  
Differential Protein

ObjectivesDark-cutting beef is a meat quality defect in which meat does not display the marketable bright-red color. Although previous studies have indicated that the ultimate pH of dark-cutting beef is greater than normal, the mechanistic basis for the occurrence is not clear. Various mitochondrial and glycolytic enzymes/proteins are involved in muscle metabolism and lowering of pH. However, limited knowledge is currently available on the muscle protein profile differences between dark-cutting and normal-pH beef. The objective of the current study was to identify proteins related to the development of the dark-cutting condition by comparing the protein expression differences between dark-cutting and normal-pH beef.Materials and MethodsDark-cutting and normal-pH beef samples were collected from six (n = 6) different animals after slaughter. Tissue samples (0.5 g) were digested in 5 mL of lysis buffer. Tissue lysates were homogenized, boiled, sonicated using a bioruptor and centrifuged at 10,000 g for 10 min. Samples were digested with trypsin/Lys-C overnight at 37°C, after which additional 2 µg/mL of protease was added and digestion was continued for another 8h. The resulting trypsinolytic peptides were acidified to 1% trifluoroacetic acid and purified by solid phase extraction with C18 affinity media. Protein expression profiles of both dark-cutting and normal-pH beef samples were determined using LC-MS/MS mass spectrometry-based proteomics. Collected raw data instrument files were searched against a bovine proteome database of 23,968 bovine proteome sequences using MaxQuant (V.1.5.3.8). Differential protein expression analysis was done in Perseus (V.1.5.1.3). Ingenuity pathway analysis (IPA) was utilized to determine the significant pathways of the differentially expressed proteins in dark-cutting and normal-pH beef. Gene ontology enrichment pathway analysis was performed to determine the main functions of the differentially expressed proteins in dark-cutting and normal-pH beef identified in our samples.ResultsMass spectrometry analysis identified 1148 proteins, and 97 of these proteins were differentially expressed between normal-pH and dark-cutting beef (P < 0.05). Fold change of 1.5 was observed for 29 proteins. Dark-cutting beef had 19 abundant proteins, while normal-pH beef had 10 abundant proteins. The majority of the upregulated proteins in dark-cutting beef were involved in mitochondrial functioning and metabolism, while the majority of the downregulated proteins were important in glycogen degradation, calcium signaling, α-adrenergic signaling, n-NOS-signaling and the proteasome pathways.ConclusionThe results identify new protein biomarkers associated with dark-cutting and suggest new mechanistic explanations for the dark-cutting phenotype.

Mass Spectrometry-based Label-free Quantitative Proteomic Analysis of CCl4-induced Acute Liver Injury in Mice Intervened by Total Glycosides from Ligustri Lucidi Fructus

2020 ◽  
Vol 17 ◽  
Author(s):  
Qian Lu ◽  
Hai-Zhu Xing ◽  
Nian-Yun Yang
Keyword(s):  
Insulin Resistance ◽  
Liver Injury ◽  
Protein Expression ◽  
Signaling Pathway ◽  
Proteomic Analysis ◽  
Acute Liver Injury ◽  
Liver Cells ◽  
Differentially Expressed ◽  
Liver Protein ◽  
Differentially Expressed Proteins

Background: CCl4 acute liver injury (ALI) is a classical model for experimental research. However, there are few reports involved in the fundamental research of CCl4-induced ALI Ligustri Lucidi Fructus (LLF) are and its prescription have been used to treat hepatitis illness clinically. LLF and its active ingredients displayed anti-hepatitis effects, but the mechanism of function has not been fully clarified Objective: To investigate the proteomic analysis of CCl4-induced ALI, and examine the effects of active total glycosides (TG) from LLF on ALI of mice4, including histopathological survey and proteomic changes of liver tissues, and delineate the possible underlying mechanism. Methods: CCl4 was used to produce ALI mice model. The model mice were intragastrically administrated with TG and the liver his-topathological changes of mice were examined. At the end of test, mice liver samples were collected, after protein denaturation, re-duction, desalination and enzymatic hydrolysis, identification was carried out by nano LC-ESI-OrbiTrap MS/MS technology. The data was processed by Maxquant software. The differentially-expressed proteins were screened and identified, and their biological information was also analyzed based on GO and KEGG analysis. Key protein expression was validated by Western blot analysis Results: A total of 705 differentially-expressed proteins were identified during the normal, model and administration group. 9 signifi-cant differential proteins were focused based on analysis. Liver protein expression changes of CCl4-induced ALI mice were mainly involved in several important signal channels, namely FoxO signaling pathway, autophagy-animal, insulin signaling pathway. TG has anti-liver damnification effect in ALI mice, the mechanism of which is related to FoxO1 and autophagy pathways Conclusion: CCl4 inhibited expression of insulin-Like growth factor 1 (Igf1) and 3-phosphoinositide-dependent protein kinase 1 (Pdpk1) in liver cells and induced insulin resistance, thus interfered with mitochondrial autophagy and regeneration of liver cells and the metabolism of glucose and lipid, and caused hepatic necrosis in mice. TG resisted liver injury in mice. TG adjusted the expression level of key proteins Igf1 and Pdpk1 after liver injury and improved insulin resistance, thus promoted autophagy and resisted the liver damage

scholarly journals Differences in metabolic biomarkers in the blood and gene expression profiles of peripheral blood mononuclear cells among normal weight, mildly obese and moderately obese subjects

2016 ◽  
Vol 116 (6) ◽  
pp. 1022-1032 ◽  
Author(s):  
Un Ju Jung ◽  
Yu Ri Seo ◽  
Ri Ryu ◽  
Myung-Sook Choi
Keyword(s):  
Gene Expression ◽  
Insulin Resistance ◽  
Peripheral Blood ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Hdl Cholesterol ◽  
Normal Weight ◽  
Peripheral Blood Mononuclear ◽  
Obese Subjects ◽  
Metabolic Biomarkers

AbstractWe compared metabolic biomarkers in the blood and peripheral blood mononuclear cell (PBMC) gene expression profiles among normal weight (BMI, 18·5–23 kg/m2), mildly obese (BMI, 25–27·5 kg/m2) and moderately obese Korean adult men (BMI, 27·5–30 kg/m2). High leptin, lipids (except LDL- and HDL-cholesterol) and apoB levels and low adiponectin and HDL-cholesterol levels were present in the plasma of both mildly and moderately obese subjects. Circulating levels of inflammatory cytokines and markers of insulin resistance, oxidative stress and liver damage were altered in moderately obese subjects but not in mildly obese subjects. PBMC transcriptome data showed enrichment of pathways involved in energy metabolism, insulin resistance, bone metabolism, cancer, inflammation and fibrosis in both mildly and moderately obese subjects. Signalling pathways involved in oxidative phosphorylation, TAG synthesis, carbohydrate metabolism and insulin production; mammalian target of rapamycin, forkhead box O, ras-proximate-1, RAS and transforming growth factor-β signalling; as well as extracellular matrix–receptor interaction were enriched only in moderately obese subjects, indicating that changes in PBMC gene expression profiles, according to metabolic disturbances, were associated with the development and/or aggravation of obesity. In particular, fourteen and fifteen genes differentially expressed only in mildly obese subjects and in both mildly and moderately obese subjects, respectively, could be used as early or stable biomarkers for diagnosing and treating obesity-associated metabolic disturbance. We characterised BMI-associated metabolic and molecular biomarkers in the blood and provided clues about potential blood-based targets for preventing or treating obesity-related complications.

scholarly journals Transcriptomic profiling of neonatal mouse granulosa cells reveals new insights into primordial follicle activation

2021 ◽  
Author(s):  
Emmalee A Ford ◽  
Emily R Frost ◽  
Emma L Beckett ◽  
Shaun D Roman ◽  
Eileen A McLaughlin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Granulosa Cells ◽  
Expression Profiles ◽  
Primordial Follicle ◽  
Differentially Expressed ◽  
Primary Follicle ◽  
Primordial Follicles ◽  
Time Points ◽  
Primordial Follicle Activation ◽  
Follicle Activation

Abstract The dormant population of ovarian primordial follicles is determined at birth and serves as the reservoir for future female fertility. Yet our understanding of the molecular, biochemical, and cellular processes underpinning primordial follicle activation remains limited. The survival of primordial follicles relies on the correct complement and morphology of granulosa cells, which provide signalling factors essential for oocyte and follicular survival. To investigate the contribution of granulosa cells in the primordial-to-primary follicle transition, gene expression profiles of granulosa cells undergoing early differentiation were assessed in a murine model. Ovaries from C57Bl/6 mice were enzymatically dissociated at time-points spanning the initial wave of primordial follicle activation. Post-natal day (PND) 1 ovaries yielded primordial granulosa cells, and PND4 ovaries yielded a mixed population of primordial and primary granulosa cells. The comparative transcriptome of granulosa cells at these time-points was generated via Illumina NextSeq 500 system which identified 131 significantly differentially expressed transcripts. The differential expression of eight of the transcripts was confirmed by RT-qPCR Following biological network mapping via Ingenuity Pathway Analysis, the functional expression of the protein products of three of the differentially expressed genes, namely FRZB, POD1 and ZFX, was investigated with in-situ immunolocalisation in PND4 mouse ovaries was investigated. Finally, evidence was provided that Wnt pathway antagonist, secreted frizzled-related protein 3 (FRZB), interacts with a suppressor of primordial follicle activation WNT3A and may be involved in promoting primordial follicle activation. This study highlights the dynamic changes in gene expression of granulosa cells during primordial follicle activation and provides evidence for a renewed focus into the Wnt signalling pathway’s role in primordial follicle activation.

scholarly journals Cite-Seq Reveals Distinct Patterns and Potential Mechanisms of Relapse in Pediatric AML

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3458-3458
Author(s):  
Tsz-Kwong Man ◽  
Mohammad Javad Najaf Panah ◽  
Jessica L. Elswood ◽  
Pavel Sumazin ◽  
Michele S. Redell
Keyword(s):  
Protein Expression ◽  
Single Cell ◽  
Actin Polymerization ◽  
Conflicts Of Interest ◽  
Limit Of Detection ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Cell Types ◽  
Differentially Expressed ◽  
Pediatric Aml

Abstract Introduction - Acute myeloid leukemia (AML) is an aggressive disease with a relapse rate of approximately 40% in children. Progress in improving cure rates has been slow, in part because AML is very heterogeneous. Molecular studies consistently show that most cases are comprised of distinct subclones that diminish or expand over the course of therapy. Single-cell profiling methods now allow parsing of the leukemic population into subsets based on gene and/or protein expression patterns. We hypothesized that comparing the features of the subsets that are dominant at relapse with those that are dominant at diagnosis would reveal mechanisms of treatment failure. Methods - We profiled diagnosis-relapse pairs from 6 pediatric AML patients by Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq). All patients were treated at Texas Children's Cancer Center and consented to banking of tissue for research. CITE-Seq was performed by Immunai (New York, NY) using a customized panel of 65 oligonucleotide-tagged antibodies, the 10x Genomics Chromium system for single-cell RNA library generation, and the Novaseq 6000 for sequencing. After data cleanup and normalization, clustering by scRNA-seq was done using the Seurat package. Cell-type identification of clusters was facilitated by published healthy bone marrow scRNA-seq datasets (van Galen et al, Cell 2019). Differentially expressed genes (DEGs) and proteins (DEPs) between diagnosis and relapse were determined using Wilcoxin ranked sum tests. Results - We generated single-cell transcriptomes for a total of 28,486 cells from 12 samples, with a mean of 2373 cells and 1416 genes per sample. Samples were integrated with batch effect correction, producing 30 distinct clusters (cell types) in total (Figure 1A). Cell types with expression profiles consistent with lymphocytes and erythroid precursors were identified in multiple patients, whereas AML cell types tended to be specific to individual patients (Figure 1B). For patients TCH1, TCH2 and TCH3, the most abundant cell types at diagnosis were rare at relapse, and cell types that were rare at diagnosis became dominant at relapse. For these 3 cases, we identified DEGs between the dominant diagnosis cell types and dominant relapse cell types. We found 18 genes that were upregulated at relapse in at least 2 of the cases. Several genes related to actin polymerization were enriched (ARPC1B, ACTB, PFN1), possibly reflecting an enhanced capacity for adhesion and migration. Also of note, macrophage migration inhibitory factor (MIF) and its receptor CD74 were upregulated at relapse, suggesting a role in chemoresistance. For patients TCH4, TCH5 and TCH6, the same cell types that were abundant at diagnosis were also abundant at relapse, and few genes were significantly altered between diagnosis and relapse in multiple cases. Only SRGN, which encodes the proteoglycan serglycin, and GAPDH were altered in 2 of these 3 cases, and both were downregulated at relapse. We performed similar comparisons to identify proteins that were differentially expressed between diagnosis and relapse pairs. The number of DEPs between the dominant diagnosis and relapse cell types ranged from 0 (TCH1 and TCH6) to 5 (TCH2). The only protein altered in more than one case was CD7, which was enriched at relapse in TCH2, TCH3 and TCH4. Conclusions - From CITE-Seq profiling of 6 pediatric AML cases we identified two distinct patterns of relapse. For 3 cases, relapse occurred by expansion of a subset that was small but present at diagnosis. Enrichment of genes associated with adhesion and survival signaling suggests that these cells survived because they were well-equipped to take advantage of interactions with the microenvironment. For 3 other cases, the population that was dominant at diagnosis persisted and expanded at relapse with few substantial changes in gene or protein expression profiles. This pattern suggests that these AML cells were a priori equipped to survive chemotherapy, even though bulk disease levels were transiently reduced below the limit of detection. Most profiled proteins did not change substantially between diagnosis and relapse. An exception is CD7, which was enriched at relapse in 50% of our cases and represents a potential therapeutic target. Analysis of more cases will refine these relapse patterns, reveal potential mechanisms of chemoresistance and inform the development of novel therapies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Tandem Mass Tag-Based Quantitative Proteomic Analysis Reveals Pathways Involved in Brain Injury Induced by Chest Exposure to Shock Waves

2021 ◽  
Vol 14 ◽  
Author(s):  
Changci Tong ◽  
Peifang Cong ◽  
Ying Liu ◽  
Xiuyun Shi ◽  
Lin Shi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Brain Injury ◽  
Signaling Pathways ◽  
Brain Damage ◽  
Western Blotting ◽  
Differentially Expressed ◽  
Tandem Mass ◽  
Differentially Expressed Proteins ◽  
Blast Exposure ◽  
Tandem Mass Tag

Recurrent chest blast exposure can lead to brain inflammation, oxidative stress, and mental disorders in soldiers. However, the mechanism that underlies brain injury caused indirectly by chest blasts remains unclear. It is urgent to find additional reliable biomarkers to reveal the intimate details of the pathogenesis of this phenomenon. We used the term tandem mass tag (TMT) labeling combined with liquid chromatography–tandem mass spectrometry (LC-MS/MS) to screen for differentially expressed proteins in rat brain at different time points after a chest blast. Data are available via ProteomeXchange with the identifier PXD025204. Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG), the Database for Annotation, Visualization and Integrated Discovery (DAVID), and Cytoscape analyses were used to analyze the proteomic profiles of blast-exposed rats. In addition, we performed Western blotting to verify protein levels. We identified 6,931 proteins, of which 255 were differentially expressed and 43, 84, 52, 97, and 49 were identified in brain tissues at 12, 24, 48, and 72 h and 1 week after chest blast exposure, respectively. In this study, the GO, KEGG, Clusters of Orthologous Groups of proteins, and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analyses indicated that brain damage caused by chest blast exposure involved many important biological processes and signaling pathways, such as inflammation, cell adhesion, phagocytosis, neuronal and synaptic damage, oxidative stress, and apoptosis. Furthermore, Western blotting confirmed that these differentially expressed proteins and affected signaling pathways were associated with brain damage caused by chest blast exposure. This study identifies potential protein biomarkers of brain damage caused indirectly by chest blast and new targets for the treatment of this condition.

scholarly journals Proteomic signatures of human visceral and subcutaneous adipocytes

2021 ◽  
Author(s):  
Pavel Hruska ◽  
Jan Kucera ◽  
Matej Pekar ◽  
Pavol Holeczy ◽  
Miloslav Mazur ◽  
...  
Keyword(s):  
Protein Expression ◽  
Expression Profiles ◽  
Protein Profile ◽  
Differential Expression Analysis ◽  
Differentially Expressed ◽  
P Value ◽  
Obese Women ◽  
Biosynthetic Activity ◽  
Mitochondrial Energy Metabolism ◽  
Key Factor

Abstract Objective Adipose tissue distribution is a key factor influencing metabolic health and risk in obesity-associated comorbidities. Here we aim to compare the proteomic profiles of mature adipocytes from different depots. Methods Abdominal subcutaneous (SA) and omental visceral adipocytes (VA) were isolated from paired AT biopsies obtained during bariatric surgery of 19 severely obese women (BMI &gt; 30 kg/m 2) and analysed using state-of-the-art mass spectrometry. Differential expression analysis and weighted gene co-expression network analysis (WGCNA) were performed to investigate proteome signature properties and to examine a possible association of the protein expression with the clinical data. Results We identified 3,686 protein groups and found 1,140 differentially expressed proteins (adj. p-value &lt; 0.05), of which 576 proteins were upregulated in SA and 564 in VA samples. We provide a global protein profile of abdominal SA and omental VA, present the most differentially expressed pathways and processes distinguishing SA from VA, and correlate them with clinical and body composition data. We show that SA are significantly more active in processes linked to vesicular transport and secretion, and to increased lipid metabolism activity. Conversely, the expression of proteins involved in the mitochondrial energy metabolism and translational or biosynthetic activity is higher in VA. Conclusion Our analysis represents a valuable resource of protein expression profiles in abdominal SA and omental VA, highlighting key differences in their role in obesity.

scholarly journals Adipose tatty acid composition and gene expression in obesity, and response to chronic marine omega-3 fatty acid supplementation.

2020 ◽  
Vol 79 (OCE2) ◽  
Author(s):  
Helena Fisk ◽  
Rob Ayres ◽  
Caroline Childs ◽  
Elizabeth Miles ◽  
Rebecca Clarke-Harris ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Fatty Acid ◽  
Inflammatory Response ◽  
Differentially Expressed Genes ◽  
Acid Composition ◽  
Normal Weight ◽  
Differentially Expressed ◽  
Omega 3 ◽  
Obese Subjects

AbstractIntroduction:Obesity is an excess of adipose tissue (AT) and is linked with increased inflammation that enhances risk of type-2 diabetes and cardiovascular disease. The BIOCLAIMS study assessed the effect of obesity on AT fatty acid composition and gene expression, and the responses of these to chronic omega-3 FA supplementation.Materials and methods:AT biopsies were collected pre- and post-12 week supplementation with 1.1 g EPA + 0.8 g DHA/day or corn oil. The composition of FA in the total lipid extract of AT from 37 normal-weight and 44 obese subjects was assessed by gas chromatography, whole AT transcriptome from 10 normal-weight and 10 obese subjects was assessed by RNA-Sequencing, and selected gene expression in AT of 27 normal-weight and 38 obese subjects was assessed by qRT-PCR.Results:789 AT genes were differentially expressed (623 upregulated, 175 downregulated) in obesity compared to normal-weight (FC > 2, P < 0.05). Differentially expressed genes included EGFL6, MMP-7 and -9, 5-LOX, WNT3 and WNT10B, DACT2, CNR1, SLC27A2 and PLA2G7, and were associated with immune and inflammatory response, cell proliferation, activation and movement, Wnt signalling, remodelling and expansion, and lipid incorporation and degradation.Chronic supplementation with EPA + DHA increased the concentration of AT EPA, DPA and DHA in normal-weight subjects (P < 0.01), and EPA in obese subjects (P = 0.006). EPA + DHA modulated the expression of 26 genes (14 upregulated, 12 downregulated) in normal-weight subjects and 7 genes (3 upregulated, 5 downregulated) in obese subjects (FC > 2, P < 0.05). Of note, EPA + DHA downregulated IGLV1-44, IGLV1-51, PROK2, and TREM1 in normal weight subjects (P < 0.05), and IGLV1-44, IGLV1-47, DACT2 and IDO1 obese subjects (P < 0.05). Genes of note upregulated by EPA + DHA included KCNH2, GCGR, SLC36A2 and FOXC2 in normal-weight subjects, and MAB21L1, LRRTM4, and COX-2, in obese subjects. Differentially expressed genes were associated with a decrease in complement activation and immunoglobulin secretion, negative regulation of cell proliferation, and positive regulation of remodelling, amino acid and glucose transport, and COX pathway metabolite synthesis.Discussion:These data indicate an altered AT transcription profile and gene expression in obesity suggesting enhanced immune and inflammatory response, tissue expansion and remodelling, and changes to lipid metabolism, as well as dysregulation in response to supplementary EPA + DHA at a gene expression level. EPA + DHA are able to modulate AT gene expression predominantly associated with reducing immune response, but obesity may involve resistance to the effects on tissue remodelling and nutrient transport.

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