protein correlation profiling
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BACKGROUND: Human colostrum and milk contain components that influence development. Our aim was to use a protein array to determine the cytokine profile of human lacteal secretions and changes that occur during the early postpartum period. METHODS: We collected 17 samples of colostrum during the first 2 days postpartum and a 2nd group of 5 sets of 2 to 3 sequential colostrum or milk samples (at 20- to 30-h intervals). We analyzed the samples with array membranes consisting of 42 or 79 antibodies directed against cytokines. RESULTS: In most samples, we detected the previously described cytokines interleukin-8 (IL-8)/CXCL8, epidermal growth factor (EGF), growth-related oncoprotein (GRO)/CXCL1-3, angiogenin, transforming growth factor beta-2, and monocyte chemotactic protein 1 (MCP-1/CCL2). In addition, we found 32 cytokines that have not been described before in colostrum. Cytokine concentrations differed among mothers, and the spectrum of cytokines changed with time after delivery. A significant decrease occurred in IL-12 and macrophage inflammatory protein-1delta/CCL15 and a significant increase in MCP-1/CCL2. The production of angiogenin, vascular endothelial growth factor, GRO/CXCL1-3, EGF, and IL-8/CXCL8 remained high throughout. The concentrations of 2 selected cytokines measured with the array technique and ELISA showed moderate to strong correlation (r = 0.63 for EGF and r = 0.84 for IL-8/CXCL8). CONCLUSION: Despite the lack of precise quantification, the protein array might be suitable for cytokine screening. It allows simultaneous detection of a broad spectrum of cytokines (including those not described before) in lacteal secretions.
- MeSH
- časové faktory MeSH
- chemokiny analýza MeSH
- čipová analýza proteinů MeSH
- cytokiny analýza MeSH
- dospělí MeSH
- ELISA MeSH
- financování organizované MeSH
- kolostrum chemie MeSH
- lidé MeSH
- mateřské mléko chemie MeSH
- mezibuněčné signální peptidy a proteiny analýza MeSH
- poporodní období MeSH
- proteomika MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- srovnávací studie MeSH
Profiling of biological relationships between different molecular layers dissects regulatory mechanisms that ultimately determine cellular function. To thoroughly assess the role of protein post-translational turnover, we devised a strategy combining pulse stable isotope-labeled amino acids in cells (pSILAC), data-independent acquisition mass spectrometry (DIA-MS), and a novel data analysis framework that resolves protein degradation rate on the level of mRNA alternative splicing isoforms and isoform groups. We demonstrated our approach by the genome-wide correlation analysis between mRNA amounts and protein degradation across different strains of HeLa cells that harbor a high grade of gene dosage variation. The dataset revealed that specific biological processes, cellular organelles, spatial compartments of organelles, and individual protein isoforms of the same genes could have distinctive degradation rate. The protein degradation diversity thus dissects the corresponding buffering or concerting protein turnover control across cancer cell lines. The data further indicate that specific mRNA splicing events such as intron retention significantly impact the protein abundance levels. Our findings support the tight association between transcriptome variability and proteostasis and provide a methodological foundation for studying functional protein degradation.
- MeSH
- alternativní sestřih MeSH
- HeLa buňky MeSH
- hmotnostní spektrometrie MeSH
- izoformy RNA genetika metabolismus MeSH
- izotopové značení metody MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- protein - isoformy analýza metabolismus MeSH
- proteiny analýza metabolismus MeSH
- proteolýza MeSH
- proteomika metody MeSH
- průběh práce MeSH
- regulace genové exprese u nádorů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Reverse transcription quantitative PCR (RT-qPCR) is already an established tool for mRNA detection and quantification. Since recently, this technique has been successfully employed for gene expression analyses, and also in individual cells (single cell RT-qPCR). Although the advantages of single cell measurements have been proven several times, a study correlating the expression measured on single cells, and in bulk samples consisting of a large number of cells, has been missing. Here, we collected a large data set to explore the relation between gene expression measured in single cells and in bulk samples, reflected by qPCR Cq values. We measured the expression of 95 genes in 12 bulk samples, each containing thousands of astrocytes, and also in 693 individual astrocytes. Combining the data, we described the relation between Cq values measured in bulk samples with either the percentage of the single cells that express the given genes, or the average expression of the genes across the single cells. We show that data obtained with single cell RT-qPCR are fully consistent with measurements in bulk samples. Our results further provide a base for quality control in single cell expression profiling, and bring new insights into the biological process of cellular expression.
- MeSH
- analýza jednotlivých buněk * MeSH
- gliový fibrilární kyselý protein genetika MeSH
- messenger RNA genetika MeSH
- myši transgenní MeSH
- myši MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- stanovení celkové genové exprese * MeSH
- zelené fluorescenční proteiny genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Proteomics profiling of intact proteins based on MALDI-TOF MS and derived platforms has been used in cancer biomarker discovery studies. This approach suffers from a number of limitations such as low resolution, low sensitivity, and that no knowledge is available on the identity of the respective proteins in the discovery mode. Nevertheless, it remains the most high-throughput, untargeted mode of clinical proteomics studies to date. Here we compare key protein separation and MS techniques available for protein biomarker identification in this type of studies and define reasons of uncertainty in protein peak identity. As a result of critical data analysis, we consider 3D protein separation and identification workflows as optimal procedures. Subsequently, we present a new protocol based on 3D LC-MS/MS with top-down at high resolution that enabled the identification of HNRNP A2/B1 intact peptide as correlating with the estrogen receptor expression in breast cancer tissues. Additional development of this general concept toward next generation, top-down based protein profiling at high resolution is discussed.
- MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- nádorové biomarkery metabolismus MeSH
- nádorové proteiny chemie izolace a purifikace metabolismus MeSH
- nádory prsu metabolismus MeSH
- peptidy chemie metabolismus MeSH
- proteomika metody MeSH
- sekvence aminokyselin MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Alzheimer's disease (AD) is the most frequent cause of dementia. Misfolded protein pathological hallmarks of AD are brain deposits of amyloid-β (Aβ) plaques and phosphorylated tau neurofibrillary tangles. However, doubts about the role of Aβ in AD pathology have been raised as Aβ is a common component of extracellular brain deposits found, also by in vivo imaging, in non-demented aged individuals. It has been suggested that some individuals are more prone to Aβ neurotoxicity and hence more likely to develop AD when aging brains start accumulating Aβ plaques. Here, we applied genome-wide transcriptomic profiling of lymphoblastoid cells lines (LCLs) from healthy individuals and AD patients for identifying genes that predict sensitivity to Aβ. Real-time PCR validation identified 3.78-fold lower expression of RGS2 (regulator of G-protein signaling 2; P=0.0085) in LCLs from healthy individuals exhibiting high vs low Aβ sensitivity. Furthermore, RGS2 showed 3.3-fold lower expression (P=0.0008) in AD LCLs compared with controls. Notably, RGS2 expression in AD LCLs correlated with the patients' cognitive function. Lower RGS2 expression levels were also discovered in published expression data sets from postmortem AD brain tissues as well as in mild cognitive impairment and AD blood samples compared with controls. In conclusion, Aβ sensitivity phenotyping followed by transcriptomic profiling and published patient data mining identified reduced peripheral and brain expression levels of RGS2, a key regulator of G-protein-coupled receptor signaling and neuronal plasticity. RGS2 is suggested as a novel AD biomarker (alongside other genes) toward early AD detection and future disease modifying therapeutics.
- MeSH
- Alzheimerova nemoc diagnóza genetika patologie MeSH
- amyloidní beta-protein genetika MeSH
- amyloidní plaky genetika patologie MeSH
- buněčné linie MeSH
- časná diagnóza MeSH
- celogenomová asociační studie * MeSH
- data mining * MeSH
- exprese genu genetika MeSH
- fenotyp MeSH
- genetické asociační studie MeSH
- genetické markery genetika MeSH
- lidé MeSH
- mozek patologie MeSH
- neurofibrilární klubka genetika patologie MeSH
- proteiny RGS genetika MeSH
- senioři MeSH
- stanovení celkové genové exprese * MeSH
- výpočetní biologie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVE: To identify novel genetic and epigenetic factors associated with Myasthenia gravis (MG) using an identical twins experimental study design. METHODS: The transcriptome and methylome of peripheral monocytes were compared between monozygotic (MZ) twins discordant and concordant for MG, as well as with MG singletons and healthy controls, all females. Sets of differentially expressed genes and differentially methylated CpGs were validated using RT-PCR for expression and target bisulfite sequencing for methylation on additional samples. RESULTS: >100 differentially expressed genes and ∼1800 differentially methylated CpGs were detected in peripheral monocytes between MG patients and controls. Several transcripts associated with immune homeostasis and inflammation resolution were reduced in MG patients. Only a relatively few genes differed between the discordant healthy and MG co-twins, and both their expression and methylation profiles demonstrated very high similarity. INTERPRETATION: This is the first study to characterize the DNA methylation profile in MG, and the expression profile of immune cells in MZ twins with MG. Results suggest that numerous small changes in gene expression or methylation might together contribute to disease. Impaired monocyte function in MG and decreased expression of genes associated with inflammation resolution could contribute to the chronicity of the disease. Findings may serve as potential new predictive biomarkers for disease and disease activity, as well as potential future targets for therapy development. The high similarity between the healthy and the MG discordant twins, suggests that a molecular signature might precede a clinical phenotype, and that genetic predisposition may have a stronger contribution to disease than previously assumed.
- MeSH
- CpG ostrůvky MeSH
- dospělí MeSH
- dvojčata monozygotní * MeSH
- epigeneze genetická MeSH
- genetická predispozice k nemoci MeSH
- genetické asociační studie MeSH
- lidé středního věku MeSH
- lidé MeSH
- metylace DNA * MeSH
- mladý dospělý MeSH
- monocyty imunologie metabolismus MeSH
- myasthenia gravis genetika metabolismus MeSH
- receptor TREM-1 genetika metabolismus MeSH
- senioři MeSH
- signální transdukce MeSH
- stanovení celkové genové exprese MeSH
- studie případů a kontrol MeSH
- transkriptom * MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- studie na dvojčatech MeSH
BACKGROUND: Yeast infections are often connected with formation of biofilms that are extremely difficult to eradicate. An excellent model system for deciphering multifactorial determinants of yeast biofilm development is the colony biofilm, composed of surface ("aerial") and invasive ("root") cells. While surface cells have been partially analyzed before, we know little about invasive root cells. In particular, information on the metabolic, chemical and morphogenetic properties of invasive versus surface cells is lacking. In this study, we used a new strategy to isolate invasive cells from agar and extracellular matrix, and employed it to perform genome wide expression profiling and biochemical analyses of surface and invasive cells. RESULTS: RNA sequencing revealed expression differences in 1245 genes with high statistical significance, indicating large genetically regulated metabolic differences between surface and invasive cells. Functional annotation analyses implicated genes involved in stress defense, peroxisomal fatty acid β-oxidation, autophagy, protein degradation, storage compound metabolism and meiosis as being important in surface cells. In contrast, numerous genes with functions in nutrient transport and diverse synthetic metabolic reactions, including genes involved in ribosome biogenesis, biosynthesis and translation, were found to be important in invasive cells. Variation in gene expression correlated significantly with cell-type specific processes such as autophagy and storage compound accumulation as identified by microscopic and biochemical analyses. Expression profiling also provided indications of cell-specific regulations. Subsequent knockout strain analyses identified Gip2p, a regulatory subunit of type 1 protein phosphatase Glc7p, to be essential for glycogen accumulation in surface cells. CONCLUSIONS: This is the first study reporting genome wide differences between surface and invasive cells of yeast colony biofilms. New findings show that surface and invasive cells display very different physiology, adapting to different conditions in different colony areas and contributing to development and survival of the colony biofilm as a whole. Notably, surface and invasive cells of colony biofilms differ significantly from upper and lower cells of smooth colonies adapted to plentiful laboratory conditions.
Sialic acids are negatively charged carbohydrates that are components of saccharide chains covalently linked to macromolecules. Sialylated glycoproteins are important for most biological processes, including reproduction, where they are associated with spermatogenesis, sperm motility, immune responses, and fertilization. Changes in the glycoprotein profile or sialylation in glycoproteins are likely to affect the quality of ejaculate. The aim of this study was to determine differences in the degree of sialylation between normozoospermic ejaculates and ejaculates with a pathological spermiogram using two lectins, Sambucus nigra (SNA) and Maackia amurensis (MAL II/MAA) recognizing α-2,6 or α-2,3 linkage of Sia to galactosyl residues. Our results show a close relationship between seminal plasma (SP) sialoproteins and the presence of anti-sperm antibodies in the ejaculate, apoptotic spermatozoa, and ejaculate quality. Using mass spectrometry, we identified SP sialoproteins such as, semenogelins, glycodelin, prolactin-inducible protein, lactotransferrin, and clusterin that are associated with spermatozoa and contribute to the modulation of the immune response and sperm apoptosis. Our findings suggest a correlation between the degree of SP glycoprotein sialylation and the existence of possible pathological states of spermatozoa and reproductive organs. Glycoproteins sialylation represents a potential parameter reflecting the overall quality of ejaculate and could potentially be utilised in diagnostics.
- MeSH
- analýza spermatu metody MeSH
- apoptóza MeSH
- ejakulace MeSH
- glykodelin metabolismus MeSH
- glykoproteiny metabolismus MeSH
- klusterin metabolismus MeSH
- kyseliny sialové metabolismus MeSH
- laktoferrin metabolismus MeSH
- lektiny metabolismus chemie MeSH
- lidé MeSH
- motilita spermií MeSH
- proteiny semenné plazmy metabolismus MeSH
- sekreční proteiny semenných váčků metabolismus MeSH
- sperma * metabolismus chemie MeSH
- spermie * metabolismus MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The ARF family of regulatory GTPases is ancient, with 16 members predicted to have been present in the last eukaryotic common ancestor. Our phylogenetic profiling of paralogues in diverse species identified four family members whose presence correlates with that of a cilium/flagellum: ARL3, ARL6, ARL13, and ARL16. No prior evidence links ARL16 to cilia or other cell functions, despite its presence throughout eukaryotes. Deletion of ARL16 in mouse embryonic fibroblasts (MEFs) results in decreased ciliogenesis yet increased ciliary length. We also found Arl16 knockout (KO) in MEFs to alter ciliary protein content, including loss of ARL13B, ARL3, INPP5E, and the IFT-A core component IFT140. Instead, both INPP5E and IFT140 accumulate at the Golgi in Arl16 KO lines, while other intraflagellar transport (IFT) proteins do not, suggesting a specific defect in traffic from Golgi to cilia. We propose that ARL16 regulates a Golgi-cilia traffic pathway and is required specifically in the export of IFT140 and INPP5E from the Golgi.
- MeSH
- cilie metabolismus MeSH
- fibroblasty * metabolismus MeSH
- fosfatasy * metabolismus MeSH
- fylogeneze MeSH
- myši MeSH
- proteiny metabolismus MeSH
- transport proteinů MeSH
- transportní proteiny genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
OBJECTIVES: The aim of this study was to evaluate the correlation of neuron specific enolase (NSE), protein S100B and time-profile of Glasgow Coma Score (GCS) development with metallothionein (MT) blood levels in patients with traumatic brain injury (TBI) during 10 days of hospitalization. Patients were divided into 2 groups with respect to NSE and S100B levels - with (group I) and without (group II) GCS improvement. METHODS: Serum NSE and S100B concentrations were measured by immunochemical methods; serum metallothionein concentration by electrochemical technique. Cortical biopsies were investigated immunohistochemically and by electron microscope. A cDNA microarray containing 700 gene probes was used to study the changes in gene expression in the ipsilateral cortex. RESULTS: Values of MT in the blood of group I showed a non-significant decrease compared to group II during 1-3 days after admission. There was an increase of MT during 4-8 days in comparison with values of 1-3 days. The highest value of MT during hospitalization was found in a patient with diffuse axonal injury (group II). The data of cDNA microarray suggested an increase in expression of gene transcripts for oxygen free radical scavenger proteins corresponding with the increase of MT during 4-8 days in both groups. CONCLUSIONS: The experimental data indicate that monitoring the content of MT in patients with trauma brain injury would be a suitable approach to evaluate the degree of injury or duration of prolonging unconsciousness, particularly in diagnosis of diffuse axonal injury.
- MeSH
- fosfopyruváthydratasa * krev metabolismus MeSH
- lidé MeSH
- magnetická rezonanční tomografie MeSH
- metalothionein * krev MeSH
- neurotrofní faktory * krev metabolismus MeSH
- poranění mozku * krev metabolismus radiografie MeSH
- proteiny S100 * krev metabolismus MeSH
- stanovení celkové genové exprese MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
