Changes in the urinary proteome before and after quadrivalent influenza vaccine and COVID-19 vaccination.

The proteome of urine samples from quadrivalent influenza vaccine cohort were analyzed with self-contrasted method. Significantly changed urine protein at 24 hours after vaccination was enriched in immune-related pathways, although each person's specific pathways varied. We speculate that this may be because different people have different immunological backgrounds associated with influenza. Then, urine samples were collected from several uninfected SARS-CoV-2 young people before and after the first, second, and third doses of the COVID-19 vaccine. The differential proteins compared between after the second dose (24h) and before the second dose were enriched in pathways involving in multicellular organismal process, regulated exocytosis and immune-related pathways, indicating no first exposure to antigen. Surprisingly, the pathways enriched by the differential urinary protein before and after the first dose were similar to those before and after the second dose. It is inferred that although the volunteers were not infected with SARS-CoV-2, they might have been exposed to other coimmunogenic coronaviruses. Two to four hours after the third vaccination, the differentially expressed protein were also enriched in multicellular organismal process, regulated exocytosis and immune-related pathways, indicating that the immune response has been triggered in a short time after vaccination. Multicellular organismal process and regulated exocytosis after vaccination may be a new indicator to evaluate the immune effect of vaccines. Urinary proteome is a terrific window to monitor the changes in human immune function.

Many kinds of oxidized proteins are present more in the urine of the elderly.

BACKGROUND: Many studies have shown an association between aging and oxidation. To our knowledge, there have been no studies exploring aging-related urine proteome modifications. The purpose of this study was to explore differences in global chemical modifications of urinary protein at different ages. METHODS: Discovery (n=38) cohort MS data including children, young and old groups were downloaded from three published studies, and this data was analyzed using open-pFind for identifying modifications. Verification cohort human samples (n=28) including young, middle-aged, and old groups, rat samples (n=7) at three-time points after birth, adulthood, and old age were collected and processed in the laboratory simultaneously based on label-free quantification combined with pFind. RESULTS: Discovery cohort: there were 28 kinds of differential oxidations in the old group that were higher than those in the young or children group in. Verification cohort: there were 17 kinds of differential oxidations of 49 oxidized proteins in the middle and old groups, which were significantly higher than those in the young group. Both oxidations and oxidized proteins distinguished different age groups well. There were also 15 kinds of differential oxidations in old age higher than others in the rat cohort. The results showed that the validation experiment was basically consistent with the results of the discovery experiment, showing that the level of oxidized proteins in urine increased significantly with age. CONCLUSIONS: Our study is the first to show that oxidative proteins occur in urine and that oxidations are higher in older than younger ages. Perhaps improving the degree of excretion of oxidative protein in vivo through the kidney is helpful for maintaining the homeostasis of the body's internal environment, delaying aging and the occurrence of senile diseases.

The ceRNA Crosstalk between mRNAs and lncRNAs in Diabetes Myocardial Infarction.

Competitive endogenous RNA regulation suggests an intricate network of all transcriptional RNAs that have the function of repressing miRNA function and regulating mRNA expression. Today, the specific ceRNA regulatory mechanisms of lncRNA-miRNA-mRNA in patients who have diabetes mellitus (DM) and myocardial infarction (MI) are still unknown. Two data sets, GSE34198 and GSE112690, were rooted in the Gene Expression Omnibus database to search for changes of lncRNA, miRNA, and mRNA in MI patients with diabetes. Weighted gene correlation network analysis (WGCNA) was used to identify the modules related to the development of diabetes in patients with MI. Target genes of miRNAs were predicted using miRWalk, TargetScan, mirDB, RNA22, and miRanda. Then, functional and enrichment analyses were performed to build the lncRNA-miRNA-mRNA interaction network. We built ceRNA regulatory networks with three lncRNAs, two miRNAs, and nine mRNAs. Differentially expressed genes enriched in biological process, including neutrophil activation, refer to immune response and positive system of defense feedback. Besides, there is significant enrichment in molecular function of calcium toll-like receptor binding, icosanoid binding, RAGE receptor binding, and arachidonic acid binding. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis enriched differentially expressed genes (DEGs) in pathways that were well known in MI, indicating inflammation and immune response. Pathways associated with diabetes were also significantly enriched. We confirmed significantly altered lncRNA, miRNA, and mRNA in MI patients with diabetes, which might serve as biomarkers for the progress and development of diabetic cardiovascular diseases. We constructed a ceRNA regulatory network of lncRNA-miRNA-mRNA, which will enable us to understand the novel molecular mechanisms included in the initiation, progression, and interaction between DM and MI, laying the foundation for clinical diagnosis and treatment.

Globular adiponectin inhibits osteoblastic differentiation of vascular smooth muscle cells through the PI3K/AKT and Wnt/ß-catenin pathway.

Lipid metabolism is closely related to the improvement of vascular calcification (VC) in chronic kidney disease (CKD). Globular adiponectin (gAd) has been reported to be involved in the development of VC in CKD, but the detailed regulatory role remains unclear. The present study is aimed to investigate the biological function and the underlying regulation mechanism of gAd in the process of VC during CKD. Vascular smooth muscle cells (VSMCs) calcification was determined by Alizarin Red S staining. Protein signaling related with VC was tested by western blotting. The expression and intracellular localization of runt-related transcription factor 2 (Runx2) was detected by immunofluorescence and uraemic rat with VC was established by a two-step nephrectomy. Combined with the results of Alizarin Red S staining, we discovered that ß-glycerophosphate (ß-Gp)-induced the osteoblastic differentiation of VSMCs was significantly reversed by gAd treatment. Along with the VSMCs calcification and the increase of Runx2 in ß-Gp-exposed VSMCs, the activities of protein kinase B (AKT) and Wnt/ß-catenin pathway were enhanced, but that were counteracted by the exposure of gAd in rat and human VSMCs. After administration with agonists of the Wnt (SKL2001) and AKT (SC79), there appeared more osteoblastic differentiation and higher expression of Runx2 in gAd-treated VSMCs, but showing lower impact in the presence of SC79 than that in the presence of SKL2001. In the in vivo experiments, intravenous injection of gAd also significantly inhibited VC and Runx2 level in uraemic rat in a dose-dependent manner, possibly through regulating Wnt/ß-catenin pathway. This study demonstrates that gAd ameliorates osteoblastic differentiation of VSMCs possibly by blocking PI3K/AKT and Wnt/ß-catenin signaling transduction. The findings provide an important foundation for gAd in treating VC in kidney diseases.

Fabrication of PAMAM antimicrobial monolayer via UV induced grafting on the surface of polyethylene terephthalate.

Poly (amidoamine) (PAMAM) with 3rd and 5th generation was covalently grafted as the contact active biocidal agent on the surface of polyethylene terephthalate (PET) with the help of UV induced carbene chemistry (PAMAM-g-PET). The graft density and the surface roughness were controlled by turning UV irradiation time and the PAMAM generation. The PAMAM graft monolayer was characterized via the contact angle, XPS, nanoIR, SEM and AFM. The antibacterial ability of PAMAM-g-PET was evaluated ex-vivo with the help of laser scanning confocal microscope (CLSM), and the results indicated that the decorated PET was able to kill both S. aureus and E. coli in the aqueous environment. Increasing the surface graft concentration and using the dendrimer with higher generation enhanced the lethality towards the bacterial. The decorated film was still able to kill the contact bacterial strain when the cationic primary amine groups were shielded by acetyl chloride, however, the bacterial in the suspension was hardly affected in this case. The un-selectivity and instantaneity of carbene chemistry endowed this grafting strategy the potential to be extended to other organic substances.

Fast fixing and comprehensive identification to help improve real-time ligands discovery based on formaldehyde crosslinking, immunoprecipitation and SDS-PAGE separation.

BACKGROUND: Fast Fixation is necessary to study real-time protein-protein interactions under physiological conditions. Fast formaldehyde cross-linking can fix transient and weak protein interactions, thereby reducing the number of false negatives but producing great complexity. To reduce this complexity, immunoaffinity purification can Fish out complexes that include particular target proteins, but affinity-based co-purification has a limited capacity to eliminate nonspecific binding to beads and/or antibodies. To Filter out these complexes, SDS-PAGE is used to disrupt non-covalent bonds, thereby eliminating uncross-linked complexes and simultaneously providing molecular weight information for identification. RESULTS: We described a 4 F strategy to help improve real-time ligands discovery based on formaldehyde crosslinking, immunoprecipitation and SDS-PAGE separation: Fast Fix, Fish, and Filter, using albumin interactome as an example. The use of gel excision without staining makes this strategy comprehensive and sensitive. The target protein must be identified in the same slice as its ligands. The ligands must be identified in slices for the experimental group but not in the corresponding control slices. Only proteins that appear in the range of molecular weights equal to or greater than the sum of the proteins' theoretical molecular weights, together with the target, are considered ligands. In this study, 5 s of cross-linking with 10% formaldehyde was achieved in human blood. The use of this strategy identified 35 ligands for albumin. Comparison with four major previous studies of the albuminome revealed that 68.57% of the 35 ligands identified in our study were identified in these other studies. CONCLUSIONS: Fast cross-linking was achieved. The 4 F strategy can be used to identify real-time in situ interactions without prior intervention and to comprehensively identify ligands of particular target proteins with fewer false positives.

Using an isolated rat kidney model to identify kidney origin proteins in urine.

The use of targeted proteomics to identify urinary biomarkers of kidney disease in urine can avoid the interference of serum proteins. It may provide better sample throughput, higher sensitivity, and specificity. Knowing which urinary proteins to target is essential. By analyzing the urine from perfused isolated rat kidneys, 990 kidney origin proteins with human analogs were identified in urine. Of these proteins, 128 were not found in normal human urine and may become biomarkers with zero background. A total of 297 proteins were not found in normal human plasma. These proteins will not be influenced by other normal organs and will be kidney specific. The levels of 33 proteins increased during perfusion with an oxygen-deficient solution compared to those perfused with oxygen. The 75 proteins in the perfusion-driven urine have a significantly increased abundance ranking compared to their ranking in normal human urine. When compared with existing candidate biomarkers, over ninety percent of the kidney origin proteins in urine identified in this study have not been examined as candidate biomarkers of kidney diseases.

Identification and characterization of cow's milk proteins from the rat intestinal lymph using a proteomic strategy.

Food proteins were considered to be absorbed into the body after being digested to amino acids, dipeptides, and tripeptides. However, there are studies indicating that some proteins can pass through the intestinal epithelium under normal physiological conditions, perhaps not in sufficient quantities to be of nutritional importance, but in quantities that may be antigenically or biologically active. In the present study, rat intestinal lymph samples were collected using a modified lymph fistula rat model in fasting and cow's milk postprandial states. Low molecular weight proteins were enriched by ultrafiltration and differential solubilization, separated by 1D-SDS-PAGE, digested in-gel based on molecular weight, and identified using nano-LC-MS/MS. In the postprandial rat intestinal lymph, nine bovine-specific proteins (false discovery rate ≤1%) were identified in different molecular weight regions. Most proteins identified in lymph were highly abundant proteins in the milk, such as ß-lactoglobulin and caseins. Seven of the nine identified bovine-specific proteins are allergens in milk. This strategy can be used to search for proteins that can enter the intestinal lymph and analyze their common features. Understanding the common features of these proteins might help to develop protein drugs taken orally, so that therapeutic proteins might embody fusion domains for cross-barrier transport or translocation.

Differential protein expression in perfusates from metastasized rat livers.

BACKGROUND: Liver perfusates exhibit theoretical advantages regarding the discovery of disease biomarkers because they contain proteins that readily enter the blood-stream, and perfusion preserves the disease state in its natural context. The purpose of the study is to explore the value of liver perfusate proteome in the biomarker discovery of liver diseases. RESULTS: In this study, 86 differentially expressed proteins were identified in perfusates from isolated rat livers metastasized by Walker-256 tumor cells. Among these proteins, 27 were predicted to be secreted, and 59 were intracellular or membrane proteins. Most of the secretory proteins (70.4%) were decreased in metastasized liver perfusates. The main canonical ingenuity pathway to which these secretory proteins belonged was acute phase response, which indicated that the liver-associated immune reaction was damaged by the metastasis. In contrast, most of the intracellular or membrane proteins (86.4%) exhibited higher relative abundances in the metastasized liver perfusates. Some of these proteins, including Rpl21, Atic, Eif3s2, Echs1, Eps15 and Ywhab, have previously been reported to be involved in cancer genesis and progression. As a member of the 14-3-3 protein family, Ywhab plays a key role in cellular proliferation and oncogenic transformation and has been reported to be involved in the development of breast cancer. Its abundance was elevated by 3.5-fold in the metastasized perfusates. Validation by Western blotting revealed a 3.7-fold increase in the abundance of this protein in metastasized plasma. CONCLUSIONS: These results show that perfusate proteome can be used as an alternative initial resource for biomarker identification, which ultimately requires validation in serum.

An individual urinary proteome analysis in normal human beings to define the minimal sample number to represent the normal urinary proteome.

BACKGROUND: The urinary proteome has been widely used for biomarker discovery. A urinary proteome database from normal humans can provide a background for discovery proteomics and candidate proteins/peptides for targeted proteomics. Therefore, it is necessary to define the minimum number of individuals required for sampling to represent the normal urinary proteome. METHODS: In this study, inter-individual and inter-gender variations of urinary proteome were taken into consideration to achieve a representative database. An individual analysis was performed on overnight urine samples from 20 normal volunteers (10 males and 10 females) by 1DLC/MS/MS. To obtain a representative result of each sample, a replicate 1DLCMS/MS analysis was performed. The minimal sample number was estimated by statistical analysis. RESULTS: For qualitative analysis, less than 5% of new proteins/peptides were identified in a male/female normal group by adding a new sample when the sample number exceeded nine. In addition, in a normal group, the percentage of newly identified proteins/peptides was less than 5% upon adding a new sample when the sample number reached 10. Furthermore, a statistical analysis indicated that urinary proteomes from normal males and females showed different patterns. For quantitative analysis, the variation of protein abundance was defined by spectrum count and western blotting methods. And then the minimal sample number for quantitative proteomic analysis was identified. CONCLUSIONS: For qualitative analysis, when considering the inter-individual and inter-gender variations, the minimum sample number is 10 and requires a balanced number of males and females in order to obtain a representative normal human urinary proteome. For quantitative analysis, the minimal sample number is much greater than that for qualitative analysis and depends on the experimental methods used for quantification.

A comparison of E15.5 fetus and newborn rat serum proteomes.

BACKGROUND: Serum proteins carry out several functions in the circulation, including transfer, immunological functions, messenger functions, coagulation, and regulation of homeostasis. To investigate changes in serum proteins that occur during development, the serum proteomes of embryonic 15.5 (E15.5) fetuses and newborn rats were compared using LC-MS/MS. RESULTS: A total of 958 proteins were identified in the serum of rats at both developmental stages. The serum proteome pattern of newborn rats was compared to E15.5 fetuses by relative quantitation. The expression patterns of hemoglobin subunits were different at the two stages, with most of the subunits having decreased expression in newborn rats compared to E15.5 fetuses. In addition, 8 of 12 apolipoproteins were significantly decreased and 10 of 11 identified complement molecules were increased, with 4 exhibiting a significant increase. Moreover, 11 of 14 of the significantly increased enzyme regulators were inhibitors. The serum proteome patterns of different littermates from both developmental stages were also compared. We found that the levels of many highly abundant serum proteins varied between littermates, and the variations were larger than the variations of the technical control. CONCLUSIONS: The serum proteomes of newborn rats and E15.5 fetuses were compared. The expression patterns of hemoglobin subunits were different at the two developmental stages, with most of the subunits having decreased expression. The majority of apolipoproteins had significantly decreased expression, while almost all identified complement proteins had increased expression. The levels of several highly abundant serum proteins also varied among littermates at these two developmental stages. This is the first study using LC-MS/MS to investigate serum proteome development.

A tool for biomarker discovery in the urinary proteome: a manually curated human and animal urine protein biomarker database.

Urine is an important source of biomarkers. A single proteomics assay can identify hundreds of differentially expressed proteins between disease and control samples; however, the ability to select biomarker candidates with the most promise for further validation study remains difficult. A bioinformatics tool that allows accurate and convenient comparison of all of the existing related studies can markedly aid the development of this area. In this study, we constructed the Urinary Protein Biomarker (UPB) database to collect existing studies of urinary protein biomarkers from published literature. To ensure the quality of data collection, all literature was manually curated. The website (http://122.70.220.102/biomarker) allows users to browse the database by disease categories and search by protein IDs in bulk. Researchers can easily determine whether a biomarker candidate has already been identified by another group for the same disease or for other diseases, which allows for the confidence and disease specificity of their biomarker candidate to be evaluated. Additionally, the pathophysiological processes of the diseases can be studied using our database with the hypothesis that diseases that share biomarkers may have the same pathophysiological processes. Because of the natural relationship between urinary proteins and the urinary system, this database may be especially suitable for studying the pathogenesis of urological diseases. Currently, the database contains 553 and 275 records compiled from 174 and 31 publications of human and animal studies, respectively. We found that biomarkers identified by different proteomic methods had a poor overlap with each other. The differences between sample preparation and separation methods, mass spectrometers, and data analysis algorithms may be influencing factors. Biomarkers identified from animal models also overlapped poorly with those from human samples, but the overlap rate was not lower than that of human proteomics studies. Therefore, it is not clear how well the animal models mimic human diseases.