Studies have shown that a low serum uric acid (SUA) level associates with Parkinson's disease (PD), but many of them did not exclude patients with impaired renal function. Studies on the association between serum bilirubin level and PD also are limited. This study determined the association between SUA level, SUA/serum creatinine (SCr) ratio and serum bilirubin levels in PD patients with normal renal and liver functions.

Glycosylation is the most common post-translational modification of serum proteins, and changes in the type and abundance of glycans in human serum have been correlated with a growing number of human diseases. While the glycosylation pattern of human serum is well studied, little is known about the profiles of other mammalian species. Here, we report detailed glycosylation profiling of canine serum by hydrophilic interaction chromatography-ultraperformance liquid chromatography (HILIC-UPLC) and mass spectrometry. The domestic dog (Canis familiaris) is a widely used model organism and of considerable interest for a large veterinary community. We found significant differences in the serum N-glycosylation profile of dogs compared to that of humans, such as a lower abundance of galactosylated and sialylated glycans. We also compare the N-glycan profile of canine serum to that of canine IgG - the most abundant serum glycoprotein. Our data will serve as a baseline reference for future studies when performing serum analyses of various health and disease states in dogs.

Endometriosis is a chronic inflammatory gynaecological disease characterized by the growth of endometrial tissue outside the uterine cavity. There are currently no definitive non-invasive diagnostic tools. Glycosylation is the most common posttranslational modification of proteins and altered glycosylation has been found in many diseases, including chronic inflammatory conditions and cancer. Sialylation and galactosylation on serum IgG have previously been found to be altered in endometriosis and serum sialylation changed after Zoladex (Goserelin Acetate) therapy. Using IgG and whole serum glycoproteins, we investigated N-glycosylation in two clinical cohorts of women with and without endometriosis. PNGase F-digested serum samples were fluorescently labelled and N-glycans were profiled by ultra-performance liquid chromatography. Clinical data was collected to link glycomic findings with metabolic and hormonal profiles. Total serum glycoprotein and IgG glycosylation differed in patients with endometriosis compared to control cases. The most significantly altered was glycan peak 3 from IgG, containing bisected biantennary glycans, which was decreased in the endometriosis cohorts (p = 0.0000005-0.018). In conclusion, this is the first pilot study to identify changes in N-glycans from whole serum glycoproteins associated with endometriosis. A larger validation study is now warranted and such studies should include the follow-up of surgically and pharmacologically treated patients.

Hypertensive disorders are the most common in pregnancy. Several studies showed a positive correlation between elevated maternal serum uric acid (UA), serum creatinine and adverse maternal and fetal outcomes, but only a few studies are available on serum cystatin C and maternal and fetal outcomes. The present study was undertaken to study the association of serum UA, creatinine and cystatin C with maternal and fetal outcomes.

Glycation of serum proteins has been proposed as an important mechanism of complications of diabetes but whether there are differences in glycation of different serum proteins and whether it has any correlation with development of microvascular complications has not been studied in depth. This study aimed to assess level of serum fructosamine, glycated albumin and glycated β-lipoprotein in type 2 diabetes mellitus patients with and without microvascular complications and to find out their correlation with diabetes complications.

The aim of this study was to investigate whether 6 candidate serum miRNAs and their interactions with serum folate level were associated with the risk for pancreatic cancer (PC).

The expression of microRNAs (miRNAs) is a promising prognostic and diagnostic tool in hepatocellular carcinoma (HCC). Here we performed small RNA sequencing (sRNA-seq) of tissue, serum and serum exosomes to investigate changes in miRNA expression between the different sample types and correlated the expression with clinical parameters. We also performed gene expression arrays on tumor and normal tissue.

Mutual clinical and molecular interactions between iron and glucose metabolism have been reported. We aimed to investigate a potential effect of glucose on iron homeostasis. We found that serum iron concentrations gradually decreased over 180 min after the administration of 75 g of glucose from 109.8 ± 45.4 mg/L to 94.4 ± 40.4 mg/L (P<.001; N=40) but remained unchanged in control subjects receiving tap water (N=21). Serum hepcidin, the key iron regulatory hormone which is mainly derived from hepatocytes but also expressed in pancreatic β-cells, increased within 120 min after glucose ingestion from 19.7 ± 9.9 nmol/L to 31.4 ± 21.0 nmol/L (P<.001). In cell culture, glucose induced the secretion of hepcidin and insulin into the supernatant of INS-1E cultures, but did not change the amount of hepcidin detectable in the hepatocyte cell culture HepG2. We additionally confirmed the expression of hepcidin in a human islet cell preparation. These results suggest that glucose acts as a regulator of serum iron concentrations, most likely by triggering the release of hepcidin from β-cells.

The inflammasome is a key contributor to the inflammatory innate immune response after stroke. We have previously shown that inflammasome proteins are released in extracellular vesicles (EV) after brain and spinal cord injury. In addition, we have shown that inflammasome proteins offer great promise as biomarkers of central nervous system (CNS) injury following brain trauma. In the present study, we used a Simple Plex Assay (Protein Simple), a novel multi-analyte automated microfluidic immunoassay platform, to analyze serum and serum-derived EV samples from stroke patients and control subjects for inflammasome protein levels of caspase-1, apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), Interleukins (IL)-1β, and (IL)-18. Receiver operator characteristic (ROC) curves with associated confidence intervals obtained from the analysis of serum samples revealed that the area under the curve (AUC) for ASC was 0.99 with a confidence interval between 0.9914 and 1.004, whereas the AUC for caspase-1, IL-1β, and IL-18 were 0.75, 0.61, and 0.67, respectively. Thus, these data indicate that ASC is a potential biomarker of stroke and highlight the role of the inflammasome in the inflammatory response after brain ischemia.

Overweight and obesity are considered as chronic low-grade inflammation accompanied by imbalanced production of adipokines. The aim of this study was to elucidate the relationship between serum bilirubin, which is an endogenous antioxidant with anti-inflammatory activity, and pro- and anti-inflammatory serum adipokines in asymptomatic normal weight and overweight individuals. Healthy men and women aged 25-49 participated in this cross-sectional study. All participants underwent fasting serological measurements of adipokines, interleukin-6, tumor necrosis factor alpha (TNF-α), C-reactive protein (CRP), total and direct serum bilirubin, and other biochemical parameters. Participants were divided into normal weight and overweight groups. We found a significant negative association between total bilirubin and CRP, TNF-α, visfatin and resistin values, and a significant positive association between total bilirubin and adiponectin values in both normal-weight and overweight groups. Importantly, after adjusting for body mass index, we also found a significant negative association between total serum bilirubin levels and both visfatin and CRP serum levels. Moreover, visfatin, resistin and CRP were predictors of the total serum bilirubin levels.

Technetium-99m human serum albumin ((99m)Tc-HSA) is an important radiopharmaceutical required in nuclear medicine studies. However, the risk of transfusion-transmitted infection remains a major safety concern. Autopreparation of serum component acquired from patient provides a "personal-exclusive" source for radiolabeling. This paper is to evaluate the practicality of on-site elusion and subsequent radiolabeling efficacy for serum albumin. Results showed that the autologous elute contained more albumin fraction than serum without extraction procedure. Good radiochemical purity and stability were demonstrated after radiolabeling. Biodistribution study showed that labeled albumin accumulated immediately in the lung, liver, and kidney. It was cleared steadily and excreted in the urine. The biologic half-life was defined, and all samples passed the pyrogenicity and sterility tests. In conclusion, autoalbumin could be extracted and radiolabeled properly in a nuclear medicine setting. Moreover, the risk of transfusion-transmitted infection associated with nonautologous, multisource (99m)Tc-HSA agents can be reduced.

Exosomes are extracellular vesicles with a diameter in the range of 50-200 nm and a double-layer lipid membrane structure that are released by various types of cells under normal or abnormal physiological conditions. At present, according to their extensive biological functions, exosomes have been used in a wide range of research fields and applications, including as potential biomarkers and drug delivery vehicles. Intrahepatic cholangiocarcinoma is a malignant tumor of the biliary epithelium with the characteristics of cholangiocellular differentiation, which accounts for 10%-15% of all types of primary liver cancer. Intrahepatic cholangiocarcinoma has no obvious clinical symptoms in the early stages, which results in a low survival rate. Imaging equipment dependent diagnostic methods and currently commonly used diagnostic markers with low sensitivity/specificity have necessitated the development of new specific markers for intrahepatic cholangiocarcinoma. In this study, exosomes were isolated from serum using a commercial kit and characterized through nanoparticle tracking analysis, Western blotting analysis, and transmission electron microscopic analysis to prove the successful isolation of exosomes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the protein profiles of the serum and serum exosome samples were significantly different. In particular, some high-abundance proteins in the serum samples were significantly reduced or disappeared in the serum exosome sample. Meanwhile, some protein bands (which may belong to exosomes) that did not appear in the serum samples appeared in the serum exosome samples, leading to the conduciveness of subsequent mass spectrometry analysis. The serum and serum exosome samples of the healthy control and intrahepatic cholangiocarcinoma groups were analyzed by liquid chromatography-mass spectrometry for label-free quantitative proteomics. In total, 547 proteins were identified in the serum exosome samples, of which 341 (more than 60%) could be found in the exosomal protein database. In addition, 271 and 430 credible proteins were screened from the serum and serum exosome samples for multi-dimensional statistical analysis and differential protein discovery. Unsupervised principal component analysis and supervised orthogonal partial least squares discriminant analysis based on the quantitative proteome of the serum and serum exosome samples could distinguish the healthy control and intrahepatic cholangiocarcinoma groups well, which illustrates that the two types of samples both have potential in the diagnosis of intrahepatic cholangiocarcinoma. There were 15 upregulated and 8 downregulated proteins screened in the intrahepatic cholangiocarcinoma group compared to the healthy control group based on the serum samples, while 33 upregulated and 18 downregulated proteins were screened in the intrahepatic cholangiocarcinoma group compared to the healthy control group based on the serum exosome samples, and only four of the differential proteins screened based on the two types of samples were duplicates. At the same time, 35 of the 51 differentially expressed proteins screened based on serum exosome samples belonged to the exosomal protein database. Finally, biological information analysis was performed according to these differential proteins. The molecular functions, biological processes, and signal pathways enriched by these differential proteins mainly involved the innate immune responses, inflammatory responses, and blood coagulation. This study provides a reference value for potential biomarker discovery and exploration of the process of occurrence, development, and metastasis of intrahepatic cholangiocarcinoma. Moreover, compared with proteomic analysis based on serum samples, proteomic analysis based on serum exosome samples can be used to identify more differential proteins and biological information, and although these differential proteins and biological information may show big differences, the specificity and sensitivity of exosome-based diagnosis and the superiority of exosomes as samples for proteomic analysis has proven the application value of exosomes.

The formation of neutrophil extracellular traps (NETs) is an immune defense mechanism of neutrophilic granulocytes. Moreover, it is also involved in the pathogenesis of autoimmune, inflammatory, and neoplastic diseases. For that reason, the process of NET formation (NETosis) is subject of intense ongoing research. In vitro approaches to quantify NET formation are commonly used and involve neutrophil stimulation with various activators such as phorbol 12-myristate 13-acetate (PMA), lipopolysaccharides (LPS), or calcium ionophores (CaI). However, the experimental conditions of these experiments, particularly the media and media supplements employed by different research groups, vary considerably, rendering comparisons of results difficult. Here, we present the first standardized investigation of the influence of different media supplements on NET formation in vitro. The addition of heat-inactivated (hi) fetal calf serum (FCS), 0.5% human serum albumin (HSA), or 0.5% bovine serum albumin (BSA) efficiently prevented NET formation of human neutrophils following stimulation with LPS and CaI, but not after stimulation with PMA. Thus, serum components such as HSA, BSA and hiFCS (at concentrations typically found in the literature) inhibit NET formation to different degrees, depending on the NETosis inducer used. In contrast, in murine neutrophils, NETosis was inhibited by FCS and BSA, regardless of the inducer employed. This shows that mouse and human neutrophils have different susceptibilities toward the inhibition of NETosis by albumin or serum components. Furthermore, we provide experimental evidence that albumin inhibits NETosis by scavenging activators such as LPS. We also put our results into the context of media supplements most commonly used in NET research. In experiments with human neutrophils, either FCS (0.5-10%), heat-inactivated (hiFCS, 0.1-10%) or human serum albumin (HSA, 0.05-2%) was commonly added to the medium. For murine neutrophils, serum-free medium was used in most cases for stimulation with LPS and CaI, reflecting the different sensitivities of human and murine neutrophils to media supplements. Thus, the choice of media supplements greatly determines the outcome of experiments on NET-formation, which must be taken into account in NETosis research.

One of the functions of Human Serum Albumin (HSA) is binding and transport of fatty acids. This ability could be altered by the presence of several blood components such as toxins or peptides - which in turn alters the functionality of the protein. We aim at characterizing HSA and its fatty acid binding in native serum environment. Native ligand binding and deviations from normal function can be monitored by electron paramagnetic resonance (EPR) spectroscopy using spin labeled fatty acids (FAs). Blood serum from healthy individuals is used to examine healthy HSA in its natural physiological conditions at different loading ratios of protein to FAs. Among the EPR spectroscopic parameters (like hyperfine coupling, line shape, rotational correlation time and population of different binding sites) the rotational correlation time is found to differ significantly between binding sites of the protein, especially at loading ratios of four FAs per HSA. Although differences are observed between individual samples, a general trend regarding the dynamics of healthy HSA at different loading ratios could be obtained and compared to a reference of purified commercially available HSA in buffer.

Bourbon virus (BRBV) was first discovered in 2014 in a fatal human case. Since then it has been detected in the tick Amblyomma americanum in the states of Missouri and Kansas in the United States. Despite the high prevalence of BRBV in ticks in these states, very few human cases have been reported, and the true infection burden of BRBV in the community is unknown. Here, we developed two virus neutralization assays, a vesicular stomatitis virus (VSV)-BRBV pseudotyped rapid assay and a BRBV focus reduction neutralization assay, to assess the seroprevalence of BRBV neutralizing antibodies in human sera collected in 2020 in St. Louis, MO. Of 440 human serum samples tested, three (0.7%) were able to potently neutralize both VSV-BRBV and wild-type BRBV. These findings suggest that human infections with BRBV are more common than previously recognized. IMPORTANCE Since the discovery of the Bourbon virus (BRBV) in 2014, a total of five human cases have been identified, including two fatal cases. BRBV is thought to be transmitted by the lone star tick, which is prevalent in the eastern, southeastern, and midwestern United States. BRBV has been detected in ticks in Missouri and Kansas, and serological evidence suggests that it is also present in North Carolina. However, the true infection burden of BRBV in humans is not known. In the present study, we developed two virus neutralization assays to assess the seroprevalence of BRBV-specific antibodies in human sera collected in 2020 in St. Louis, MO. We found that a small subset of individuals are seropositive for neutralizing antibodies against BRBV. Our data suggest that BRBV infection in humans is more common than previously thought.

Microenvironment plays an important role for stem cell proliferation and differentiation. Macromolecular crowding (MMC) was recently shown to assist stem cells in forming their own matrix microenvironment in vitro. The ability of MMC to support adipose stem cell (ASC) proliferation, metabolism, and multilineage differentiation was studied under different conditions: fetal bovine serum- (FBS-) and human serum- (HS-) based media and xeno- and serum-free (XF/SF) media. Furthermore, the immunophenotype of ASCs under MMC was evaluated. The proliferative capacity of ASCs under MMC was attenuated in each condition. However, osteogenic differentiation was enhanced under MMC, shown by increased deposition of mineralized matrix in FBS and HS cultures. Likewise, significantly greater lipid droplet accumulation and increased collagen IV deposition indicated enhanced adipogenesis under MMC in FBS and HS cultures. In contrast, chondrogenic differentiation was attenuated in ASCs expanded under MMC. The ASC immunophenotype was maintained under MMC with significantly higher expression of CD54. However, MMC impaired metabolic activity and differentiation capacity of ASCs in XF/SF conditions. Both the supportive and inhibitory effects of MMC on ASC are culture condition dependent. In the presence of serum, MMC maintains ASC immunophenotype and enhances adipogenic and osteogenic differentiation at the cost of reduced proliferation.

Chemical cross-linking combined with mass spectrometry has proven useful for studying protein-protein interactions and protein structure, however the low density of cross-link data has so far precluded its use in determining structures de novo. Cross-linking density has been typically limited by the chemical selectivity of the standard cross-linking reagents that are commonly used for protein cross-linking. We have implemented the use of a heterobifunctional cross-linking reagent, sulfosuccinimidyl 4,4'-azipentanoate (sulfo-SDA), combining a traditional sulfo-N-hydroxysuccinimide (sulfo-NHS) ester and a UV photoactivatable diazirine group. This diazirine yields a highly reactive and promiscuous carbene species, the net result being a greatly increased number of cross-links compared with homobifunctional, NHS-based cross-linkers. We present a novel methodology that combines the use of this high density photo-cross-linking data with conformational space search to investigate the structure of human serum albumin domains, from purified samples, and in its native environment, human blood serum. Our approach is able to determine human serum albumin domain structures with good accuracy: root-mean-square deviation to crystal structure are 2.8/5.6/2.9 Å (purified samples) and 4.5/5.9/4.8Å (serum samples) for domains A/B/C for the first selected structure; 2.5/4.9/2.9 Å (purified samples) and 3.5/5.2/3.8 Å (serum samples) for the best out of top five selected structures. Our proof-of-concept study on human serum albumin demonstrates initial potential of our approach for determining the structures of more proteins in the complex biological contexts in which they function and which they may require for correct folding. Data are available via ProteomeXchange with identifier PXD001692.

Early prognostication of neurological outcome in comatose patients after cardiac arrest (CA) is vital for clinicians when assessing the survival time of sufferers and formulating appropriate treatment strategies to avoid the withdrawal of life-sustaining treatment (WLST) from patients. However, there is still a lack of sensitive and specific serum biomarkers for early and accurate identification of these patients. Using an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic approach, we discovered 55 differentially expressed proteins, with 39 up-regulated secreted serum proteins and 16 down-regulated secreted serum proteins between three comatose CA survivors with good versus poor neurological recovery. Then, four proteins were selected and were validated via an enzyme-linked immunosorbent assay (ELISA) approach in a larger-scale sample containing 32 good neurological outcome patients and 46 poor neurological outcome patients, and it was confirmed that serum angiotensinogen (AGT) and alpha-1-antitrypsin (SERPINA1) were associated with neurological function and prognosis in CA survivors. A prognostic risk score was developed and calculated using a linear and logistic regression model based on a combination of AGT, SERPINA1 and neuron-specific enolase (NSE) with an area under the curve of 0.865 (P < .001), and the prognostic risk score was positively correlated with the CPC value (R = 0.708, P < .001). We propose that the results of the risk score assessment not only reveal changes in biomarkers during neurological recovery but also assist in enhancing current therapeutic strategies for comatose CA survivors.

Zn-alpha2-glycoprotein (ZAG) is a serum protein implicated in cancer cachexia and lipolysis. Our aim was to investigate serum levels of ZAG and polymorphisms in the ZAG gene in relation to serum lipids in man. Serum levels of ZAG correlated with serum levels of cholesterol (P = .00088) in healthy subjects and during weight loss (P = .059). The ZAG genotype was associated with total cholesterol (P = .014) and low-density lipoprotein cholesterol (P = .026) in healthy subjects, and the associations were replicated in an additional cohort (P = .0017 and P = .060, respectively). Our data indicate that ZAG plays a role in lipid metabolism.

In the research field of extracellular vesicles (EVs), the use of fetal bovine serum (FBS) depleted of EVs for in vitro studies is advocated to eliminate the confounding effects of media derived EVs. EV-depleted FBS may either be prepared by ultracentrifugation or purchased commercially. Nevertheless, these preparations do not guarantee an RNA-free FBS for in vitro use. In this study we address the RNA contamination issue, of small non-coding (nc)RNA in vesicular or non-vesicular fractions of FBS, ultracentrifugation EV-depleted FBS, commercial EV-depleted FBS, and in our recently developed filtration based EV-depleted FBS. Commercially available serum- and xeno-free defined media were also screened for small ncRNA contamination. Our small ncRNA sequencing data showed that all EV-depleted media and commercially available defined media contained small ncRNA contaminants. Out of the different FBS preparations studied, our ultrafiltration-based method for EV depletion performed the best in depleting miRNAs. Certain miRNAs such miR-122 and miR-203a proved difficult to remove completely and were found in all media. Compared to miRNAs, other small ncRNA (snRNA, Y RNA, snoRNA, and piRNA) were difficult to eliminate from all the studied media. Additionally, our tested defined media contained miRNAs and other small ncRNAs, albeit at a much lower level than in serum preparations. Our study showed that no media is free of small ncRNA contaminants. Therefore, in order to screen for baseline RNA contamination in culturing media, RNA sequencing data should be carefully controlled by adding a media sample as a control. This should be a mandatory step before performing cell culture experiments in order to eliminate the confounding effects of media.