Search Results (16)

Encephalitozoon cuniculi causes both clinical and subclinical infections in rabbits, complicating a diagnosis due to the limitations of conventional tools like ELISA. This study analyzes serum proteomic profiles across clinical, subclinical, and healthy rabbits to identify discriminatory biomarkers. Serum from 90 pet rabbits (30 per group) was pooled (10 samples per pool, 3 pools per group) and analyzed using one-dimensional gel electrophoresis and mass spectrometry. The proteomic analysis revealed 109, 98, and 74 proteins expressed in healthy, subclinical, and clinical groups, respectively. Of these, 50, 40, and 33 proteins were unique to the healthy, subclinical, and clinical groups, respectively, with only 10 proteins shared across all. A total of 88 proteins were differentially expressed in infected groups compared to healthy controls. Importantly, 12 proteins were consistently upregulated in both subclinical and clinical infections. These include markers related to the immune response (beta-2-microglobulin, alpha-2-HS-glycoprotein), coagulation (antithrombin-III, alpha-1-antiproteinase S-1), vitamin A transport (retinol-binding proteins), lipid metabolism (apolipoprotein C-III), cytoskeletal regulation (actin-depolymerizing factor), extracellular matrix integrity (fibrillin 2), and oxidative stress (monooxygenase DBH-like 1). Additionally, Gc-globulin and ER lipid-raft-associated 1 were linked to immune modulation and signaling. These findings identify specific serum proteins as promising biomarkers for distinguishing subclinical from clinical encephalitozoonosis in rabbits, enabling an early diagnosis and effective disease monitoring. Full article

Metacestodes of Echinococcus multilocularis are the causative agents of alveolar echinococcosis, a neglected, life-threatening, zoonotic disease. To study these metacestodes in vitro, a model system using a culture medium conditioned by rat hepatoma cells is available. A key question is how the parasite interacts with the host and, in particular, which host-derived compounds are taken up. In this study, we focus on the uptake of host-derived proteins. Studies with artificially labeled proteins suggest that this uptake may occur independently of protein size or charge. Closer investigation using proteomics draws, however, a different picture. Of 1170 host (i.e., rat or bovine) proteins as identified by LC-MS/MS-based proteomics present in the culture medium, only 225 are found in metacestode vesicle tissue or fluid. Moreover, their relative abundances differ. Serum albumin, the most abundant culture medium host protein, is only the third most abundant protein in vesicle fluid, where Alpha-2-HS-glycoprotein becomes the most abundant protein. In vesicle fluid obtained ex vivo from experimentally infected mice, the situation is again different, with histone isoforms as the most abundant proteins. This suggests that while maintaining their internal milieu constant, metacestodes may adjust the spectrum of host proteins taken up. Potential uptake mechanisms and functions are discussed. Full article

Obesity is defined as the excessive accumulation of adipose tissue and is currently the most common disease in cats. Similarly to humans, obesity negatively impacts the health and welfare of cats, predisposing them to many other disorders. The objective of this study was to compare the serum proteomes of normal-weight and overweight/obese cats, aiming to gain insights into the physiopathology of feline obesity and potentially identify new biomarkers. For this, serum samples from a total of 20 adult neutered domestic shorthair client-owned cats, ten normal weight and ten overweight/obese, were submitted to tandem mass tags labelling and liquid chromatography-mass spectrometry (LC-MS/MS) analysis. A total of 288 proteins were detected in the serum samples. Out of these, 12 proteins showed statistically significant differences in abundance between control cats and cats with obesity, namely Ig-like domain-containing protein, Alpha-2-HS-glycoprotein, Complement C8 gamma chain, An-tithrombin-III, Serpin family A member 1, Complement factor H, C3-beta-c, Albumin, C4b-binding protein alpha chain, Alpha-1-B glycoprotein, Solute carrier family 12 member 4, and Fibronectin. Overall this report identifies new proteins involved and provides additional knowledge about the physiopathological changes related to feline obesity. Full article

Background/Objectives: Survival prospects following SARS-CoV-2 infection may extend beyond the acute phase, influenced by various factors including age, health conditions, and infection severity; however, this topic has not been studied in detail. Therefore, within this study, the mortality risk post-acute COVID-19 in the CRIT-COV-U cohort was investigated. Methods: Survival data from 651 patients that survived an acute phase of COVID-19 were retrieved and the association between urinary peptides and future death was assessed. Data spanning until December 2023 were collected from six countries, comparing mortality trends with age- and sex-matched COVID-19-negative controls. A death prediction classifier was developed and validated using pre-existing urinary peptidomic datasets. Results: Notably, 13.98% of post-COVID-19 patients succumbed during the follow-up, with mortality rates significantly higher than COVID-19-negative controls, particularly evident in younger individuals (<65 years). These data for the first time demonstrate that SARS-CoV-2 infection highly significantly increases the risk of mortality not only during the acute phase of the disease but also beyond for a period of about one year. In our study, we were further able to identify 201 urinary peptides linked to mortality. These peptides are fragments of albumin, alpha-2-HS-glycoprotein, apolipoprotein A-I, beta-2-microglobulin, CD99 antigen, various collagens, fibrinogen alpha, polymeric immunoglobulin receptor, sodium/potassium-transporting ATPase, and uromodulin and were integrated these into a predictive classifier (DP201). Higher DP201 scores, alongside age and BMI, significantly predicted death. Conclusions: The peptide-based classifier demonstrated significant predictive value for mortality in post-acute COVID-19 patients, highlighting the utility of urinary peptides in prognosticating post-acute COVID-19 mortality, offering insights for targeted interventions. By utilizing these defined biomarkers in the clinic, risk stratification, monitoring, and personalized interventions can be significantly improved. Our data also suggest that mortality should be considered as one possible symptom or a consequence of post-acute sequelae of SARS-CoV-2 infection, a fact that is currently overlooked. Full article

Identifying the potential presence of stress at the pig farm is fundamental since it affects pig welfare. As a result, a reliable and straightforward tool to monitor stress could record the welfare status of the animals. Although numerous methods to assess the welfare of pigs have been developed in the past, no gold standard has been established yet. Recently, the value of saliva as a tool to identify chronic stress in piglets was explored, as it can be collected fast and non-invasively. Since the protein composition, i.e., the proteome of porcine saliva, responds to stress, the affected proteins could be used as salivary stress biomarkers. The present review first defines stress and its relationship with welfare. Next, the porcine gland-specific salivary proteome is characterized. Finally, six potential salivary biomarkers for stress are proposed, i.e., odorant-binding protein, vomeromodulin-like protein, chitinase, lipocalin-1, long palate lung and nasal epithelium protein, and alpha-2-HS-glycoprotein. Full article

Laminins are trimeric glycoproteins with important roles in cell-matrix adhesion and tissue organization. The laminin α, ß, and γ-chains have short N-terminal arms, while their C-termini are connected via a triple coiled-coil domain, giving the laminin molecule a well-characterized cross-shaped morphology as a result. The C-terminus of laminin alpha chains contains additional globular laminin G-like (LG) domains with important roles in mediating cell adhesion. Dynamic conformational changes of different laminin domains have been implicated in regulating laminin function, but so far have not been analyzed at the single-molecule level. High-speed atomic force microscopy (HS-AFM) is a unique tool for visualizing such dynamic conformational changes under physiological conditions at sub-second temporal resolution. After optimizing surface immobilization and imaging conditions, we characterized the ultrastructure of laminin-111 and laminin-332 using HS-AFM timelapse imaging. While laminin-111 features a stable S-shaped coiled-coil domain displaying little conformational rearrangement, laminin-332 coiled-coil domains undergo rapid switching between straight and bent conformations around a defined central molecular hinge. Complementing the experimental AFM data with AlphaFold-based coiled-coil structure prediction enabled us to pinpoint the position of the hinge region, as well as to identify potential molecular rearrangement processes permitting hinge flexibility. Coarse-grained molecular dynamics simulations provide further support for a spatially defined kinking mechanism in the laminin-332 coiled-coil domain. Finally, we observed the dynamic rearrangement of the C-terminal LG domains of laminin-111 and laminin-332, switching them between compact and open conformations. Thus, HS-AFM can directly visualize molecular rearrangement processes within different laminin isoforms and provide dynamic structural insight not available from other microscopy techniques. Full article

To assess effects of environmental heat stress (HS) on the local and systemic inflammatory responses to lipopolysaccharide (LPS), broilers were reared under thermoneutral (TN) or cyclic HS conditions. Thermoneutral temperatures followed commercial production settings, with HS broilers exposed to 35 °C for 14 h/day from 4 days onward. At 37 days, HS- and TN-broilers were assigned to either LPS (100 μg/mL) or endotoxin-free phosphate-buffered saline (PBS; vehicle) treatments, eight each to HS- and TN-LPS, four each to HS- and TN-PBS. Treatments were administered by intradermal injection of growing feather (GF) pulps; 10 μL/GF; 12 GF/broiler. Blood and GF were collected before and at 6 and 24 h post-injection to assess leukocyte population changes in GF-pulps and blood, reactive oxygen species (ROS) generation and cytokine expression in GF-pulps, and plasma concentrations of alpha-1 acid glycoprotein (AGP-1). HS-LPS broilers had lower (p ≤ 0.05) infiltration of heterophils and macrophages, ROS generation, and inflammatory cytokine expression in GF-pulps, and lacked the increases in heterophil, monocyte, and plasma AGP-1 concentrations observed in TN-LPS broilers. HS-broilers had similar or greater drops in blood lymphocytes 6 h post-LPS or -PBS injection, respectively, and lower baseline levels (p ≤ 0.05) of circulating T- and B-lymphocytes than TN-broilers. Results indicated that cyclic HS reduced the local and systemic acute inflammatory responses to LPS in broilers, likely impairing their innate defense against microbial infection. Full article

Background: In recent years, various biomarkers of ulcerative colitis (UC) have emerged; however, few studies have simultaneously examined the utility of multiple biomarkers for monitoring disease activity. Additionally, serum leucine-rich alpha-2 glycoprotein (LRG), a new biomarker, may show a blunt response to anti-TNF antibody therapy. This prospective study explored effective biomarkers that could monitor disease activity changes in patients with UC. In addition, we examined the effect of anti-TNF antibody therapy on changes in LRG. Methods: Blood and stool samples were collected twice from patients with UC: at baseline and at least 8 weeks later. Changes in serum LRG, interleukin (IL)-6, prealbumin (pre-Alb), high-sensitivity C-reactive protein (hs-CRP), CRP, and fecal calprotectin (FC) were measured and correlated with changes in disease activity. The relationship between anti-TNF antibody therapy and LRG levels was also examined in patients with the same disease activity. Results: Forty-eight patients with UC (96 samples) were analyzed. ΔLRG and ΔIL-6 correlated strongly with the change in the partial Mayo (pMayo) score between the two time points (ΔpMayo) (r = 0.686, 0.635, respectively). In contrast, FC and IL-6 were particularly accurate predictors of clinical remission, and their area under the curves (AUCs) were significantly higher than that of CRP (AUC: 0.81, 0.76 vs. 0.50; p = 0.001, 0.005). No association was found between the administration of anti-TNF antibody preparations and the LRG values. Conclusions: Correlations were found between changes in UC disease activity and LRG, IL-6, pre-Alb, hs-CRP, CRP, and FC. LRG reflects disease activity during anti-TNF antibody therapy. Full article

Sepsis is a crucial public health problem with a high mortality rate caused by a dysregulated host immune response to infection. Vascular endothelial cell injury is an important hallmark of sepsis, which leads to multiple organ failure and death. Early biomarkers to diagnose sepsis may provide early intervention and reduce risk of death. Damage-associated molecular patterns (DAMPs) are host nuclear or cytoplasmic molecules released from cells following tissue damage. We postulated that DAMPs could potentially be a novel sepsis biomarker. We used an in vitro model to determine suitable protein–DAMPs biomarkers for early sepsis diagnosis. Low and high lipopolysaccharide (LPS) doses were used to stimulate the human umbilical vein endothelial cell line EA.hy926 for 24, 48, and 72 h. Results showed that cell viability was reduced in both dose-dependent and time-dependent manners. Cell injury was corroborated by a significant increase in lactate dehydrogenase (LDH) activity within 24 h in cell-conditioned medium. Secreted protein–DAMPs in the supernatant, collected at different time points within 24 h, were characterized using shotgun proteomics LC-MS/MS analysis. Results showed that there were 2233 proteins. Among these, 181 proteins from the LPS-stimulated EA.hy926 at 1, 12, and 24 h were significantly different from those of the control. Twelve proteins were up-regulated at all three time points. Furthermore, a potential interaction analysis of predominant DAMPs-related proteins using STITCH 5.0 revealed the following associations with pathways: response to stress; bacterium; and LPS (GO:0080134; 0009617; 0032496). Markedly, alpha-2-HS-glycoprotein (AHSG or fetuin-A) and lactotransferrin (LTF) potentially presented since the first hour of LPS stimulation, and were highly up-regulated at 24 h. Taken together, we reported proteomic profiling of vascular endothelial cell-specific DAMPs in response to early an in vitro LPS stimulation, suggesting that these early damage-response protein candidates could be novel early biomarkers associated with sepsis. Full article

The objectives of the study were: (1) the evaluation of the blood serum concentration of ZAG (Zinc-alpha 2 Glycoprotein) in girls with anorexia nervosa, as well as in girls with simple obesity and healthy girls; and (2) the valuation of the relationship between the blood level of ZAG and the duration of AN and anthropometric parameters, parameters of the lipid and carbohydrate metabolism, thyroid hormones, and cortisol in the blood in all study subjects. Materials and methods: The study covered 87 girls (aged 11–17.9 years). The studied group (AN) contained 30 girls suffering from anorexia nervosa, and the control groups contained 30 healthy girls (H) and 27 girls with simple obesity (OB), respectively. Results: The mean concentration of ZAG in the blood serum in the AN group was significantly higher than in the OB and H groups. Accumulatively, the level of ZAG in the entire studied group correlated negatively with the parameters of their nutritional status. The mean concentrations of ZAG in the entire group correlated positively with the concentrations of HDL and cortisol and negatively with insulin, HOMA-IR, triglycerides, and hsCRP. Conclusions: The higher blood concentrations of ZAG in girls with AN compared to healthy subjects seemed to constitute a secondary adaptation mechanism in response to the undernourishment status. ZAG blood concentration values correlated negatively with body mass, BMI, Cole’s index, the level of insulin, and the HOMA-IR score, whereas they correlated positively with the level of cortisol. Increased ZAG levels in AN patients may result from increased levels of cortisol, manifesting in malfunction along the hypothalamic–pituitary–adrenal axis, which in effect can promote body weight loss. Full article

Metastasis is a serious and often life-threatening condition, representing the leading cause of death among women with breast cancer (BC). Although the current clinical classification of BC is well-established, the addition of minimally invasive laboratory tests based on peripheral blood biomarkers that reflect pathological changes in the body is of utmost importance. In the current study, the serum proteome and lipidome profiles for 50 BC patients with (25) and without (25) metastasis were studied. Targeted proteomic analysis for concertation measurements of 125 proteins in the serum was performed via liquid chromatography–multiple reaction monitoring mass spectrometry (LC–MRM MS) using the BAK 125 kit (MRM Proteomics Inc., Victoria, BC, Canada). Untargeted label-free lipidomic analysis was performed using liquid chromatography coupled to tandem mass-spectrometry (LC–MS/MS), in both positive and negative ion modes. Finally, 87 serum proteins and 295 lipids were quantified and showed a moderate correlation with tumor grade, histological and biological subtypes, and the number of lymph node metastases. Two highly accurate classifiers that enabled distinguishing between metastatic and non-metastatic BC were developed based on proteomic (accuracy 90%) and lipidomic (accuracy 80%) features. The best classifier (91% sensitivity, 89% specificity, AUC = 0.92) for BC metastasis diagnostics was based on logistic regression and the serum levels of 11 proteins: alpha-2-macroglobulin, coagulation factor XII, adiponectin, leucine-rich alpha-2-glycoprotein, alpha-2-HS-glycoprotein, Ig mu chain C region, apolipoprotein C-IV, carbonic anhydrase 1, apolipoprotein A-II, apolipoprotein C-II and alpha-1-acid glycoprotein 1. Full article

The present studies were conducted to evaluate key serum proteins and other components that mediate anchorage-independent growth (3-D growth) of LNCaP prostate cancer cells as spheroids. The cells were cultured on ultra-low attachment plates in the absence and presence of fetuin-A and with or without extracellular vesicles. The data show that fetuin-A (alpha 2HS glycoprotein) is the serum protein that mediates 3-D growth in these cells. It does so by sequestering extracellular vesicles of various sizes on the surfaces of rounded cells that grow as spheroids. These vesicles in turn transmit growth signals such as the activation of AKT and MAP kinases in a pattern that differs from the activation of these key growth signaling pathways in adherent and spread cells growing in 2-D. In the process of orchestrating the movement and disposition of extracellular vesicles on these cells, fetuin-A is readily internalized in adhered and spread cells but remains on the surfaces of non-adherent cells. Taken together, our studies suggest the presence of distinct signaling domains or scaffolding platforms on the surfaces of prostate tumor cells growing in 3-D compared to 2-D. Full article

(1) Background: The aim of this dynamic-LC/MS-human-serum-proteomic-study was to identify potential proteins-candidates for biomarkers of acute ischemic stroke, their changes during acute phase of stroke and to define potential novel drug-targets. (2) Methods: A total of 32 patients (29–80 years) with acute ischemic stroke were enrolled to the study. The control group constituted 29 demographically-matched volunteers. Subjects with stroke presented clinical symptoms lasting no longer than 24 h, confirmed by neurological-examination and/or new cerebral ischemia visualized in the CT scans (computed tomography). The analysis of plasma proteome was performed using LC-MS (liquid chromatography–mass spectrometry). (3) Results: Ten proteins with significantly different serum concentrations between groups volunteers were: complement-factor-B, apolipoprotein-A-I, fibronectin, alpha-2-HS-glycoprotein, alpha-1B-glycoprotein, heat-shock-cognate-71kDa protein/heat-shock-related-70kDa-protein-2, thymidine phosphorylase-2, cytoplasmic-tryptophan-tRNA-ligase, ficolin-2, beta-Ala-His-dipeptidase. (4) Conclusions: This is the first dynamic LC-MS study performed on a clinical model which differentiates serum proteome of patients in acute phase of ischemic stroke in time series and compares to control group. Listed proteins should be considered as risk factors, markers of ischemic stroke or potential therapeutic targets. Further clinical validation might define their exact role in differential diagnostics, monitoring the course of the ischemic stroke or specifying them as novel drug targets. Full article

Cataracts are one of the most common eye diseases that can cause blindness. Discovering susceptibility factors in the proteome that contribute to cataract development would be helpful in gaining new insights in the molecular mechanisms of the cataract process. We used label-free nanoflow ultra-high-performance liquid chromatography–tandem mass spectrometry to compare aqueous humor protein expressions in cataract patients with different cataract risk factors such as diabetes mellitus (DM) and smoking and in controls (with cataract) without risk exposure. Eight patients with diabetes and who smoked (with double risk factors), five patients with diabetes and five patients who smoked (both with a single risk factor), and nine aged-matched cataract controls patients (non-risk exposure) were enrolled. In total, 136 aqueous humor proteins were identified, of which only alpha-2-Heremans–Schmid (HS)-glycoprotein was considered to be significantly risk-associated because it was differentially expressed in these three groups and exhibited increased expression with increasing risk factors. Significant changes in the aqueous humor level of alpha-2-HS-glycoprotein between DM and control samples and between smoking and control samples were confirmed using ELISA. The alpha-2-HS-glycoprotein, called fetuin-a, could be a potential aqueous biomarker associated with DM and smoking, which were cataract risk factors. Full article

Preeclampsia (PE) is a severe pregnancy complication, which may be considered as a systemic response in the second half of pregnancy to physiological failures in the first trimester, and can lead to very serious consequences for the health of the mother and fetus. Since PE is often associated with proteinuria, urine proteomic assays may represent a powerful tool for timely diagnostics and appropriate management. High resolution mass spectrometry was applied for peptidome analysis of 127 urine samples of pregnant women with various hypertensive complications: normotensive controls (n = 17), chronic hypertension (n = 16), gestational hypertension (n = 15), mild PE (n = 25), severe PE (n = 25), and 29 patients with complicated diagnoses. Analysis revealed 3869 peptides, which mostly belong to 116 groups with overlapping sequences. A panel of 22 marker peptide groups reliably differentiating PE was created by multivariate statistics, and included 15 collagen groups (from COL1A1, COL3A1, COL2A1, COL4A4, COL5A1, and COL8A1), and single loci from alpha-1-antitrypsin, fibrinogen, membrane-associated progesterone receptor component 1, insulin, EMI domain-containing protein 1, lysine-specific demethylase 6B, and alpha-2-HS-glycoprotein each. ROC analysis of the created model resulted in 88% sensitivity, 96.8% specificity, and receiver operating characteristic curve (AUC) = 0.947. Obtained results confirm the high diagnostic potential of urinary peptidome profiling for pregnancy hypertensive disorders diagnostics. Full article