Search Results (23)

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

Several biologically active compounds involved in the green synthesis of silver and gold nanoparticles have been isolated from snail mucus and characterized. This paper presents a successful method for the application of snail mucus from Cornu aspersum as a bioreducing agent of copper sulfate and as a biostabilizer of the copper oxide nanoparticles (CuONPs-Muc) obtained. The synthesis at room temperature and neutral pH yielded nanoparticles with a spherical shape and an average diameter of 150 nm. The structure and properties of CuONPs-Muc were characterized using various methods and techniques, such as ultraviolet–visible spectroscopy (UV–vis), high-performance liquid chromatography (HPLC), one-dimensional polyacrylamide gel electrophoresis (1D-PAGE), up-conversion infrared spectroscopy Fourier transform (FTIR), scanning electron microscopy combined with energy dispersive spectroscopy (SEM/EDS), Raman spectroscopy and imaging, thermogravimetric analysis (TG-DSC), etc. Mucus proteins with molecular weights of 30.691 kDa and 26.549 kDa were identified, which are involved in the biogenic production of CuONPs-Muc. The macromolecular shell of proteins formed around the copper ions contributes to a higher efficiency of the synthesized CuONPs-Muc in inhibiting the bacterial growth of several Gram-positive (Bacillus subtilis NBIMCC2353, Bacillus spizizenii ATCC 6633, Staphylococcus aureus ATCC 6538, Listeria innocua NBIMCC8755) and Gram-negative (Escherichia coli ATCC8739, Salmonella enteitidis NBIMCC8691, Salmonella typhimurium ATCC 14028, Stenotrophomonas maltophilia ATCC 17666) bacteria compared to baseline mucus. The bioorganic synthesis of snail mucus presented here provides CuONPs-Muc with a highly pronounced antimicrobial effect. These results will expand knowledge in the field of natural nanomaterials and their role in emerging dosage forms. Full article

Four new metal–organic frameworks based on cobalt(II) salts and 1,4-diazabicyclo[2.2.2]octane N,N’-dioxide (odabco) were obtained. Their crystallographic formulae are [Co₃(odabco)₂(OAc)₆] (1, OAc⁻ = acetate), [Co(H₂O)₂(HCOO)₂]·odabco (2), [Co₂(H₂O)(NO₃)(odabco)₅](NO₃)₃·3.65H₂O (3), and [Co₂(DMF)₂(odabco)₄](NO₃)₄·3H₂O (4; DMF = N,N-dimethylformamide). Crystal structures of 1–4 were determined by single-crystal X-ray crystallography. Coordination polymer 1 comprises binuclear and mononuclear metal–acetate blocks alternating within uncharged one-dimensional chains, in which odabco acts as a bridging ligand. A layered Co(II) formate 2 contains odabco only as guest molecules located in the interlayer space. Layered compound 3 and three-dimensional 4 have cationic coordination frameworks with 26% and 34% specific void volumes, respectively, unveiling high structural diversity of Co(II)-odabco MOFs based on quite a rare aliphatic moiety. Magnetization measurements were performed for 1, 3, and 4 and the obtained data were interpreted on the basis of their crystal structures. A strong (J/k_B~100 K) antiferromagnetic coupling was found within binuclear metal blocks in 1. Ion exchange experiments revealed a considerable iodide uptake by 3 resulting in an up to 75% guest nitrate substitution within the voids of a coordination framework, found by capillary zone electrophoresis data and confirmed by single-crystal XRD. A preservation of 3 crystallinity during the exchange allowed for the guest I⁻ positions within a new adduct with the formula [Co₂(H₂O)(NO₃)(odabco)₅]I₂(NO₃)·1.85H₂O (3-I) to be successfully determined and the odabco aliphatic core to be revealed as a main adsorption center for quite large and easily polarizable iodide anions. In summary, this work presents a comprehensive study for a series of 1,4-diazabicyclo[2.2.2]octane N,N’-dioxide-based MOFs of cobalt(II) within the framework of magnetic properties and reports the first example of anion exchange in odabco-based coordination networks, supported by direct X-ray structural data. The reported results unveil promising applications of such frameworks bearing ligands with an aliphatic core in the diverse structural design of selective adsorbents and other types of functional materials. Full article

Listeria monocytogenes is a foodborne pathogen that is ubiquitous and largely distributed in food manufacturing environments. It is responsible for listeriosis, a disease that can lead to significant morbidity and fatality in immunocompromised patients, pregnant women, and newborns. Few reports have been published about proteome adaptation when L. monocytogenes is cultivated in stress conditions. In this study, we applied one-dimensional electrophoresis and 2D-PAGE combined with tandem mass spectrometry to evaluate proteome profiling in the following conditions: mild acid, low temperature, and high NaCl concentration. The total proteome was analyzed, also considering the case of normal growth-supporting conditions. A total of 1,160 proteins were identified and those related to pathogenesis and stress response pathways were analyzed. The proteins involved in the expression of virulent pathways when L. monocytogenes ST7 strain was grown under different stress conditions were described. Certain proteins, particularly those involved in the pathogenesis pathway, such as Listeriolysin regulatory protein and Internalin A, were only found when the strain was grown under specific stress conditions. Studying how L. monocytogenes adapts to stress can help to control its growth in food, reducing the risk for consumers. Full article

The potential development of a rapid and highly sensitive breast cancer (BC) diagnostic method has been increasingly investigated by many researchers in order to significantly improve the diagnosis of this disease that affects millions of women worldwide. Thus, this investigation aimed to establish a potential BC urinary peptidomic pattern using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D SDS-PAGE) coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a useful approach for BC diagnosis. The results of Lowry’s assay demonstrated that the total protein concentration increased after precipitation and that the healthy control group (HCs, 160 to 318 µg/mL, 142 ± DD µg/mL, on average) presented higher total protein content than the BC patients (140 to 311 µg/mL, 115 ± DD µg/mL, on average). Related to MALDI-TOF MS analysis, the results revealed that four peptide ion biosignatures (m/z 1046.5, 1062.5, 1237.7 and 1727.9) allowed the discrimination between BC patients and HCs. The distinction efficiency and accuracy of BC urine peptides were determined by receiver operating characteristic (ROC) curve analysis that enabled the recognition of some features with great sensitivity (88%) and specificity (98%). Therefore, the obtained data revealed MALDI-TOF MS as a powerful tool to explore peptidomic biosignatures due to its speed, sensitivity, and mass accuracy, which allow the establishment of novel disease biomarkers. Full article

The fact that cancer is one of the leading causes of death requires researchers to create new systems of effective treatment for malignant tumors. One promising area is genetic therapy that uses small interfering RNA (siRNA). These molecules are capable of blocking mutant proteins in cells, but require specific systems that will deliver RNA to target cells and successfully release them into the cytoplasm. Dendronized and PEGylated silver nanoparticles as potential vectors for proapoptotic siRNA (siMCL-1) were used here. Using the methods of one-dimensional gel electrophoresis, the zeta potential, dynamic light scattering, and circular dichroism, stable siRNA and AgNP complexes were obtained. Data gathered using multicolor flow cytometry showed that AgNPs are able to deliver (up to 90%) siRNAs efficiently to some types of tumor cells, depending on the degree of PEGylation. Analysis of cell death showed that complexes of some AgNP variations with siMCL-1 lead to ~70% cell death in the populations that uptake these complexes due to apoptosis. Full article

It is thought that accurate risk assessment and early diagnosis of breast cancer (BC) can help reduce cancer-related mortality. Proteomics analysis of breast milk may provide biomarkers of risk and occult disease. Our group works on the analysis of human milk samples from women with BC and controls to investigate alterations in protein patterns of milk that could be related to BC. In the current study, we used mass spectrometry (MS)-based proteomics analysis of 12 milk samples from donors with BC and matched controls. Specifically, we used one-dimensional (1D)-polyacrylamide gel electrophoresis (PAGE) coupled with nanoliquid chromatography tandem MS (nanoLC-MS/MS), followed by bioinformatics analysis. We confirmed the dysregulation of several proteins identified previously in a different set of milk samples. We also identified additional dysregulations in milk proteins shown to play a role in cancer development, such as Lactadherin isoform A, O-linked N-acetylglucosamine (GlcNAc) transferase, galactosyltransferase, recoverin, perilipin-3 isoform 1, histone-lysine methyltransferase, or clathrin heavy chain. Our results expand our current understanding of using milk as a biological fluid for identification of BC-related dysregulated proteins. Overall, our results also indicate that milk has the potential to be used for BC biomarker discovery, early detection and risk assessment in young, reproductively active women. Full article

Theileria orientalis is an emerging apicomplexan pathogen of cattle occurring in areas populated by the principal vector tick, Haemaphysalis longicornis. Unlike transforming Theileria spp. that induce cancer-like proliferation of lymphocytes via their schizont stage, T. orientalis destroys host erythrocytes during its piroplasm phase resulting in anaemia. The underlying pathogenic processes of T. orientalis infection are poorly understood; consequently, there are no vaccines for prevention of T. orientalis infection and chemotherapeutic options are limited. To identify antigens expressed during the piroplasm phase of T. orientalis, including those which may be useful targets for future therapeutic development, we examined the proteome across three common genotypes of the parasite (Ikeda, Chitose and Buffeli) using preparations of piroplasms purified from bovine blood. A combination of Triton X-114 extraction, one-dimensional electrophoresis and LC-MS/MS identified a total of 1113 proteins across all genotypes, with less than 3% of these representing host-derived proteins. Just over three quarters of T. orientalis proteins (78%) identified were from the aqueous phase of the TX-114 extraction representing cytosolic proteins, with the remaining 22% from the detergent phase, representing membrane-associated proteins. All enzymes involved in glycolysis were expressed, suggesting that this is the major metabolic pathway used during the T. orientalis piroplasm phase. Proteins involved in binding and breakdown of haemoglobin were also identified, suggesting that T. orientalis uses haemoglobin as a source of amino acids. A number of proteins involved in host cell interaction were also identified which may be suitable targets for the development of chemotherapeutics or vaccines. Full article

Gestational diabetes mellitus (GDM), a glucose intolerance developing or first recognized during pregnancy, leads to a series of short- and long-term maternal and fetal complications, somehow related to placenta structural and functional changes. The focus and the objective of the present review are to discuss the results which can be obtained by different mass spectrometric approaches in the study of placenta protein profile. Thus, matrix-assisted laser desorption/ionization mass spectrometry (MALDI) has been applied on placenta omogenates before and after one-dimensional electrophoretic separation, followed by tryptic digestion. MALDI imaging was used for direct investigation on the placenta tissue (both maternal and fetal sides). The results showed that some differences among the absolute abundances of some proteins are present for placenta samples from GDM patients. The majority of investigations were carried out by two-dimensional electrophoresis (2DE) followed by LC-MS/MS or, directly by the label-free LC-MS^E approach. It should be emphasized that all these techniques were showed differences in the protein expression between the placenta samples from healthy or GDM subjects. 2DE was also employed to separate and compare placental protein levels from GDM and the control groups: differentially expressed proteins between the two groups were identified by MALDI-TOF/TOF mass spectrometry and were further confirmed by Western blotting. The physiopathological significance of the obtained results are reported and discussed in this narrative review. The experimental data obtained until now show that the newest, mass spectrometric approaches can be considered a valid tool to investigate the possible changes of placenta in the presence of GDM. Full article

Magnetic levitation (MagLev) has recently emerged as a powerful method to develop diagnostic technologies based on the exploitation of the nanoparticle (NP)–protein corona. However, experimental procedures improving the robustness, reproducibility, and accuracy of this technology are largely unexplored. To contribute to filling this gap, here, we investigated the effect of total flow rate (TFR) and flow rate ratio (FRR) on the MagLev patterns of microfluidic-generated graphene oxide (GO)–protein complexes using bulk mixing of GO and human plasma (HP) as a reference. Levitating and precipitating fractions of GO-HP samples were characterized in terms of atomic force microscopy (AFM), bicinchoninic acid assay (BCA), and one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis (1D SDS-PAGE), and nanoliquid chromatography–tandem mass spectrometry (nano-LC-MS/MS). We identified combinations of TFR and FRR (e.g., TFR = 35 μL/min and FRR (GO:HP) = 9:1 or TFR = 3.5 μL/min and FRR (GO:HP) = 19:1), leading to MagLev patterns dominated by levitating and precipitating fractions with bulk-like features. Since a typical MagLev experiment for disease detection is based on a sequence of optimization, exploration, and validation steps, this implies that the optimization (e.g., searching for optimal NP:HP ratios) and exploration (e.g., searching for MagLev signatures) steps can be performed using samples generated by bulk mixing. When these steps are completed, the validation step, which involves using human specimens that are often available in limited amounts, can be made by highly reproducible microfluidic mixing without any ex novo optimization process. The relevance of developing diagnostic technologies based on MagLev of coronated nanomaterials is also discussed. Full article

Colorectal cancer (CRC) is one of the leading causes of cancer-related death over the world. There is a great need for biomarkers capable of early detection and as targets for treatment. Differential protein expression was investigated with two-dimensional gel electrophoresis (2D-PAGE) followed by identification with liquid chromatography–tandem mass spectrometry (LC-MS/MS) in CRC patient tissue from (i) the peripheral part of the tumor, (ii) the central part of the tumor as well as from (iii) a non-involved part of the colorectal tissue. The expression patterns of six identified proteins were further evaluated by one-dimensional Western blot (1D-WB) analysis of the CRC tissue. Proteins that were perturbed in expression level in the peripheral or in the central part of the tumor as compared with the non-involved part included S100A11, HNRNPF, HNRNPH1 or HNRNPH2, GSTP1, PKM and FABP1. These identified markers may have future diagnostic potential or may be novel treatment targets after further evaluation in larger patient cohorts. Full article

Chlorhexidine (CHX) is considered the gold standard for the chemical control of bacterial plaque and is often used after surgical treatment. However, CHX employment over an extended time is responsible for side effects such as the appearance of pigmentations on the teeth and tongue; the discoloration effects are less pronounced when using a CHX-based mouthwash with added an anti-discoloration system (ADS). The aim of this study was to evaluate, using one- and two-dimensional gel electrophoresis combined with mass spectrometry, the possible proteomic changes induced by CHX and CHX+ADS in the supragingival dental sites susceptible to a discoloration effect. The tooth surface collected material (TSCM) was obtained by curettage after resective bone surgery from three groups of patients following a supportive therapy protocol in which a mechanical control was combined with placebo rinses or CHX or a CHX+ADS mouthwash. The proteomic analysis was performed before surgery (basal conditions) and four weeks after surgery when CHX was used (or not) as chemical plaque control. Changes in the TSCM proteome were only revealed following CHX treatment: glycolytic enzymes, molecular chaperones and elongation factors were identified as more expressed. These changes were not detected after CHX+ADS treatment. An ADS could directly limit TSCM forming and also the CHX antiseptic effect reduces its ability to alter bacterial cell permeability. However, Maillard’s reaction produces high molecular weight molecules that change the surface properties and could facilitate bacterial adhesion. Full article

Dirofilariarepens is a parasitic nematode causing a vector-borne zoonotic infection (dirofilariosis), considered an emerging problem in human and veterinary medicine. Currently, diagnosis is based on the detection of the adult parasite and microfilariae in the host tissues. However, the efficacy of tests relying on microfilariae detection is limited by microfilariae periodic occurrence. Therefore, a new reliable and affordable serological diagnostic method is needed. Better characteristic of the parasite biology and its interaction with host immune system should help to achieve this goal. This study analyzes adult and microfilariae proteomes, and the use of one-dimensional electrophoresis (1-DE) and two-dimensional electrophoresis (2-DE) proteomics, immunoproteomics, and LC-MS/MS mass spectrometry allowed us to identify 316 potentially immunogenic proteins (75 belong to adult stage, 183 to microfilariae, and 58 are common for both). Classified by their ontology, the proteins showed important similarities and differences between both parasite stages. The most frequently identified proteins are structural, metabolic, and heat shock proteins. Additionally, real-time PCR analysis of some immunogenic targets revealed significant differences between microfilariae and adult life stages. We indicated molecules involved in parasite-host interactions and discussed their importance in parasite biology, which may help to reveal potential diagnostic antigens or select drug and vaccine targets. Full article

We aimed to analyse the effects of maternal intake of an unbalanced diet during lactation in the composition and the levels of proteins present in milk. Milk samples from control nursing dams (C-dams) or from nursing dams fed a cafeteria diet during lactation (CAF-dams) were obtained. We conducted a proteomic approach to identify significantly altered proteins in breast milk of C- and CAF-dams, and evaluated the levels of leptin, adiponectin and irisin for their implication in energy homeostasis. One-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), revealed that the bands that presented a lower intensity in CAF-dams than control contain some caseins (α-S1-casein, α-S2-casein like B, and β-casein), α-lactalbumin and haptoglobin. Leptin and adiponectin levels were greater in the breast milk of CAF-dams than in controls, while levels of irisin were lower. In summary, the relative concentration of bioactive peptides was influenced by maternal diet consumption during lactation; these changes at early stages of life could influence the phenotypic traits of the offspring. Full article

Urine is a biological diagnostic material suitable not only for the analysis of kidney and urinary tract functions but also the function of other tissues and organs. The urine proteome of adult mammals differs from the urine proteome of neonatal ones. The establishment of urinary protein maps of healthy newborn calves is important for diagnosing and monitoring the progression of various diseases. The experiment was carried out on a Polish-Friesian var. of Black-and-White male calves in the sixth day of postnatal life. The two proteomics approaches used for separation and identification of urinary proteins were: 2-DE with MALDI-TOF-TOF-MS/MS and 1-DE with LC-MS/MS. This resulted in the identification of 692 urinary proteins. The majority of them were classified as extracellular proteins (40.32%), as well as proteins involved in regulation of major cellular processes (31.07%). We have observed the presence of unique proteins associated with embryonic (ameloblastin, alpha-fetoprotein, Delta-like protein, embryo-specific fibronectin 1 transcript variant, Indian hedgehog homolog) and kidney development (angiotensin-converting enzyme, angiotensinogen, aquaporin-1, calbindin, glypican 3, nidogen 1, pro-cathepsin H). Additionally, proteins involved in the renal regulation of water and electrolyte balance (angiotensinogen, angiotensin-converting enzyme, aquaporin-1, ezrin, uromodulin) were detected. Presented in the current study 1-D and 2-D urinary proteomic maps are the basis for the identification and detection of prognostic biomarkers important for defining a calf’s health status. Full article