Peripheral Blood CD8+ T-Lymphocyte Immune Response in Benign and Subpopulations of Breast Cancer Patients.

Peripheral blood CD8+ T lymphocytes play a crucial role in cell-mediated immunity and tumor-related immune responses in breast cancer. In this study, label-free quantification analysis and gene set enrichment analysis (GSEA) of CD8+ T lymphocytes in the peripheral blood of benign patients and patients with different breast cancer (BC) subtypes, i.e., luminal A, luminal B, and triple-negative breast cancer (TNBC), were performed using nano-UHPLC and Orbitrap mass spectrometry. Differential protein expression in CD8+ T lymphocytes revealed significant downregulation (log2 FC ≥ 0.38 or ≤-0.38, adj. p < 0.05), particularly in proteins involved in cytotoxicity, cytolysis, and proteolysis, such as granzymes (GZMs) and perforin 1 (PRF1). This downregulation was observed in the benign group (GZMH, GZMM, and PRF1) and luminal B (GZMA, GZMH) subtypes, whereas granzyme K (GZMK) was upregulated in TNBC in comparison to healthy controls. The RNA degradation pathway was significantly downregulated (p < 0.05, normalized enrichment score (NES) from -1.47 to -1.80) across all BC subtypes, suggesting a potential mechanism for regulating gene expression during T cell activation. Also, the Sm-like proteins (LSM2, LSM3, and LSM5) were significantly downregulated in the RNA degradation pathway. Proteomic analysis of CD8+ T lymphocytes in peripheral blood across different breast cancer subtypes provides a comprehensive view of the molecular mechanisms of the systemic immune response that can significantly contribute to advancements in the diagnosis, treatment, and prognosis of this disease.

Release of Monomers from Dental Composite Materials into Saliva and the Possibility of Reducing the Toxic Risk for the Patient.

Background and Objectives: The objective of this study was (1) to measure the amount of monomers released into the saliva depending on the time elapsed after the hardening of the composite and on the type of monomer used; and (2) with the prolongation of the light-curing procedure, to publish information on whether it would be possible to influence the level of leached monomers. Materials and Methods: HPLC technique was used to monitor the levels of the unpolymerized monomers Bis-GMA, Bis/EMA, TEGDMA, and UDMA from the four commonly used composite materials, released into the saliva of a volunteer with intact dentition. The levels were monitored in 3 time periods during 24 h after composite hardening. From every composite material, 4 samples were formed and cured with an LED lamp for 10 s, 20 s, 40 s, and 60 s. After the light curing, the same polishing procedure was used and the samples were leached in blank saliva samples. Results: We observed that every monitored composite material eluted monomers into the saliva after its application. The amount of monomers depended on the time elapsed after the curing of the composite and on the type of composite used. A 40 s LED curing procedure can reduce the amount of leached monomers in comparison with the standard 20 s procedure, especially for monomers of higher molecular weight. Conclusions: Our study confirmed the hypothesis that the release of monomers gradually decreases with increasing time after the hardening of the composite filling.

Experimental Analysis of Tear Fluid and Its Processing for the Diagnosis of Multiple Sclerosis.

A pilot analysis of the tear fluid of patients with multiple sclerosis (MS) collected by glass microcapillary was performed using various experimental methods: liquid chromatography-mass spectrometry, Raman spectroscopy, infrared spectroscopy, and atomic-force microscopy. Infrared spectroscopy found no significant difference between the tear fluid of MS patients and the control spectra; all three significant peaks were located at around the same positions. Raman analysis showed differences between the spectra of the tear fluid of MS patients and the spectra of healthy subjects, which indicated a decrease in tryptophan and phenylalanine content and changes in the relative contributions of the secondary structures of the polypeptide chains of tear proteins. Atomic-force microscopy exhibited a surface fern-shaped dendrite morphology of the tear fluid of patients with MS, with less roughness on both oriented silicon (100) and glass substrates compared to the tear fluid of control subjects. The results of liquid chromatography-mass spectrometry showed downregulation of glycosphingolipid metabolism, sphingolipid metabolism, and lipid metabolism. Proteomic analysis identified upregulated proteins in the tear fluid of patients with MS such as cystatine, phospholipid transfer protein, transcobalamin-1, immunoglobulin lambda variable 1-47, lactoperoxidase, and ferroptosis suppressor protein 1; and downregulated proteins such as haptoglobin, prosaposin, cytoskeletal keratin type I pre-mRNA-processing factor 17, neutrophil gelatinase-associated lipocalin, and phospholipase A2. This study showed that the tear proteome in patients with MS is modified and can reflect inflammation. Tear fluid is not a commonly used biological material in clinico-biochemical laboratories. Experimental proteomics has the potential to become a promising contemporary tool for personalized medicine, and it might be applied in clinical practice by providing a detailed analysis of the tear-fluid proteomic profile of patients with MS.

Differential Urinary Proteomic Analysis of High-Risk Cervical Intraepithelial Neoplasia.

Human papillomavirus (HPV)-associated lesions and malignancies exhibit alterations in the composition and functionality of the extracellular matrix (ECM) that represent the complex molecular pathways present between infection and disease. A total of 20 urine samples were used, including from 10 patients with cervical intraepithelial neoplasia grade 3 (CIN3) and 10 healthy controls to perform the label-free quantitative analysis using the nano-HPLC and ESI-MS ion trap mass analyzer and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) fast screening. Among 476 identified/quantified proteins, 48 were significantly changed (log2-fold change ≥1.0 or ≤-1.0, -log10 (bbinominal, p-value ≥ 1.3), of which were 40 proteins (down-regulated) and 8 proteins (up-regulated) in CIN3, in comparison to healthy controls. The biological function and key pathway enrichment of the gene set using gen set enrichment analysis (GSEA) were analyzed. The ECM-receptor interaction pathway (NES = -1.64, p = 0.026) was down-regulated by 13 proteins (HSPG2, COL6A1, COL6A3, SPP1, THBS1, TNC, DAG1, FN1, COMP, GP6, VTN, SDC1, and CD44; log2 FC range from -0.03 to -1.48) for the CIN3 group in the KEGG database. The MALDI-TOF/MS screening showed the difference of protein profiles between the control and CIN3 groups, i.e., using the scatter plot with a well-separated shape, as well as effectively distinguishing both groups (control and CIN3) using genetic algorithms (GA) with cross-validation (51.56%) and recognition capability (95.0%). Decreased levels of ECM-receptor interaction proteins may cause disturbances in the interactions of cells with the ECM and play an important role in the development and progression of cervical cancer.

Delayed bradykinin postconditioning modulates intrinsic neuroprotective enzyme expression in the rat CA1 region after cerebral ischemia: a proteomic study.

Pyramidal cells in the CA1 brain region exhibit an ischemic tolerance after delayed postconditioning; therefore, this approach seems to be a promising neuroprotective procedure in cerebral postischemic injury improvement. However, little is known about the effect of postconditioning on protein expression patterns in the brain, especially in the affected hippocampal neurons after global cerebral ischemia. This study is focused on the examination of the ischemia-vulnerable CA1 neuronal layer and on the acquisition of protection from delayed neuronal death after ischemia. Ischemic-reperfusion injury was induced in Wistar rats and bradykinin was applied 2 days after the ischemic insult in an attempt to overcome delayed cell death. Analysis of complex peptide CA1 samples was performed by automated two dimensional liquid chromatography (2D-LC) fractionation coupled to tandem matrix assisted laser desorption/ionization time-of-flight (MALDI TOF/TOF) mass spectrometry instrumentation. We devoted our attention to differences in protein expression mapping in ischemic injured CA1 neurons in comparison with equally affected neurons, but with bradykinin application. Proteomic analysis identified several proteins occurring only after postconditioning and control, which could have a potentially neuroprotective influence on ischemic injured neurons. Among them, the prominent position occupies a regulator of glutamate level aspartate transaminase AATC, a scavenger of glutamate in brain neuroprotection after ischemia-reperfusion. We identified this enzyme in controls and after postconditioning, but AATC presence was not detected in the ischemic injured CA1 region. This finding was confirmed by two-dimensional differential electrophoresis followed by MALDI-TOF/TOF MS identification. Results suggest that bradykinin as delayed postconditioning may be associated with modulation of protein expression after ischemic injury and thus this procedure can be involved in neuroprotective metabolic pathways.

Determination of lasalocid residues in the tissues of broiler chickens by liquid chromatography-tandem mass spectrometry.

Lasalocid is a polyether ionophoric coccidiostat used for the prevention of coccidiosis in poultry at a prescribed concentration and during a certain time interval. Due to a public health concern about the presence of coccidiostat residues in poultry, the aim of the present study was to determine the levels of lasalocid residues in the edible tissues of broiler chickens (breast muscle, thigh muscle, heart, liver, gizzard, kidneys and skin/fat) fed commercially produced feed containing 100 mg kg⁻¹) of lasalocid in complete feed throughout the 5-day withdrawal period (WP). The residues were investigated by liquid chromatography coupled with electrospray ionisation (ESI) tandem mass spectrometry (MS/MS) with triple quadrupole. The limit of detection (LOD) and the limit of quantification (LOQ) of the method were 0.47 and 1.44 µg kg⁻¹, respectively. The average recovery based on the matrix-fortified calibrations for chicken tissues ranged between 79% and 98%. Lasalocid was found to accumulate in the liver, followed by the heart, skin/fat, kidneys, thigh muscle and gizzard. The lowest concentrations of lasalocid residues were found in the breast muscle. On day 5 of the WP, residue concentrations of lasalocid did not decline below the LOQ of the method, but were far below the maximum residue level (MRL) established for lasalocid in poultry from 20 to 100 µg kg⁻¹ by European Commission Regulation (EU) No. 37/2010. The results confirmed that the WP established for lasalocid is sufficient to ensure the decline of its residues in the tissues of broiler chickens to the safe residue level.

Liquid chromatography tandem mass spectrometry determination of maduramycin residues in the tissues of broiler chickens.

Maduramycin is a polyether ionophoric coccidiostat used to prevent coccidiosis in poultry at a prescribed concentration over a certain time interval. Due to public health concerns about the presence of coccidiostat residues in poultry, the aim of the present study was to determine the level of maduramycin residues in the tissues of broiler chickens fed commercially produced feed containing 5 mg kg(-1) of maduramycin in complete feed throughout the 5-day withdrawal period (WP). The residues were investigated by liquid chromatography (LC) coupled with electrospray ionisation (ESI) tandem mass spectrometry (MS/MS). The limit of detection (LOD) and limit of quantification (LOQ) of the method were 0.3 and 0.8 microg kg(-1), respectively. The average recovery based on matrix-fortified calibrations for chicken tissues was 90%. Maduramycin was found to be rapidly distributed in all tissues. The highest concentrations of maduramycin residues were found in the heart followed by the skin, liver, gizzard, kidneys and, finally, muscle (thigh and breast). On day 5 of the WP, residue concentrations of maduramycin did not decline below the LOQ of the method. Our results emphasize the need to establish a maximum residue limit (MRL) for maduramycin to control its residue levels in edible tissues from chickens before slaughter.

Evaluation of three different microbial inhibition tests for the detection of sulphamethazine residues in the edible tissues of rabbit.

The aim of the present study was to evaluate three microbial inhibition tests (MIT) based on inhibition of growth of the test organisms: (a) four plate test (FPT) containing Bacillus subtilis BGA, (b) screening test for antibiotic residues (STAR) containing Bacillus stearothermophilus var. calidolactis_ATCC 10149 and (c) the Premi(R)Test containing Bacillus stearothermophilus var. calidolactis. The tests were used to determine sulphamethazine (SMZ) residues in edible tissues of rabbit after oral administration up to day 15 of the withdrawal period (WP). A solvent extraction procedure was used to enhance the capability of the tests to detect SMZ residues at or below the maximum residue limit (MRL). Para-aminobenzoic acid (PABA) was employed to previously identify SMZ residues in the first stage of the residue screening. The presence of SMZ residues in the samples was confirmed and quantified by a validated HPLC method. The Premi(R)Test detected SMZ residues in the muscle and heart tissue up to day 9 of the WP, and in the liver, lungs and kidneys up to day 10 of the WP. The STAR detected SMZ residues in the edible organs of rabbits up to day 8 of the WP. The kidneys were positive up to day 5 of the WP, the liver until day 4 of the WP and the lungs until day 3 of the WP. No SMZ residues were detected in the muscle and heart. By using the solvent extraction procedure, SMZ residues were detected in the muscle extract up to day 10 of the WP and the muscle was positive until day 6 of the WP. No detection sensitivity was observed using the FPT. After solvent extraction, SMZ residues were detected in the muscle extract until day 8 of the WP and the muscle was positive until day 3 of the WP. No positive results were detected after the addition of PABA into/onto the agar medium. PABA at a concentration of 10 microg ml(-1) completely reversed the inhibitory activity of SMZ and enabled reliable identification of SMZ in the examined samples. Using HPLC, SMZ was detected in the muscle samples until day 10 of WP (0.02 mg kg(-1)) and in the liver until day 12 of the WP (0.09 mg kg(-1)). The results obtained by the HPLC method and the limit of detection (LOD) of screening tests for SMZ (FPT 0.4 microg ml(-1), STAR 0.2 microg ml(-1), Premi(R) Test 0.05 microg ml(-1)) allowed us to state that the most suitable screening tests for the detection of SMZ residues in the edible tissues of rabbits at level corresponding to the MRL of 0.1 mg kg(-1), established for sulphonamides, are the Premi(R)Test and STAR in conjunction with the solvent-extraction procedure.