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.

B Cell Lymphocytes as a Potential Source of Breast Carcinoma Marker Candidates.

Despite advances in the genomic classification of breast cancer, current clinical tests and treatment decisions are commonly based on protein-level information. Nowadays breast cancer clinical treatment selection is based on the immunohistochemical (IHC) determination of four protein biomarkers: Estrogen Receptor 1 (ESR1), Progesterone Receptor (PGR), Human Epidermal Growth Factor Receptor 2 (HER2), and proliferation marker Ki-67. The prognostic correlation of tumor-infiltrating T cells has been widely studied in breast cancer, but tumor-infiltrating B cells have not received so much attention. We aimed to find a correlation between immunohistochemical results and a proteomic approach in measuring the expression of proteins isolated from B-cell lymphocytes in peripheral blood samples. Shotgun proteomic analysis was chosen for its key advantage over other proteomic methods, which is its comprehensive and untargeted approach to analyzing proteins. This approach facilitates better characterization of disease-associated changes at the protein level. We identified 18 proteins in B cell lymphocytes with a significant fold change of more than 2, which have promising potential to serve as breast cancer biomarkers in the future.

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.

The Role of ARHGAP1 in Rho GTPase Inactivation during Metastasizing of Breast Cancer Cell Line MCF-7 after Treatment with Doxorubicin.

Breast cancer is the most prevalent cancer type in women worldwide. It proliferates rapidly and can metastasize into farther tissues at any stage due to the gradual invasiveness and motility of the tumor cells. These crucial properties are the outcome of the weakened intercellular adhesion, regulated by small guanosine triphosphatases (GTPases), which hydrolyze to the guanosine diphosphate (GDP)-bound conformation. We investigated the inactivating effect of ARHGAP1 on Rho GTPases involved signaling pathways after treatment with a high dose of doxorubicin. Label-free quantitative proteomic analysis of the proteome isolated from the MCF-7 breast cancer cell line, treated with 1 µM of doxorubicin, identified RAC1, CDC42, and RHOA GTPases that were inactivated by the ARHGAP1 protein. Upregulation of the GTPases involved in the transforming growth factor-beta (TGF-beta) signaling pathway initiated epithelial-mesenchymal transitions. These findings demonstrate a key role of the ARHGAP1 protein in the disruption of the cell adhesion and simultaneously allow for a better understanding of the molecular mechanism of the reduced cell adhesion leading to the subsequent metastasis. The conclusions of this study corroborate the hypothesis that chemotherapy with doxorubicin may increase the risk of metastases in drug-resistant breast cancer cells.

Human peripheral blood mononuclear cells: A review of recent proteomic applications.

Human peripheral blood mononuclear cells (PBMCs) represent a sentinel blood sample which reacts to different pathophysiological stimuli in the form of immunological responses/immunophenotypic changes. The study of molecular content of PBMCs can provide better understanding of immune processes giving the possibility of monitoring the health conditions of the host organism. Proteomic analysis of PBMCs can achieve mentioned goal as important immune-related biomarkers are easily accessible for analysis. PBMCs have been gaining attention in different research areas including preclinical or clinical investigations. In this review, recent applications of proteomic analysis of PBMCs are described and discussed. Approaches are divided based on different proteomic workflows such as in-gel, in-solution and on-filter modes. The effect of various diseases such as autoimmune, cancer, neurodegenerative, viral, metabolic, and various immune stimulations such as radiation, vaccine, corticosteroids over PBMCs proteome, are described with emphasis on promising protein biomarker candidates.

The unfolded protein response controls endoplasmic reticulum stress-induced apoptosis of MCF-7 cells via a high dose of vitamin C treatment.

Recent in vitro studies have shown that vitamin C (Vit C) with pro-oxidative properties causes cytotoxicity of breast cancer cells by selective oxidative stress. However, the effect of Vit C in itself at different concentration levels on MCF-7 breast cancer cell line after 24 h, has not yet been described. We aimed to examine the effect of Vit C on the viability and signalling response of MCF-7/WT (MCF-7 wild-type) cells that were exposed to various concentrations (0.125-4 mM) of Vit C during 24 h. The cytotoxic effect of Vit C on MCF-7/VitC (MCF-7/WT after added 2 mM Vit C) was observed, resulting in a decrease of cell index after 12 h. Also, the cytotoxicity of Vit C (2 mM) after 24 h was confirmed by flow cytometry, i.e., increase of dead, late apoptotic, and depolarized dead MCF-7/VitC cells compared to MCF-7/WT cells. Moreover, changes in proteomic profile of MCF-7/VitC cells compared to the control group were investigated via label-free quantitative mass spectrometry and post-translational modification. Using bioinformatics assessment (i.e., iPathwayGuide and SPIA R packages), a significantly impacted pathway in MCF-7/VitC was identified, namely the protein processing in endoplasmic reticulum. The semi-quantitative change (log2fold change = 4.5) and autophosphorylation at Thr-446 of protein kinase (PKR) (involved in this pathway) indicates that PKR protein could be responsible for the unfolded protein response and inhibition of the cell translation during endoplasmic reticulum stress, and eventually, for cell apoptosis. These results suggest that increased activity of PKR (Thr-446 autophosphorylation) related to cytotoxic effect of Vit C (2 mM) may cause the MCF-7 cells death.

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.

MALDI-TOF/MS Profiling of Whole Saliva and Gingival Crevicular Fluid in Patients with the Invisalign System and Fixed Orthodontic Appliances.

The movement of teeth by orthodontic treatment with the Invisalign (IN) system and fixed orthodontic appliances (FOA) is characterized by the reconstruction of periodontal ligaments, alveolar bone, and gingiva. A reflection of these phenomena can be found in the composition of gingival crevicular fluid (GCF). A total of 90 samples from 45 participants (45 whole saliva and 45 GCF), including 15 patients with FOA, 15 patients with IN, and 15 patients with oral health, were subjected to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF/MS) analysis. Mass fingerprints were generated for each sample. Three models were tested: a quick classifier (QC), a genetic algorithm (GA), and a supervised neural network (SNN). For both groups of samples (saliva and GCF), the GA model showed the highest recognition abilities of 88.89% (saliva) and 95.56% (GCF). Differences between the treated (FOA and IN) groups and the control group in saliva and GCF samples were determined using cluster analysis. In addition, we monitored the effect of long-term orthodontic treatment (after 6 months) in the lag phase of orthodontic tooth movement. The results show increased levels of inflammatory markers (α-defensins), which may indicate an ongoing inflammatory process even after 21 days from force application.

The Performance of CR180IF and DP600 Laser Welded Steel Sheets under Different Strain Rates.

The presented research background is a car body manufacturer's request to test the car body's components welded from dissimilar steel sheets. In view of the vehicle crew's protection, it is necessary to study the static and dynamic behavior of welded steels. Therefore, the influence of laser welding on the mechanical and dynamical properties, microstructure, microhardness, and welded joint surface roughness of interstitial free CR180IF and dual-phase DP600 steels were investigated. Static tensile tests were carried out by using testing machine Zwick 1387, and dynamic test used rotary hammer machine RSO. Sheet steel was tested at different strain rates ranging from 10-3 to 103 s-1. The laser welds' microstructure and microhardness were evaluated in the base metal, heat-affected zone, and fusion zone. The comprehensive analysis also included chemical analysis, fracture surface analysis, and roughness measurement. The research results showed that the strain rate had an influence on the mechanical properties of base materials and welded joints. The dynamic loading increases the yield stress more than the ultimate tensile strength for the monitored steels, while the most significant increase was recorded for the welded material.

Diagnostic Test Accuracy of First-Void Urine Human Papillomaviruses for Presence Cervical HPV in Women: Systematic Review and Meta-Analysis.

First-void urine usually contains exfoliated cells of the debris and mucus from the female genital organs and cervix, i.e., high concentration of human papillomavirus deoxyribonucleic acid (HPV DNA). We conducted a meta-analysis of published data and determined an accuracy of HPV detection in first-void urine compared to the women's cervix. According to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, we carried out a comprehensive literature search. Eligible articles published from 2011 until 2021 were gathered by searching Embase, PubMed and Cochrane Library Central databases. The patient selection, index test, standard test, and patient flow were the factors involved in quality evaluation. A meta-analysis of 15 studies (3412 women) based on 5054 potential records was conducted. Pooled sensitivity for high-risk HPV detection in urine of 78% (70-84%) and specificity of 89% (81-94%) were calculated. Any HPV detection in urine of 87% (74-94%) and 91% (83-96%) were pooled sensitivity and specificity, respectively. HPV 16 and 18 had a pooled sensitivity of 77% (76-77%) and specificity of 98% (98-98%). Meta-analysis indicated variations between the pooled specificities and sensitivities. In meta-regression analysis, a heterogeneity in accuracy by using covariates (bias in patient selection, purpose, sample timing, storage temperature and HPV detection method) were not detected. Our meta-analysis demonstrates the accuracy of detection of HPV in urine for the presence of cervical HPV. Although progress is continuously made in urinary HPV detection, further studies are needed to evaluate and to improve the accuracy of the first-void urine test in order to be comparable with other screening methods.

3D Printing Optimization for Environmental Sustainability: Experimenting with Materials of Protective Face Shield Frames.

The motivation for research on 3D printing of protective face shields was the urgent societal demand for healthcare in the fight against the spread of COVID19 pandemic. Research is based on a literature review that shows that objects produced by additive technologies do not always have consistent quality suitable for the given purpose of use. Besides, they have different effects on the environment and leave different footprints. The overall goal of the research was to find out the most suitable thermoplastic material for printing shield frames in terms of mechanical properties, geometric accuracy, weight, printing time, filament price, and environmental sustainability. Fused deposition modeling (FDM) technology was used for 3D printing, and three different filaments were investigated: polylactic acid (PLA), polyethylene terephthalate (PETG), and polyhydroxyalkanoate (PHA). The weighted sum method for multi-objective optimization was used. Finally, PHA material was chosen, mainly due to its environmental sustainability, as it has the most negligible impact on the environment.

RHOA and mDia1 Promotes Apoptosis of Breast Cancer Cells Via a High Dose of Doxorubicin Treatment.

BACKGROUND: Transforming RhoA proteins (RHOA) and their downstream Diaphanous homolog 1 proteins (DIAPH1) or mDia1 participate in the regulation of actin cytoskeleton which plays critical role in cells, i.e., morphologic changes and apoptosis. METHODOLOGY: To determine the cell viability the real time cell analysis (RTCA) and flow cytometry were used. To perform proteomic analysis, the label-free quantitative method and post-translation modification by the nano-HPLC and ESI-MS ion trap mass analyser were used. RESULTS: The results of the cell viability showed an increase of dead cells (around 30 %) in MCF-7/DOX-1 (i.e., 1µM of doxorubicin was added to MCF-7/WT breast cancer cell line) compared to MCF-7/WT (control) after 24 h doxorubicin (DOX) treatment. The signalling pathway of the Regulation of actin cytoskeleton (p<0.0026) was determined, where RHOA and mDia1 proteins were up-regulated. Also, post-translational modification analysis of these proteins in MCF-7/DOX-1 cells revealed dysregulation of the actin cytoskeleton, specifically the collapse of actin stress fibbers due to phosphorylation of RHOA at serine 188 and mDia1 at serine 22, resulting in their deactivation and cell apoptosis. CONCLUSION: These results pointed to an assumed role of DOX to dysregulation of actin cytoskeleton and cell death.

Achievements in robotic automation of solvent extraction and related approaches for bioanalysis of pharmaceuticals.

Currently, the growing demand on quick, easy and ecological sample pretreatment methods is unquestionable. Such challenge involves also approaches focusing on the analysis of pharmaceuticals and other endogenous compounds in biological matrices, termed as Bioanalysis. Solvent extraction such as liquid-liquid extraction (LLE), derived liquid phase microextraction (LPME) and related approaches such as solid liquid extraction (SLE), proved to be applicable in bioanalysis, as numerous papers have been published in this field. However, their manual performances may suffer from a long-term and laborious preparation, due to the inherent complexity of the biological samples. A high sample-throughput (enabling measurement of tens or hundreds of samples on a daily basis) can be achieved when automation of sample pretreatment is performed, resulting in decreased imprecision and low waste production of hazardous solvents and risky biological materials. Here, robotic systems have a key role, especially when multiple processing (e.g., 96-well plate format) and coupling to modern analytical instrumentation (e.g. LC-MS) are combined. A thorough overview on the up-to-date automations of LLE, LPME, SLE and solid LLE via robotics, is therefore presented. Pharmaceuticals and related compounds determined in classical liquid biological samples (i.e. plasma/serum, whole blood, urine, saliva etc.) and modern dried matrix spots (DMS) were considered as analytes of interest. The methodologies were critically compared to manual setups and among themselves.

Proteomic analysis of the vitamin C effect on the doxorubicin cytotoxicity in the MCF-7 breast cancer cell line.

PURPOSE: Doxorubicin is an anthracycline drug which inhibits the growth of breast cancer cell lines. However, a major factor limiting its use is a cumulative, dose-dependent cardiotoxicity, resulting in a permanent loss of cardiomyocytes. Vitamin C was found to potentiate the cytotoxic effects of a variety of chemotherapeutic drugs including doxorubicin. The aim of the study was to describe the changes in protein expression and proliferation of the MCF-7 cells induced by the vitamin C applied with doxorubicin. METHODS: Label-free quantitative proteomics and real-time cell analysis methods were used to search for proteome and cell proliferation changes. These changes were induced by the pure DOX and by DOX combined with vitamin C applied on the MCF-7 cell line. RESULTS: From the real-time cell analysis experiments, it is clear that the highest anti-proliferative effect occurs with the addition of 200 µM of vitamin C to 1 µM of doxorubicin. By applying both the label-free protein quantification method and total ion current assay, we found statistically significant changes (p ≤ 0.05) of 26 proteins induced by the addition of vitamin C to doxorubicin on the MCF-7 cell line. These differentially expressed proteins are involved in processes such as structural molecule activity, transcription and translation, immune system process and antioxidant, cellular signalling and transport. CONCLUSION: The detected proteins may be capable of predicting response to DOX therapy. This is a key tool in the treatment of breast cancer, and the combination with vit C seems to be of particular interest due to the fact that it can potentiate anti-proliferative effect of DOX.