Two Decades of Progress in Personalized Medicine of Colorectal Cancer in Serbia-Insights from the Institute for Oncology and Radiology of Serbia.

BACKGROUND: It is projected that, by 2040, the number of new cases of colorectal cancer (CRC) will increase to 3.2 million, and the number of deaths to 1.6 million, highlighting the need for prevention strategies, early detection and adequate follow-up. In this study, we aimed to provide an overview of the progress in personalized medicine of CRC in Serbia, with results and insights from the Institute for Oncology and Radiology of Serbia (IORS), and to propose guidance for tackling observed challenges in the future. METHODS: Epidemiological data were derived from official global and national cancer registries and IORS electronic medical records. Germline genetic testing for Lynch syndrome was performed by Next Generation Sequencing. RAS and BRAF mutation analyses were performed using qPCR diagnostic kits. RESULTS: Epidemiology and risk factors, prevention and early detection programs, as well as treatment options and scientific advances have been described in detail. Out of 103 patients who underwent germline testing for Lynch syndrome, 19 (18.4%) showed a mutation in MMR genes with pathogenic or likely pathogenic significance and 8 (7.8%) in other CRC-associated genes (APC, CHEK2, MUTYH). Of 6369 tested patients, 50.43% had a mutation in KRAS or NRAS genes, while 9.54% had the V600 mutation in the BRAF gene. CONCLUSIONS: Although significant improvements in CRC management have occurred globally in recent years, a strategic approach leading to population-based systemic solutions is required. The high incidence of young-onset CRC and the growing elderly population due to a rise in life expectancy will be especially important factors for countries with limited healthcare resources like Serbia.

CHARMM at 45: Enhancements in Accessibility, Functionality, and Speed.

Since its inception nearly a half century ago, CHARMM has been playing a central role in computational biochemistry and biophysics. Commensurate with the developments in experimental research and advances in computer hardware, the range of methods and applicability of CHARMM have also grown. This review summarizes major developments that occurred after 2009 when the last review of CHARMM was published. They include the following: new faster simulation engines, accessible user interfaces for convenient workflows, and a vast array of simulation and analysis methods that encompass quantum mechanical, atomistic, and coarse-grained levels, as well as extensive coverage of force fields. In addition to providing the current snapshot of the CHARMM development, this review may serve as a starting point for exploring relevant theories and computational methods for tackling contemporary and emerging problems in biomolecular systems. CHARMM is freely available for academic and nonprofit research at https://academiccharmm.org/program.

EGFR mutation testing from pleural effusions of non-small cell lung cancer patients at the institute for oncology and radiology of Serbia.

BACKGROUND: The use tumor-derived cell-free DNA extracted from body fluids is being evaluated for genetic testing in lung cancer. The aim of this study was to explore the feasibility and utility of implementation of EGFR molecular testing from pleural effusions in non-small cell lung cancer in the clinical diagnostics workflow. PATIENTS AND METHODS: This study included patients diagnosed with primary lung adenocarcinoma in the period July 2016 to June 2023. EGFR mutation testing was performed by qPCR (Cobas®) and dPCR. Testing was performed from 211 plasma samples when tissue was unavailable at diagnosis, and from 301 plasma samples and 18 pleural effusions at progression on first/second generation of EGFR TKIs. Descriptive methods of statistical analysis were used to summarize the sample data. Fisher's exact test, McNemar's test, Cohen's kappa tests were used for statistical analyses. Two-sided p-values <0.05 were considered statistically significant. RESULTS: A significantly higher detection rate of the T790M mutation in pleural effusion was obtained compared to blood (50% and 20%, p=0.047). When comparing the detection success rate of the resistant T790M mutation in pleural effusion and blood, a statistically significant difference was obtained in favor of pleural effusion (50% vs. 21.87%, p=0.01). CONCLUSIONS: Superior performance of pleural effusions compared to blood plasma was shown both in the analysis of success rate and in the detection of the resistant T790M mutation, at progression on EGFR TKIs. Pleural effusion should be considered in this setting whenever available, especially in countries with limited health resources.

Ion channel selectivity through ion-modulated changes of selectivity filter pKa values.

In bacterial voltage-gated sodium channels, the passage of ions through the pore is controlled by a selectivity filter (SF) composed of four glutamate residues. The mechanism of selectivity has been the subject of intense research, with suggested mechanisms based on steric effects, and ion-triggered conformational change. Here, we propose an alternative mechanism based on ion-triggered shifts in pKa values of SF glutamates. We study the NavMs channel for which the open channel structure is available. Our free-energy calculations based on molecular dynamics simulations suggest that pKa values of the four glutamates are higher in solution of K+ ions than in solution of Na+ ions. Higher pKa in the presence of K+ stems primarily from the higher population of dunked conformations of the protonated Glu sidechain, which exhibit a higher pKa shift. Since pKa values are close to the physiological pH, this results in predominant population of the fully deprotonated state of glutamates in Na+ solution, while protonated states are predominantly populated in K+ solution. Through molecular dynamics simulations we calculate that the deprotonated state is the most conductive, the singly protonated state is less conductive, and the doubly protonated state has significantly reduced conductance. Thus, we propose that a significant component of selectivity is achieved through ion-triggered shifts in the protonation state, which favors more conductive states for Na+ ions and less conductive states for K+ ions. This mechanism also suggests a strong pH dependence of selectivity, which has been experimentally observed in structurally similar NaChBac channels.

Intrinsically disordered interaction network in an RNA chaperone revealed by native mass spectrometry.

RNA-binding proteins contain intrinsically disordered regions whose functions in RNA recognition are poorly understood. The RNA chaperone Hfq is a homohexamer that contains six flexible C-terminal domains (CTDs). The effect of the CTDs on Hfq's integrity and RNA binding has been challenging to study because of their sequence identity and inherent disorder. We used native mass spectrometry coupled with surface-induced dissociation and molecular dynamics simulations to disentangle the arrangement of the CTDs and their impact on the stability of Escherichia coli Hfq with and without RNA. The results show that the CTDs stabilize the Hfq hexamer through multiple interactions with the core and between CTDs. RNA binding perturbs this network of CTD interactions, destabilizing the Hfq ring. This destabilization is partially compensated by binding of RNAs that contact multiple surfaces of Hfq. By contrast, binding of short RNAs that only contact one or two subunits results in net destabilization of the complex. Together, the results show that a network of intrinsically disordered interactions integrate RNA contacts with the six subunits of Hfq. We propose that this CTD network raises the selectivity of RNA binding.

Protein pKa Prediction by Tree-Based Machine Learning.

Protonation states of ionizable protein residues modulate many essential biological processes. For correct modeling and understanding of these processes, it is crucial to accurately determine their pKa values. Here, we present four tree-based machine learning models for protein pKa prediction. The four models, Random Forest, Extra Trees, eXtreme Gradient Boosting (XGBoost), and Light Gradient Boosting Machine (LightGBM), were trained on three experimental PDB and pKa datasets, two of which included a notable portion of internal residues. We observed similar performance among the four machine learning algorithms. The best model trained on the largest dataset performs 37% better than the widely used empirical pKa prediction tool PROPKA and 15% better than the published result from the pKa prediction method DelPhiPKa. The overall root-mean-square error (RMSE) for this model is 0.69, with surface and buried RMSE values being 0.56 and 0.78, respectively, considering six residue types (Asp, Glu, His, Lys, Cys, and Tyr), and 0.63 when considering Asp, Glu, His, and Lys only. We provide pKa predictions for proteins in human proteome from the AlphaFold Protein Structure Database and observed that 1% of Asp/Glu/Lys residues have highly shifted pKa values close to the physiological pH.

Determinants of conductance of a bacterial voltage-gated sodium channel.

Through molecular dynamics (MD) and free energy simulations in electric fields, we examine the factors influencing conductance of bacterial voltage-gated sodium channel NavMs. The channel utilizes four glutamic acid residues in the selectivity filter (SF). Previously, we have shown, through constant pH and free energy calculations of pKa values, that fully deprotonated, singly protonated, and doubly protonated states are all feasible at physiological pH, depending on how many ions are bound in the SF. With 173 MD simulations of 450 or 500 ns and additional free energy simulations, we determine that the conductance is highest for the deprotonated state and decreases with each additional proton bound. We also determine that the pKa value of the four glutamic residues for the transition between deprotonated and singly protonated states is close to the physiological pH and that there is a small voltage dependence. The pKa value and conductance trends are in agreement with experimental work on bacterial Nav channels, which show a decrease in maximal conductance with lowering of pH, with pKa in the physiological range. We examine binding sites for Na+ in the SF, compare with previous work, and note a dependence on starting structures. We find that narrowing of the gate backbone to values lower than the crystal structure's backbone radius reduces the conductance, whereas increasing the gate radius further does not affect the conductance. Simulations with some amount of negatively charged lipids as opposed to purely neutral lipids increases the conductance, as do simulations at higher voltages.

White blood cell subsets in HER2-positive breast cancer patients treated with trastuzumab in relation to clinical outcome.

To examine whether HER2+ breast cancer patients who have decreased immune effector cells could respond well to trastuzumab, we evaluated the alterations in circulating immune system cell subsets: CD16+ and/or CD56+ lymphocytes, lymphocytes and granulocytes in these patients before and after treatment with trastuzumab-based regimens in relation to clinical response to therapy. The study involved 55 patients with HER2+ breast cancer before and 2 months after the initiation of the therapy. Progressive disease was confirmed in nine out of 55 patients (non-responders), while other patients achieved complete or partial response, or stable disease (responders). Control group consisted of up to 52 healthy individuals. Significantly lower percentages of total lymphocytes, CD16+, CD56+, and CD16+CD56+ lymphocytes as well as higher percentage of granulocytes and a higher ratio of granulocyte to lymphocyte percentages were found in patients before therapy and 2 months after the initiation of the therapy, compared with those in healthy individuals. Responder subgroup showed significantly lower percentages of CD16+, CD56+, and CD16+CD56+ lymphocytes before therapy, compared with those in healthy controls. Two months after the initiation of the therapy, the percentages of immune cell subsets remained significantly lower in responders in comparison with those in the healthy donors, while a significantly decreased percentages of CD56+ and CD16+CD56+ lymphocytes were observed in non-responders, in comparison with those in healthy controls. Our study demonstrated that HER2+ breast cancer patients who have decreased percentages of CD16+, CD56+, and CD16+CD56+ lymphocytes may achieve response to trastuzumab-containing treatment.

Cytotoxic activities of Hypericum perforatum L. extracts against 2D and 3D cancer cell models.

Six extracts were obtained from plant species Hypericum perforatum L., collected at Samsun in Turkey. The aim of this study was to examine the mechanisms of the anticancer activity of these extracts. Methanol, ethyl-acetate and hexane were used as a solvents for extraction from both branch-body part of the plant (extracts 1, 2 and 3) and from plant flowers (extracts 4, 5 and 6). The cytotoxic effects of the extracts were determined against 2D and 3D cancer cell models. Cell cycle changes of treated HeLa cells were analyzed by flow cytometry. Measurements of gene and microRNA expression levels in treated HeLa cells were done by quantitative real time PCR. Five examined extracts (2-6) exerted selective concentration-dependent cytotoxic effects on HeLa, K562, and A549 cancer cells, while the extract 1 exhibited very weak cytotoxicity. The extract 6 showed the highest intensity of cytotoxic activity. All tested extracts (2-6) demonstrated the ability to induce apoptosis in HeLa cells through activation of caspase-3. These extracts remarkably decreased gene expression levels of MMP2, MMP9, TIMP3, and VEGFA in HeLa cells. Flower extracts might have stronger effects on miR128/193a-5p/335 level changes than branch-body extracts. Hypericum perforatum extracts exerted weaker cytotoxic effects on 3D HeLa spheroids when compared with their effects on 2D monolayer HeLa cells. Taken together, results of our research may suggest the promising anticancer properties of the Hypericum perforatum extracts.

3D HeLa spheroids as a model for investigating the anticancer activity of Biginelli-hybrids.

In previous study, we examined the anticancer effects of novel Biginelli-hybrids against HeLa cell line on 2D monolayer culture. The five most effective compounds were chosen for further analysis of their anticancer activity against HeLa spheroids. Using the 3D models implies the possible differences in anticancer effects and mechanisms of activity of tested compounds. The compounds 4c and 4d exerted the strongest activity against 3D HeLa spheroids and induced to some extent loosened cell-cell contacts in spheroids, leading to the largest reduction in the diameter of the spheroids. Additionally, the highest accumulation of the cells in the subG1 phase of the cell cycle was observed after the treatment with compounds 4d and 4c, while the compound 4f led to the G2/M arrest. The invasion potential of treated HeLa cells in spheroids was monitored by imaging of spheroids embedded in a matrix made of matrigel and collagen and by determination of MMP2, MMP9, and VEGF gene expression levels. The compound 4l did not show invasion-suppressive activity, while the compounds 4c and 4d exerted the strongest anti-invasive activity.

Influence of selective acid-etching on functionality of halloysite-chitosan nanocontainers for sustained drug release.

The functionality of halloysite (Hal) nanotubes as drug carriers can be improved by lumen enlargement and polymer modification. This study investigates the influence of selective acid etching on Hal functionalization with cationic biopolymer chitosan. Hal was subjected to lumen etching under mild conditions, loaded under vacuum with nonsteroidal antiinflammatory drug aceclofenac, and incubated in an acidic solution of chitosan. The functionality of pristine and etched Hal before and upon polymer functionalization was assessed by ζ-potential measurements, structural characterization (FT-IR, DSC and XRPD analysis), cell viability assay, drug loading and drug release studies. Acid etching increased specific surface area, pore volume and pore size of Hal, decreased ζ-potential and facilitated binding of the cationic polymer. XRPD and DSC analysis revealed crystalline structure of etched Hal. Successful chitosan binding and drug entrapment were further confirmed by FT-IR and DSC studies. XRPD showed surface polymer binding. DSC and FT-IR analyses confirmed the presence of the entrapped drug in its crystalline form. Drug loading was increased for ≈81% by selective lumen etching. Slight decrease of drug content occurred during chitosan functionalization due to aceclofenac diffusion in the polymer solution. The drug release was more sustained from etched Hal nanocomposites (up to ≈87% for 12 h) than from pristine Hal (up to ≈97% for 12 h) due to more intensive chitosan binding. High human fibroblast survival rates upon exposure to pristine and etched Hal before and after chitosan functionalization (>90% in the concentration of 1000 µg/mL) confirmed that both lumen etching under mild conditions and polymer functionalization had no significant effect on cytocompatibility. Based on these findings, selective lumen etching in combination with polycation modification appears to be a promising approach for improvement of Hal nanotubes functionality by increasing payload, polymer binding capacity, and sustained release properties with no significant effect on their cytocompatibility.

Exploring the real-world effect of the SARS-CoV-2 pandemic on the molecular diagnostics for cancer patients and high-risk individuals.

Background: The SARS-CoV-2 pandemic introduced a global distraction effect in cancer patients' care. The aim of this study was to explore the effect of the pandemic on the largest molecular diagnostics center for cancer patients and high-risk individuals in Serbia.Research design and methods: EGFR, KRAS/NRAS, BRAF, and BRCA1/2 mutation testing were performed by qPCR and NGS. NGS was used for panel testing of hereditary breast/ovarian cancer and cancers associated with Lynch syndrome. The analytical output during the state of emergency (SoE) was compared to the period before and after the outbreak using one-way ANOVA. Statistical significance was set at p < 0.05.Results: A 38% reduction in the number of analysis was detected during the SoE. After the SoE, a 19% reduction was noted compared to SoE and 50% compared to the period before the SoE (p = 0.038). Three of the 48 scheduled appointments for pretest genetic counseling were carried out during the SoE, but the number of NGS tests increased by 50%.Conclusions: The SARS-CoV-2 pandemic had a profound negative effect on the diagnostic output of our centralized molecular diagnostics center. The only positive effect was shortening of waiting lists for hereditary cancer patients and high-risk individuals.

Mahonia aquifolium Extracts Promote Doxorubicin Effects against Lung Adenocarcinoma Cells In Vitro.

Mahonia aquifolium and its secondary metabolites have been shown to have anticancer potential. We performed MTT, scratch, and colony formation assays; analyzed cell cycle phase distribution and doxorubicin uptake and retention with flow cytometry; and detected alterations in the expression of genes involved in the formation of cell-cell interactions and migration using quantitative real-time PCR following treatment of lung adenocarcinoma cells with doxorubicin, M. aquifolium extracts, or their combination. MTT assay results suggested strong synergistic effects of the combined treatments, and their application led to an increase in cell numbers in the subG1 phase of the cell cycle. Both extracts were shown to prolong doxorubicin retention time in cancer cells, while the application of doxorubicin/extract combination led to a decrease in MMP9 expression. Furthermore, cells treated with doxorubicin/extract combinations were shown to have lower migratory and colony formation potentials than untreated cells or cells treated with doxorubicin alone. The obtained results suggest that nontoxic M. aquifolium extracts can enhance the activity of doxorubicin, thus potentially allowing the application of lower doxorubicin doses in vivo, which may decrease its toxic effects in normal tissues.

Integration of dry-column flash chromatography with NMR and FTIR metabolomics to reveal cytotoxic metabolites from Amphoricarpos autariatus.

Metabolomics generate a profile of small molecules from plant extracts, which could be directly responsible for bioactivity effects. Using dry-column flash chromatography enabled a rapid and inexpensive method for the very efficient separation of plant extract with a high resolution. This separation method coupled to NMR and FTIR-based metabolomics is applied to identify bioactive natural products. OPLS multivariate analysis method, was used for correlation the chemical composition of the plant extracts, Amphoricarpos autariatus, with the results of cytotoxic activity against Human cervical adenocarcinoma cell line (HeLa) and epithelial lung cancer cell line (A549). In this way, the highest contribution to the cytotoxic activity was recorded for the guaianolide sesquiterpene lactones named amphoricarpolides. The compounds indicated as bioactive after metabolomics analysis were tested, and their cytotoxic activity were confirmed.

Protonation state of the selectivity filter of bacterial voltage-gated sodium channels is modulated by ions.

The selectivity filter (SF) of bacterial voltage-gated sodium channels consists of four glutamate residues arranged in a C4 symmetry. The protonation state population of this tetrad is unclear. To address this question, we simulate the pore domain of bacterial voltage-gated sodium channel of Magnetococcus sp. (Nav Ms) through constant pH methodology in explicit solvent and free energy perturbation calculations. We find that at physiological pH the fully deprotonated as well as singly and doubly protonated states of the SF appear feasible, and that the calculated pKa decreases with each additional bound ion, suggesting that a decrease in the number of ions in the pore can lead to protonation of the SF. Previous molecular dynamics simulations have suggested that protonation can lead to a decrease in the conductance, but no pKa calculations were performed. We confirm a decreased ionic population of the pore with protonation, and also observe structural symmetry breaking triggered by protonation; the SF of the deprotonated channel is closest to the C4 symmetry observed in crystal structures of the open state, while the SF of protonated states display greater levels of asymmetry which could lead to transition to the inactivated state which possesses a C2 symmetry in the crystal structure. We speculate that the decrease in the number of ions near the mouth of the channel, due to either random fluctuations or ion depletion due to conduction, could be a self-regulatory mechanism resulting in a nonconducting state that functionally resembles inactivated states.

EGFR mutation testing from liquid biopsy of non-small cell lung cancer at the Institute for Oncology and Radiology of Serbia.

PURPOSE: Resistance to tyrosine kinase inhibitors (TKIs) in lung cancer often occurs, so mutation testing from liquid biopsy is the method of choice as a minimally invasive approach that quickly provides information for additional therapeutic options. The purpose of this study was to assess the success rate and usefulness of EGFR testing from liquid biopsy at the Institute for Oncology and Radiology of Serbia (IORS). METHODS: EGFR mutation testing was performed by real-time qPCR in 4750 tumor samples using the Cobas® EGFR Mutation Test v2. EGFR testing from 104 liquid biopsy samples was used to track the resistance on first-line EGFR-TKIs as well as for initial testing of 124 patients without tissue biopsies. RESULTS: Liquid biopsy samples were tested in cases with inadequate material for DNA isolation or without tissue biopsy at diagnosis. Nine mutated samples were detected (7.3 %) with a 99.2 % testing success rate. Testing liquid biopsy samples of patients who progressed on EGFR-TKIs showed an accordance rate of 67% with driver mutations, and 49% of mutated patients had the T790M mutation which rendered them eligible for third-generation EGFR-TKIs. An additional 5 patients tested EGFR wild type from plasma after progression were rebiopsied and 3 of them had the T790M mutation. CONCLUSIONS: EGFR mutation testing from liquid biopsy has been successfully implemented in Serbia and has proven invaluable for detecting molecular resistance mechanisms to EGFR-TKIs and as an alternative sample source for patients with scarce biopsy material or without any at all.

Biological Potential of Novel Methoxy and Hydroxy Substituted Heteroaromatic Amides Designed as Promising Antioxidative Agents: Synthesis, 3D-QSAR Analysis, and Biological Activity.

This paper discusses antioxidative and biological activities of 25 novel amidino substituted benzamides with a variety of heteroaromatic nuclei attached to the benzamide moiety and with a variable number of methoxy or hydroxy substituents. Targeted compounds, bearing either amidino or 2-imidazolinyl substituent, were obtained in the Pinner reaction from cyano precursors. 3D-QSAR models were generated to predict antioxidative activity of the 25 novel aromatic and heteroaromatic benzamide derivatives. The compounds were tested for antioxidative activity using in vitro spectrophotometric assays. Direct validation of 3D-QSAR approach for predicting activities of novel benzamide derivatives was carried out by comparing experimental and computationally predicted antioxidative activity. Experimentally determined activities for all novel compounds were found to be within a standard deviation of error of the models. Following this, structure-activity relationships among the synthesized compounds are discussed. Furthermore, antiproliferative activity in vitro against HeLa cells as well as antibacterial and antifungal activity was tested to confirm the other biological activities of the prepared compounds.

Mechanisms of Chemotherapy Resistance in Triple-Negative Breast Cancer-How We Can Rise to the Challenge.

Triple-negative (TNBC) is the most lethal subtype of breast cancer owing to high heterogeneity, aggressive nature, and lack of treatment options. Chemotherapy remains the standard of care for TNBC treatment, but unfortunately, patients frequently develop resistance. Accordingly, in recent years, tremendous effort has been made into elucidating the mechanisms of TNBC chemoresistance with the goal of identifying new molecular targets. It has become evident that the development of TNBC chemoresistance is multifaceted and based on the elaborate interplay of the tumor microenvironment, drug efflux, cancer stem cells, and bulk tumor cells. Alterations of multiple signaling pathways govern these interactions. Moreover, TNBC's high heterogeneity, highlighted in the existence of several molecular signatures, presents a significant obstacle to successful treatment. In the present, in-depth review, we explore the contribution of key mechanisms to TNBC chemoresistance as well as emerging strategies to overcome them. We discuss novel anti-tumor agents that target the components of these mechanisms and pay special attention to their current clinical development while emphasizing the challenges still ahead of successful TNBC management. The evidence presented in this review outlines the role of crucial pathways in TNBC survival following chemotherapy treatment and highlights the importance of using combinatorial drug strategies and incorporating biomarkers in clinical studies.

Association of uPA and PAI-1 tumor levels and 4G/5G variants of PAI-1 gene with disease outcome in luminal HER2-negative node-negative breast cancer patients treated with adjuvant endocrine therapy.

BACKGROUND: The aim of this study was to evaluate the prognostic potential of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) tumor tissue levels and examine the association between these biomarkers and classical prognostic factors in early node-negative luminal breast cancer patients. The clinical value of 4G/5G variants of PAI-1 gene was evaluated. PATIENTS AND METHODS: This study involved 81 node-negative, estrogen receptor-positive and/or progesterone receptor-positive and human epidermal growth factor receptor 2-negative operable breast cancer patients who underwent radical surgical resection and received adjuvant endocrine therapy. Determination of uPA and PAI-1 concentrations in the breast cancer tissue extracts was performed using FEMTELLE® uPA/PAI-1 ELISA. An insertion (5G)/deletion (4G) polymorphism at position - 675 of the PAI-1 gene was detected by PCR-RFLP analysis. RESULTS: Our research showed that patients with uPA tumor tissue levels higher than 3 ng/mg of protein had significantly reduced disease-free survival (DFS) and overall survival (OS) when compared to patients with uPA tumor tissue levels lower or equal to 3 ng/mg of protein. Patients with PAI-1 tumor tissue levels higher than 14 ng/mg of protein had significantly decreased OS in comparison with patients with PAI-1 tumor tissue levels lower or equal to 14 ng/mg of protein. ROC analysis confirmed the uPA and PAI-1 discriminative potential for the presence/absence of relevant events in these patients and resulted in higher cut-off values (5.65 ng/mg of protein for uPA and 27.10 ng/mg of protein for PAI-1) than standard reference cut-off values for both biomarkers. The prognostic importance of uPA and PAI-1 ROC cut-off values was confirmed by the impact of uPA higher than 5.65 ng/mg of protein and PAI-1 higher than 27.10 ng/mg of protein on poorer DFS, OS and event-free survival (EFS). We observed that patients with dominant allele in PAI-1 genotype (heterozygote and dominant homozygote, - 675 4G/5G and - 675 5G/5G) had significantly increased DFS, OS and EFS when compared with patients with recessive homozygote genotype (- 675 4G/4G). CONCLUSION: Our study indicates that uPA and PAI-1 tumor tissue levels and 4G/5G variants of PAI-1 gene might be of prognostic significance in early node-negative luminal HER2-negative breast cancer patients treated with adjuvant endocrine therapy.

Comparative in vitro studies of the biological potential and chemical composition of stems, leaves and berries Aronia melanocarpa's extracts obtained by subcritical water extraction.

Preparation of functional products as well as natural-based products requires non-toxic but effective extraction techniques. In this study, subcritical water extraction was used for the extraction of different aronia parts in order to explore their potential. Stems, leaves and berries of Aronia melanocarpa were extracted under the following conditions: temperature 130 °C; pressure 35 bar; time 20 min. The total phenols and flavonoid contents of the produced extracts were evaluated by conventional spectrophotometric methods. Additionally, the main phenolic compounds were also identified and quantified by high performance liquid chromatography with diode array detection (HPLC-DAD). The biological potential of the extracts was evaluated by determining their antioxidant (DPPH, ABTS and lipid peroxidation assays), antimicrobial, enzyme inhibitory (cholinesterase and elastase) and cytotoxic effects (HeLa, A-549, LS-174T, MRC-5 cell lines). The results indicate that leaves and berries extracts exhibited stronger antioxidant action when compared with stems. The strongest cholinesterase and elastase inhibitory activity was also found in berries extract. Similarly, the extracts obtained from leaves and berries showed considerable cytotoxic effects against tested cell lines. A moderate antimicrobial effects was observed too. Demonstrated biological potential of all three aronia parts can trace a new road map for developing newly designed functional products.