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In calcium electroporation (CaEP), electroporation enables the cellular uptake of supraphysiological concentrations of Ca , causing the induction of cell death. The effectiveness of CaEP has already been evaluated in clinical trials; however, confirmatory preclinical studies are still needed to further elucidate its effectiveness and underlying mechanisms. Here, we tested and compared its efficiency on two different tumor models to electrochemotherapy (ECT) and in combination with gene electrotransfer (GET) of a plasmid encoding interleukin-12 (IL-12). We hypothesized that IL-12 potentiates the antitumor effect of local ablative therapies as CaEP and ECT. The effect of CaEP was tested as well as in murine melanoma B16-F10 and murine mammary carcinoma 4T1 in comparison to ECT with bleomycin. Specifically, the treatment efficacy of CaEP with increasing calcium concentrations alone or in combination with IL-12 GET in different treatment protocols was investigated. We closely examined the tumor microenvironment by immunofluorescence staining of immune cells, as well as blood vessels and proliferating cells. , CaEP and ECT with bleomycin reduced cell viability in a dose-dependent manner. We observed no differences in sensitivity between the two cell lines. A dose-dependent response was also observed ; however, the efficacy was better in 4T1 tumors than in B16-F10 tumors. In 4T1 tumors, CaEP with 250 mM Ca resulted in more than 30 days of growth delay, which was comparable to ECT with bleomycin. In contrast, adjuvant peritumoral application of IL-12 GET after CaEP prolonged the survival of B16-F10, but not 4T1-bearing mice. Moreover, CaEP with peritumoral IL-12 GET modified tumor immune cell populations and tumor vasculature. Mice bearing 4T1 tumors responded better to CaEP than mice bearing B16-F10 tumors, even though a similar response was observed . Namely, one of the most important factors might be involvement of the immune system. This was confirmed by the combination of CaEP or ECT with IL-12 GET, which further enhanced antitumor effectiveness. However, the potentiation of CaEP effectiveness was also highly dependent on tumor type; it was more pronounced in poorly immunogenic B16-F10 tumors compared to moderately immunogenic 4T1 tumors.

Background Disruption of Ca 2+ homeostasis after calcium electroporation (CaEP) in tumors has been shown to elicit an enhanced antitumor effect with varying impacts on healthy tissue, such as endothelium. Therefore, our study aimed to determine differences in Ca 2+  kinetics and gene expression involved in the regulation of Ca 2+  signaling and homeostasis, as well as effects of CaEP on cytoskeleton and adherens junctions of the established endothelial cell lines EA.hy926 and HMEC-1. Methods CaEP was performed on EA.hy926 and HMEC-1 cells with increasing Ca 2+ concentrations. Viability after CaEP was assessed using Presto Blue, while the effect on cytoskeleton and adherens junctions was evaluated via immunofluorescence staining (F-actin, α-tubulin, VE-cadherin). Differences in intracellular Ca 2+ regulation ([Ca 2+ ] i ) were determined with spectrofluorometric measurements using Fura-2-AM, exposing cells to DPBS, ionomycin, thapsigargin, ATP, bradykinin, angiotensin II, acetylcholine, LaCl 3 , and GdCl 3 . Molecular distinctions were identified by analyzing differentially expressed genes and pathways related to the cytoskeleton and Ca 2+ signaling through RNA sequencing. Results EA.hy926 cells, at increasing Ca 2+ concentrations, displayed higher CaEP susceptibility and lower survival than HMEC-1. Immunofluorescence confirmed CaEP-induced, time- and Ca 2+ -dependent morphological changes in EA.hy926’s actin filaments, microtubules, and cell–cell junctions. Spectrofluorometric Ca 2+ kinetics showed higher amplitudes in Ca 2+ responses in EA.hy926 exposed to buffer, G protein coupled receptor agonists, bradykinin, and angiotensin II compared to HMEC-1. HMEC-1 exhibited significantly higher [Ca 2+ ] i changes after ionomycin exposure, while responses to thapsigargin, ATP, and acetylcholine were similar in both cell lines. ATP without extracellular Ca 2+ ions induced a significantly higher [Ca 2+ ] i rise in EA.hy926, suggesting purinergic ionotropic P2X and metabotropic P2Y receptor activation. RNA-sequencing analysis showed significant differences in cytoskeleton- and Ca 2+ -related gene expression, highlighting upregulation of ORAI2 , TRPC1 , TRPM2 , CNGA3 , TRPM6 , and downregulation of TRPV4 and TRPC4 in EA.hy926 versus HMEC-1. Moreover, KEGG analysis showed upregulated Ca 2+ import and downregulated export genes in EA.hy926. Conclusions Our finding show that significant differences in CaEP response and [Ca 2+ ] i regulation exist between EA.hy926 and HMEC-1, which may be attributed to distinct transcriptomic profiles. EA.hy926, compared to HMEC-1, displayed higher susceptibility and sensitivity to [Ca 2+ ] i changes, which may be linked to overexpression of Ca 2+ -related genes and an inability to mitigate changes in [Ca 2+ ] i . The study offers a bioinformatic basis for selecting EC models based on research objectives.

Background: Electrochemotherapy (ECT) is a clinically validated local ablative treatment increasingly recognized for its ability to induce immunogenic cell death and stimulate antitumor immunity. Its combination with immune checkpoint inhibitors, such as anti-PD-1 antibodies, may enhance systemic immune responses and improve therapeutic efficacy, particularly in poorly immunogenic tumors. Methods: We evaluated the antitumor effectiveness of ECT combined with a murine analog of the anti-PD-1 antibody in four syngeneic murine tumor models with differing histology and immune status: WEHI fibrosarcoma, CT26 and MC38 colorectal carcinoma, and 4T1 mammary carcinoma. In vitro cytotoxicity assays assessed tumor cell sensitivity to ECT, while in vivo experiments evaluated complete response (CR) rates, immune cell infiltration, and long-term immune memory through secondary tumor challenge. Immunohistochemical analysis of CD4+, CD8+, and granzyme B+ effector cells. Results: In vitro, WEHI cells exhibited the highest sensitivity to ECT. In vivo, ECT monotherapy induced CRs in 100% of WEHI tumors, 60% of CT26, 17% of 4T1, and 15% of MC38. The addition of anti-PD-1 significantly enhanced outcomes in less responsive models, increasing CRs to 90% in CT26, 91% in MC38, and 53% in 4T1. Combination therapy promoted pronounced infiltration of CD4+, CD8+, and granzyme B+ T cells and the formation of tertiary lymphoid structure, particularly in MC38 tumors. Secondary challenge experiments confirmed long-term immune memory in CT26 and MC38 models and induced memory in 4T1, which was absent following monotherapy. Conclusions: ECT synergizes with PD-1 blockade to potentiate local and systemic antitumor immunity, overcoming immune resistance in poorly immunogenic tumors. These findings support further clinical development of ECT in combination with immune checkpoint inhibitors as a component of personalized cancer immunotherapy.

Disruption of Ca homeostasis after calcium electroporation (CaEP) in tumors has been shown to elicit an enhanced antitumor effect with varying impacts on healthy tissue, such as endothelium. Therefore, our study aimed to determine differences in Ca  kinetics and gene expression involved in the regulation of Ca signaling and homeostasis, as well as effects of CaEP on cytoskeleton and adherens junctions of the established endothelial cell lines EA.hy926 and HMEC-1. CaEP was performed on EA.hy926 and HMEC-1 cells with increasing Ca concentrations. Viability after CaEP was assessed using Presto Blue, while the effect on cytoskeleton and adherens junctions was evaluated via immunofluorescence staining (F-actin, α-tubulin, VE-cadherin). Differences in intracellular Ca regulation ([Ca ] ) were determined with spectrofluorometric measurements using Fura-2-AM, exposing cells to DPBS, ionomycin, thapsigargin, ATP, bradykinin, angiotensin II, acetylcholine, LaCl , and GdCl . Molecular distinctions were identified by analyzing differentially expressed genes and pathways related to the cytoskeleton and Ca signaling through RNA sequencing. EA.hy926 cells, at increasing Ca concentrations, displayed higher CaEP susceptibility and lower survival than HMEC-1. Immunofluorescence confirmed CaEP-induced, time- and Ca -dependent morphological changes in EA.hy926's actin filaments, microtubules, and cell-cell junctions. Spectrofluorometric Ca kinetics showed higher amplitudes in Ca responses in EA.hy926 exposed to buffer, G protein coupled receptor agonists, bradykinin, and angiotensin II compared to HMEC-1. HMEC-1 exhibited significantly higher [Ca ] changes after ionomycin exposure, while responses to thapsigargin, ATP, and acetylcholine were similar in both cell lines. ATP without extracellular Ca ions induced a significantly higher [Ca ] rise in EA.hy926, suggesting purinergic ionotropic P2X and metabotropic P2Y receptor activation. RNA-sequencing analysis showed significant differences in cytoskeleton- and Ca -related gene expression, highlighting upregulation of ORAI2, TRPC1, TRPM2, CNGA3, TRPM6, and downregulation of TRPV4 and TRPC4 in EA.hy926 versus HMEC-1. Moreover, KEGG analysis showed upregulated Ca import and downregulated export genes in EA.hy926. Our finding show that significant differences in CaEP response and [Ca ] regulation exist between EA.hy926 and HMEC-1, which may be attributed to distinct transcriptomic profiles. EA.hy926, compared to HMEC-1, displayed higher susceptibility and sensitivity to [Ca ] changes, which may be linked to overexpression of Ca -related genes and an inability to mitigate changes in [Ca ] . The study offers a bioinformatic basis for selecting EC models based on research objectives.

To search for new targets of anticancer therapies using phytoestrogens we performed comparative metabolic profiling of the breast cancer cell line MCF-7 and the non-tumorigenic breast cell line MCF-12A. Application of gas chromatography-mass spectrometry (GC-MS) revealed significant differences in the metabolic levels after exposure with 17ß-estradiol, genistein or a composition of phytoestrogens within a native root flax extract. We observed the metabolites 3-(4-hydroxyphenyl)-lactic acid, cis-aconitic acid, 11-beta-hydroxy-progesterone, chenodeoxycholic acid and triacontanoic acid with elevated levels due to estrogen action. Particularly highlighted were metabolites of the sphingolipid metabolism. Sphingosine and its dihydro derivate as well as ethanolaminephosphate were significantly altered after exposure with 1 nM 17ß-estradiol in the cell line MCF-7, while MCF-12A was not affected. Treatment with genistein and the flax extract normalized the sphingosine concentrations to the basic levels found in MCF-12A cells. We could further demonstrate that the expression levels of the sphingosine metabolizing enzymes: sphingosine-1-phosphate kinase (Sphk) and lyase (S1P lyase) were significantly influenced by estrogens as well as phytoestrogens. The isoform Sphk2 was overexpressed in the tumorigenic cell line MCF-7, while S1P lyase was predominantly expressed in the non-tumorigenic cell line MCF-12A. Importantly, in MCF-7 the weak S1P lyase expression could be significantly increased after exposure with 10 µM genistein and 1 µg/ml root flax extract. Here, we present, for the first time, an analysis of metabolic response of phytoestrogens to breast cancer cell lines. The contrasting regulation of sphingolipid enzymes in MCF-7 and MCF-12A render them as preferred targets for future anticancer strategies.

The main purpose of this research is to evaluate the improvement in positional accuracy (PAI) of cadastral boundary points’ coordinates through the adjustment of a large set of digital cadastral index maps of rural regions based on traditional Franciscan-origin maps of heterogeneous geometric quality. The distribution of residuals of local coordinates of reference points onto the as yet unconnected neighboring points is researched. In this article, we use the adjustment method based on neighborhood transformation with a mechanical membrane model deriving from Hooke’s Law and consider a general case study of a Slovenian traditional cadastral graphic database of various historical origins. The number of geometric errors in fieldbook information from outdated measurement technologies and inappropriate implementations of cadastral index map geometric maintenance reduces the number of complying datasets of relative geometry by 50%. Previous experiments in traditional cadastral index maps of rural regions, with triangle-based piecewise affine plane transformation (RMSE = 2.4 m), have been improved by the membrane method (RMSE = 1.0 m), based on tests at 623 control points. Positional accuracy improvement of cadastral geospatial data and the integration of geometric subsystems provided recognizable benefits for the future maintenance of a unique, integrated, centralized graphical cadastral subsystem, which is in the testing phase in Slovenia.

The main objective of this study was to identify genomic regions involved in biomass heterosis using QTL, generation means, and mode-of-inheritance classification analyses. In a modified North Carolina Design III we backcrossed 429 recombinant inbred line and 140 introgression line populations to the two parental accessions, C24 and Col-0, whose F ₁ hybrid exhibited 44% heterosis for biomass. Mid-parent heterosis in the RILs ranged from −31 to 99% for dry weight and from −58 to 143% for leaf area. We detected ten genomic positions involved in biomass heterosis at an early developmental stage, individually explaining between 2.4 and 15.7% of the phenotypic variation. While overdominant gene action was prevalent in heterotic QTL, our results suggest that a combination of dominance, overdominance and epistasis is involved in biomass heterosis in this Arabidopsis cross.

To search for new targets of anticancer therapies using phytoestrogens we performed comparative metabolic profiling of the breast cancer cell line MCF-7 and the non-tumorigenic breast cell line MCF-12A. Application of gas chromatography-mass spectrometry (GC-MS) revealed significant differences in the metabolic levels after exposure with 17ß-estradiol, genistein or a composition of phytoestrogens within a native root flax extract. We observed the metabolites 3-(4-hydroxyphenyl)-lactic acid, cis-aconitic acid, 11-beta-hydroxy-progesterone, chenodeoxycholic acid and triacontanoic acid with elevated levels due to estrogen action. Particularly highlighted were metabolites of the sphingolipid metabolism. Sphingosine and its dihydro derivate as well as ethanolaminephosphate were significantly altered after exposure with 1 nM 17ß-estradiol in the cell line MCF-7, while MCF-12A was not affected. Treatment with genistein and the flax extract normalized the sphingosine concentrations to the basic levels found in MCF-12A cells. We could further demonstrate that the expression levels of the sphingosine metabolizing enzymes: sphingosine-1-phosphate kinase (Sphk) and lyase (S1P lyase) were significantly influenced by estrogens as well as phytoestrogens. The isoform Sphk2 was overexpressed in the tumorigenic cell line MCF-7, while S1P lyase was predominantly expressed in the non-tumorigenic cell line MCF-12A. Importantly, in MCF-7 the weak S1P lyase expression could be significantly increased after exposure with 10 [micro]M genistein and 1 [micro]g/ml root flax extract. Here, we present, for the first time, an analysis of metabolic response of phytoestrogens to breast cancer cell lines. The contrasting regulation of sphingolipid enzymes in MCF-7 and MCF-12A render them as preferred targets for future anticancer strategies.

Fontan survivors have depressed cardiac index that worsens over time. Serum biomarker measurement is minimally invasive, rapid, widely available, and may be useful for serial monitoring. The purpose of this study was to identify biomarkers that correlate with lower cardiac index in Fontan patients. Methods and results This study was a multi-centre case series assessing the correlations between biomarkers and cardiac magnetic resonance-derived cardiac index in Fontan patients ⩾6 years of age with biochemical and haematopoietic biomarkers obtained ±12 months from cardiac magnetic resonance. Medical history and biomarker values were obtained by chart review. Spearman's Rank correlation assessed associations between biomarker z-scores and cardiac index. Biomarkers with significant correlations had receiver operating characteristic curves and area under the curve estimated. In total, 97 cardiac magnetic resonances in 87 patients met inclusion criteria: median age at cardiac magnetic resonance was 15 (6-33) years. Significant correlations were found between cardiac index and total alkaline phosphatase (-0.26, p=0.04), estimated creatinine clearance (0.26, p=0.02), and mean corpuscular volume (-0.32, p<0.01). Area under the curve for the three individual biomarkers was 0.63-0.69. Area under the curve for the three-biomarker panel was 0.75. Comparison of cardiac index above and below the receiver operating characteristic curve-identified cut-off points revealed significant differences for each biomarker (p<0.01) and for the composite panel [median cardiac index for higher-risk group=2.17 L/minute/m2 versus lower-risk group=2.96 L/minute/m2, (p<0.01)]. Higher total alkaline phosphatase and mean corpuscular volume as well as lower estimated creatinine clearance identify Fontan patients with lower cardiac index. Using biomarkers to monitor haemodynamics and organ-specific effects warrants prospective investigation.