Polish National Science Centre Research Articles

Abstract LB197: Methylation patterns at adjacent CpG sites within enhancers are part of cell identity and are disrupted during malignant transformation

Abstract Background: Following the principles of physiology of DNA methylation, it is generally accepted that methylation status of CpG sites spaced up to 50 bp is correlated and accumulation of locally disordered methylation at CpG sites is a part of neoplastic transformation, acting in similar way as stochastic accumulation of mutations during malignant transformation. Methods: We developed a new algorithm allowing to analyze methylation status at the adjacent CpG sites. We then, used EPIC microarray data to analyze genome wide methylation patterns at the adjacent CpG sites in almost 600 samples, representing 12 healthy tissues and cell types as well as 572 cancer specimens. We also used bisulfite next generation sequencing data and Sanger sequencing to validate our findings. Results: Our analysis, identified in human genome a subset of loci with adjacent CpG sites spaced less than 50bp (average 28bp), harboring one cytosine methylated and the other devoid of methyl group. These loci are enriched in enhancers that are targeted by families of transcription factors involved in cell differentiation. The methylation patterns at these loci can differ between alleles within a cell, what allows for remarkable level of heterogeneity of methylation patterns. However, our analysis also showed that, different types of specialized cells, acquire during differentiation only one specific and remarkably stable pattern of methylation at each of these loci and that pattern is to a large extent lost during neoplastic transformation. Conclusions: Contrary to generally accepted principle of identical methylation status of the adjacent CpG sites being essential for regulatory function of DNA methylation. We have shown that methylation at a subset of adjacent CpG sites in human genome located within important functional genomic elements differs in healthy cells. These CpG sites acquire remarkably stable and tissue specific patterns of methylation during cell differentiation. Moreover, the methylation patterns at these CpG sites, are lost during malignant transformation suggesting the involvement of identified phenomenon in cancer pathology. Funding: This work was funded by grant OPUS22 from Polish National Science Centre (grant number: 2021/43/B/NZ2/02979) and Polish National Agency for Academic Exchange (grant number: PPN/PPO/2018/1/00088/U). Citation Format: Olga Taryma-Leśniak, Jan Bińkowski, Patrycja K. Przybylowicz, Katarzyna E. Sokolowska, Konrad Borowski, Tomasz K. Wojdacz. Methylation patterns at adjacent CpG sites within enhancers are part of cell identity and are disrupted during malignant transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB197.

History of the Bantu Peoples by Credo Mutwa– Zulu Tradition or Mutwa’s Invention?

Vusamazulu Credo Mutwa (1921–2020) was one the most famous and controversial sangomas – sages, clairvoyants, healers and diviners – in South Africa. He was also the first sangoma who published books revealing sangomas’ esoteric calling, vocation and cooperation with spirits. He wrote extensively about Zulu mythology. A part of his version of mythology is the ‘history’ of the Bantu languages speaking peoples, their divine origins and the conquest of the lands in the South of Africa. Mutwa also wrote about relationships between Zulus and other ethnic groups, focusing on their mythological beginnings. The aim of this article is to analyse Mutwa’s myths and stories about the history of Bantu languages speaking peoples, relationships between Zulus and other peoples, and place it all within historical facts and religious beliefs known to academics. The article is part of the project ‘Esoteric landscape of Southern Africa’ financed by the Polish National Science Centre (Narodowe Centrum Nauki), Poland, project no. 2017/25/N/HS1/02500.

Towards High-Quality Broad-Range Luminescence Thermometry - the Case of Pr3+-Activated Garnets

Accurate and sensitive remote temperature measurement over a wide range of temperatures, presumably 1000 degrees, is required in many existing and emerging applications. Novel and emerging technologies pose new requirements on the quality of temperature measuring, the resistance to disturbance by external electric and/or magnetic fields, spatial resolution, and the operating range of thermometers. Luminescence thermometry has the potential to become the technology of choice when classic methods become non-practical and/or do not offer the expected quality.Designing a luminescence thermometer capable of measuring temperature reliably over, for example, 1000 degrees range is challenging, as managing luminescence processes in such a range is extremely difficult. Yet, wide-range luminescence thermometers are of interest in such fields as aviation, space, nuclear plant, and others. Fast, accurate, and credible measurements of quickly changing temperatures are critical in these areas.In this presentation, we shall report on the thermometric competencies of a series of garnet phosphors, typically activated with Pr3+. We shall focus on the possibility of managing the physics related to energy flow between the excited levels of Pr3+ and thermal quenching of its three emissions resulting from the 5d®4f, 3PJ®3HJ and 1D2®3HJ transitions. Not only shall we demonstrate that the sensitivity of such sensors may be deliberately tuned to reach its maximum at temperatures where it is most needed, but we will also show that garnets nowadays may offer temperature measuring over a range of 800 degrees at least. This research was supported by the Polish National Science Center (NCN) under the grant #UMO2018/29/B/ST5/00420. Figure 1

SrAl12O19:Eu,Cr As Luminescence Thermometers

Luminescent nanothermometers offer several approaches to determine the absolute temperature. Mostly, temperature-stimulated changes in the intensity ratio of emission bands, emission kinetics, band/emission line width, or luminescence band/line peak position are explored. The list is longer and is still growing. In our project, the Cr3+, Eu2+-based aluminates materials were designed taking into account the possibility to work as non-contact luminescent thermometers using mentioned above approaches. In Figure 1 the luminescence spectra of SrAl12O19:Eu,Cr phosphor are shown in the temperature range of 15-500 K. The emission comes from both the directly excited ions, Eu2+ and Cr3+. Particularly strong changes with temperature can be seen in the Eu2+ emission around 350 - 470 nm and Cr3+ above 670 nm. Luminescent mechanisms of energy transfer between specific activators of the aluminates lattice will be presented, the consequence of which is obtaining by the material sophisticated thermometric properties in a wide range of measured temperature, from cryogenic to around 500 K. The research finances by the Polish National Science Centre under grant #UMO2018/31/B/ST4/00924 and partially by program “Excellence Initiative – Research University” (IDUB) for years 2020-2026 for University of Wrocław (grant # BPIDUB.4610.678.2021) Figure 1

Lower blood levels of miR‐29b‐3p in healthy middle‐aged AD risk gene carriers

AbstractBackgroundA number of late‐onset Alzheimer’s disease (AD) risk genes have been discovered, among which the most studied but not fully explored is the apolipoprotein E gene (APOE). PICALM (coding Phosphatidylinositol Binding Clathrin Assembly Protein) is another well‐recognized risk‐gene, shown to interact with the APOE gene in conferring AD risk. MicroRNAs (miRNAs) are post‐transcriptional regulators of mRNAs, some of which are known to be involved in AD pathology. The purpose of this study was to investigate in middle‐aged, healthy participants the association between AD risk‐genes (APOE and PICALM) and circulating miRNAs that were previously identified as altered in AD patients.MethodBlood samples were collected from 76 healthy individuals (36 F, 36 M; age: 56.38±3.16), assigned to three groups: A+P+ (APOE/PICALM risk), A+P‐ (APOE risk, PICALM neutral alleles) and N group (APOE/PICALM neutral alleles). RT‐qPCR was used to compare the levels of 11 microRNA identified by us in previous studies as candidate early AD biomarkers in blood plasma (hsa‐miR‐29b‐3p, hsa‐miR‐34a‐5p, hsa‐miR‐125b, hsa‐miR‐146a, hsa‐miR‐135a, hsa‐miR‐30b‐5p, hsa‐miR‐142‐3p, hsa‐miR‐200a‐3p, hsa‐miR‐483‐5p, hsa‐miR‐486‐5p, hsa‐miR‐502‐3p) [1, 2]. We followed standard procedures of blood assays.ResultSingle gene risk (APOE) carriers had lower levels of miR‐29b‐3p than non‐carriers and double risk carriers (F(2,73) = 12.75, p < 0.001 with Welch homogeneity correction; post‐hoc with Tukey correction: p < 0.01 for A+P‐ versus N, p < 0.001 for A+P‐ versus A+P+). No other changes in miRNA panels were discovered.ConclusionDownregulation of miR‐29b‐3p in blood was detected repeatedly before in patients with AD, in 4 independent studies [1]. Here we show the same effect in a subpopulation of middle‐age, healthy individuals with APOE related genetic risk for AD. Further research is needed to understand the interaction reversing this effect in PICALM risk carriers. Longitudinal follow‐up may validate this blood‐based miRNA not only as an easy‐to‐access diagnostic biomarker, but also as a risk‐assessment biomarker for AD prevention.Funding: This work was funded by the Polish National Science Centre (NCN) grant no. 2018/31/N/HS6/03551, 2016/20/W/NZ4/00354 and 2018/29/B/NZ7/02757.[1], [2] https://doi.org/10.1016/j.arr.2018.10.008; 10.18632/oncotarget.15109.

Epigenetic and Transcriptional Consequences of PIM Kinase Activity and Inhibition in Diffuse Large B-Cell Lymphoma (DLBCL)

The PIM kinase family consists of 3 proto-oncogenic proteins: PIM1, PIM2 and PIM3, expressed in numerous malignancies, including DLBCL. PIM kinases regulate crucial processes, such as proliferation, apoptosis, metabolism, or migration, therefore their inhibition is of great interest as a potential therapeutic strategy. Although recent studies have shed new light on the biological role of PIMs in lymphoid cancers, the details and mechanisms of PIM's oncogenic effects and the consequences of their inhibition in DLBCL remain insufficiently understood. Earlier studies have demonstrated a potential epigenetic role of PIM through histone H3S10 phosphorylation, however, described only at a single locus. The broad genomic role of PIM in modulating transcription in lymphoma cells remains unclear. We confirmed the effect of PIM inhibition on histone modifications using small molecule inhibitors as well as genetic silencing of PIM kinases in DLBCL cell lines. PIM kinase inhibition changed the global amounts of histone modifications (including histone H4 pan-acetylation), accompanied by lower phosphorylation of RNA polymerase II, suggesting an involvement of PIMs in the RNA polymerase II pause release/elongation phase of transcription. Local histone acetylation changes in the enhancer (H3K27ac) and promoter (H3K9ac) regions in response to treatment with the pan-PIM inhibitor were also identified, coupled with gene expression profiling in DLBCL cell lines. PIM inhibition decreased the expression of genes controlled by super-enhancers (SE), i.e. wide regulatory regions responsible for the high level of expression of the most important genes for the cell, including oncogenes. The integrated results of epigenomic and transcriptomic analyses explain the changes in expression of genes associated with many pathways, including transcription, inflammatory response, apoptosis, epigenetic mechanisms or DNA damage. The induction of the latter was further experimentally confirmed by the examination of DNA breaks using γH2AX, the activating phosphorylation of CHK2 or comet assays following PIM inhibition. The conducted studies document an epigenetic function of PIM kinases, and suggest that inhibition of PIM activity leads to alterations in the DLBCL epigenetic landscape and associated changes in transcription. They also indicate that PIM inhibitors can be a therapeutic option in DLBCL. Study supported aby Foundation For Polish Science TEAM Grant # POIR.04.04.00-00-5C84/17 and Polish National Science Centre Grant # 2018/29/B/NZ5/01471.

Formation of denser plasma fibrin clots is associated with coronary artery ectasias and aneurysms - preliminary results from the CARE-ANEURYSM study

Abstract Background Coronary artery ectasias and aneurysms (CAEAs) are defined as diffused or localized coronary artery dilations, that exceed diameter of the adjacent segment by at least 50%. In the Caucasian population, CAEAs are present in 2-5% of all-comer patients undergoing coronary angiography and are associated with increased risk of myocardial infarction and death. Purpose To determine whether CAEAs are associated with increased thrombin generation potential and unfavorably modified plasma fibrin clot properties. Methods In this prospective study 19 patients with CAEAs, 19 sex-, age-, fibrinogen- and diabetes mellitus-matched patients with coronary artery disease (CAD) but without CAEAs, and 20 healthy control subjects were assessed. Plasma fibrin clot permeability (Ks) reflecting an average pore size within fibrin network, plasma fibrinolytic potential expressed as clot lysis time (CLT), and thrombin generation capacity defined as endogenous thrombin potential (ETP) were assessed. The CARE-ANEURYSM study, founded by the Polish National Science Centre (no. UMO-2020/39/O/NZ5/02863), was approved by the Ethical Committee and conducted in accordance with the Declaration of Helsinki. Patients provided written informed consent. Results As many as 84.2% of CAEA patients were men, mean age was 69.6 years, and 73.7% individuals had CAD. On average, CAEA affected 3 coronary segments and exceeded vessels referral diameter by 2.15-fold. Patients with CAEA had 46.5% reduced Ks (median, 3.98 [3.30-4.63] vs 7.43 [6.15-8.88] x10-9cm2, p<0.001), a tendency to prolonged CLT (112 [103-116.5] vs 99 [89-114] min, p=0.07), and 51% higher ETP (1685 [1502-1786] vs 1119 [1025-1189] nM×min, p<0.001) compared to healthy control (Fig. 1A-C). Moreover, individuals with CAEA compared to CAD patients had 19.5% lower Ks (median 3.98 [3.30-4.63] vs 5.33 [4.86-6.78] x10-9cm2, p=0.016) and 21% longer CLT (112 [103-116.50] vs 92.52 [81.50-97.50] min, p=0.015) and (1685 [1502-1786] vs 1119 [1693-1966] nM×min, p<0.001) (Fig. 1A-C). In the CAEA group, Ks was associated with the CAEA maximal diameter (R=-0.595, p=0.007) and the ratio of CAEA maximal diameter to referral vessel diameter (R=0.743, p<0.001; Figu.1D). There were no associations between the number of coronary artery segments affected by the lesion and fibrin clot properties or ETP. However, CAEA patients in whom the ratio of the CAEA maximal diameter to the referral vessel diameter exceed 2 (n=9) had 24.5% lower Ks (3.45 [1.88-3.68] vs 4.57 [4.08-5.35] x10-9cm2, p=0.004) and 14.7% higher ETP (1779 [1685-1812] vs 1515 [1431-1674] nM×min, p=0.028) with no difference in CLT (p>0.05). Conclusion CAEAs are associated with denser fibrin clot structure and enhanced thrombin generation, which indicate that CAEA patients are characterized by prothrombotic state. Further studies are needed to elucidate whether prothrombotic clot phenotype may be associated with increased risk of thromboembolic events in CAEA patients.Figure 1.

Valvular presence of coagulation factor XI correlates with the severity of aortic stenosis

Abstract Background The role of coagulation system as a factor involved in the progression of aortic stenosis (AS) has been proven. Considering recent studies about oral factor XIa (FXIa) inhibitor – asundexian with antithrombotic efficacy, our aim was to investigate whether FXI is present within stenotic leaflets in patients with severe AS and whether its expression correlates with valvular calcification. Methods We recruited 20 patients aged 66 ±9 years with severe AS defined as mean transvalvular pressure gradient (PGmean) ≥ 40mmHg, peak transvalvular velocity (Vmax) of ≥4.0 m/s and aortic valve area (AVA) <1 cm2. Stenotic aortic valves were obtained during valve replacement surgery. Aortic leaflets obtained from AS patients and from age-matched apparently healthy autopsy donors (n=3) were used to evaluate valvular expression of FXI, FIX, tissue factor (TF) and bone morphogenetic protein 2 (BMP2) by single and double-labeled immunostaining. The immunopositive valve area was computed as the ratio (%) of positively and negatively stained areas. The Ethical Committee approved the study and all participants provided written informed consent. Results AS patients with median PGmean of 54 [46-67] mmHg, Vmax of 4.5 [4.4-5.0] m/s and AVA of 0.8 [0.7-0.9] cm2 were treated with angiotensin converting enzyme inhibitors (80%), beta-blockers (90%), statins (70%) and acetylsalicylic acid (80%). In loco analysis revealed valvular expression of FXI within all studied stenotic valves, but not within control ones (Figure 1 A and B). The FXI-immunopositive valve area constituted 21.4±4.5% of the total leaflet area, while these for FIX and TF were 17.2 ±3.8% and 26.5±5.1%, respectively. The mean BMP2-positive area was 22.9±4.1%. The expression of studied proteins was observed at the aortic side of the stenotic leaflets and presented a condensed pattern of fluorescence. Moreover, the expression of FXI co-expressed with FIX in 76% and TF in 71%, suggesting local activation of FXI (Figure 1 D and E). Moreover, FXI co-expressed with BMP2 in 83% (Figure C), which may indicate that FXI activation occurs at the site of leaflet calcification. Interestingly, valvular amounts of FXI correlated with AS severity measured as PGmean (r=0.49, p=0.03), PGmax (r=0.53, p=0.02), Vmax (r=0.54, p=0.01) and AVA (r=-0.53, p=0.02). Conclusions We are the first to show the valvular presence of FXI, co-expressing with FIX and TF, which indicates FXI in loco activation. A strong co-expression of FXI with BMP2 and associations with disease severity highlight the impact of coagulation on valve calcification. It is tempting to speculate that FXIa inhibition could attenuate not only coagulation activation but also valvular calcification and thus retard AS progression. Clinical relevance of these findings requires further studies. This work was supported by the Polish National Science Centre (UMO-2021/41/N/NZ5/03323).Figure 1_ESC_2023

White Izangoma: The Creation of New Significance or New Members of Traditional Healing-Divining Practice?

One of the social changes with the collapse of Apartheid in the South African society was the emergence of so-called 'white isangomas' or 'white izangoma'. This was not the first time that people of European origins were called by amadlozi (ancestral spirits) to ubungoma2. The first records are dating back to the beginning of the 20th century, but the social situation (colonialism and then Apartheid) made it impossible for Whites to be trained. However, with the growing awareness of the importance of African cultures, white people who felt the calling, started looking for answers. The calling is characterized by a sickness of body and mind that shows itself in having visions and an overwhelming weakness. It is widely believed that it can be healed only through the training by a fully-fledged isangoma. This essay analyses white izangoma vocation and work, establishing if they really are part of ubungo-ma, and if they influence and change black izangoma'steachings and work. The information originates from the author's field study, written izangoma'stestimonials, and other academic research. The field studies that allowed gathering first-hand information for this essay were financed by the Polish National Science Centre (Narodowe Centrum Nauki), Poland, project no. 2017/25/N/HS1/02500.

Cele mediacji a dobór i stosowanie strategii i technik mediacyjnych przez mediatorów w sporach cywilnych – sprawozdanie z badań (część III – wywiady)

Studies of understanding and identification of mediation objectives, strategies and techniques and the effectiveness of mediation proceedings are justified from the cognitive and practical perspectives. The aim of this paper is to present the report of an empirical study, devoted to the subject matter mentioned above, conducted by the author as part of the scientific activity financed by the Polish National Science Centre. The paper is complex in nature – it deals with the research, concept and methodological threads. The empirical study was conducted in Poland, with Polish mediators participating in it, providing mediation services mainly in one of the Mediation Centres operating at District Chambers of Legal Advisors, making up the National Network of Legal Advisor Mediation Centres. However, considering the universal and utilitarian nature of the issue in hand, the comparative potential, originality and cognitive value of the study findings may be of interest to both Polish and international scientists and practitioners of mediation as an amicable form of holistic legal dispute management. Given the scope and depth of the issues addressed, the article is divided into three parts. This text (part III) contains significant, selected data gleaned from in-depth interviews with mediators, as well as their concise discussion and major conclusions and the summary of the whole scientific activity.

Normalization methods for RT‐qPCR assessment of circulating microRNAs in Alzheimer’s and other aging‐related diseases

AbstractBackgroundCirculating miRNAs in blood plasma present significant potential for use as minimally‐invasive biomarkers for diagnosis of aging‐related diseases, such as cancer or Alzheimer’s disease (AD). The most sensitive and inexpensive analysis method of circulating miRNA is RT‐qPCR. However, lack of RT‐qPCR standardisation is one of the major causes of data inconsistency across different studies and it hinders progress towards widespread implementation of miRNA biomarkers in clinical use. Normalisation of RT‐qPCR data to stably expressed miRNAs is a key step in data analysis. Current methods of identifying optimal normalisers are lacking in their evaluation of the stability of normalisers and their combinations in aging populations. To address these needs, we created a novel, transparent, method for selecting optimal normalisers in aging populations, using the Python programming language (v. 3.7.3).MethodOur method uses the Python packages: pandas (v. 1.2.3), numpy (v. 1.17.1), scipy (v. 1.3.1). The method uses three measures to evaluate the miRNA Cq values for potential normalisers: Kolmogorov‐Smirnov score (KS‐score), average displacement from a mean of zero and mean standard deviation. In each case, the comparators are two distinct biological groups of interest (such as AD and cognitively normal controls). Before comparison, the group of potential normalisers were normalised according to the log2(2−ΔΔCq) method. The miRNA data were collected from Polish subjects including 44 AD patients and 70 similar‐aged, cognitively normal, controls). Circulating miRNA molecules were isolated from plasma, followed by RT‐qPCR with a selection of 7 miRNAs tested as potential normalisers, along with suitable controls for mRNA isolation and reverse transcription.ResultWe provide a novel method for identification of optimal normalisers with the advantages of: assessments of a greater number of combined potential normalisers (7 in this study), transparency of decision making and the ability for end users to weight that decision making according to their judgment of relative importance.ConclusionWe recommend assessment of plasma miRNA levels in an aging population employing a novel set of normalisers.Work supported by the Polish National Science Centre grant OPUS 2018/29/B/NZ7/02757and by the EU Horizon2020 FETOPEN grant no 737390 (ArrestAD).

Modeling Contingent Decision Behavior: A Bayesian Nonparametric Preference-Learning Approach

We propose a preference-learning algorithm for uncovering Decision Makers’ (DMs’) contingent evaluation strategies in the context of multiple criteria sorting. We assume the preference information in the form of holistic assignment examples derived from the analysis of alternatives’ performance vectors and textual descriptions. We characterize the decision policies using a mixture of threshold-based, value-driven preference models and associated latent topics. The latter serve as the stimuli underlying the contingency in decision behavior. Such a probabilistic model is constructed by using a flexible and nonparametric Bayesian framework. The proposed method adopts a hierarchical Dirichlet process as the prior so that a group of DMs can share a countably infinite number of contingent models and topics. For all DMs, it automatically identifies the components representing their evaluation strategies adequately. The posterior is summarized by using the Hamiltonian Monte Carlo sampling method. We demonstrate the method’s practical usefulness in a real-world recruitment problem considered by a Chinese IT company. We also compare the approach with counterparts that use a single preference model, implement the parametric framework, or consider each DM’s preferences individually. The results indicate that our approach performs favorably in both interpreting DMs’ contingent decision behavior and recommending decisions on new alternatives. Furthermore, the approach’s performance and robustness are investigated through a computational experiment involving real-world data sets. History: Accepted by Ram Ramesh, Area Editor for Data Science and Machine Learning. Funding: J. Liu received financial support from the National Natural Science Foundation of China [Grants 72071155 and 71701160]. M. Kadziński received financial support from the Polish National Science Center under the SONATA BIS project [Grant DEC-2019/34/E/HS4/00045]. X. Liao received financial support from the National Natural Science Foundation of China [Grant 71872144]. Supplemental Material: The software that supports the findings of this study is available within the paper and its Supplemental Information ( https://pubsonline.informs.org/doi/suppl/10.1287/ijoc.2023.1292 ) as well as from the IJOC GitHub software repository ( https://github.com/INFORMSJoC/2021.0328 ) at ( http://dx.doi.org/10.5281/zenodo.7608750 ).

PIM Kinases Regulate Super-Enhancer-Dependent Gene Expression in Diffuse Large B-Cell Lymphoma

The PIM kinase family consists of 3 proto-oncogenic proteins: PIM1, PIM2 and PIM3, expressed in numerous cancers, including diffuse large B-cell lymphoma (DLBCL). PIM kinases regulate crucial processes, such as proliferation, apoptosis, metabolism, and migration, therefore their inhibition is of great interest as a potential therapeutic strategy. However, the detailed mechanisms of PIM's oncogenic effects and the consequences of their inhibition in DLBCL remain insufficiently understood. Our earlier studies (Szydłowski et al., Blood 2017, Szydłowski et al., Cancer Res. 2021) highlighted immunomodulatory functions of PIM kinases in lymphoid malignancies, but also indicated that their inhibition has a profound impact on gene expression. Although earlier studies indicated that PIMs regulate certain transcription factors and/or regulate gene expression via epigenetic mechanisms through histone H3S10 phosphorylation, the detailed genomic functions of PIM kinases in modulating transcription in DLBCL remains unclear. In order to investigate the mechanisms and consequences of PIM kinase inhibition on gene expression in DLBCL cells, we first performed RNA sequencing-based transcriptome profiling in DLBCL cells treated with the pan-PIM and FLT3 inhibitor MEN1703, which is currently in clinical development. MEN1703 markedly downregulated the expression of transcripts controlled by super-enhancers (SE), i.e. large regulatory regions responsible for high levels of transcript expression of the key essential genes for the cell, including oncogenes. The analysis also suggested new biological processes controlled by PIMs, including the reduction of non-coding RNAs generated from the SE regions. To understand the mechanisms linking PIM inhibition with decreased SE-driven gene expression, we assessed PIM-dependent H3S10 phosphorylation in lymphoma cells. Since H3S10 phosphorylation facilitates MYST family KAT8 (MOF) acetyltransferase recruitment to certain promoters/enhancers, we evaluated the histone H3 and H4 acetylations in DLBCL cells following PIM inhibition. MEN1703 markedly reduced the H3K9Ac, H3K14Ac and H4 pan-acetylation, which was associated with reduced BRD4 binding to the SE regions and reduced RNAPII serine 5 (S5) phosphorylation, but not S3 or S7 phosphorylations. Together, these observations suggest the participation of PIM in the elongation phase of SE-dependent gene transcription via H3S10-dependent epigenetic mechanism. The effects of PIM inhibition on histone modifications and proliferation were confirmed by genetic silencing or CRISPR-Cas9 deletion of individual PIM kinases. To better characterize the function of H3S10 phosphorylation in DLBCL, non-phosphorylatable H3S10A mutants were introduced to DLBCL cells. Compared to wild type counterparts, cells with the mutated H3S10 exhibited decreased proliferation, indicating a significant biological role of this modification in DLBCL. The effect was potentiated in cells exposed to metabolic stress (starvation) conditions. Together, these studies confirm the epigenetic role of PIM kinases, especially in the regulation of SE-dependent gene transcription and suggest that inhibition of PIM activity leads to disturbances of SE integrity and DLBCL transcription. The results document new, epigenetic function of PIM kinases and present a novel cytotoxicity mechanism of their inhibition in DLBCL. They also indicate that PIM inhibition with MEN1703 can be a therapeutic option in DLBCL. Study supported aby Foundation For Polish Science TEAM Grant # POIR.04.04.00-00-5C84/17 and Polish National Science Centre Grant # 2018/29/B/NZ5/01471.

The cardioprotective and anti-inflammatory effect of inhaled nitric oxide during Fontan surgery in patients with single ventricle congenital heart defects: a prospective randomized study

BackgroundFontan surgery with cardiopulmonary bypass (CPB) causes tremendous systemic stress and inflammatory responses, affecting postoperative organ function, morbidity, and mortality. Although this reaction triggers partially protective anti-inflammatory responses, it is harmful in patients with single ventricle congenital heart defects. Despite decades of research, an effective anti-inflammatory and stress defense strategy is lacking. This study investigated the influence of inhaled nitric oxide (NO) during CPB on early clinical results, including the duration of postoperative respiratory support as a primary outcome and a panel of laboratory analytes.MethodsIn this study, 115 patients were randomized to the Fontan-NO group (n = 48) and the Fontan group (n = 49). Eighteen patients were excluded from the study. The Fontan-NO group received NO inhaled directly into the oxygenator during CPB. Clinical data were collected, and blood samples were drawn for analysis at repeated intervals. Multiplex assays were used to analyze a proteome profile of molecules involved in stress response, inflammation, metabolic reactions, as well as heart and lung protection.ResultsFontan-NO patients had significantly shorter respiratory support time with a median of 9.3 h (7.0; 13,2) vs 13.9 h (3.7; 18.5) by the absolute difference of 4.6 h [95% confidence interval, − 30.9 to 12.3; (p = 0.03)]. In addition, they have a shorter time in intensive care (p = 0.04) and lower pulmonary artery pressure after CPB discontinuation (p = 0.04), 4 h (p = 0.03) and 8 h (p = 0.03) after surgery. Fontan-NO patients also had a lower concentration of lactates (p = 0.04) and glucose after separation from CPB (p = 0.02) and lower catecholamine index (p = 0.042). Plasma factors analysis has shown a significantly higher concentration of interleukin-10, and a lower concentration of interleukin-6, interleukin-8, interleukin-1β, pentraxin, matrix metalloproteinase-8, troponin-I, creatine kinase myocardial band (CK-MB), and insulin in Fontan-NO group.ConclusionsNO inhaled into the oxygenator during CPB can improve short-term clinical outcomes. It shortens intubation time and intensive care time. It reduces inflammatory response, improves myocardial and lung protection, and diminishes metabolic stress in patients with a single ventricle undergoing Fontan surgery.Trial registration number: The trial was preregistered, supervised, and supported by The Polish National Science Center (NCN/01/B/NZ5/04246).

(Invited) Combining the 5d→4f and 4f→4f Luminescence of Lanthanides for High-Quality Broad-Range Luminescence Thermometry

Accurate and sensitive remote temperature measurement is required in a growing number of existing and emerging applications. As the technology advances and nanotechnology enters still new fields, the size of objects whose temperature needs to be controlled gets smaller and the non-contact temperature reading is the only reasonable approach. Luminescence thermometry is believed to become the technology of choice for such purposes, as it is resistant to disturbances by an electromagnetic field and the sensitivity and accuracy of measurements may be tuned for the specific requirements. Thus, whether it comes to reading temperature at cryo-range, around the room, or at high temperatures, one may design a luminescent thermometer offering high-quality measurements. This is not to say that such designing is easy to do. Even under laboratory conditions, highly-sensitive and accurate luminescent thermometers are not that frequent.Additional problems appear when it comes to measurements over a broad range of temperatures. At first, the basic physics of luminescence processes are not conducive to the design of a luminescence thermometer offering very sensitive and accurate measurements over a broad range of temperature (a few hundred degrees, for example). On the other hand, wide-range luminescence thermometers are of interest for such important fields as aviation and space industries. Fast, accurate, and credible measurements of quickly changing temperatures are of special importance in these areas.In this presentation, we shall report a new approach toward wide-range high-quality luminescence thermometry. Phosphors activated with either Pr3+ or Eu2+ will be examined for that purpose. Both these ions are capable of generating two types of emissions: (i) intra-configurational 5f→4f and inter-configurational 4f→4f ones. We shall prove that such phosphors may offer both broad-range and high-accuracy and high-sensitivity temperature measurements. Furthermore, we will demonstrate that the sensitivity of such sensors may be deliberately tuned to reach its maximum at temperatures where it is most needed. A few examples of such phosphors will be discussed and possible expectations for further development using this approach will be discussed.This research was supported by the Polish National Science Center (NCN) under the grant #UMO-2018/29/B/ST5/00420. Figure 1

(Invited) Operando EPR Study on Radicals in Anion-Exchange Membrane Fuel Cells

In the rapidly developing modern society, there is an urgent need for the wide-ranging availability of advanced and eco-friendly energy sources. One of the possible alternatives is the application of anion exchange membrane fuel cells (AEMFCs) with a catalyst reducing dioxygen efficiently. These promising devices can revolutionize the energy sector since they practically produce no pollution. However, to make them more widely used, several obstacles must be overcome. As a result, vast-ranging investigations focus on improving the properties of conductive polymer membranes [1] and catalysts for the oxygen reduction reaction (ORR) [2]. The durability of the membrane electrode assembly (MEA) is one of the critical requirements for the successful commercialization of anion exchange membrane fuel cells (AEMFCs). Despite significant impacts of nucleophilic degradation on ion-exchange capability and the anionic conductivity of investigated membranes, it is believed to affect only cationic sites of membrane polymers and thus cannot explain the reported loss in the mechanical strength of degraded AEMs. Such a phenomenon might be related to polymer backbone degradation caused by free radicals. This was widely described in the literature in the case of fuel cells using proton-conducting membranes [3] but barely mentioned for AEMFCs [4]. Since the oxidative degradation of hydrocarbon polymers is very well known, we aimed to comprehensively investigate the formation of the short-lived species generated during the operation of AEMFCs as well as stable radicals present in the polymer membranes.We investigated the LDPE-base membranes with Pt black, Pd black, PdAg, and Ag as the ORR catalysts, whereas for HOR the Pt black, Pd black, and NiFe catalysts were used. The in-situ measurements are performed with a micro-AEMFC inserted into a resonator of an electron paramagnetic resonance (EPR) spectrometer, which enables separate monitoring of radicals formed on the anode and cathode sides. The creation of radicals was monitored by the EPR spin trapping technique. In Figure 1 the EPR spectra of DMPO spin adducts trapped during operation of micro-fuel cell placed in EPR spectrometer cavity are presented. In this experiment, the LDPE-base membrane with platinum catalysts on both sides was used. The main detected adducts during the operation of the micro-AEMFC were DMPO-OOH and DMPO-OH on the cathode side and DMPO-H on the anode side. Additionally, we clearly show the formation and presence of stable radicals in AEMs during and after long-term AEMFC operation [5]. Preliminary results suggest that the creation of the short-living radicals during AEMFCs operation is independent of the used membrane. However, the applied catalysts determine the number of detected radicals. The EPR investigations indicate that, in addition to the known chemical degradation mechanisms of the cationic ammonium groups of the membrane, oxidative degradation, including radical reactions, has to be taken into account when the stability of an anion conductive polymer for AEMFCs is investigated. The formation of stable radicals in AEMs was proven for the first time in this study. All short-living radicals formed during the AEMFC operation were fully identified. The presence of radicals in the AEM after AEMFC testing indicates that reactive oxygen species may play a very important role in the degradation mechanism of the anion conducting polymers. Results from this study shed light on the understanding of radical formation and presence in the membranes during AEMFC tests, which in turn may help to solve the challenge of anion exchange membrane stability. Acknowledgments. This work was supported by the Polish National Science Centre (NCN) project OPUS-14, No. 2017/27/B/ST5/01004.

Non-vitamin K antagonist oral anticoagulants (NOACs) attenuate valvular calcification in patients with severe aortic stenosis

Abstract Background In vitro studies has demonstrated that non-vitamin K antagonist oral anticoagulants (NOACs) down-regulated the expression of proteins responsible for valve calcification and inflammation in valve interstitial cells (VICs) cultures. Aims Our goal was to evaluate whether long-term anticoagulation with NOACs, including dabigatran (thrombin inhibitor), rivaroxaban and apixaban (FXa inhibitors) can impair the valvular calcification and inflammation in patients with severe AS. Methods We enrolled 38 patients with severe AS aged 70±5.9 years (mean gradient - PGmean: 50mmHg, max gradient - PGmax: 80mmHg), including 18 individuals with AS and concomitant atrial fibrillation taking NOACs for at least 3 years (51±10 months) (AS-NOACs group). Stenotic aortic valves were obtained during valve replacement surgery. Valvular calcification was estimated ex vivo using micro-computed tomography (micro-CT). Total volume of each calcification (bone volume - BV), the distribution of clods sizes of calcification parts (trabecular thickness -Tbth) and the volume of the hardest / most calcified part (hard CT) of each valve were assessed. Valvular expression of bone morphogenetic protein 2 (BMP-2) and NFκB was evaluated by immunostaining. The fluorescence intensity (FI) was computed as the ratio (%) of positively and negatively stained areas. Results AS patients did not differ from AS-NOACs participants with regard to demographic or risk factors. Micro-CT analysis showed 65.5% lower BV in AS-NOACs valves compared to patients not taking NOACs (87±47 mm3 vs. 252±146 mm3, p=0.0017). Of note, in AS-NOACs patients Tbth showed strong association with transvalvular pressure gradients (PGmean: r=0.76, p=0.033; PGmax: r=0.77, p=0.01), as clinical measures of the AS severity. The duration of anticoagulation was associated with the lower Tbth (from 0.35 mm for 60 months to 0.51 mm for 36 months, p<0.01), which suggests that long NOACs treatment changed the pattern of valvular calcification, leading to formation of calcium deposits with reduced volume. Moreover, in AS-NOACs patients BV correlated with hard CT (r=−0.69, p=0.027), suggesting that the lesions increase their volume but do not ossify. Valvular expression of BMP-2 and NFκB was 44% and 33% decreased in AS-NOACs patients, compared to those without such treatment and the expression of both proteins was positively associated (r=0.34, p=0.001). Moreover, in AS-NOACs patients the expression of BMP-2 and NFκB was observed mainly in the fibrosa layer of the leaflets, but not in the deeper layers of tissue, as in AS patients without NOACs treatment. Conclusions NOACs can inhibit the increase of valvular calcium volume and its ossification via suppressed NFkB expression. Our study may suggest that long-term anticoagulation with NOACs could slow down the rate of AS progression, at least in patients with an indication for anticoagulant therapy. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Polish National Science Centre

Discovery of new characterizations of parameter distributions in a semi- empirical mass model: an investigation by dividing an overdetermined system into numerous balanced subsystems* *Supported by the National Natural Science Foundation of China (11975209, U2032211, 12075287), the Physics Research and Development Program of Zhengzhou University (32410017), the Project of Youth Backbone Teachers of Colleges and Universities of Henan Province (2017GGJS008)

We propose and test a new method of estimating the model parameters of the phenomenological Bethe-Weizsäcker mass formula. Based on the Monte Carlo sampling of a large dataset, we obtain, for the first time, a Cauchy-type parameter distribution formed by the exact solutions of linear equation systems. Using the maximum likelihood estimation, the location and scale parameters are evaluated. The estimated results are compared with those obtained by solving overdetermined systems, e.g., the solutions of the traditional least-squares method. Parameter correlations and uncertainty propagation are briefly discussed. As expected, it is also found that improvements in theoretical modeling (e.g., considering microscopic corrections) decrease the parameter and propagation uncertainties.

(Invited) Oxygen Reduction Reaction in Alkaline Media: The Effect of Carbon Support Crystallinity, Conductivity, and Doping

The rapid development of energy consumption combined with growing environmental awareness is conducive to the wide-ranging search for ecological energy sources. In the recent decade, a significant amount of research has focused on the development of anion-exchange membrane (AEM) fuel cells (AEMFCs), as such approach exhibits faster oxygen reduction reaction (ORR) kinetics in the alkaline environment of the cell [1]. However, to become a serious alternative to the current mainstream acidic fuel cells, alkaline systems should overcome current limitations of performance and stability [2]. The design and synthesis of high-performance electrocatalysts facilitating the slow oxygen reduction reaction (ORR) is, therefore, a key issue in the context of the full commercialization of fuel cells. Transition metal oxides with spinel structure constitute an interesting group of potential catalysts that can replace the widely used materials based on Pt [3, 4]. However, due to the low electrical conductivity and the tendency to undesirable aggregation of spinel nanoparticles, it is important to support them on appropriate carbon carriers. Since the nature of the carbon carrier plays a significant role in the ORR activity, we aimed to comprehensively investigate the influence of the electric conductivity of carbon supports, the role of the surface functional oxygen-bearing groups, and doping with heteroatoms on the performance of the bare and cobalt-manganese spinels deposited on the functionalized carbon supports, acting as ORR electrocatalysts in the alkaline media [5].We investigated commercially available Vulcan XC-72, Printex85, MWCNT, amorphous carbon (C-am.), and carbons synthesized by structural nano-replication using the mesoporous molecular sieves MCM-48 (carbon denoted CMK-1) and the spherical silica calcinated at temperature range 650-1050 oC (SPH-650-1050). To investigate the effect of doping on the ORR catalytical activity the MWCNT was modified by nitrogen and sulfur heteroatoms. Additionally, the doped supports were then treated by plasma to introduce surface oxygen groups. On all investigated carbon supports the nanoparticles of manganese-cobalt spinel were dispersed. They were prepared at 150oC by a microwave-assisted hydrothermal route. Comprehensive physicochemical characterization of carbon support was performed using Raman and UV-Vis spectroscopies, XPS, TPD, XRD, TGA techniques, electron microscopy (TEM), and N2 adsorption (BET) (Figure 1). The relative electric conductivity measurements were performed using the contactless microwave technique. The electrochemical properties of the obtained materials were determined by cyclic voltammetry (CV), whereas the electrocatalytic activity of the obtained materials towards ORR was determined using the RDE and RRDE techniques.It was found that the nature of the carbon carrier plays a triple role in the ORR activity, determining dispersion and faceting of the spinel nanoparticles, as well as the extent of the undesired 2e– reduction pathway, controlled by the fraction of an amorphous component. In the case of SPH samples, it was shown that with the increasing carbonization temperature of the carbon support the electric conductivity increase in parallel to the crystallization extent. The electrocatalytic performance of the carbon carriers and the supported cobalt manganese spinels exhibits a volcano-type shape reaching the maximum for carbon materials obtained at 850°C. Such dependence results from two descriptors changing oppositely with the carbonization temperature: an increasing electric conductivity and a decreasing amount of the carbonyl and quinone groups in the sample. The electric conductivity multiplied by the amount of the carboxyl and quinone groups exhibits the same variation with the carbon calcination temperature as the number of electrons exchanged in ORR, confirming that optimal conjunction of these both properties plays a decisive role in this process. The dispersed spinel active phase favors the direct 4e− pathway of ORR and improves the value of the E onset potential distinctly. To the best of our knowledge, the role of electric conductivity and surface oxygen functionalities in the ORR activity over carbon materials and supported spinel electrocatalysts was disentangled for the first time in this study. Acknowledgments: This work was supported by the Polish National Science Centre (NCN) project OPUS-14, No. 2017/27/B/ST5/01004.

Abstract 3324: Optimizing the therapeutic potential of tyrosine kinase inhibitors in chemo-immunotherapy of B-cell acute lymphoblastic leukemia involving rituximab

Abstract B-cell acute lymphoblastic leukemia (B-ALL) is a malignancy caused by accumulation of immature B cells. Advances in the field, especially in genomic studies and new treatment development, significantly improved the prognosis of B-ALL over the last decades. However, the B-ALL subtype harboring Philadelphia chromosome t(9;22)(q34;q11) (Ph+ B-ALL) still remains a therapeutic challenge despite available targeted treatment with tyrosine kinase inhibitors (TKIs). The novel therapeutic regimens for adult B-ALL often include rituximab (RTX) - a monoclonal anti-CD20 antibody - for patients with >20% CD20+ blasts. Moreover, drug combinations including approved TKIs and antibodies are being tested in clinical trials. Since some TKIs display immune effector cell inhibition, investigating their immunomodulatory effects in the context of combined chemo-immunotherapies is clinically relevant for the rational combination design. We found that blasts isolated from Ph+ B-ALL patients showed the highest CD20 expression on mRNA level among defined B-ALL genetic subtypes. As Ph+ CD20+ patients may be qualified for treatment with TKIs along with RTX, this result prompted the need of investigating the effects of TKIs on immune effectors. We therefore compared how first-, second- and third-generation TKIs (imatinib, dasatinib, nilotinib, bosutinib, ponatinib) and allosteric inhibitor asciminib recently approved for the treatment of chronic myeloid leukemia (CML) affect innate and antibody-mediated cytotoxicity. B-ALL cell lines, patient-derived lymphoblasts propagated in NSG mice (primografts) and ex vivo whole blood assays were used for testing the efficacy of effector cell-mediated mechanisms in the presence of TKIs. The TKIs were compared in NK-cell based natural cytotoxicity tests, antibody-dependent cytotoxicity (ADCC) tests and phagocytosis assays with monocyte-derived macrophages. Dasatinib was also tested using whole blood ex vivo ADCC assays before and after administration of the TKI. Out of six tested TKIs, asciminib presented the most favorable profile, causing slight or no effector cell inhibition in both innate and antibody-mediated responses. In contrast, dasatinib significantly decreased the immune cells activity in all tests, reducing natural and RTX-mediated cytotoxicity of NK cells against B-ALL blasts. Dasatinib and ponatinib significantly inhibited in vitro phagocytosis of primograft B-ALL cells. To a lesser extent, cytotoxicity and phagocytosis were suppressed by imatinib, bosutinib and nilotinib. Importantly, we confirmed that the orally administered dasatinib impairs the NK cell activity in patients’ blood. Our findings indicate that the TKIs differentially impact RTX-mediated effector mechanisms. The novel, allosteric TKI asciminib could be preferentially used in combination with RTX-based immunotherapy. This work was supported by the Polish National Science Centre grant 2019/35/B/NZ5/01428 and by the Ministry of Education and Science within “Regional Initiative of Excellence” program 013/RID/2018/19." Citation Format: Krzysztof Domka, Martyna Poprzeczko, Zuzanna Urbanska, Lukasz Komorowski, Agata Pastorczak, Klaudyna Fidyt, Agnieszka Dabkowska, Mieszko Lachota, Magdalena Winiarska, Karolina Siudakowska, Elzbieta Patkowska, Łukasz Sędek, Bartosz Perkowski, Beata Krzymieniewska, Malgorzata Firczuk. Optimizing the therapeutic potential of tyrosine kinase inhibitors in chemo-immunotherapy of B-cell acute lymphoblastic leukemia involving rituximab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3324.