Polish National Science Centre Grant Research Articles

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.

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).

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.

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.

Abstract 106: Diclofenac hybrids with oleanolic acid oximes modulate key signaling pathways in pancreatic cancer cells

Abstract Pancreatic cancer is characterized by constitutive activation of mitogen-activated protein kinases (MAPKs) like JNK and p38 determining its malignant phenotypes. Overexpression of NF-κB and COX-2 involved in inflammation contributes to increased cell growth and invasiveness. The latter might be related to the activated AKT pathway. Nonsteroidal anti-inflammatory drugs were suggested for pancreas cancers prophylaxis. Naturally occurring oleanolic acid (OA) was shown to possess anti-inflammatory and anti-tumorigenic activities. Coupling its analogs with NSAIDs may enhance this effect. Our study aimed to evaluate the effect of two oleanolic acid oximes derivatives conjugated with diclofenac (DCL), 4c (3-diclofenacoxyiminoolean-12-en-28-oic acid benzyl ester) and 4d (3-diclofenacoxyiminoolean-12-en-28-oic acid morpholide) on inflammation-related pathways, MAPK and NF-κB, cell cycle arrest and ROS formation in PSN-1 pancreatic cancer cells. PSN-1 cells were incubated with DCL, 4c or 4d at the concentration of 30µM. Activation of NF-κB was assessed by measuring binding of its p50 and p65 subunits to DNA using ELISA method. Their nuclear concentrations were determined by Western blot. RT-PCR was used to evaluate NF-κB subunits and COX-2 mRNA levels. Bead-based immunoassay determined the cytosolic levels of p38, JNK, CREB, AKT, P70S6K and STAT3 proteins. Cell cycle distribution and ROS formation were assessed using flow cytometry. Conjugate 4d significantly reduced nuclear level of p65 subunit and its mRNA expression, although compound 4c had more significant impact on COX-2 expression. Both derivatives significantly diminished p65 DNA binding, but only 4d had significant effect on p50. Both compounds reduced ROS production, but neither of them affected the cell cycle arrest. We observed decreased cytosolic AKT level along with increased levels of unphosphorylated JNK and p38 kinases which may have an antagonistic effect on proliferation by reducing activity of inflammatory mediators acting as tumor promoters. AKT decrease after treatment with 4d derivative might be the cause of increased unphosphorylated CREB level. Accumulation of p38 and JNK in cytosol could be a result of decreased ROS production and/or cause increased CREB protein level. Additionally, increased STAT3 cytosolic level suggests inhibition of mTORC1 pathway that explains reduced p70S6 kinase. Overall, these results suggest that OAO-DCL morpholide conjugate may affect both NF-κB and MAPK signaling pathways in pancreatic cancer cells. Further studies are required to explain detailed mechanism of these modifications. Funding: Studies were supported by Polish National Science Centre grant 2016/21/B/NZ7/01758. Acknowledgments: Authors thank Dr. Gary Gorbsky, Chair of Cell Cycle & Cancer Biology Program in Oklahoma Medical Research Foundation for supporting financing Maria Narozna conference participation. Citation Format: Maria Narozna, Violetta Krajka-Kuzniak, Robert Kleszcz, Wanda Baer-Dubowska. Diclofenac hybrids with oleanolic acid oximes modulate key signaling pathways in pancreatic cancer cells [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 106.

Association between APOE gene and stress coping strategies, memory, and family history of AD in a healthy middle‐aged Polish population: Preliminary results

One of the best-known genes associated with late-onset Alzheimer's disease (AD) is the apolipoprotein E (APOE). It is thought that the ε4 allele of this gene increases the risk of developing AD. The aim of this study was to investigate the preclinical influence of APOE-ε4 on brain cognitive functions in a healthy population. 200 (F:108, M: 92) healthy middle-aged (50-64 years old; M=55) right-handed adults completed a series of psychometric questionnaires: RAVEN (Raven's Progressive Matrices, classical version), BDI (Beck Depression Inventory), Mini-COPE (Mini-COPE Questionnaire) and CVLT (California Verbal Learning Test). We also collected information about demography, family history of AD and health. APOE alleles were determined by sequencing their chosen variants (APOE SNPs rs429358/rs7412) using a traditional Sanger sequencing protocol. The group was divided according to the results of the genetic test for ε4-non carriers (150) and ε4-carriers (50). Both groups were equivalent in age, gender, and years of completed education. There were no differences in intelligence (RAVEN scores), depressive symptoms (BDI) nor memory test results (CVLT). More ε4-carriers had a family (parents) history of AD (Welch's t-test, t(75.21)=-2.11, p<0.05). In the Mini-COPE questionnaire ε4-carriers group also scored less points on two (out of 3) scales rating problem-focused (adaptation) strategies: Planning (2. scale) (trend: Welch's t-test t(77.45)=1.84, p=0.07) and Using Instrumental Support (8. scale) (Welch's t-test t(77.40)=2.07, p<0.05). Despite overall comparable memory performance, there was also a tendency for ε4-carriers to be statistically better at recalling words from List A (5) (Welch's t-test, t(112.72)=-2.14, p<0.05) in the CVLT test. These preliminary results indicate that even in a healthy middle-aged population APOE genotype could influence cognitive abilities and alter stress-coping strategies. Further research is needed to determine if better memory recall is present also in younger ε4-carriers or whether it is a (transient) benefit of mnemonics techniques developed later in life to cope with a slowly starting memory decline. Supported by Polish National Science Centre (NCN) grant no. 2018/31/N/HS6/03551.

Dynamics at the threshold for blowup for supercritical wave equations outside a ball**This research was supported by the Polish National Science Centre Grant No. 2017/26/A/ST2/00530. Computations have been performed on Minerva cluster of the Max-Planck Institute for Gravitational Physics.

We consider spherically symmetric supercritical focusing wave equations outside a ball. Using mixed analytical and numerical methods, we show that the threshold for blowup is given by a codimension-one stable manifold of the unique static solution with exactly one unstable direction. We analyse in detail the convergence to this critical solution for initial data fine-tuned to the threshold.

Model-data comparison of sound propagation in a glacierized fjord with a variable ice top-boundary layer

Transmission loss measurements were conducted in the meltwater-modified surface layer near Hansbreen Glacier in Hornsund Fjord in southwestern Svalbard in September 2017 [Deane and Glowacki, JASA 143, 1711 (2018)]. An m-sequence source signal (149 dB re 1 μPa, 11 kHz carrier frequency) was tethered at 7 m depth to a boat drifting from 0 to 200 m. This signal was received by two Hitech HTI-96 hydrophones at 8 and 17 m depth deployed from a stationary boat anchored 500 m from the glacier. Within this environment, and typical for a glacierized fjord, regular calving events contributed to an ice melange top boundary layer with larger icebergs occasionally obstructing the signal transmission path. The propagation environment was upward refracting, causing propagation sound to repeatedly reflect from the surface layer. A ray-based approach was applied to model the measured data. The variability of the top boundary was included in the model by incorporating surface scattering and inserting icebergs. Comparisons between several increasingly complex iterations of this model with the collected data will be presented. [Work supported by the NDSEG Fellowship, ONR Grant Nos. N00014-17-1-2633 and N00014-14-1-0213, and the Polish National Science Centre Grant No. 2013/11/N/ST10/01729.]

Histone deacetylase 4 protects from denervation and skeletal muscle atrophy in a murine model of amyotrophic lateral sclerosis

BackgroundHistone deacetylase 4 (HDAC4) has been proposed as a target for Amyotrophic Lateral Sclerosis (ALS) because it mediates nerve-skeletal muscle interaction and since its expression in skeletal muscle correlates with the severity of the disease. However, our recent studies on the skeletal muscle response upon long-term denervation highlighted the importance of HDAC4 in maintaining muscle integrity.MethodsTo fully identify the yet uncharacterized HDAC4 functions in ALS, we genetically deleted HDAC4 in skeletal muscles of a mouse model of ALS. Body weight, skeletal muscle, innervation and spinal cord were analyzed over time by morphological and molecular analyses. Transcriptome analysis was also performed to delineate the signaling modulated by HDAC4 in skeletal muscle of a mouse model of ALS.FindingsHDAC4 deletion in skeletal muscle caused earlier ALS onset, characterized by body weight loss, muscle denervation and atrophy, and compromised muscle performance, although the main catabolic pathways were not activated. Transcriptome analysis identified the gene networks modulated by HDAC4 in ALS, revealing UCP1 as a top regulator that may be implicated in worsening ALS features.InterpretationHDAC4 plays an important role in preserving innervations and skeletal muscle in ALS, likely by modulating the UCP1 gene network. Our study highlights a possible risk in considering HDAC inhibitors for the treatment of ALS.FundThis work was supported by FIRB grant (RBFR12BUMH) from Ministry of Education, Universities and Research, by Fondazione Veronesi, by Sapienza research project 2017 (RM11715C78539BD8) and Polish National Science Center grant (UMO-2016/21/B/NZ3/03638).

PO-512 Protein markers of progression risk in patients with squamous cell vulvar carcinoma

IntroductionThe application of the concept of tumour-specific markers to assess a possible disease outcome and to choose appropriate treatment options, is still obstructed by the limited knowledge on vulvar carcinoma (VC) biology. We aimed to identify protein markers of VC that would be indicative of a tumour that is more likely to progress.Material and methodsPrimary tumour samples from 28 patients with early stage squamous cell VC and 14 samples of normal vulvar tissue were studied using iTRAQ analysis. The results obtained for tumour samples of VC patients that progressed during 8–12 years of follow-up period (‘progVC’, n=14) were be compared to those obtained for samples of patients who were disease-free at the time of last observation (‘d-fVC’, n=14). The differentially expressed proteins were subsequently validated using targeted proteomics methods (PRM) and immunohistochemistry using a larger sample set.Results and discussions5510 proteins were identified in the analysed samples. Gene Ontology (GO) analysis of the proteins differentially expressed in progVC and d-fVC tumours proteins indicated an immune response as the most over-represented GO category in this sample comparison. The top 47 candidate markers were chosen for further validation. Correlation of the validation results with clinical parameters of the enrolled VC patients indicated that HMGA2, ANO1, PRTN3, UBE2C, ABI3BP and PRELP should be considered as potential protein markers for the prediction of progression in VC patients.ConclusionOur findings provide evidence showing that deregulation of eight proteins’ abundance is significantly associated with aggressive phenotype of VC. Their immunohistochemical assessment hold promise as a patient stratification tool.AcknowledgementThis work was supported by the Polish National Science Centre grant No. 2013/10/E/NZ5/00663.

Mapping direct and indirect activation of cortical neurons by weak electrical pulses with massively parallel MEA-based electrical stimulation and recording

Mapping direct and indirect activation of cortical neurons by weak electrical pulses with massively parallel MEA-based electrical stimulation and recording

Cholesterol Hydroperoxide Trafficking: Impairment of Macrophage Cholesterol Efflux with Implications for Atherogenesis under Oxidative Stress

This study describes a type of “stealthy” delivery of cholesterol 7α/β hydroperoxides (7‐OOH) via a natural cholesterol trafficking pathway. In vascular macrophages this pathway normally delivers cholesterol to mitochondria to initiate export signaling when its level becomes excessive. Because 7‐OOHs are structurally similar to cholesterol, they can be recognized by steroidogenic acute regulatory (StAR) proteins and trafficked to mitochondria alongside cholesterol.Oxidative stress associated with cardiovascular disease can produce various oxidized lipids, including cholesterol oxides such as 7‐OOH, 7‐hydroxide (7‐OH), and 7‐ketone (7=O). Unlike 7=O and 7‐OH, 7‐OOH is redox‐active, giving rise to the others via potentially toxic free radical reactions. In the present study we tested the novel hypothesis that under oxidative stress conditions, StAR family proteins not only deliver cholesterol to/into mitochondria of vascular macrophages, but also 7‐OOH, which induces peroxidative damage that impairs early stage reverse cholesterol transport. Stimulation of human monocyte‐derived THP‐1 macrophages with dibutyryl‐cAMP resulted in substantial upregulation of StarD1 and plasma membrane ABCA1 which exports cholesterol to acceptors such as ApoA1. siRNA‐induced StarD1 knockdown (kd) prior to stimulation had no effect on StarD4, but reduced ABCA1 upregulation, linking the latter to StarD1 functionality. Mitochondria in stimulated StarD1‐kd cells internalized 7‐OOH slower than non‐stimulated controls and underwent less 7‐OOH‐induced lipid peroxidation and membrane depolarization, as probed with C11‐BODIPY and JC‐1, respectively. Major functional consequences of 7‐OOH exposure were (i) loss of mitochondrial CYP27A1 activity, (ii) reduced 27‐hydroxycholesterol (27‐OH) output, and (iii) down‐regulation of ABCA1 and ABCG1. Consistently, 7‐OOH‐challenged macrophages exported less cholesterol to apoA‐I or HDL than did non‐challenged controls. StarD1‐mediated 7‐OOH transport was also found to be highly cytotoxic, whereas 7=O and 7‐OH were minimally toxic. These findings support our hypothesis that 7‐OOH found in oxidized low density lipoprotein gets delivered to macrophage mitochondria and promotes atherogenesis through impairment of cholesterol homeostasis.Support or Funding InformationThis work was supported by Polish National Science Center grant 2014/13/B/NZ3/00833 (WK) and by National Institutes of Health grants HL85677 (AWG) and HL58012 (DS). Support of American Heart Association Fellowship 14PRE185800221 (ACC) is also acknowledged

Self-injective artin algebras without short cycles in the component quiver

The project was supported by the Polish National Science Center grant awarded on the basis of the decision number DEC-2011/01/N/ST1/02064.

Fully Automatic 2D hp -adaptive Finite Element Method for Non-stationary Heat Transfer

Abstract In this paper we present a fully automatic hp adaptive finite element method code for non-stationary two dimensional problems. The code utilizes the α -scheme for time discretization and fully automatic hp adaptive finite element method discretization for numerical solution of each time step. The code is verified on the examplary non-stationary problem of heat transfer over the L-shape domain. Acknowledgements The work of MP is supported by Polish National Science Center grant no. 2012/07/B/ST6/01229. The work of MS is supported by a Dean's grant.

Evolution of magnetic and crystal structure of FeTe doped with Cr and Ni

Due to inherent phase separation, it has been so far impossible to grow ideally stoichiometric (1:1) tetragonal (P 4/n m m) iron telluride. The excess iron ions are located in the inter-planar positions and usually represented as a fraction x in a general formula Fe1+xTe [1], where x ranges from about 4% to 17%. The additional iron has been found to negatively correlate with the level of anion site doping and subsequently with hindering the superconductivity (SC), for example in the Fe(Te,Se,S) [2] series, where SC can be induced by doping with selenium or small amounts of sulfur. A binary Fe1+xTe orders magnetically into incommensurate magnetic structure with the transition temperature and crystal structure in the magnetic state depending on the excess iron. For x&lt;0.12 a monoclinic (P 21/m) distortion was observed and for x&gt;0.12 an orthorhombic one (P m m n). In our work, we attempted to create and investigate compounds electronically equivalent to variable iron stoichometry by substituting Fe with chromium (3d electron deficient) or nickel (3d electron rich). Single crystal samples several millimeter in size were grown by solidification from melt method in the substitution range 0.025 to 0.1 and only in the case of nickel the incorporation of dopant into host lattice was confirmed. Despite low effective Cr content in the single crystal form [3], neutron powder diffraction (NPD) of polycrystaline specimens revealed systematic decrease of long range magnetic moment and gradual suppression of monoclinic (M) distortion in both series. In the Cr doped series, the structure evolved through a mixed phase region into orthorhombic (O) one (Fig. 1), whereas in the nickel system a complete restoration of tetragonal symmetry was found. The suppression of magnetic ordering and lack of the structural distortion did not result in the SC. This work is supported by the Polish National Science Centre grant No 2011/01/B/ST3/00425

Crystal structures of 2D coordination networks in [M(bbtr)3](ClO4)2(M=Mn, Cd)

[Fe(bbtr)3](ClO4)2 (bbtr=1,4-di(1,2,3-triazol-1-yl)butane) represents a spin crossover (SCO) system where the first coordination sphere consists of 1,2,3-triazole rings coordinated by exodentate nitrogen atoms [1]. Iron(II) ion is linked to six other iron(II) ions by bbtr ligands. This creates two dimensional (2D) polymeric layers. SCO is abrupt, accompanied by hysteresis loop. In the cooling mode P-3 -&gt; P-1 structural phase transition precedes SCO. The non-magnetic structural transformation is accompanied by reorganization of weak intermolecular interactions and shift of 2D layers with respect to each other. Surprisingly, an analog [Fe(bbtr)3](BF4)2, does not exhibit in cooling mode neither thermally SCO nor structural phase transition [2]. To clarify the role of structural phase transition on SCO we have performed structural modifications by exchanging the kind of anions and/or metal ions. An exchange of perchlorate on triflate anion involves deeper structural changes. A topology of the polymeric layer remains the same, but the SCO is shifted to higher temperature and structural phase transition is not observed. The studies of isostructural zinc(II) analogs confirmed the crucial role of anion in the occurrence of non-magnetic structural phase transition. The [Zn(bbtr)3](ClO4)2 exhibites P-3 -&gt; P-1 structural phase transition which is not present in tetrafluoroborate analog [2]. We expand studies on other hexacoordinating metal(II) ions. Reactions between manganese(II) or cadmium(II) perchlorates and bbtr in acetonitrile lead to [M(bbtr)3](ClO4)2 (M=Mn, Cd) complexes. Single crystal X-ray diffraction studies revealed that both compounds create a 2D polymeric networks. The temperature dependence of lattice parameters for these complexes showed that, in contrast to [Fe(bbtr)3](ClO4)2 and [Zn(bbtr)3](ClO4)2 systems, the structural phase transition is not present. This work was funded by the Polish National Science Centre Grant No. DEC-2011/01/B/ST5/06311.

Correction: Chemerin Is an Antimicrobial Agent in Human Epidermis

An organization providing funding the grant to the last author (JC) were incorrectly omitted, and a funding organization for that grant was also incorrectly given in the Funding Statement. The first sentence of the Funding Statement should read: “This work was supported in part by the grant from the Foundation for Polish Science TEAM/2010-5/1, co-financed by the European Union within European Regional Development Fund and Polish National Science Center grant 2011/02/A/NZ5/00337 (to JC).

Variability of gene expression profile in breast cancer core biopsy: Impact on prognostic and predictive multigene signatures.

e21049 Background: Gene expression profiling has been shown to be effective in the analysis of postoperative tumor samples in breast cancer. However, when small specimens like core biopsy samples are considered, the small size of the collected material makes it difficult to obtain high tumor percentage in the specimen. Thus, we tested the strategy of obtaining three independent cores for molecular analysis and assessed the influence of the stability of different prognostic and predictive signatures. Methods: We prospectively collected breast core biopsies from 82 consecutive patients treated by pre-operative chemotherapy. In each patient, three cores from different tumor areas were sampled for molecular testing, independent of the cores collected for histopathological analysis. RNA was isolated by QIAGEN RNeasy kits, gene expression profiling was carried out using Affymetrix HG-U133 Plus 2.0 microarrays. Results: In 27 patients, three independent specimens were analyzed by microarray gene expression profiling (in total 81 samples). When this training set was analyzed by unsupervised Principal Component Analysis, the overall concordance between the three tumor cores was observed, but no such concordance was seen for three tumors (for each with one significant outlier). When we assessed the set of chosen 20 prognostic and predictive signatures, we confirmed that the homogeneity of gene expression within the signatures is significantly better than for random sets of genes, but we observed the notable differences between the signatures. The heterogeneity decreased when genes with low between-patient variance were excluded; they also showed a higher intra-patient variability of gene expression. We also assessed the heterogeneity of single expression markers and furher validated our findings on an independent set of 55 patients. Conclusions: The use of three independent cores for gene expression testing in breast cancer may be a successful strategy to overcome tumor heterogeneity and sampling error and result in more stable results of prognostic and/or predictive signatures. Supported by Polish National Science Center grant N402 6861 40