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The FAS ligand (FASLG) is expressed on lymphocytes, which employ it to activate death receptors on target cells. Cancer cells are generally resistant to apoptosis triggered by FASLG. In this work, we found a way to circumvent this resistance by treatment with actinomycin D (ActD) and nutlin-3a (Nut3a). We selected this drug combination based on our transcriptomic data showing strong activation of proapoptotic genes, including those for receptor-mediated apoptosis, in cells exposed to actinomycin D and nutlin-3a. To test our hypothesis, we pre-exposed cancer cell lines to this drug combination for 45 h and then treated them with recombinant FASLG. This almost instantaneously killed most cells. Actinomycin D and nutlin-3a strongly cooperated in the sensitization because the effect of the drugs acting solo was not as spectacular as the drug combination, which together with FASLG killed more than 99% of cells. Based on the caspase activation pattern (caspase-8, caspase-9, caspase-10), we conclude that both extrinsic and intrinsic pro-apoptotic pathways were engaged. In engineered p53-deficient cells, this pro-apoptotic effect was completely abrogated. Therefore, the combination of ActD + Nut3a activates p53 in an extraordinary way, which overcomes the resistance of cancer cells to apoptosis triggered by FASLG. Interestingly, other combinations of drugs, e.g., etoposide + nutlin-3a, actinomycin D + RG7112, and actinomycin D + idasanutlin had a similar effect. Moreover, normal human fibroblasts are less sensitive to death induced by ActD + Nut3a + FASLG. Our findings create the opportunity to revive the abandoned attempts of cancer immunotherapy employing the recombinant FAS ligand., (© 2024. The Author(s).)

Background: Given the notable prevalence of temporomandibular disorders (TMD) in the Polish population, there is a clear need for the use of simple, reliable questionnaires as screening tools to facilitate the referral of patients to TMD specialists., Objectives: The aim of the study was to translate and adapt the Fonseca Anamnestic Index (FAI) into Polish and assess its reliability and validity in identifying TMD symptoms., Material and Methods: The Polish adaptation of the FAI (FAI-PL) was developed in accordance with the international guidelines, including the translation and evaluation of the psychometric properties of the questionnaire. Every patient received a standardized assessment, which involved history taking and clinical examination, including the Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) and the FAI questionnaire. The psychometric analyses included an evaluation of the questionnaire's reliability and validity, as well as an exploratory factor analysis (EFA)., Results: Of the 122 individuals enrolled in the study, 63.9% were female. The mean age of the participants was 28.1 years (standard deviation (SD): 6.3). According to the RDC/TMD standards, 40.9% of patients had no TMD, while the FAI assessment indicated that 27% of patients had no TMD. The Cronbach's alpha coefficient for the FAI-PL was 0.75. The exploratory factor analysis revealed 3 factors, accounting for 55.2% of the total variation. The diagnostic sensitivity of the FAI-PL was 98.6%, while the diagnostic specificity reached a level of 65.3%., Conclusions: The Polish version of the FAI is a reliable and valid tool for the screening of TMD symptoms in the Polish-speaking population.

The p53 tumor suppressor protein activates various sets of genes depending on its covalent modifications, which are controlled by the nature and intensity of cellular stress. We observed that actinomycin D and nutlin-3a (A + N) collaborate in inducing activating phosphorylation of p53. Our recent transcriptomic data demonstrated that these substances strongly synergize in the upregulation of DUSP13 , a gene with an unusual pattern of expression, coding for obscure phosphatase having two isoforms, one expressed in the testes and the other in skeletal muscles. In cancer cells exposed to A + N, DUSP13 is expressed from an alternative promoter in the intron, resulting in the expression of an isoform named TMDP-L1. Luciferase reporter tests demonstrated that this promoter is activated by both endogenous and ectopically expressed p53. We demonstrated for the first time that mRNA expressed from this promoter actually produces the protein, which can be detected with Western blotting, in all examined cancer cell lines with wild-type p53 exposed to A + N. In some cell lines, it is also induced by clinically relevant camptothecin, by nutlin-3a acting alone, or by a combination of actinomycin D and other antagonists of p53-MDM2 interaction-idasanutlin or RG7112. This isoform, fused with green fluorescent protein, localizes in the perinuclear region of cells.

The p53 tumor suppressor protein is best known as an activator of cell cycle arrest and apoptosis. Only a fraction of p53-activated genes encode proteins affecting cellular replication and various forms of cell death (apoptosis, ferroptosis, autophagy). The p53-regulated genes can be divided into so-called the core transcriptional program, which comprises genes activated in most cell types by most activators, and into the group of genes activated in in cell- or stress-specific manner. Activation of p53 occurs via the extensive set of posttranslational modifications, which adjust its stability, interaction with other transcription regulators, and its ability to form a tetramer. Surprisingly, in mouse models, the activation of the best-studied p53 target genes encoding the inhibitor of the cell cycle (CDKN1A) or the inducers of apoptosis (e.g. NOXA, PUMA) is dispensable for protection against cancers. Thus, the non-classical functions of p53 must be studied to better understand its tumor suppressive mechanisms.

Transcriptomic analyses have revealed hundreds of p53-regulated genes; however, these studies used a limited number of cell lines and p53-activating agents. Therefore, we searched for candidate p53-target genes by employing stress factors and cell lines never before used in a high-throughput search for p53-regulated genes. We performed RNA-Seq on A549 cells exposed to camptothecin, actinomycin D, nutlin-3a, as well as a combination of actinomycin D and nutlin-3a (A + N). The latter two substances synergise upon the activation of selected p53-target genes. A similar analysis was performed on other cell lines (U-2 OS, NCI-H460, A375) exposed to A + N. To identify proteins in cell lysates or those secreted into a medium of A549 cells in control conditions or treated with A + N, we employed mass spectrometry. The expression of selected genes strongly upregulated by A + N or camptothecin was examined by RT-PCR in p53-deficient cells and their controls. We found that p53 participates in the upregulation of: ACP5, APOL3, CDH3, CIBAR2, CRABP2, CTHRC1, CTSH, FAM13C, FBXO2, FRMD8, FRZB, GAST, ICOSLG, KANK3, KCNK6, KLRG2, MAFB, MR1, NDRG4, PTAFR, RETSAT, TMEM52, TNFRSF14, TRANK1, TYSND1, WFDC2, WFDC5, WNT4 genes. Twelve of these proteins were detected in the secretome and/or proteome of treated cells. Our data generated new hypotheses concerning the functioning of p53. Many genes activated by A + N or camptothecin are also activated by interferons, indicating a noticeable overlap between transcriptional programs of p53 and these antiviral cytokines. Moreover, several identified genes code for antagonists of WNT/β-catenin signalling pathways, which suggests new connections between these two cancer-related signalling systems. One of these antagonists is DRAXIN. Previously, we found that its gene is activated by p53. In this study, using mass spectrometry and Western blotting, we detected expression of DRAXIN in a medium of A549 cells exposed to A + N. Thus, this protein functions not only in the development of the nervous system, but it may also have a new cancer-related function., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: Targeting tumor vasculature is an efficient weapon to fight against cancer; however, activation of alternative pathways to rebuild the disrupted vasculature leads to rapid tumor regrowth. Immunotherapy that exploits host immune cells to elicit and sustain potent antitumor response has emerged as one of the most promising tools for cancer treatment, yet many treatments fail due to developed resistance mechanisms. Therefore, our aim was to examine whether combination of immunotherapy and anti-vascular treatment will succeed in poorly immunogenic, difficult-to-treat melanoma and triple-negative breast tumor models., Methods: Our study was performed on B16-F10 melanoma and 4T1 breast tumor murine models. Mice were treated with the stimulator of interferon genes (STING) pathway agonist (cGAMP) and vascular disrupting agent combretastatin A4 phosphate (CA4P). Tumor growth was monitored. The tumor microenvironment (TME) was comprehensively investigated using multiplex immunofluorescence and flow cytometry. We also examined if such designed therapy sensitizes investigated tumor models to an immune checkpoint inhibitor (anti-PD-1)., Results: The use of STING agonist cGAMP as monotherapy was insufficient to effectively inhibit tumor growth due to low levels of STING protein in 4T1 tumors. However, when additionally combined with an anti-vascular agent, a significant therapeutic effect was obtained. In this model, the obtained effect was related to the TME polarization and the stimulation of the innate immune response, especially activation of NK cells. Combination therapy was unable to activate CD8 + T cells. Due to the lack of PD-1 upregulation, no improved therapeutic effect was observed when additionally combined with the anti-PD-1 inhibitor. In B16-F10 tumors, highly abundant in STING protein, cGAMP as monotherapy was sufficient to induce potent antitumor response. In this model, the therapeutic effect was due to the infiltration of the TME with activated NK cells. cGAMP also caused the infiltration of CD8 + PD-1 + T cells into the TME; hence, additional benefits of using the PD-1 inhibitor were observed., Conclusion: The study provides preclinical evidence for a great influence of the TME on the outcome of applied therapy, including immune cell contribution and ICI responsiveness. We pointed the need of careful TME screening prior to antitumor treatments to achieve satisfactory results., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Czapla, Drzyzga, Matuszczak, Cichoń, Rusin, Jarosz-Biej, Pilny and Smolarczyk.)

Acute promyelocytic leukemia (APL) is an aggressive subtype of acute myeloid leukemia (AML) in which the PML/RARα fusion protein exerts oncogenic activities by recruiting repressive complexes to the promoter of specific target genes. Other epigenetic perturbations, as alterations of histone H3 lysine 9 trimethylation (H3K9me3), have been frequently found in AMLs and are associated with leukemogenesis and leukemia progression. Here, we characterized the epigenomic effects of maltonis, a novel maltol-derived molecule, in APL cells. We demonstrate that maltonis treatments induce a profound remodulation of the histone code, reducing global H3K9me3 signal and modulating other histone post-translational modifications. Transcriptomic and epigenomic analyses revealed that maltonis exposure induces changes of genes expression associated with a genomic redistribution of histone H3 lysine 4 trimethylation (H3K4me3) and lysine 27 acetylation (H3K27ac). Upregulation of interferon alpha and gamma response and downregulation of c-MYC target genes, in function of c-MYC reduced expression (monitored in all the hematopoietic neoplasms tested), represent the most significant modulated pathways. These data demonstrate the ability of maltonis to epigenetically reprogram the gene expression profile of APL cells, inducing an intriguing antiviral-like response, concomitantly with the downregulation of c-MYC-related pathways, thus making it an attractive candidate for antileukemic therapy., (© 2022. The Author(s).)

The p53 tumor suppressor protein is best known as an inhibitor of the cell cycle and an inducer of apoptosis. Unexpectedly, these functions of p53 are not required for its tumor suppressive activity in animal models. High-throughput transcriptomic investigations as well as individual studies have demonstrated that p53 stimulates expression of many genes involved in immunity. Probably to interfere with its immunostimulatory role, many viruses code for proteins that inactivate p53. Judging by the activities of immunity-related p53-regulated genes it can be concluded that p53 is involved in detection of danger signals, inflammasome formation and activation, antigen presentation, activation of natural killer cells and other effectors of immunity, stimulation of interferon production, direct inhibition of virus replication, secretion of extracellular signaling molecules, production of antibacterial proteins, negative feedback loops in immunity-related signaling pathways, and immunologic tolerance. Many of these p53 functions have barely been studied and require further, more detailed investigations. Some of them appear to be cell-type specific. The results of transcriptomic studies have generated many new hypotheses on the mechanisms utilized by p53 to impact on the immune system. In the future, these mechanisms may be harnessed to fight cancer and infectious diseases.

One of the current challenges facing researchers is the search for alternative biological material, as opposed to routinely and invasively collected (such as blood), as the analysis of the former would provide information about the state of human health, allowing for the diagnosis of diseases in their early stages. With the search for disease biomarkers in alternative materials, the development of newer analytical solutions has been observed. This study aims to develop a reliable analytical method using the capillary isotachophoresis technique for the determination of organic acids in children's saliva, the presence/elevation of which can be used in the future for diagnostic purposes. Organic acids such as formic, lactic, acetic, propionic, and butyric acid, were determined in the saliva of healthy children without carious lesions. The limit of quantification determined in the validation process was found to vary from 0.05 to 1.56 mg/L, the recoveries at the two levels were determined to vary between 90% and 110% for level I, while for level II the corresponding values of 75% and 106% were found; the presentation, expressed as relative standard deviation values (RSD), did not exceed 5%. The parameters determined while validating the results method indicated that the obtained are reliable. The Red-Green-Blue (RGB) additive color model was used for the evaluation of the method. This comparative analysis allowed us to define the color of the method, which expresses whether it meets the given assumptions and requirements. According to the RGB model, the isotachophoresis method developed requires less reagent input, shorter sample preparation times, and results with lower energy consumption. Thus, the subject procedure may provide an alternative, routine tool for determining organic acids in human saliva, to be applied in the diagnosing of diseases of various etiological origins.

To detect the epigenetic drift of time passing, we determined the genome-wide distributions of mono- and tri-methylated lysine 4 and acetylated and tri-methylated lysine 27 of histone H3 in the livers of healthy 3, 6 and 12 months old C57BL/6 mice. The comparison of different age profiles of histone H3 marks revealed global redistribution of histone H3 modifications with time, in particular in intergenic regions and near transcription start sites, as well as altered correlation between the profiles of different histone modifications. Moreover, feeding mice with caloric restriction diet, a treatment known to retard aging, reduced the extent of changes occurring during the first year of life in these genomic regions.

Background: The dataset includes georeferenced occurrences of species listed in Annex I of Resolution 6 of the Bern Convention and, partly, in the Red Data Book of Ukraine. The dataset was compiled within the work of NGO "Ukrainian Nature Conservation Group" aimed to prepare a Shadow list of Emerald Network (European network Areas of Special Conservation Interest) in Ukraine - newly proposed territories aimed at conservation of particular species and habitats mentioned in Resolution 4 and 6 of the Bern Convention. The list was prepared in 2017-2020 for expanding the already existing Emerald Network of Ukraine. Based on actual registrations of flora and fauna collected and gathered by scientists and naturalists in a form of dataset, which is described in the following paper., New Information: This dataset provides information about 29,938 occurrences of species from the territory of Ukraine listed in Annex I of Resolution 6 of the Bern Convention, as well as in the Red Data Book of Ukraine. This is the largest public dataset on occurrences of rare and endangered species from Ukraine till now. Data presented here laid the foundations for the proposal of 106 approved Emerald Network sites (2019), as well as for 148 Emerald Network sites that were nominated in 2020. New insights on the endemic species Centaureapseudoleucolepis Kleopow is provided, which was previously considered to be extinct, according to the IUCN Red List., (Oleksii Vasyliuk, Oleh Prylutskyi, Oleksii Marushchak, Anna Kuzemko, Iuliia Kutsokon, Oksana Nekrasova, Niels Raes, Mikhail Rusin.)

Background: Foot problems may have a substantial negative impact on rheumatoid arthritis (RA) patients' mobility. They affect walking and the functional capacity to perform daily tasks., Methods: This study included 61 patients with RA and foot pain or swelling. The study group comprised 37 patients (aged 54.3 ± 9.5 years) with foot lesions, as demonstrated in an ultrasound, and the control group comprised 24 patients (aged 57.3 ± 11.5 years) without foot lesions. The patients' health statuses were evaluated with the Foot Function Index-Revised Short Form (FFI-RS), the Polish version of the Health Assessment Questionnaire-Disability Index (HAQ-DI), and the Disease Activity Score 28 (DAS 28)., Results: The FFI-RS showed significant differences between the study and control groups in total results, as well as in the pain and stiffness subscales. Subsequent analyses showed numerous significant correlations. The FFI-RS total results correlated with the HAQ's standing up, walking, and total results. The FFI-RS pain results correlated with the social issues and HAQ's total results. The FFI-RS difficulty results correlated with the disease's duration. In the study group, there were significant correlations of the FFI-RS stiffness, difficulty, and social issues results with the HAQ's standing up, walking, and total results, and also of the FFI-RS activity limitation results with the HAQ's standing up results. In the control group, there were correlations of the FFI-RS stiffness, difficulty, and activity limitation results with the HAQ's walking and total results. Finally, in the study group, we also found correlations of the FFI-RS total, pain, stiffness, difficulty, and social issues results with the Visual Analog Scale (VAS) results, as well as of the FFI-RS total results with the DAS 28 results., Conclusions: The FFI-RS is an effective tool for assessing RA patients' functional status and can be used to evaluate treatment effects. The FFI-RS detected RA-related changes in the foot joint function in patients without foot lesions, as assessed by ultrasound.

Background: Epigenetic remodeling is emerging as a critical process for both the onset and progression of Alzheimer's disease (AD), the most common form of neurodegenerative dementia. However, it is not clear to what extent the distribution of histone modifications is involved in AD., Methods: To investigate histone H3 modifications in AD, we compared the genome-wide distributions of H3K4me3 and H3K27me3 in entorhinal cortices from severe sporadic AD patients and from age-matched healthy individuals of both sexes., Results: AD samples were characterized by typical average levels and distributions of the H3K4me3 and H3K27me3 signals. However, AD patients showed a lower H3K4me3 and higher H3K27me3 signal, particularly in males. Interestingly, the genomic sites found differentially trimethylated at the H3K4 between healthy and AD samples involve promoter regions of genes belonging to AD-related pathways such as glutamate receptor signaling., Conclusions: The signatures of H3K4me3 and H3K27me3 identified in AD patients validate the role of epigenetic chromatin remodeling in neurodegenerative disease and shed light on the genomic adaptive mechanisms involved in AD.

DNA double-strand breaks induced by ionizing radiation can activate the atypical NF-κB pathway via ATM-mediated phosphorylation of NEMO/IKKγ. We aimed to determine whether the status of p53 influenced the activation of this particular NF-κB pathway. The NF-κB signaling was activated either by irradiation with a single 8 Gy dose or by TNFα cytokine in p53-proficient and p53-deficient variants of HCT116, RKO, and U2-OS human cancer cell lines. To assess pathway activation the kinetics of phosphorylation (Ser32) and proteolytic degradation of IκBα inhibitor and phosphorylation (Ser536) of RelA(p65) NF-κB subunit were analyzed. Though activation of the radiation-induced atypical pathway was delayed and weakened when compared to the cytokine-induced canonical pathway, no significant differences were noted between p53-proficient and p53-deficient variants, which indicated that activation of both NF-κB pathways was not affected by the p53 status. In marked contrast, the presence of p53 significantly affected downstream effects of NF-κB activation, i.e. transcription of NF-κB-dependent genes. However, different patterns of such interference were observed, which indicated gene-specific and cell-specific mechanisms of interactions between NF-κB and p53 at the transcription regulation level.

It seems extremely important to know the biomechanical properties of the orofacial tissues among patients with increased activity of masticatory muscles, such as bruxism. The aim of this study was to evaluate biomechanical properties of the masseter muscle by using MyotonPRO in adults with probable bruxism and to define gender relations. This study was conducted in the Temporomandibular Disorders Department at Poznan University of Medical Sciences, Poland (June 2021-November 2021) among patients that reported bruxism symptoms The patients underwent a clinical examination, then the biomechanical properties of the masseter muscles were assessed. The MyotonPro measured masseter tone, stiffness and elasticity in 36 patients with a self-assessment of bruxism (18 women and 18 men). Data were collected from relaxed and contracted muscles. In relaxed masseter muscles there were no statistically significant differences between the sexes in muscle tone, stiffness and elasticity. During contraction significant differences were found between the sexes in the tension and the stiffness of the masseter muscles. Moreover, women often experienced headaches in the temporal region and pain in the masseter muscles during palpation. Among patients with a self-assessment of bruxism, accompanying pain was significantly more frequent in women. Male gender was associated with increased muscle tension and stiffness of the contracted masseter muscle.

BACKGROUND This study aimed to compare the effects of myotherapy using sublingual relaxation splints and stretching exercises in 110 patients with myofascial pain with and without self-reported sleep bruxism using The Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) Axis I Questionnaire. MATERIAL AND METHODS The study involved 110 patients with myofascial pain. The diagnosis was based on the RDC/TMD questionnaire. The number of painful muscle sites (PMS) and the range of maximum mouth opening (MMO) were assessed 2 times - at the first visit and after 3 months. Then, the influence of possible bruxism on the treatment was assessed. RESULTS The mean age of the patients was 26.8 years (SD 5.4); 89% of the subjects were women; and 60.9% of the patients reported bruxism. Each patient was instructed to perform muscle stretching at the first visit and after 1 week all patients received a sublingual relaxation splint. The number of PMS decreased and the range of MMO increased in both groups after a period of 3 months of treatment (P<0.05). Significant differences were observed in the obtained treatment effects between the patients with and without possible bruxism. CONCLUSIONS This study evaluated the effectiveness of the sublingual relaxation splint and stretching exercises in patients with myofascial pain. Patients at a single center in Poland who reported myofascial pain that was not associated with self-reported sleep bruxism had a significantly better response to myotherapy when compared to patients with self-reported sleep bruxism.

Background: Women represent the majority of Alzheimer's disease patients and show typical symptoms. Genetic, hormonal, and behavioral mechanisms have been proposed to explain sex differences in dementia prevalence. However, whether sex differences exist in the epigenetic landscape of neuronal tissue during the progression of the disease is still unknown., Methods: To investigate the differences of histone H3 modifications involved in transcription, we determined the genome-wide profiles of H3K4me3, H3K27ac, and H3K27me3 in brain cortexes of an Alzheimer mouse model (PSAPP). Gastrocnemius muscles were also tested since they are known to be different in the two sexes and are affected during the disease progression., Results: Correlation analysis distinguished the samples based on sex for H3K4me3 and H3K27me3 but not for H3K27ac. The analysis of transcription starting sites (TSS) signal distribution, and analysis of bounding sites revealed that gastrocnemius is more influenced than brain by sex for the three histone modifications considered, exception made for H3K27me3 distribution on the X chromosome which showed sex-related differences in promoters belonging to behavior and cellular or neuronal spheres in mice cortexes., Conclusions: H3K4me3, H3K27ac, and H3K27me3 signals are slightly affected by sex in brain, with the exception of H3K27me3, while a higher number of differences can be found in gastrocnemius.

The present pilot study investigates whether an abnormal miRNA profile in NIPT plasma samples can explain the finding of a low cell-free DNA (cfDNA) fetal fraction (cfDNAff) in euploid fetuses and non-obese women. Twelve women who underwent neoBona ® NIPT with a normal fetal karyotype were studied. Six with a cfDNAff < 4% were matched with a control group with normal levels of cfDNAff > 4%. Samples were processed using the nanostring nCounter ® platform with a panel of 800 miRNAs. Four of the maternal miRNAs, miR-579, miR-612, miR-3144 and miR-6721, had a significant abnormal expression in patients. A data filtering analysis showed that miR-579, miR-612, miR-3144 and miR-6721 targeted 169, 1, 48 and 136 placenta-specific genes, respectively. miR-579, miR-3144 and miR-6721 shared placenta-specific targeted genes involved in trophoblast invasion and migration pathways (IGF2R, PTCD2, SATB2, PLAC8). Moreover, the miRNA target genes encoded proteins localized in the placenta and involved in the pathogenesis of pre-eclampsia, including chorion-specific transcription factor GCMa, PRG2, Lin-28 Homolog B and IGFBP1. In conclusion, aberrant maternal miRNA expression in circulating plasma could be a source of dysregulating trophoblast invasion and migration and could represent a novel cause of a low cfDNAff in the sera of pregnant women at the time of NIPT analysis.