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Michal Gorzkiewicz,1,2 Monika Marcinkowska,1 Maciej Studzian,3,4 Iwona Karwaciak,4,5 Lukasz Pulaski,3,4 Barbara Klajnert-Maculewicz1 1Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland; 2Department of Molecular Medicine II, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany; 3Department of Oncobiology and Epigenetics, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland; 4Laboratory of Transcriptional Regulation, Institute of Medical Biology PAS, Lodz, Poland; 5Laboratory of Epigenetics, Institute of Medical Biology PAS, Lodz, PolandCorrespondence: Michal Gorzkiewicz, Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, 141/143 Pomorska Street, Lodz, 90-236, Poland, Tel +48 42 635 41 47, Email michal.gorzkiewicz@biol.uni.lodz.plBackground: Mesalazine is one of the main drugs used to treat inflammatory bowel diseases. However, its applicability is limited by its rapid inactivation and removal from the organism, as well as the need for its membrane transporter-dependent cellular uptake to exert therapeutic effect. The present study involved the development of an innovative nanocarrier, based on poly(amidoamine) (PAMAM) dendrimer of the 4th generation, to obtain higher concentrations of the drug in the intestinal epithelial cells, thus increasing its anti-inflammatory potential. The work involved synthesis and in vitro characterization of covalent PAMAM-mesalazine conjugate with succinic linker.Results: PAMAM-mesalazine conjugate was synthesized and characterized by 1H NMR, 13C NMR, FTIR and MALDI-TOF MS. This allowed to confirm the purity of the obtained compound and intermediates. Based on the analyses, it was found that ~45 drug molecules were successfully attached to one molecule of PAMAM dendrimer. The conjugate was then characterized in terms of hydrodynamic diameter, zeta potential, spectral properties, drug release from the carrier, as well as cellular uptake and cytotoxicity in two in vitro models of gastrointestinal epithelium (CaCo-2 and HT-29 human cell lines). Analyzing cellular parameters related to the specific mechanism of action of mesalazine (inhibition of NF-κB signaling, decrease in interleukin and prostaglandin synthesis, and ROS scavenging), we showed that such a dendrimer-based carrier may enhance cellular uptake of the drug, which translated into its improved anti-inflammatory efficacy.Conclusion: The use of PAMAM macromolecule as a carrier for mesalazine increases the bioavailability of the drug, ensuring enhanced cellular uptake and bypassing the need to utilize mesalazine-specific membrane transporters. All these characteristics translate into an improved anti-inflammatory activity of mesalazine in vitro. In conjunction with appropriately designed in vivo studies, such a compound may prove to be a promising alternative to the therapeutics currently used in inflammatory bowel diseases.Graphical Abstract: Keywords: PAMAM dendrimer, mesalazine, inflammatory bowel disease, drug delivery

Bisphenol A (BPA, a plastic polymer component) and tetrabromobisphenol A (TBBPA, a brominated flame retardant) are industrial compounds and representative persistent organic pollutants (POPs) with similar chemical structure. We studied their impact on biological membrane dynamics, which is an emerging and understudied target for environmental contaminants, using a set of state-of-the-art methods. We found that exposure to TBBPA, but not to BPA, leads to disruption of biophysical homeostasis of the plasma membrane in myeloid cell lines HL-60, THP-1 and Mono Mac 6. Applied methods include: pyrene excimer formation, fluorescence anisotropy, solvatochromic shift ratiometry (using di-4-ANEPPDHQ, NR12A and laurdan) and fluorescence recovery after photobleaching. TBBPA increased rotational and lateral mobility and decreased general polarity and lipid order in plasma membranes of myeloid cells, but decreased mobility and increased rigidity in internal membranes of the same cells. Strikingly, BPA had no significant membrane effects in these cells, suggesting a specific molecular interaction mechanism of TBBPA action which may potentially lead to disruption of immune function. Identification of this novel threat from an established pollutant with documented exposure pathways highlights the possibility that immunotoxicity of POPs may contribute to their environmental toxicity burden., 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 The Author(s). Published by Elsevier B.V. All rights reserved.)

Nonprocessed foodstuffs of plant origin, especially whole-grain cereals, are considered to be health-promoting components of the human diet. While most of their well-studied effects derive from their high fiber content and low glycemic index, the presence of underrated phenolic phytonutrients has recently been brought to the attention of nutritionists. In this review, we report and discuss findings on the sources and bioactivities of 3,5-dihydroxybenzoic acid (3,5-DHBA), which is both a direct dietary component (found, e.g., in apples) and, more importantly, a crucial metabolite of whole-grain cereal-derived alkylresorcinols (ARs). 3,5-DHBA is a recently described exogenous agonist of the HCAR1/GPR81 receptor. We concentrate on the HCAR1-mediated effects of 3,5-DHBA in the nervous system, on the maintenance of cell stemness, regulation of carcinogenesis, and response to anticancer therapy. Unexpectedly, malignant tumors take advantage of HCAR1 expression to sense 3,5-DHBA to support their growth. Thus, there is an urgent need to fully identify the role of whole-grain-derived 3,5-DHBA during anticancer therapy and its contribution in the regulation of vital organs of the body via its specific HCAR1 receptor. We discuss here in detail the possible consequences of the modulatory capabilities of 3,5-DHBA in physiological and pathological conditions in humans.

Flame retardants (FRs) are chemical substances used to inhibit the spread of fire in numerous industrial applications, and their abundance in modern manufactured products in the indoor and outdoor environment leads to extensive direct and food chain exposure of humans. Although once considered relatively non-toxic, FRs are demonstrated by recent literature to have disruptive effects on many biological processes, including signaling pathways, genome stability, reproduction, and immune system function. This review provides a summary of research investigating the impact of major groups of FRs, including halogenated and organophosphorus FRs, on animals and humans in vitro and/or in vivo. We put in focus those studies that explained or referenced the modes of FR action at the level of cells, tissues and organs. Since FRs are highly hydrophobic chemicals, their biophysical and biochemical modes of action usually involve lipophilic interactions, e.g. with biological membranes or elements of signaling pathways. We present selected toxicological information about these molecular actions to show how they can lead to damaging membrane integrity, damaging DNA and compromising its repair, changing gene expression, and cell cycle as well as accelerating cell death. Moreover, we indicate how this translates to deleterious bioactivity of FRs at the physiological level, with disruption of hormonal action, dysregulation of metabolism, adverse effects on male and female reproduction as well as alteration of normal pattern of immunity. Concentrating on these subjects, we make clear both the advances in knowledge in recent years and the remaining gaps in our understanding, especially at the mechanistic level., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Lukasz Pulaski reports financial support was provided by National Science Centre Poland., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: To evaluate corneal sensitivity and corneal nerve morphology among patients with Wolfram syndrome (WFS)., Design: An observational clinical case series with confirmatory experiments., Methods: We included a group of 12 patients with biallelic mutations in the WFS1 gene and a control group composed of 30 individuals with type 1 diabetes (T1D). All participants (n = 42) underwent a complete ophthalmic examination, esthesiometry, and retinal nerve fiber layer assessment using optical coherence tomography. Morphologic assessment of corneal neuropathy by in vivo corneal confocal microscopy was conducted in 11 patients with WFS (both eyes) and 1 WFS patient (1 eye) as well as in 24 patients with T1D (both eyes in 6 patients and 1 eye in 18 patients). Additionally, corneas from Wfs1KO mice and their wild-type littermates were subjected to laser scanning confocal microscopy., Results: Corneal sensitivity was significantly reduced in patients with WFS compared with patients with T1D (4.50 cm [interquartile range, 3.50-5.50 cm] vs 6.00 cm [interquartile range, 6.00-6.00 cm]; P < 10 -5 ). Additionally, corneal nerve fiber and branch density as well as nerve fiber length were low among patients with WFS. Corneal sensitivity correlated with macular average thickness (R = 0.6928; P = .039) and best-corrected visual acuity (R = -0.61; P = .002) in the WFS group. Similarly, Wfs1 knockout mice also presented corneal neurodegeneration changes when corneal nerve fiber density and length were measured using laser scanning confocal microscopy., Conclusions: Decreased corneal sensitivity and corneal nerve degeneration are observed in WFS. Corneal sensitivity is linked with the degree of disease progression as measured by visual acuity and retinal thinning., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cyanobacterial blooms constitute a recognized danger to aquatic environment and public health not only due to presence of main group of cyanotoxins, such as microcystins, cylindrospermopsin or anatoxin-a, but also other emerging bioactivities. An innovative approach identifying such bioactivities is the application of cellular biosensors based on reporter genes which detect the impact of cyanobacterial cells and components on actual human cells in a physiological-like setting. In the present study biosensor cell lines detecting four different types of bioactivities (ARE - oxidative stress, NFKBRE - immunomodulatory pathogen-associated molecular patterns, AHRE - persistent organic pollutants, GRE - endocrine disruptors) were exposed to concentrated cyanobacterial cells from 21 environmental bloom samples and from eight cultures (Microcystis aeruginosa, Aphanizomenon flos-aquae, Planktothrix agardhii and Raphidiopsis raciborskii). The AHRE and GRE biosensors did not detect any relevant bioactivity. In turn, ARE biosensors were significantly activated by bloom samples from Jeziorsko (180-250%) and Sulejów (250-400%) reservoirs with the highest cyanobacterial biomass, while activation by cultures was weak/undetectable. The same biosensors were stimulated by microcystin-LR (250%) and anatoxin-a (150%). The NFKBRE biosensors were activated to varying extent (140-650%) by most bloom and culture samples, pointing to potential immunomodulatory toxic effects on humans. Lipopolysaccharide and lipoproteins were identified as responsible for NFKBRE activation (probably via pattern recognition receptors), while peptidoglycan had no bioactivity in this assay. Thus, the holistic approach to sample analysis with the application of cellular biosensors geared towards 4 separate pathways/bioactivities was validated for identification of novel bioactivities in organisms with recognized public health significance (e.g. this study is the first to describe cyanobacterial lipoproteins as potential environmental immunomodulators). Moreover, the ability of cellular biosensors to be activated by intact cyanobacterial cells from blooms provides proof of concept of their direct application for environmental monitoring, especially comparison of potential threats without need for chemical analysis and identification of toxicants., Competing Interests: Declaration of competing interest Authors declare that this research was funded by the National Science Centre, project no. UMO-2012/07/B/NZ8/03991 - “Application of reporter cell biosensors in ecotoxicology of cyanobacteria: new targets for bioactivity” and partly from EEA/Norway Grant Mechanism research grant PL0107 “Cellular biosensors for automated monitoring of environmental pollution”. 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 © 2021. Published by Elsevier B.V.)

The increased level of hydrogen peroxide accompanies some modes of macrophage specification and is linked to ROS-based antimicrobial activity of these phagocytes. In this study, we show that activation of toll-like receptors with bacterial components such as LPS is accompanied by the decline in transcription of hydrogen peroxide decomposing enzyme-catalase, suppression of which facilitates the polarization of human macrophages towards the pro-inflammatory phenotype. The chromatin remodeling at the CAT promoter involves LSD1 and HDAC1, but activity of the first enzyme defines abundance of the two proteins on chromatin, histone acetylation status and the CAT transcription. LSD1 inhibition prior to macrophage activation with LPS prevents CAT repression by enhancing the LSD1 and interfering with the HDAC1 recruitment to the gene promoter. The maintenance of catalase level with LSD1 inhibitors during M1 polarization considerably limits LPS-triggered expression of some pro-inflammatory cytokines and markers such as IL1β , TNFα , COX2 , CD14 , TLR2 , and IFNAR , but the effect of LSD1 inhibitors is lost upon catalase deficiency. Summarizing, activity of LSD1 allows for the CAT repression in LPS stimulated macrophages, which negatively controls expression of some key pro-inflammatory markers. LSD1 inhibitors can be considered as possible immunosuppressive drugs capable of limiting macrophage M1 specialization.

New therapeutic strategies for personalized medicine need to involve innovative pharmaceutical tools, for example, modular nanoparticles designed for direct immunomodulatory properties. We synthesized mannose-functionalized poly(propyleneimine) glycodendrimers with a novel architecture, where freely accessible mannose moieties are presented on poly(ethylene glycol)-based linkers embedded within an open-shell maltose coating. This design enhanced glycodendrimer bioactivity and led to complex functional effects in myeloid cells, with specific induction of interleukin-8 expression by mannose glycodendrimers detected in HL-60 and THP-1 cells. We concentrated on explaining the molecular mechanism of this phenomenon, which turned out to be different in both investigated cell lines: in HL-60 cells, transcriptional activation via AP-1 binding to the promoter predominated, while in THP-1 cells (which initially expressed less IL-8), induction was mediated mainly by mRNA stabilization. The success of directed immunomodulation, with synthetic design guided by assumptions about mannose-modified dendrimers as exogenous regulators of pro-inflammatory chemokine levels, opens new possibilities for designing bioactive nanoparticles.

Two immortalized brain microvascular endothelial cell lines (hCMEC/D3 and RBE4, of human and rat origin, respectively) were applied as an in vitro model of cellular elements of the blood-brain barrier in a nanotoxicological study. We evaluated the impact of CdSe/ZnS core-shell-type quantum dot nanoparticles on cellular homeostasis, using gold nanoparticles as a largely bioorthogonal control. While the investigated nanoparticles had surprisingly negligible acute cytotoxicity in the evaluated models, a multi-faceted study of barrier-related phenotypes and cell condition revealed a complex pattern of homeostasis disruption. Interestingly, some features of the paracellular barrier phenotype (transendothelial electrical resistance, tight junction protein gene expression) were improved by exposure to nanoparticles in a potential hormetic mechanism. However, mitochondrial potential and antioxidant defences largely collapsed under these conditions, paralleled by a strong pro-apoptotic shift in a significant proportion of cells (evidenced by apoptotic protein gene expression, chromosomal DNA fragmentation, and membrane phosphatidylserine exposure). Taken together, our results suggest a reactive oxygen species-mediated cellular mechanism of blood-brain barrier damage by quantum dots, which may be toxicologically significant in the face of increasing human exposure to this type of nanoparticles, both intended (in medical applications) and more often unintended (from consumer goods-derived environmental pollution).

Tecto(dendrimers) are well-defined, dendrimer cluster type covalent structures. In this article, we present the synthesis of such a PAMAM [G5:G3-(TREN)]- N -(4-carbomethoxy) pyrrolidone terminated tecto(dendrimer). This tecto(dendrimer) exhibits nontraditional intrinsic luminescence (NTIL; excitation 376 nm; emission 455 nm) that has been attributed to three fluorescent components characterized by different fluorescence lifetimes. Furthermore, it has been shown that this PAMAM [G5:G3-(TREN)]- N -(4-carbomethoxy) pyrrolidone terminated tecto(dendrimer) is able to form a polyplex with double stranded DNA, and is nontoxic for HeLa and HMEC-1 cells up to a concentration of 10 mg/mL, even though it accumulates in endosomal compartments as demonstrated by its unique NTIL emission properties. Many of the above features would portend the proposed use of this tecto(dendrimer) as an efficient transfection agent. Quite surprisingly, transfection activity could not be demonstrated in HeLa cells, and the possible reasons are discussed in the article.

The aim of the present study was to define the mtDNA variability of Polish population and to visualize the genetic relations between Poles. For the first time, the study of Polish population was conducted on such a large number of individuals (5852) representing administrative units of both levels of local administration in Poland (voivodeships and counties). Additionally, clustering was used as a method of population subdivision. Performed genetic analysis, included F ST , MDS plot, AMOVA and SAMOVA. Haplogroups were classified and their geographical distribution was visualized using surface interpolation maps. Results of the present study showed that Poles are characterized by the main West Eurasian mtDNA haplogroups. Furthermore, the level of differentiation within the Polish population was quite low but the existing genetic differences could be explained well with geographic distances. This may lead to a conclusion that Poles can be considered as genetically homogenous but with slight differences, highlighted at the regional level. Some patterns of variability were observed and could be explained by the history of demographic processes in Poland such as resettlements and migrations of women or relatively weaker urbanisation and higher rural population retention of some regions.

Background: Recent advances in ancient DNA studies, especially in increasing isolated DNA yields and quality, have opened the possibility of analysis of ancient host microbiome. However, such pitfalls as spurious identification of pathogens based on fragmentary data or environmental contamination could lead to incorrect epidaemiological conclusions. Within the Mycobacterium genus, Mycobacterium tuberculosis complex members responsible for tuberculosis share up to ∼99% genomic sequence identity, while other more distantly related Mycobacteria other than M. tuberculosis can be causative agents for pulmonary diseases or soil dwellers. Therefore, reliable determination of species complex is crucial for interpretation of sequencing results., Results: Here we present a novel bioinformatical approach, used for screening of ancient tuberculosis in sequencing data, derived from 28 individuals (dated 4400-4000 and 3100-2900 BC) from central Poland. We demonstrate that cost-effective next-generation screening sequencing data (∼20M reads per sample) could yield enough information to provide statistically supported identification of probable ancient disease cases., Conclusions: Application of appropriate bioinformatic tools, including an unbiased selection of genomic alignment targets for species specificity, makes it possible to extract valid data from full-sample sequencing results (without subjective targeted enrichment procedures). This approach broadens the potential scope of palaeoepidaemiology both to older, suboptimally preserved samples and to pathogens with difficult intrageneric taxonomy., (© The Author(s) 2019. Published by Oxford University Press.)

Malignant melanoma is the most aggressive skin cancer and can only be cured if detected early. Unfortunately, later stages of the disease do not guarantee success due to the rapid rate of melanoma cell metastasis and their high resistance to applied therapies. The search for new molecular targets and targeted therapy may represent the future in the development of effective methods for combating this cancer. SIRT2 is a promising target; thus, we downregulated SIRT2 expression in melanoma cells in vertical growth and metastatic phases and demonstrated that sirtuin acts as regulator of the basic functions of melanoma cells. A detailed transcriptomic analysis showed that SIRT2 regulates the expression of multiple genes encoding the tyrosine kinase pathways that are molecular targets of dasatinib. Indeed, cells with low SIRT2 expression were more susceptible to dasatinib, as demonstrated by multiple techniques, e.g., neutral red uptake, 3/7 caspase activity, colony formation assay, and in vitro scratch assay. Furthermore, these cells showed an altered phosphorylation profile for proteins playing roles in the response to dasatinib. Thus, our research indicates new, previously unknown SIRT2 functions in the regulation of gene expression, which is of key clinical significance., Competing Interests: The authors declare no conflicts of interest.

Fludarabine is an anticancer antimetabolite essential for modern chemotherapy, but its efficacy is limited due to the complex pharmacokinetics. We demonstrated the potential use of maltose-modified poly(propyleneimine) dendrimer as drug delivery agent to improve the efficiency of therapy with fludarabine. In this study, we elaborated a novel synthesis technique for radioactively labeled fludarabine triphosphate to prove for the first time the direct ability of nucleotide-glycodendrimer complex to enter and kill leukemic cells, without the involvement of membrane nucleoside transporters and intracellular kinases. This will potentially allow to bypass the most common drug resistance mechanisms observed in the clinical setting. Further, we applied surface plasmon resonance and molecular modeling to elucidate the properties of the drug-dendrimer complexes. We showed that clofarabine, a more toxic nucleoside analogue drug, is characterized by significantly different molecular interactions with poly(propyleneimine) dendrimers than fludarabine, leading to different cellular outcomes (decreased rather than increased treatment efficiency). The most probable mechanistic explanation of uniquely dendrimer-enhanced fludarabine toxicity points to a crucial role of both an alternative cellular uptake pathway and the avoidance of intracellular phosphorylation of nucleoside drug form.

Differentiation of human macrophages predisposes these cells to numerous tasks, i.e. killing invading pathogens, and this entails the need for enhanced intracellular defences against stress, including conditions that may increase DNA damage. Our study shows that expression of DNA repair enzymes, such as PARP1, BRCA1 and XRCC1, are activated during macrophage development by the SWI/SNF chromatin remodelling complex, which serves as a histone acetylation sensor. It recognises and displaces epigenetically marked nucleosomes, thereby enabling transcription. Acetylation is controlled both in monocytes and macrophages by the co-operation of EP300 and HDAC1 activities. Differentiation modulates the activities of individual components of EP300-HDAC1-SWI/SNF functional unit and entails recruitment of PBAF to gene promoters. In monocytes, histone-deacetylated promoters of repressed PARP1, BRCA1 and XRCC1 respond only to HDAC inhibition, with an opening of the chromatin structure by BRM, whereas in macrophages both EP300 and HDAC1 contribute to the fine-tuning of nucleosomal acetylation, with HDAC1 remaining active and the balance of EP300 and HDAC1 activities controlling nucleosome eviction by BRG1-containing SWI/SNF. Since EP300-HDAC1-SWI/SNF operates at the level of gene promoters characterized simultaneously by the presence of E2F binding site(s) and CpG island(s), this allows cells to adjust PARP1, BRCA1 and XRCC1 transcription to the differentiation mode and to restart cell cycle progression. Thus, mutual interdependence between acetylase and deacetylase activities defines the acetylation-dependent code for regulation of histone density and gene transcription by SWI/SNF, notably on gene promoters of DNA repair enzymes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Adenosine analogue drugs (such as fludarabine or cladribine) require transporter-mediated uptake into cells and subsequent phosphorylation for anticancer activity. Therefore, application of nanocarrier systems for direct delivery of active triphosphate forms has been proposed. Here, we applied isothermal titration calorimetry and zeta potential titration to determine the stoichiometry and thermodynamic parameters of interactions between 4th generation poly(propyleneimine) dendrimers (unmodified or sugar-modified for increased biocompatibility) and ATP as a model adenosine nucleotide. We showed that glycodendrimers have the ability to efficiently interact with nucleoside triphosphates and to form stable complexes via electrostatic interactions between the ionized phosphate and amino groups on the nucleotide and the dendrimer, respectively. The complexation process is spontaneous, enthalpy-driven and depends on buffer composition (strongest interactions in organic buffer) and pH (more binding sites in acidic pH). These properties allow us to consider maltose-modified dendrimers as especially promising carriers for adenosine analogues., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Fludarabine, a nucleoside analogue antimetabolite, has complicated pharmacokinetics requiring facilitated transmembrane transport and intracellular conversion to triphosphate nucleotide form (Ara-FATP), causing it to be susceptible to emergence of drug resistance. We are testing a promising strategy to improve its clinical efficacy by direct delivery of Ara-FATP utilizing a biocompatible glycodendrimer nanocarrier system. Here, we present results of a proof-of-concept experiment in several in vitro-cultured leukemic cell lines (CCRF, THP-1, U937) using noncovalent complexes of maltose-modified poly(propyleneimine) dendrimer and fludarabine triphosphate. We show that Ara-FATP has limited cytotoxic activity toward investigated cells relative to free nucleoside (Ara-FA), but complexation with the glycodendrimer (which does not otherwise influence cellular metabolism) drastically increases its toxicity. Moreover, we show that transport via hENT1 is a limiting step in Ara-FA toxicity, while complexation with dendrimer allows Ara-FATP to kill cells even in the presence of a hENT1 inhibitor. Thus, the use of glycodendrimers for drug delivery would allow us to circumvent naturally occurring drug resistance due to decreased transporter activity. Finally, we demonstrate that complex formation does not change the advantageous multifactorial intracellular pharmacodynamics of Ara-FATP, preserving its high capability to inhibit DNA and RNA synthesis and induce apoptosis via the intrinsic pathway. In comparison to other nucleoside analogue drugs, fludarabine is hereby demonstrated to be an optimal candidate for maltose glycodendrimer-mediated drug delivery in antileukemic therapy.

Poly(propyleneimine) dendrimers of fourth generation partially modified with maltose (open shell structure, PPI-m OS) have been proposed as carriers for nucleotide anticancer drugs. The aim of this work was to provide basic insight into interactions between fluorescently labeled PPI-m dendrimer and two distinct leukemia cell models: CCRF-1301 lymphoid cell line and HL-60 myeloid cell line. We applied qualitative confocal imaging and quantitative flow cytometry, as well as trypan blue quenching and pharmacological inhibition, to investigate the course, kinetics, and molecular mechanisms of internalization of nanoparticles. CCRF-1301 cells take up glycodendrimer macromolecules via a relatively slow, adsorptive endocytosis process, which is cholesterol-dependent, clathrin- and caveolin-independent, and not followed by recycling or exocytosis. Morphological features of this phenomenon point to the involvement of aggregation-induced cell polarity changes (capping). In HL-60 cells, internalization is very fast, independent of binding to the cell surface, and proceeds from the fluid phase via a classical clathrin-dependent mechanism, ending up in an endolysosomal compartment from which it is not further released. This substantial difference in internalization rate and mechanism between two cell types has important repercussions for potential application of this class of glycodendrimers as drug delivery agents.

Aims: Cancer cells, due to the Warburg effect, are more dependent on glycolysis than normal cells, so glycolytic inhibitor 3-bromopyruvic acid (3-BP) was proposed as a promising candidate for anticancer therapy. Overexpression of multidrug transporters is the main reason of resistance of cancer cells to chemotherapy. As the activity of multidrug transporters imposes an energetic burden on the cells, it can be expected that inhibition of ATP generation may exert a selective cytotoxicity to cells overexpressing multidrug transporters. The aim of this study was to compare the effect of 3-BP on the survival and ATP level in MDCK-II cells and MDCK-II cells overexpressing ABCB1 (Pgp) or ABCG2 (BCRP)., Main Methods: Cell survival was measured with resazurin and with neutral red. ATP level was assayed with luciferin/luciferase kit. Luteolin transport was measured by an original method described in the paper., Key Findings: 3-BP (10-200μM) induced a decrease of ATP level after 1-h incubation in all cell lines studied, more drastically in ABCB1-overexpressing cells. 50 and 200μM 3-BP significantly decreased cell viability; the effect was more pronounced for ABCB1-overexpressing cells. PSC833, inhibitor of ABCB1, ameliorated the toxic effect of 3-BP on MDCK-II ABCB1 cells and MDCK-II cells. 3-BP inhibited luteolin transport in MDCK-II ABCG2 cells., Significance: These results indicate that 3-BP shows selective toxicity against ABCB1- but not ABCG2-overexpressing cells, apparently due to enhanced ATP depletion but in a manner independent of the transport activity of Pgp, suggesting a novel mechanism of hypersensitivity of ABCB1-overexpressing cells to 3-BP., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Despite the focus of most ecotoxicological studies on cyanobacteria on a select group of cyanotoxins, especially microcystins, a growing body of evidence points to the involvement of other cyanobacterial metabolites in deleterious health effects. In the present study, original, self-developed reporter gene-based cellular biosensors, detecting activation of the main human xenobiotic stress response pathways, PXR and NFkappaB, were applied to detect novel potentially toxic bioactivities in extracts from freshwater microcystin-producing cyanobacterial blooms. Crude and purified extracts from cyanobacteria containing varying levels of microcystins, and standard microcystin-LR were tested. Two cellular biosensor types applied in this study, called NHRTOX (detecting PXR activation) and OXIBIOS (detecting NFkappaB activation), successfully detected potentially toxic or immunomodulating bioactivities in cyanobacterial extracts. The level of biosensor activation was comparable to control cognate environmental toxins. Despite the fact that extracts were derived from microcystin-producing cyanobacterial blooms and contained active microcystins, biosensor-detected bioactivities were shown to be unrelated to microcystin levels. Experimental results suggest the involvement of environmental toxins (causing a response in NHRTOX) and lipopolysaccharides (LPS) or other cell wall components (causing a response in OXIBIOS) in the potentially harmful bioactivity of investigated extracts. These results demonstrate the need for further identification of cyanobacterial metabolites other than commonly studied cyanotoxins as sources of health risk, show the usefulness of cellular biosensors for this purpose and suggest a novel, more holistic approach to environmental monitoring., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The HMC-1 cell line represents the phenotype of immature mast cells. The HIF1A gene product HIF-1alpha plays key roles in maintaining oxygen homeostasis in eukaryotic organisms and is involved in many processes, including immune response and hematopoiesis. In this study we investigated HIF1A expression in HMC-1 immature hematopoietic cells and CD34+ hematopoietic progenitors. HMC-1 cells exhibited exceptionally low levels of HIF1A expression compared to other cell lines as determined by real-time PCR, and multipotent CD34+ hematopoietic progenitors in bone marrow exhibited significantly lower levels of HIF1A mRNA compared to mature blood cells in peripheral blood. We searched for the mechanisms responsible for suppression of HIF1A expression in HMC-1 cells and obtained evidence for a DNA methylation-dependent process. In vitro methylation of the HIF1A promoter resulted in a decrease in its transcriptional activity and the level of DNA methylation in the HIF1A promoter region in analyzed cell lines was negatively correlated with HIF1A expression. Furthermore, the DNA demethylating agent 5'-azacytidine increased HIF1A expression, and MeCP2 protein was preferentially associated with the HIF1A promoter in vivo. In conclusion, we report that the HIF1A gene in HMC-1 immature hematopoietic cells is suppressed by a process dependent on DNA methylation, and we present evidence indicating downregulation of HIF1A expression in multipotent CD34+ hematopoietic progenitors., (Copyright 2009 Elsevier Inc. All rights reserved.)

The erythrocytes of 12-month old Sod1 (-/-) mice showed an increased level of reactive oxygen species (ROS), as estimated by the degree of dihydroethidine and dihydrorhodamine oxidation, and the increased level of Heinz bodies. No indices of severe oxidative stress were found in the red blood cells and blood plasma of Sod1 (-/-) mice as judged from the lack of significant changes in the levels of erythrocyte and plasma glutathione, plasma protein thiol and carbonyl groups and thiobarbituric-acid reactive substances in the blood plasma. However, a decreased erythrocyte lifespan, increased reticulocyte count and splenomegaly were noted, indicating the importance of superoxide dismutase for maintaining erythrocyte viability. The levels of erythrocyte ROS and Heinz bodies and the reticulocyte count were indistinguishable in Sod1 (+/+) and Sod1 (+/-) mice, suggesting that a superoxide dismutase activity decrease to half of its normal value may be sufficient to secure the protective effects of the enzyme.