RESUMO
Alveolar macrophages (AMs) act as gatekeepers of the lung's immune responses, serving essential roles in recognizing and eliminating pathogens. The transcription factor (TF) Early Growth Response 2 (EGR2) has been recently described as required for mature AMs in mice; however, its mechanisms of action have not been explored. Here, we identified EGR2 as an epigenomic regulator and likely direct proximal transcriptional activator in AMs using epigenomic approaches (RNA-sequencing, ATAC-sequencing, and CUT&RUN). The predicted direct proximal targets of EGR2 included a subset of AM identity genes, and ones related to pathogen recognition, phagosome maturation, and adhesion, such as Clec7a, Atp6v0d2, Itgb2, Rhoc, and Tmsb10. We provided evidence that EGR2 deficiency led to impaired zymosan internalization and reduced the capacity to respond to Aspergillus fumigatus. Mechanistically, the lack of EGR2 altered the transcriptional response, secreted cytokines (i.e., CXCL11), and inflammation-resolving lipid mediators (i.e., RvE1) of AMs during in vivo zymosan-induced inflammation, which manifested in impaired resolution. Our findings demonstrated that EGR2 is a key proximal transcriptional activator and epigenomic bookmarker in AMs responsible for select, distinct components of cell identity and a protective transcriptional and epigenomic program against fungi.
RESUMO
The emergence of drug-resistant bacteria and fungi represents a serious health problem worldwide. It has long been known that cationic compounds can inhibit the growth of bacteria and fungi by disrupting the cell membrane. The advantage of using such cationic compounds is that the microorganisms would not become resistant to cationic agents, since this type of adaptation would mean significantly altering the structure of their cell walls. We designed novel, DBU (1,8-diazabicyclo[5.4.0]undec-7-ene)-derived amidinium salts of carbohydrates, which may be suitable for disturbing the cell walls of bacteria and fungi due to their quaternary ammonium moiety. A series of saccharide-DBU conjugates were prepared from 6-iodo derivatives of d-glucose, d-mannose, d-altrose and d-allose by nucleophilic substitution reactions. We optimized the synthesis of a d-glucose derivative, and studied the protecting group free synthesis of the glucose-DBU conjugates. The effect of the obtained quaternary amidinium salts against Escherichia coli and Staphylococcus aureus bacterial strains and Candida albicans yeast was investigated, and the impact of the used protecting groups and the sugar configuration on the antimicrobial activity was analyzed. Some of the novel sugar quaternary ammonium compounds with lipophilic aromatic groups (benzyl and 2-napthylmethyl) showed particularly good antifungal and antibacterial activity.
Assuntos
Antifúngicos , Sais , Antifúngicos/farmacologia , Sais/farmacologia , Relação Estrutura-Atividade , Antibacterianos/farmacologia , Fungos , Bactérias , Compostos de Amônio Quaternário/química , Carboidratos/farmacologia , Glucose/farmacologia , Açúcares/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
BACKGROUND/AIM: Conventional viability tests, help to screen the cellular effects of candidate molecules, but the endpoint of these measurements lacks sufficient information regarding the molecular aspects. A non-invasive, easy-to-setup live-cell microscopic method served to in-depth analysis of mechanisms of potential anticancer drugs. MATERIALS AND METHODS: The proposed method combining the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test with time-lapse scanning microscopy (TLS), provided additional data related to the cell-cycle and the dynamic properties of cell morphology. Apoptotic and necrotic events became detectable with these methods. RESULTS: Quantification of the results was assisted by image analysis of the acquired image sequences. After demonstrating the potential of the TLS method, a series of experiments compared the in vitro effect of a known and a newly synthesized nucleoside analogue. CONCLUSION: The proposed approach provided a more in-depth insight into the cellular processes that can be affected by known chemotherapeutic agents including nucleoside analogues rather than applying repeated individual treatments.
Assuntos
Antineoplásicos/farmacologia , Nucleosídeos/farmacologia , Sais de Tetrazólio , Tiazóis , Imagem com Lapso de Tempo , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Microscopia , Nucleosídeos/análogos & derivados , Imagem com Lapso de Tempo/métodosRESUMO
A small library of 3'-deoxy-C3'-substituted xylofuranosyl-pyrimidine nucleoside analogues were prepared by photoinduced thiol-ene addition of various thiols, including normal and branched alkyl-, 2-hydroxyethyl, benzyl-, and sugar thiols, to 3'-exomethylene derivatives of 2',5'-di-O-tert-butyldimethylsilyl-protected ribothymidine and uridine. The bioactivity of these derivatives was studied on tumorous SCC (mouse squamous carcinoma cell) and immortalized control HaCaT (human keratinocyte) cell lines. Several alkyl-substituted analogues elicited promising cytostatic activity in low micromolar concentrations with a slight selectivity toward tumor cells. Near-infrared live-cell imaging revealed SCC tumor cell-specific mitotic blockade via genotoxicity of analogue 10, bearing an n-butyl side chain. This analogue essentially affects the chromatin structure of SCC tumor cells, inducing a condensed nuclear material and micronuclei as also supported by fluorescent microscopy. The results highlight that thiol-ene chemistry represents an efficient strategy to discover novel nucleoside analogues with non-natural sugar structures as anticancer agents.
Assuntos
Citostáticos/síntese química , Citostáticos/farmacologia , Conformação Molecular , Nucleosídeos/síntese química , Nucleosídeos/farmacologia , Xilose/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dimetil Sulfóxido/farmacologia , Humanos , Concentração Inibidora 50 , Espectroscopia de Prótons por Ressonância Magnética , Compostos de Sulfidrila/químicaRESUMO
The toxicity of gadolinium is reduced by chelating agents that render this heavy metal into contrast complexes used for medical magnetic resonance imaging. However, the dissociation of gadolinium chelates is known to generate Gd3+ ions, the cellular toxicity of which has not been tested in details. The cytotoxic effects of Gd(III) ions were evaluated by monitoring the proliferation, measuring the cellular motility and following chromatin changes in various cell lines upon Gd3+ treatment. Measurements applied long-term scanning microscopy and a perfusion platform that replaced the medium with test solutions, bypassed physical contact with the cell culture during experiments, and provided uninterrupted high time-resolution time-lapse photomicrography for an extended period of time. Genotoxicity specific chromatin changes characteristic to Gd(III) were distinguished in human skin keratinocytes (HaCaT), human limbal stem cells (HuLi), colorectal adenocarcinoma (CaCO2), murine squamous carcinoma (SCC) and Indian muntjac (IM) cell lines. Characteristic features of Gd(III) toxicity were: loss of cellular motility, irreversible attachment of cells to the growth surface and cell death. Injury-specific chromatin changes manifested at micromolar Gd3+ concentrations as premature chromatin condensation and highly condensed sticky chromatin patches. Gd(III) concentration- and cell type-dependent reduction of normal adherence, as well as premature chromatin condensation confirmed apoptosis. The risk related to the release of toxic Gd3+ ions from gadolinium complexes and their effects on mono- and multi-layer cellular barriers have to be reconsidered when these chelated complexes are used as contrasting agents especially in relation to possible blood-brain barrier damages.