Bovine serum albumin-embedded Au nanoclusters (BSA-AuNCs) are thoroughly probed by continuous wave electron paramagnetic resonance (CW-EPR), light-induced EPR (LEPR), and sequences of microscopic investigations performed via high-resolution transmission electron microscopy (HR-TEM), scanning transmission electron microscopy (STEM), and energy dispersive X-ray analysis (EDS). To the best of our knowledge, this is the first report analyzing the BSA-AuNCs by CW-EPR/LEPR technique. Besides the presence of Au(0) and Au(I) oxidation states in BSA-AuNCs, the authors observe a significant amount of Au(II), which may result from a disproportionation event occurring within NCs: 2Au(I) → Au(II) + Au(0). Based on the LEPR experiments, and by comparing the behavior of BSA versus BSA-AuNCs under UV light irradiation (at 325 nm) during light off-on-off cycles, any energy and/or charge transfer event occurring between BSA and AuNCs during photoexcitation can be excluded. According to CW-EPR results, the Au nano assemblies within BSA-AuNCs are estimated to contain 6–8 Au units per fluorescent cluster. Direct observation of BSA-AuNCs by STEM and HR-TEM techniques confirms the presence of such diameters of gold nanoclusters in BSA-AuNCs. Moreover, in situ formation and migration of Au nanostructures are observed and evidenced after application of either a focused electron beam from HR-TEM, or an X-ray from EDS experiments.

Plasmonic photothermal therapy (PPTT) employing plasmonic gold nanorods (GNRs) presents a potent strategy for eradication of tumors including aggressive brain gliomas. Despite its promise, there is a pressing need for a more comprehensive evaluation of PPTT using sophisticated in vitro models that closely resemble tumor tissues, thereby facilitating the elucidation of therapeutic mechanisms. In this study, we exposed 3D glioma spheroids (tumoroids) to (16-mercaptohexadecyl)trimethylammonium bromide-functionalized gold nanorods (MTABGNRs) and a near-infrared (NIR) laser. We demonstrate that the photothermal effect can be fine-tuned by adjusting the nanoparticle concentration and laser power. Depending on the selected parameters, the laser can trigger either regulated or non-regulated cell death (necrosis) in both mouse GL261 and human U-87 MG glioma cell lines, accompanied by translocation of phosphatidylserine in the membrane. Our investigation into the mechanism of regulated cell death induced by PPTT revealed an absence of markers associated with classical apoptosis pathways, such as cleaved caspase 3. Instead, we observed the presence of cleaved caspase 1, gasdermin D, and elevated levels of NLRP3 in NIR-irradiated tumoroids, indicating the activation of pyroptosis. This finding correlates with previous observations of lysosomal accumulation of MTAB-GNRs and the known lysosomal pathway of pyroptosis activation. We further confirmed the absence of toxic breakdown products of GNRs using electron microscopy, which showed no melting or fragmentation of gold nanoparticles under the conditions causing regulated cell death. In conclusion, PPTT using coated gold nanorods offers significant potential for glioma cell elimination occurring through the activation of pyroptosis rather than classical apoptosis pathways.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known for its multifunctionality in several pathogenic bacteria. Our previously reported data suggest that the GAPDH homologue of Francisella tularensis, GapA, might also be involved in other processes beyond metabolism. In the present study, we explored GapA’s potential implication in pathogenic processes at the host cell level. Using immunoelectron microscopy, we demonstrated the localization of this bacterial protein inside infected macrophages and its peripheral distribution in bacterial cells increasing with infection time. A quantitative proteomic approach based on stable isotope labeling of amino acids in cell culture (SILAC) combined with pull-down assay enabled the identification of several of GapA’s potential interacting partners within the host cell proteome. Two of these partners were further confirmed by alternative methods. We also investigated the impact of gapA deletion on the transcription of selected cytokine genes and the activation of the main signaling pathways. Our results show that ∆gapA-induced transcription of genes encoding several cytokines whose expressions were not affected in cells infected with a fully virulent wild-type strain. That might be caused, at least in part, by the detected differences in ERK/MAPK signaling activation. The experimental observations together demonstrate that the F. tularensis GAPDH homologue is directly implicated in multiple host cellular processes and, thereby, that it participates in several molecular mechanisms of pathogenesis.

IL-6 signaling is involved in the pathogenesis of a number of serious diseases, including chronic inflammation and cancer. Targeting of IL-6 receptor (IL-6R) by small molecules is therefore an intensively studied strategy in cancer treatment. We describe the design, synthesis, and characteristics of two new bis-pentamethinium salts 5 and 6 (meta and para) bearing indole moieties. Molecular docking studies showed that both compounds have the potential to bind IL-6R (free energy of binding −9.5 and −8.1 kcal/mol). The interaction with IL-6R was confirmed using microscale thermophoresis analyses, which revealed that both compounds had strong affinity for the IL-6R (experimentally determined dissociation constants 26.5 ± 2.5 nM and 304 ± 27.6 nM, respectively). In addition, both compounds were cytotoxic for a broad spectrum of cancer cell lines in micromolar concentrations, most likely due to their accumulation in mitochondria and inhibition of mitochondrial respiration. In summary, the structure motif of bis-pentamethinium salts represents a promising starting point for the design of novel multitargeting compounds with the potential to inhibit IL-6 signaling and simultaneously target mitochondrial metabolism in cancer cells.

Dendritic cells (DCs) hijacked by intracellular bacteria contribute to pathogen dissemination and immunopathology. How bacteria achieve DC subversion remains largely unknown. Here, we describe the mechanism used by tularemia agent Francisella tularensis exploiting host mitochondrial anaplerosis. Shortly after internalization, Francisella associates with DC mitochondria, which leads to the rapid repurposing of their oxidative metabolism for production of mitochondrial reactive oxygen species (mtROS). Mitochondrial metabolic rewiring is orchestrated by the intramitochondrial signaling mediated by protein acetylation and involves switching to glutamate as the primary substrate for DC tricarboxylic acid cycle. Rather than killing the bacterium, glutamate-fueled mtROS production activates p38-dependent proinflammatory gene expression. Blocking of glutamate utilization prevents DC activation and bacterial dissemination and alleviates inflammation in vivo. Our findings underscore the importance of metabolic plasticity in antibacterial DC response and open up potential avenues for therapies targeting host metabolism.

Lamins, the nuclear intermediate filaments, are important regulators of nuclear structural integrity as well as nuclear functional processes such as DNA transcription, replication and repair, and epigenetic regulations. A portion of phosphorylated lamin A/C localizes to the nuclear interior in interphase, forming a lamin A/C pool with specific properties and distinct functions. Nucleoplasmic lamin A/C molecular functions are mainly dependent on its binding partners; therefore, revealing new interactions could give us new clues on the lamin A/C mechanism of action. In the present study, we show that lamin A/C interacts with nuclear phosphoinositides (PIPs), and with nuclear myosin I (NM1). Both NM1 and nuclear PIPs have been previously reported as important regulators of gene expression and DNA damage/repair. Furthermore, phosphorylated lamin A/C forms a complex with NM1 in a phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent manner in the nuclear interior. Taken together, our study reveals a previously unidentified interaction between phosphorylated lamin A/C, NM1, and PI(4,5)P2 and suggests new possible ways of nucleoplasmic lamin A/C regulation, function, and importance for the formation of functional nuclear microdomains.