PAMAM dendrimers are distinguished by their capacity for functionalization, which enhances the properties of the compounds they transport, rendering them highly versatile nanoparticles with extensive applications in the biomedical domain, including drug, vaccine, and gene delivery. These dendrimers can be internalized into cells through various endocytic mechanisms, such as passive diffusion, clathrin-mediated endocytosis, and caveolae-mediated endocytosis, allowing them to traverse the cytoplasm and reach intracellular targets, such as the mitochondria or nucleus. Despite the significant challenge posed by the cytotoxicity of these nanoparticles, which is contingent upon the dendrimer size, surface charge, and generation, numerous strategies have been documented to modify the dendrimer surface using polyethylene glycol and other chemical groups to temporarily mitigate their cytotoxic effects. The potential of PAMAM dendrimers in cancer therapy and other biomedical applications is substantial, owing to their ability to enhance bioavailability, pharmacokinetics, and pharmacodynamics of active ingredients within the body. This underscores the necessity for further investigation into the optimization of internalization pathways and cytotoxicity of these nanoparticles. This review offers a comprehensive synthesis of the current literature on the diverse cellular internalization pathways of PAMAM dendrimers and their cargo molecules, emphasizing the mechanisms of entry, intracellular trafficking, and factors influencing these processes.

Pannexin 1 channels located in the cell membrane are permeable to ions, metabolites, and signaling molecules. While the activity of these channels is known to be modulated by phosphorylation on T198, T308, and S206, the possible involvement of other putative phosphorylation sites remains unknown. Here, we describe that the activity of Panx1 channels induced by mechanical stretch is reduced by adenosine via a PKA-dependent pathway. The mechanical stretch-induced activity—measured by changes in DAPI uptake—of Panx1 channels expressed in HeLa cell transfectants was inhibited by adenosine or cAMP analogs that permeate the cell membrane. Moreover, inhibition of PKA but not PKC, p38 MAPK, Akt, or PKG prevented the effects of cAMP analogs, suggesting the involvement of Panx1 phosphorylation by PKA. Accordingly, alanine substitution of T302 or S328, two putative PKA phosphorylation sites, prevented the inhibitory effect of cAMP analogs. Moreover, phosphomimetic mutation of either T302 or S328 to aspartate prevented the mechanical stretch-induced activation of Panx1 channels. A molecular dynamics simulation revealed that T302 and S328 are located in the water–lipid interphase near the lateral tunnel of the intracellular region, suggesting that their phosphorylation could promote conformational changes in lateral tunnels. Thus, Panx1 phosphorylation via PKA could be modulated by G protein-coupled receptors associated with the Gs subunit.

The role of ventral hippocampus (vHipp) astroglial gliotransmission in depression was studied using chronic restraint stress (CRS) and chronic unpredictable mild stress (CUMS) rodent models. CRS increased Cx43 hemichannel activity and extracellular glutamate levels in the vHipp and blocking astroglial Cx43 hemichannel-dependent gliotransmission during CRS prevented the development of depression and glutamate buildup. Moreover, the acute blockade of Cx43 hemichannels induced antidepressant effects in rats previously subjected to CRS or CUMS. This antidepressant effect was prevented by coinjection of glutamate and D-serine. Furthermore, Cx43 hemichannel blockade decreased postsynaptic NMDAR currents in vHipp slices in a glutamate and D-serine-dependent manner. Notably, chronic microinfusion of glutamate and D-serine, L-serine, or the NMDAR agonist NMDA, into the vHipp induced depressive-like symptoms in nonstressed rats. We also identified a small molecule, cacotheline, which blocks Cx43 hemichannels and its systemic administration induced rapid antidepressant effects, preventing stress-induced increases in astroglial Cx43 hemichannel activity and extracellular glutamate in the vHipp, without sedative or locomotor side effects. In conclusion, chronic stress increases Cx43 hemichannel-dependent release of glutamate and D-/L-serine from astrocytes in the vHipp, overactivating postsynaptic NMDARs and triggering depressive-like symptoms. This study highlights the critical role of astroglial gliotransmitter release in chronic stress-induced depression and suggests it can be used as a target for the prevention and treatment of depression.

The coagulation cascade is the process of the conversion of soluble fibrinogen to insoluble fibrin that terminates in production of a clot. Factor Xa (FXa) is a serine protease involved in the blood coagulation cascade. Moreover, FXa plays a vital role in the enzymatic sequence which ends with the thrombus production. Thrombosis is a common causal pathology for three widespread cardiovascular syndromes: acute coronary syndrome (ACS), venous thromboembolism (VTE), and strokes. In this research a series of N-propargyltetrahydroquinoline and 1,2,3-triazole derivatives as a potential factor Xa (FXa) inhibitor were designed, synthesized, and evaluated for their FXa inhibitor activity, cytotoxicity activity and coagulation parameters. Rational design for the desired novel molecules was performed through protein-ligand complexes selection and ligand clustering. The microwave-assisted synthetic strategy of selected compounds was carried out by using Ullmann-Goldberg, N-propargylation, Mannich addition, Friedel-Crafts, and 1,3-dipolar cycloaddition type reactions under microwave irradiation. The microwave methodology proved to be an efficient way to obtain all novel compounds in high yields (73–93%). Furthermore, a thermochemical analysis, optimization and reactivity indexes such as electronic chemical potential (µ), chemical hardness (η), and electrophilicity (ω) were performed to understand the relationship between the structure and the energetic behavior of all the series. Then, in vitro analysis showed that compounds 27, 29–31, and 34 exhibited inhibitory activity against FXa and the corresponding half maximal inhibitory concentration (IC₅₀) values were calculated. Next, a cell viability assay in HEK293 and HepG2 cell lines, and coagulation parameters (anti FXa, Prothrombin time (PT), activated Partial Thromboplastin Time (aPTT)) of the most active novel molecules were performed to determine the corresponding cytotoxicity and possible action on clotting pathways. The obtained results suggest that compounds 27 and 29 inhibited FXa targeting through coagulation factors in the intrinsic and extrinsic pathways. However, compound 34 may target coagulation FXa mainly by the extrinsic and common pathway. Interestingly, the most active compounds in relation to the inhibition activity against FXa and coagulation parameters did not show toxicity at the performed coagulation assay concentrations. Finally, docking studies confirmed the preferential binding mode of N-propargyltetrahydroquinoline and 1,2,3-triazole derivatives inside the active site of FXa.

Salicylic acid (SA) is a hormone that modulates plant defenses by inducing changes in gene expression. The mechanisms that control SA accumulation are essential for understanding the defensive process. TGA transcription factors from clade II in Arabidopsis, which include the proteins TGA2, TGA5, and TGA6, are known to be key positive mediators for the transcription of genes such as PR-1 that are induced by SA application. However, unexpectedly, stress conditions that induce SA accumulation, such as infection with the avirulent pathogen P. syringae DC3000/AvrRPM1 and UV-C irradiation, result in enhanced PR-1 induction in plants lacking the clade II TGAs (tga256 plants). Increased PR-1 induction was accompanied by enhanced isochorismate synthase-dependent SA production as well as the upregulation of several genes involved in the hormone’s accumulation. In response to avirulent P. syringae, PR-1 was previously shown to be controlled by both SA-dependent and -independent pathways. Therefore, the enhanced induction of PR-1 (and other defense genes) and accumulation of SA in the tga256 mutant plants is consistent with the clade II TGA factors providing negative feedback regulation of the SA-dependent and/or -independent pathways. Together, our results indicate that the TGA transcription factors from clade II negatively control SA accumulation under stress conditions that induce the hormone production. Our study describes a mechanism involving old actors playing new roles in regulating SA homeostasis under stress.