The L467F-F508del Complex Allele Hampers Pharmacological Rescue of Mutant CFTR by Elexacaftor/Tezacaftor/Ivacaftor in Cystic Fibrosis Patients: The Value of the Ex Vivo Nasal Epithelial Model to Address Non-Responders to CFTR-Modulating Drugs

Loss-of-function mutations of the CFTR gene cause cystic fibrosis (CF) through a variety of molecular mechanisms involving altered expression, trafficking, and/or activity of the CFTR chloride channel. The most frequent mutation among CF patients, F508del, causes multiple defects that can be, however, overcome by a combination of three pharmacological agents that improve CFTR channel trafficking and gating, namely, elexacaftor, tezacaftor, and ivacaftor. This study was prompted by the evidence of two CF patients, compound heterozygous for F508del and a minimal function variant, who failed to obtain any beneficial effects following treatment with the triple drug combination. Functional studies on nasal epithelia generated in vitro from these patients confirmed the lack of response to pharmacological treatment. Molecular characterization highlighted the presence of an additional amino acid substitution, L467F, in cis with the F508del variant, demonstrating that both patients were carriers of a complex allele. Functional and biochemical assays in heterologous expression systems demonstrated that the double mutant L467F-F508del has a severely reduced activity, with negligible rescue by CFTR modulators. While further studies are needed to investigate the actual prevalence of the L467F-F508del allele, our results suggest that this complex allele should be taken into consideration as plausible cause in CF patients not responding to CFTR modulators.     

Influence of the alkyl chain length of HSO3-R-MCM-41 on the esterification of glycerol with fatty acids

Ordered mesoporous materials functionalized with alkyl sulfonic acid groups have been proved to be efficient catalysts for esterification. In this paper we report on the synthesis and characterization of new materials containing simultaneously alkyl groups (vinyl, chloromethyl), that have been sulfonated in a post-synthesis step, and methyl groups. Different techniques have been used to characterize the obtained materials: XRD, TEM, elemental analysis, nitrogen adsorption, TGA, ²⁹Si and ¹³C MAS NMR. Finally, the influence of the different synthesis parameters and functional groups on the behavior of the catalysts in the reaction of esterification of glycerol with lauric and oleic acids has been studied. The catalytic performance of the resulting HSO₃-ethyl-MCM-41, HSO₃-ethyl/methyl-MCM-41 and HSO₃-methyl-MCM-41 new materials is also compared to those of HSO₃-phenyl/methyl-MCM-41 and HSO₃-propyl/methyl-MCM-41 synthesized previously. It has been demonstrated that an optimum balance among parameters such as nature of the organic group which support the sulfonic acid, distance between the sulfonic group and the silica surface, average pore size of the material, etc., allows to obtain catalysts with improved activity and selectivity for these reactions.     

Caffeic Acid Enhances the Anti-Leukemic Effect of Imatinib on Chronic Myeloid Leukemia Cells and Triggers Apoptosis in Cells Sensitive and Resistant to Imatinib

Among the phenolic acids tested on the K562 cell line, a model of chronic myeloid leukemia (CML), caffeic acid (CA) was biologically active on sensitive and imatinib (IM)-resistant cells at micro-molar concentration, either in terms of reduction of cell proliferation or triggering of apoptosis. The CA treatment provoked mitochondrial membrane depolarization, genomic DNA fragmentation and phosphatidylserine exposure, hallmarks of apoptosis. Cell cycle analysis following the treatment with comparable cytotoxic concentrations of IM or CA showed marked differences in the distribution profiles. The reduction of cell proliferation by CA administration was associated with increased expression of two cell cycle repressor genes, CDKN1A and CHES1, while IM at a cytotoxic concentration increased the CHES1 but not the CDKN1A expression. In addition, CA treatment affected the proliferation and triggered the apoptosis in IM-resistant cells. Taken together, these data suggested that CA induced the anti-proliferative effect and triggered apoptosis of CML cells by a different mechanism than IM. Finally, the combined administration of IM and CA at suboptimal concentrations evidenced a synergy of action in determining the anti-proliferative effect and triggering apoptosis. The ability of CA to potentiate the anti-leukemic effect of IM highlighted the nutraceutical potential of CA in CML.     

Structural Cues for Understanding eEF1A2 Moonlighting

Spontaneous mutations in the EEF1A2 gene cause epilepsy and severe neurological disabilities in children. The crystal structure of eEF1A2 protein purified from rabbit skeletal muscle reveals a post-translationally modified dimer that provides information about the sites of interaction with numerous binding partners, including itself, and maps these mutations onto the dimer and tetramer interfaces. The spatial locations of the side chain carboxylates of Glu301 and Glu374, to which phosphatidylethanolamine is uniquely attached via an amide bond, define the anchoring points of eEF1A2 to cellular membranes and interorganellar membrane contact sites. Additional bioinformatic and molecular modeling results provide novel structural insight into the demonstrated binding of eEF1A2 to SH3 domains, the common MAPK docking groove, filamentous actin, and phosphatidylinositol-4 kinase IIIβ. In this new light, the role of eEF1A2 as an ancient, multifaceted, and articulated G protein at the crossroads of autophagy, oncogenesis and viral replication appears very distant from the “canonical” one of delivering aminoacyl-tRNAs to the ribosome that has dominated the scene and much of the thinking for many decades.     

Modulating drug release from poly(lactic-co-glycolic) acid microparticles by the addition of alginate and pectin

The aim of the present work is the characterization of PLGA microparticles including biopolymers for the controlled release of tilmicosin, a broad-spectrum antibiotic. Microparticles were prepared using the double-emulsion solvent evaporation technique. The effect of alginate and pectin incorporation over particle size and porosity, encapsulation efficiency (EE) and pH-responsive drug release was evaluated. Formulations presented a mean particle size of 5.5 μm approximately and a drug EE ranged from 22%–57%. PLGA-Alginate particles showed an increased porosity. Tilmicosin release profiles from PLGA and PLGA-biopolymer microparticles were affected by the particular combination of polymers and the pH of the release medium. The experimental data was simulated using a mathematical model, which takes into account the autocatalytic polymer degradation and the different mechanisms of drug transport. The combination of PLGA and biopolymers strongly influenced the morphology of the particles, offering the possibility of controlling the drug release profiles according to the therapy.     

Biohybrid Bovine Bone Matrix for Controlled Release of Mesenchymal Stem/Stromal Cell Lyosecretome: A Device for Bone Regeneration

SmartBone^® (SB) is a biohybrid bone substitute advantageously proposed as a class III medical device for bone regeneration in reconstructive surgeries (oral, maxillofacial, orthopedic, and oncology). In the present study, a new strategy to improve SB osteoinductivity was developed. SB scaffolds were loaded with lyosecretome, a freeze-dried formulation of mesenchymal stem cell (MSC)-secretome, containing proteins and extracellular vesicles (EVs). Lyosecretome-loaded SB scaffolds (SBlyo) were prepared using an absorption method. A burst release of proteins and EVs (38% and 50% after 30 min, respectively) was observed, and then proteins were released more slowly with respect to EVs, most likely because they more strongly adsorbed onto the SB surface. In vitro tests were conducted using adipose tissue-derived stromal vascular fraction (SVF) plated on SB or SBlyo. After 14 days, significant cell proliferation improvement was observed on SBlyo with respect to SB, where cells filled the cavities between the native trabeculae. On SB, on the other hand, the process was still present, but tissue formation was less organized at 60 days. On both scaffolds, cells differentiated into osteoblasts and were able to mineralize after 60 days. Nonetheless, SBlyo showed a higher expression of osteoblast markers and a higher quantity of newly formed trabeculae than SB alone. The quantification analysis of the newly formed mineralized tissue and the immunohistochemical studies demonstrated that SBlyo induces bone formation more effectively. This osteoinductive effect is likely due to the osteogenic factors present in the lyosecretome, such as fibronectin, alpha-2-macroglobulin, apolipoprotein A, and TGF-β.