Residual Cystine Transport Activity for Specific Infantile and Juvenile CTNS Mutations in a PTEC-Based Addback Model.

Cystinosis is a rare, autosomal recessive, lysosomal storage disease caused by mutations in the gene CTNS, leading to cystine accumulation in the lysosomes. While cysteamine lowers the cystine levels, it does not cure the disease, suggesting that CTNS exerts additional functions besides cystine transport. This study investigated the impact of infantile and juvenile CTNS mutations with discrepant genotype/phenotype correlations on CTNS expression, and subcellular localisation and function in clinically relevant cystinosis cell models to better understand the link between genotype and CTNS function. Using CTNS-depleted proximal tubule epithelial cells and patient-derived fibroblasts, we expressed a selection of CTNSmutants under various promoters. EF1a-driven expression led to substantial overexpression, resulting in CTNS protein levels that localised to the lysosomal compartment. All CTNSmutants tested also reversed cystine accumulation, indicating that CTNSmutants still exert transport activity, possibly due to the overexpression conditions. Surprisingly, even CTNSmutants expression driven by the less potent CTNS and EFS promoters reversed the cystine accumulation, contrary to the CTNSG339R missense mutant. Taken together, our findings shed new light on CTNS mutations, highlighting the need for robust assessment methodologies in clinically relevant cellular models and thus paving the way for better stratification of cystinosis patients, and advocating for the development of more personalized therapy.

Statin-Sensitive Akt1/Src/Caveolin-1 Signaling Enhances Oxidative Stress Resistance in Rhabdomyosarcoma.

Identifying the molecular mechanisms underlying radioresistance is a priority for the treatment of RMS, a myogenic tumor accounting for approximately 50% of all pediatric soft tissue sarcomas. We found that irradiation (IR) transiently increased phosphorylation of Akt1, Src, and Cav1 in human RD and RH30 lines. Synthetic inhibition of Akt1 and Src phosphorylation increased ROS levels in all RMS lines, promoting cellular radiosensitization. Accordingly, the elevated activation of the Akt1/Src/Cav1 pathway, as detected in two RD lines characterized by overexpression of a myristoylated Akt1 form (myrAkt1) or Cav1 (RDCav1), was correlated with reduced levels of ROS, higher expression of catalase, and increased radioresistance. We found that treatment with cholesterol-lowering drugs such as lovastatin and simvastatin promoted cell apoptosis in all RMS lines by reducing Akt1 and Cav1 levels and increasing intracellular ROS levels. Combining statins with IR significantly increased DNA damage and cell apoptosis as assessed by γ histone 2AX (γH2AX) staining and FACS analysis. Furthermore, in combination with the chemotherapeutic agent actinomycin D, statins were effective in reducing cell survival through increased apoptosis. Taken together, our findings suggest that the molecularly linked signature formed by Akt1, Src, Cav1, and catalase may represent a prognostic determinant for identifying subgroups of RMS patients with higher probability of recurrence after radiotherapy. Furthermore, statin-induced oxidative stress could represent a treatment option to improve the success of radiotherapy.

Investigation on Electrospun and Solvent-Casted PCL-PLGA Blends Scaffolds Embedded with Induced Pluripotent Stem Cells for Tissue Engineering.

The design, production, and characterisation of tissue-engineered scaffolds made of polylactic-co-glycolic acid (PLGA), polycaprolactone (PCL) and their blends obtained through electrospinning (ES) or solvent casting/particulate leaching (SC) manufacturing techniques are presented here. The polymer blend composition was chosen to always obtain a prevalence of one of the two polymers, in order to investigate the contribution of the less concentrated polymer on the scaffolds' properties. Physical-chemical characterization of ES scaffolds demonstrated that tailoring of fibre diameter and Young modulus (YM) was possible by controlling PCL concentration in PLGA-based blends, increasing the fibre diameter from 0.6 to 1.0 µm and reducing the YM from about 22 to 9 MPa. SC scaffolds showed a "bubble-like" topography, caused by the porogen spherical particles, which is responsible for decreasing the contact angles from about 110° in ES scaffolds to about 74° in SC specimens. Nevertheless, due to phase separation within the blend, solvent-casted samples displayed less reproducible properties. Furthermore, ES samples were characterised by 10-fold higher water uptake than SC scaffolds. The scaffolds suitability as iPSCs culturing support was evaluated using XTT assay, and pluripotency and integrin gene expression were investigated using RT-PCR and RT-qPCR. Thanks to their higher wettability and appropriate YM, SC scaffolds seemed to be superior in ensuring high cell viability over 5 days, whereas the ability to maintain iPSCs pluripotency status was found to be similar for ES and SC scaffolds.