A cellular disease model toward gene therapy of TGM1-dependent lamellar ichthyosis.

Lamellar ichthyosis (LI) is a chronic disease, mostly caused by mutations in the TGM1 gene, marked by impaired skin barrier formation. No definitive therapies are available, and current treatments aim at symptomatic relief. LI mouse models often fail to faithfully replicate the clinical and histopathological features of human skin conditions. To develop advanced therapeutic approaches, such as combined ex vivo cell and gene therapy, we established a human cellular model of LI by efficient CRISPR-Cas9-mediated gene ablation of the TGM1 gene in human primary clonogenic keratinocytes. Gene-edited cells showed complete absence of transglutaminase 1 (TG1) expression and recapitulated a hyperkeratotic phenotype with most of the molecular hallmarks of LI in vitro. Using a self-inactivating γ-retroviral (SINγ-RV) vector expressing transgenic TGM1 under the control of its own promoter, we tested an ex vivo gene therapy approach and validate the model of LI as a platform for pre-clinical evaluation studies. Gene-corrected TGM1-null keratinocytes displayed proper TG1 expression, enzymatic activity, and cornified envelope formation and, hence, restored proper epidermal architecture. Single-cell multiomics analysis demonstrated proviral integrations in holoclone-forming epidermal stem cells, which are crucial for epidermal regeneration. This study serves as a proof of concept for assessing the potential of this therapeutic approach in treating TGM1-dependent LI.

Anakinra-Loaded Sphingomyelin Nanosystems Modulate In Vitro IL-1-Dependent Pro-Tumor Inflammation in Pancreatic Cancer.

Pancreatic cancer is a very aggressive disease with a dismal prognosis. The tumor microenvironment exerts immunosuppressive activities through the secretion of several cytokines, including interleukin (IL)-1. The IL-1/IL-1 receptor (IL-1R) axis is a key regulator in tumor-promoting T helper (Th)2- and Th17-type inflammation. Th2 cells are differentiated by dendritic cells endowed with Th2-polarizing capability by the thymic stromal lymphopoietin (TSLP) that is secreted by IL-1-activated cancer-associated fibroblasts (CAFs). Th17 cells are differentiated in the presence of IL-1 and other IL-1-regulated cytokines. In pancreatic cancer, the use of a recombinant IL-1R antagonist (IL1RA, anakinra, ANK) in in vitro and in vivo models has shown efficacy in targeting the IL-1/IL-1R pathway. In this study, we have developed sphingomyelin nanosystems (SNs) loaded with ANK (ANK-SNs) to compare their ability to inhibit Th2- and Th17-type inflammation with that of the free drug in vitro. We found that ANK-SNs inhibited TSLP and other pro-tumor cytokines released by CAFs at levels similar to ANK. Importantly, inhibition of IL-17 secretion by Th17 cells, but not of interferon-γ, was significantly higher, and at lower concentrations, with ANK-SNs compared to ANK. Collectively, the use of ANK-SNs might be beneficial in reducing the effective dose of the drug and its toxic effects.

Biochemical role of FOXM1-dependent histone linker H1B in human epidermal stem cells.

Epidermal stem cells orchestrate epidermal renewal and timely wound repair through a tight regulation of self-renewal, proliferation, and differentiation. In culture, human epidermal stem cells generate a clonal type referred to as holoclone, which give rise to transient amplifying progenitors (meroclone and paraclone-forming cells) eventually generating terminally differentiated cells. Leveraging single-cell transcriptomic data, we explored the FOXM1-dependent biochemical signals controlling self-renewal and differentiation in epidermal stem cells aimed at improving regenerative medicine applications. We report that the expression of H1 linker histone subtypes decrease during serial cultivation. At clonal level we observed that H1B is the most expressed isoform, particularly in epidermal stem cells, as compared to transient amplifying progenitors. Indeed, its expression decreases in primary epithelial culture where stem cells are exhausted due to FOXM1 downregulation. Conversely, H1B expression increases when the stem cells compartment is sustained by enforced FOXM1 expression, both in primary epithelial cultures derived from healthy donors and JEB patient. Moreover, we demonstrated that FOXM1 binds the promotorial region of H1B, hence regulates its expression. We also show that H1B is bound to the promotorial region of differentiation-related genes and negatively regulates their expression in epidermal stem cells. We propose a novel mechanism wherein the H1B acts downstream of FOXM1, contributing to the fine interplay between self-renewal and differentiation in human epidermal stem cells. These findings further define the networks that sustain self-renewal along the previously identified YAP-FOXM1 axis.

AlPaCas: allele-specific CRISPR gene editing through a protospacer-adjacent-motif (PAM) approach.

Gene therapy of dominantly inherited genetic diseases requires either the selective disruption of the mutant allele or the editing of the specific mutation. The CRISPR-Cas system holds great potential for the genetic correction of single nucleotide variants (SNVs), including dominant mutations. However, distinguishing between single-nucleotide variations in a pathogenic genomic context remains challenging. The presence of a PAM in the disease-causing allele can guide its precise targeting, preserving the functionality of the wild-type allele. The AlPaCas (Aligning Patients to Cas) webserver is an automated pipeline for sequence-based identification and structural analysis of SNV-derived PAMs that satisfy this demand. When provided with a gene/SNV input, AlPaCas can: (i) identify SNV-derived PAMs; (ii) provide a list of available Cas enzymes recognizing the SNV (s); (iii) propose mutational Cas-engineering to enhance the selectivity towards the SNV-derived PAM. With its ability to identify allele-specific genetic variants that can be targeted using already available or engineered Cas enzymes, AlPaCas is at the forefront of advancements in genome editing. AlPaCas is open to all users without a login requirement and is freely available at https://schubert.bio.uniroma1.it/alpacas.

Static Behavior of a 3D-Printed Short Carbon Fiber Polyamide: Influence of the Meso-Structure and Water Content.

The knowledge of the mechanical behavior of a 3D-printed material is fundamental for the 3D printing outbreaking technology to be considered for a range of applications. In this framework, the significance, reliability, and accuracy of the information obtained by testing material coupons assumes a pivotal role. The present work focuses on an evaluation of the static mechanical properties and failure modes of a 3D-printed short carbon fiber-reinforced polyamide in relation to the specimen's unique meso-structural morphology and water content. Within the manufacturing limitations of a commercially available printer, specimens of dedicated combinations of geometry and printing patterns were specifically conceived and tested. The specimens' meso-structure morphologies were investigated by micro-computed tomography. The material failure mechanisms were inferred from an analysis of the specimens' fracture surfaces and failure morphologies. The outcomes of the present analysis indicate that each test specimen retained proper mechanical properties, thereby suggesting that they should be accurately designed to deliver representative information of the underlying material beads or of their deposition layout. Suggestions on the adoption of preferred test specimens for evaluating specific material properties were proposed.

Enhancing the nitric oxide inhibitory activity using a combination of plant essential oils and mixture design approach.

The synergistic effects of essential oils (EOs) from three aromatic plant species, Foeniculum vulgare subsp. piperitum (C.Presl) Bég. (FV), Origanum heracleoticum L. (OH) and Lavandula austroapennina N.G.Passal., Tundis & Upson. (LA), were evaluated for their inhibitory properties on nitric oxide production in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS). We utilized a Design of Experiments (DoE) methodology to optimize a formulation by combining three Essential Oils (EOs), while simultaneously taking into account two response variables, maximization of NO inhibition with minimum cytotoxicity. The optimal blend of components was predicted, and the statistical outcome's efficacy was experimentally verified. The combination corresponding to 87.7 % FV, 12.3 % LA and 0.0 % OH showed high inhibitory effect (76.3 %) with negligible cytotoxicity (4.5 %). This research provides new information on the interactions among fennel, oregano and lavender essential oils and shows how they can synergistically inhibit in vitro LPS-induced NO production.

Dual-responsive chondroitin sulfate self-assembling nanoparticles for combination therapy in metastatic cancer cells.

In this study, we developed self-assembling nanoparticles (LCPs) able to trigger the release of Chlorambucil (Chl) and Doxorubicin (DOX) to MDA-MB-231 cells by exploiting the enzyme and redox signals. The DOX loaded LCPs was prepared by the self-assembly of two chondroitin sulphate (CS) derivatives, obtained by the covalent conjugation of Lipoic Acid (LA) and Chlorambucil (Chl) to the CS backbone. After the physic-chemical characterization of the conjugates by FT-IR, 1H NMR, and determination of the critical aggregation concentration, spherical nanoparticles with mean hydrodynamic diameter of 45 nm (P.D.I. 0.24) and Z-potential of - 44 mV were obtained by water addition/solvent evaporation method. In vitro experiments for the release of Chl and DOX were performed in healthy and cancer cells, using a cell culture media to maintain the physiological intracellular conditions (pH 7.4) (and concentration of esterase and GSH. The results allowed the selective release of the payloads to be detected: Chl release of 0 and 41% were obtained after 2 h incubation in normal and in cancer cells respectively, while values of 35 (in healthy cells) and 60% (in cancer cells) were recorded for DOX release after 96 h. Finally, viability studies proved the ability of the newly proposed nanosystem to enhance the cytotoxic activity of the two drugs against cancer cells.

Bread or roses? Trade unions, female employment and the expansion of work-family policies.

In the Fordist era, trade unions promoted welfare state expansion and coverage against risks for the broader workforce. With the shift to the post-industrial economy, however, new economic groups have been left without representation. This is particularly evident for women: despite a rapid increase in female employment since the 1980s, unions' membership base remains anchored in the male, old and industrial working class. Without the crucial pressure of labour, welfare systems have failed to enhance the reconciliation of work and family life. Under which conditions do unions support the expansion of work-family policies? Marshalling evidence from 20 OECD countries in the 1980-2010 period, this paper investigates the role of political actors in family policy reform. Findings suggest that unions promote the expansion of work-family packages when they are gender-inclusive and have institutional access to policy-making.

Multivariate Approaches in Quantitative Structure-Property Relationships Study for the Photostability Assessment of 1,4-Dihydropyridine Derivatives.

1,4-dihydropyridines (1,4-DHPs) are widely recognized as highly effective L-type calcium channel blockers with significant therapeutic benefits in the treatment of cardiovascular disorders. 1,4-DHPs can also target T-type calcium channels, making them promising drug candidates for neurological conditions. When exposed to light, all 1,4-DHPs tend to easily degrade, leading to an oxidation product derived from the aromatization of the dihydropyridine ring. Herein, the elaboration of a quantitative structure-property relationships (QSPR) model was carried out by correlating the light sensitivity of structurally different 1,4-DHPs with theoretical molecular descriptors. Photodegradation experiments were performed by exposing the drugs to a Xenon lamp following the ICH rules. The degradation was monitored by spectrophotometry, and experimental data were elaborated by Multivariate Curve Resolution (MCR) methodologies to assess the kinetic rates. The results were confirmed by the HPLC-DAD method. PaDEL-Descriptor software was used to calculate molecular descriptors and fingerprints related to the chemical structures. Seventeen of the 1875 molecular descriptors were selected and correlated to the photodegradation rate by means of the Ordinary Least Squares (OLS) algorithm. The chemometric model is useful to predict the photosensitivity of other 1,4-DHP derivatives with a very low relative error percentage of 5.03% and represents an effective tool to design new analogs characterized by higher photostability.

Viability assessment of livers donated after circulatory determination of death during normothermic regional perfusion.

BACKGROUND: Abdominal normothermic regional perfusion (A-NRP) allows in-situ reperfusion and recovery of abdominal organs metabolism in donors after circulatory death (DCD). Besides improving liver transplantation outcomes, liver injury and function can be assessed during A-NRP. METHODS: To refine liver viability assessment during A-NRP, prospectively collected data of controlled DCD donors managed at our Institution between October 2019 and May 2022 were retrospectively analyzed. Baseline characteristics, procedural variables and A-NRP parameters of donors whose liver was successfully transplanted were compared to those of donors whose liver was discarded. RESULTS: Twenty-seven donors were included and in 20 (74%) the liver was accepted (positive outcome). No differences between study groups were observed concerning baseline characteristics and warm ischemia times (WIT). Initial lactate levels were positively correlated with functional WIT (r2 = 0.4, p = 0.04), whereas transaminase levels were not. Blood flow during A-NRP was comparable, whereas oxygen consumption (VO2 ) was significantly higher in the positive outcome group after 1 h. Time courses of lactate, AST and ALT were significantly different between study groups (p < 0.001). Donors whose liver was accepted showed faster lactate clearance, a difference which was amplified by normalizing lactate clearance to oxygen delivery (DO2 ) and VO2 . Lactate clearance was correlated to transaminase levels and DO2 -normalized lactate clearance was the parameter best discriminating between study groups. CONCLUSIONS: DO2 -normalized lactate clearance may represent an element of liver viability assessment during A-NRP.