Analysis of biomechanical properties of mouse skin dermis through atomic force microscopy: Application to demonstrate a sexual dimorphism.

An in-depth understanding of the mechanical properties of the dermis is indispensable to improve wound healing or slow-down skin ageing. Despite crucial research issues for dermatological and cosmetic industries, very little is known about the mechanical behaviour of the dermis at nanoscale level. This knowledge is relevant not only to human skin but also to mouse skin since this animal model is widely used in basic and preclinical studies for skin biology and health. Here, we describe an original protocol that we developed to specifically measure the mechanical properties of mouse dermis using atomic force microscopy-based nano-indentation approach. Using horizontal cryosections (i.e. parallel to the skin surface) performed at different depths through the dermis of dorsal skin, our protocol allowed us to detect nanoscale mechanical changes between female and male dermis samples. We found that the dermis was softer (i) in females than in males and (ii) with depth within the dermis of male mice. We also quantified compositional differences between female and male skin dermis and found that increased extracellular matrix gene expression and type V collagen staining were associated with increased dermal stiffness in male mice, compared with females. Our results demonstrating a sexual dimorphism in the nanomechanical properties and molecular composition of mouse dermis, open the way to better consider sex-related cutaneous differences to understand skin disease and to stimulate the development of female versus male-specific products with more appropriate dermatological treatments and cosmetic interventions.

Combining an optimized mRNA template with a double purification process allows strong expression of in vitro transcribed mRNA.

mRNA is a blooming technology for vaccination and has gained global attention during the SARS-CoV-2 pandemic. However, the recent clinical trials have highlighted increased reactogenicity when using high mRNA doses. Intending to increase the potency of mRNA therapeutics and to decrease the therapeutic dose, we designed a mRNA backbone and optimized the mRNA purification process. We used the enhanced green fluorescent protein (eGFP) reporter gene flanked by one 5' untranslated region (UTR) and two 3' UTRs of the human ß-globin as a reference mRNA and identified the most promising mRNA sequence using in vitro and in vivo models. First, we assessed the impact of different poly(A) sizes on translation and selected the most optimal sequence. Then, we selected the best 5' UTR among synthetic sequences displaying a high ribosome loading. Finally, we evaluated the transfection efficiency of our standard mRNA template after two capping strategies and purification using either double-stranded RNA (dsRNA) depletion or dephosphorylation of 5'PPP RNA or both combined. Double purification was shown to give the best results. Altogether, the use of a newly defined 5' UTR coupled to post-transcriptional treatments will be of great interest in the mRNA vaccine field, by limiting the amount of the antigen-coding transcript and subsequently the formulation components needed for an efficient vaccination.

Latent TGF-ß Activation Is a Hallmark of the Tenascin Family.

Transforming growth factor-ß (TGF-ß) isoforms are secreted as inactive complexes formed through non-covalent interactions between bioactive TGF-ß entities and their N-terminal pro-domains called latency-associated peptides (LAP). Extracellular activation of latent TGF-ß within this complex is a crucial step in the regulation of TGF-ß activity for tissue homeostasis and immune cell function. We previously showed that the matrix glycoprotein Tenascin-X (TN-X) interacted with the small latent TGF-ß complex and triggered the activation of the latent cytokine into a bioactive TGF-ß. This activation most likely occurs through a conformational change within the latent TGF-ß complex and requires the C-terminal fibrinogen-like (FBG) domain of the glycoprotein. As the FBG-like domain is highly conserved among the Tenascin family members, we hypothesized that Tenascin-C (TN-C), Tenascin-R (TN-R) and Tenascin-W (TN-W) might share with TN-X the ability to regulate TGF-ß bioavailability through their C-terminal domain. Here, we demonstrate that purified recombinant full-length Tenascins associate with the small latent TGF-ß complex through their FBG-like domains. This association promotes activation of the latent cytokine and subsequent TGF-ß cell responses in mammary epithelial cells, such as cytostasis and epithelial-to-mesenchymal transition (EMT). Considering the pleiotropic role of TGF-ß in numerous physiological and pathological contexts, our data indicate a novel common function for the Tenascin family in the regulation of tissue homeostasis under healthy and pathological conditions.

Stroma Involvement in Pancreatic Ductal Adenocarcinoma: An Overview Focusing on Extracellular Matrix Proteins.

Pancreatic cancer is the seventh leading cause of cancer-related deaths worldwide and is predicted to become second in 2030 in industrialized countries if no therapeutic progress is made. Among the different types of pancreatic cancers, Pancreatic Ductal Adenocarcinoma (PDAC) is by far the most represented one with an occurrence of more than 90%. This specific cancer is a devastating malignancy with an extremely poor prognosis, as shown by the 5-years survival rate of 2-9%, ranking firmly last amongst all cancer sites in terms of prognostic outcomes for patients. Pancreatic tumors progress with few specific symptoms and are thus at an advanced stage at diagnosis in most patients. This malignancy is characterized by an extremely dense stroma deposition around lesions, accompanied by tissue hypovascularization and a profound immune suppression. Altogether, these combined features make access to cancer cells almost impossible for conventional chemotherapeutics and new immunotherapeutic agents, thus contributing to the fatal outcomes of the disease. Initially ignored, the Tumor MicroEnvironment (TME) is now the subject of intensive research related to PDAC treatment and could contain new therapeutic targets. In this review, we will summarize the current state of knowledge in the field by focusing on TME composition to understand how this specific compartment could influence tumor progression and resistance to therapies. Attention will be paid to Tenascin-C, a matrix glycoprotein commonly upregulated during cancer that participates to PDAC progression and thus contributes to poor prognosis.

Poly(lactic acid) nanoparticles and cell-penetrating peptide potentiate mRNA-based vaccine expression in dendritic cells triggering their activation.

Messenger RNA-based vaccines have the potential to trigger robust cytotoxic immune responses, which are essential for fighting cancer and infectious diseases like HIV. Dendritic Cells (DCs) are choice targets for mRNA-based vaccine strategies, as they link innate and adaptive immune responses and are major regulators of cytotoxic and humoral adaptive responses. However, efficient delivery of antigen-coding mRNAs into DC cytosol has been highly challenging. In this study, we developed an alternative to lipid-based mRNA delivery systems, using poly(lactic acid) nanoparticles (PLA-NPs) and cationic cell-penetrating peptides as mRNA condensing agent. The formulations are assembled in two steps: (1) formation of a polyplex between mRNAs and amphipathic cationic peptides (RALA, LAH4 or LAH4-L1), and (2) adsorption of polyplexes onto PLA-NPs. LAH4-L1/mRNA polyplexes and PLA-NP/LAH4-L1/mRNA nanocomplexes are taken up by DCs via phagocytosis and clathrin-dependent endocytosis, and induce strong protein expression in DCs in vitro. They modulate DC innate immune response by activating both endosome and cytosolic Pattern Recognition Receptors (PRRs), and induce markers of adaptive responses in primary human DCs in vitro, with prevalent Th1 signature. Thus, LAH4-L1/mRNA and PLA-NP/LAH4-L1/mRNA represent a promising platform for ex vivo treatment and mRNA vaccine development.