Mycobacterium avium subsp. paratuberculosis (MAP) is known to cause paratuberculosis. One notable protein, MAP3773c, plays a critical role in iron metabolism as a transcription factor. This study aims to investigate the binding affinity of MAP3773c to the chromatin of the Ferroportin1 (FPN1) gene in murine macrophage J774 A.1. We conducted a sequence alignment to identify potential interaction sites for MAP3773c. Following this, we used in silico analysis to predict binding interactions, complemented by electrophoretic mobility shift assay (EMSA) to confirm in vitro binding of MAP3773c. The map3773c gene was cloned into the pcDNA3.1 vector, with subsequent expression analysis carried out via Western blotting and real-time PCR. Chromatin immunoprecipitation (CHiP) assays were performed on transfected macrophages to confirm binding in the native chromatin context. Our in silico and in vitro analysis indicated that MAP3773c interacts with two binding motifs within the FPN1 coding region. The ChiP results provided additional validation, demonstrating the binding of MAP3773c to the FPN1 chromatin through successful amplification of the associated chromatin fragment via PCR. Our study demonstrated that MAP3773c binds to FPN1 and provides insight into the role of MAP3773c and its effect on host iron transport.

(1) Background: Mycobacterium avium subsp. paratuberculosis (MAP) is a bacterium that causes paratuberculosis disease. One of its proteins, MAP3773c, is involved in iron metabolism and acts as a transcription factor. Our goal is to investigate the binding of the MAP3773c protein in 2 sites of the chromatin of the ferroportin1 gene of mouse macrophages J774 A.1. (2) Methods: We performed a sequence alignment, then a docking, then EMSA to analyze in vitro the binding of MAP3773c, then we performed the cloning of map3773c in pCDNA3.1 to perform transfection in macrophages, then we performed the expression by Western blotting and PCR in real time then a CHiP of transfected macrophages. (3) Results: Our results showed in silico that MAP3773c binds to two boxes in the FPN1 coding sequence, by EMSA, MAP3773c is glued at 2 sites: EBOX and MBOX, equally with iron, zinc or EDTA. In CHiP, we also determined the binding of MAP3773c in the chromatin of FPN1 by PCR amplifying the chromatin fragment. (4) Conclusions: Our in silico, in vitro and in vivo study showed that MAP3773c binds to FPN1. Overall, our study provides information on the role of MAP3773c and its effect on host iron transport.

Acute and chronic wounds remain a major clinical burden, motivating the design of multilayer biomedical composite systems capable of combining structural support, antimicrobial protection, and controlled release of therapeutic agents within a single device. These architectures integrate natural and synthetic biomaterials, hydrogels, electrospun membranes, bioactive nanoparticles, and 3D-printed scaffolds to reproduce the multifunctionality of the native extracellular matrix. This review examines how layer-by-layer design and biomaterial selection govern mechanical strength, as well as bioactivity, and how these parameters can be tuned to the distinct phases of wound repair. Particular emphasis is placed on strategies for incorporating growth factors, antimicrobial agents, metal/metal-oxide nanoparticles to enhance re-epithelialization, angiogenesis, and infection control; emerging gene-based delivery strategies are also discussed. The main technologies and biomaterial combinations reported to date are compared, alongside a critical overview of their in vitro and in vivo performance. Reproducibility, scalability, and regulatory standardization remain the main barriers to clinical translation. We conclude by outlining priority research directions to advance multilayer composites from bench-scale prototypes toward approved wound-care products.