The contamination of both soil and water by nitrobenzene (NB) is a problem that has been studied, where several reactive agents have been developed for the degradation of this compound as well as different methods. Nanoparticles with semiconductive properties have been studied for organic compounds photodegradation due to their assistance in optimizing the degradation processes. Two of the most promising photocatalysts are ZnO and TiO₂ because of their optimal results. In the present work the performance of the zinc peroxide (ZnO₂) nanoparticles was evaluated. ZnO₂ nanoparticles were synthesized from zinc acetate and hydrogen peroxide using the Sol-Gel method under ultrasound assistance. The characterization was carried out by UV–Vis spectroscopy, infrared Fourier transform total reflectance (ATR-FT-IR) spectroscopy, Raman spectroscopy, X-ray diffraction (XRD), Zeta potential, dynamic light scattering (DLS), field emission scanning electron microscopy (FE-SEM), and Energy Dispersive X-ray spectroscopy (EDX). The experiments for the degradation of NB were carried out in a photoreactor with UV lamps of 254 nm at 25 °C, using a solution of nitrobenzene with the nanoparticles. The best conditions for NB photodegradation were 30 ppm (ZnO₂) and 15 ppm (NB) at pH 2, reaching up to 90% degradation in 2 h. The intermediates formed during the photodegradation of NB were identified by gas chromatography mass spectrometry.

Paratuberculosis is a disease caused by Mycobacterium avium subsp. paratuberculosis (MAP). It is of great interest to better understand the proteins involved in the pathogenicity of this organism in order to be able to identify potential therapeutic targets and design new vaccines. The protein of interest–MAP3773c–was investigated, and molecular modeling in silico, docking, cloning, expression, purification, and partial characterization of the recombinant protein were achieved. In the in silico study, it was shown that MAP3773c of MAP has 34% sequence similarity with Mycobacterium tuberculosis (MTB) FurB, which is a zinc uptake regulator (Zur) protein. The docking data showed that MAP3773c exhibits two metal-binding sites. The presence of structural Zn²⁺ in the purified protein was confirmed by SDS-PAGE PAR staining. The purification showed one band that corresponded to a monomer, which was confirmed by liquid chromatography–mass spectrometry (LC-MS). The presence of a monomer was verified by analyzing the native protein structure through BN-SDS-PAGE (Native Blue (BN) Two-Dimensional Electrophoresis) and BN–Western blotting. The MAP3773c protein contains structural zinc. In conclusion, our results show that MAP3773c displays the features of a Fur-type protein with two metal-binding sites, one of them coordinating structural Zn²⁺.

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.