Direct Genetic and Enzymatic Evidence for Oxidative Cyclization in Hygromycin B Biosynthesis

Hygromycin B is an aminoglycoside antibiotic with a structurally distinctive orthoester linkage. Despite its long history of use in industry and in the laboratory, its biosynthesis remains poorly understood. We show here, by in-frame gene deletion <i>in vivo</i> and detailed enzyme characterization <i>in vitro</i>, that formation of the unique orthoester moiety is catalyzed by the α-ketoglutarate- and non-heme iron-dependent oxygenase HygX. In addition, we identify HygF as a glycosyltransferase adding UDP-hexose to 2-deoxystreptamine, HygM as a methyltransferase responsible for N-3 methylation, and HygK as an epimerase. These experimental results and bioinformatic analyses allow a detailed pathway for hygromycin B biosynthesis to be proposed, including the key oxidative cyclization reactions

Structural Basis of the Selectivity of GenN, an Aminoglycoside <i>N</i>‑Methyltransferase Involved in Gentamicin Biosynthesis

Gentamicins are heavily methylated, clinically valuable pseudotrisaccharide antibiotics produced by <i>Micromonospora echinospora</i>. GenN has been characterized as an <i>S</i>-adenosyl-l-methionine-dependent methyltransferase with low sequence similarity to other enzymes. It is responsible for the 3″-<i>N</i>-methylation of 3″-dehydro-3″-amino-gentamicin A2, an essential modification of ring III in the biosynthetic pathway to the gentamicin C complex. Purified recombinant GenN also efficiently catalyzes 3″-<i>N</i>-methylation of related aminoglycosides kanamycin B and tobramycin, which both contain an additional hydroxymethyl group at the C5″ position in ring III. We have obtained eight cocrystal structures of GenN, at a resolution of 2.2 Å or better, including the binary complex of GenN and <i>S</i>-adenosyl-l-homocysteine (SAH) and the ternary complexes of GenN, SAH, and several aminoglycosides. The GenN structure reveals several features not observed in any other <i>N-</i>methyltransferase that fit it for its role in gentamicin biosynthesis. These include a novel N-terminal domain that might be involved in protein:protein interaction with upstream enzymes of the gentamicin X2 biosynthesis and two long loops that are involved in aminoglycoside substrate recognition. In addition, the analysis of structures of GenN in complex with different ligands, supported by the results of active site mutagenesis, has allowed us to propose a catalytic mechanism and has revealed the structural basis for the surprising ability of native GenN to act on these alternative substrates

Visualizing miR-155 To Monitor Breast Tumorigenesis and Response to Chemotherapeutic Drugs by a Self-Assembled Photoacoustic Nanoprobe

MicroRNA-155 (miR-155), which facilitates breast tumor growth and invasion by promoting tumor cell proliferation and inhibiting cell apoptosis, is considered an ideal early diagnostic and prognostic marker. Herein, we developed a self-assembled hybridization chain reaction (HCR)-based photoacoustic (PA) nanoprobe for highly sensitive in situ monitoring of dynamic changes in miR-155 expression during breast tumorigenesis and chemotherapy. The PA nanoprobes (Au-H1/PEG and Au-H2/PEG) were constructed by linking poly­(ethylene glycol) (PEG) and two hairpin DNA strands (H1 and H2, respectively) to the surface of gold nanoparticles (AuNPs). In the presence of miR-155, the PA nanoprobes self-assembled into Au aggregates via HCR between H1, H2, and miR-155. The decreased interparticle distance in these aggregates enhanced the surface plasmon resonance (SPR) in the AuNPs. Consequently, the absorption peak of the PA nanoprobes red-shifted, and strong PA signals were generated. This strategy enabled the sensitive and quantitative detection of miR-155 with a low detection limit of 0.25 nM. As a result, PA signals of miR-155 were captured on the second day after tumor inoculation when the solid tumor had not yet formed. Dynamic changes in miR-155 during tumor growth and chemotherapy were also monitored in real time to assess the therapeutic effects via PA imaging. By virtue of these advantages, the PA nanoprobes may provide a powerful platform for in situ detection of miR-155 and thus real-time monitoring of tumorigenesis and drug response in breast cancer

Preparation of a Novel Formaldehyde-Free Impregnated Decorative Paper Containing MnO₂ Nanoparticles for Highly Efficient Formaldehyde Removal

The loading of catalytic manganese dioxide (MnO2) nanoparticles onto an impregnated decorative paper has been an effective method for the removal of indoor formaldehyde (HCHO) pollutants. However, its preparation can present numerous challenges, including instability in dipping emulsions and leaching. In this investigation, a novel and stable formaldehyde-free polyacrylate dipping emulsion containing MnO2 particles was prepared and then back-coated on a decorative paper. To improve the dispersion and fixation, the MnO2 was modified with silane. HCHO can undergo physical adsorption on the cellulosic fibers present in the paper, while it can also undergo chemical degradation into CO2 within the MnO2 groups. The silane not only enhanced the interfacial adhesion to a polyacrylate resin but also increased the interlayer distance, thereby creating a larger space for HCHO absorption. The impregnated decorative paper back-coated with 10 wt % of silane-modified MnO2 exhibited a removal efficiency of approximately 90% for HCHO at 20 °C. The removal rate further improved to approximately 100% when the temperature was increased to 60 °C. Moreover, it is worth noting that the release of volatile organic compounds was exceptionally minimal. Additionally, the particleboard bonded with this impregnated decorative paper exhibited an extremely low emission of HCHO, with a value that approached 0 mg·L–1. Furthermore, the bonding strength of the surface remained unaffected. Therefore, this study provides a simple and eco-friendly method for effectively removing HCHO, which can enhance indoor air quality

Visualization and Inhibition of Mitochondria-Nuclear Translocation of Apoptosis Inducing Factor by a Graphene Oxide-DNA Nanosensor

High concentrations of oxidized low density lipoprotein (oxLDL) induce aberrant apoptosis of vascular smooth muscle cells (VSMCs) in atherosclerotic plaques. This apoptosis cannot be blocked completely by the inhibition of caspase, and it eventually potentiates plaque disruption and risk for cardiovascular disease. Given the important role of apoptosis inducing factor (AIF) in caspase-independent apoptosis, here we develop an AIF-targeting nanosensor by the assembly of graphene oxide (GO) nanosheets and dye-labeled DNA hybrid structures. This nanosensor selectively localizes in the cytosol of VSMCs, where it exhibits a “turn-off” fluorescence signal. Under oxLDL stimuli, the release of AIF from mitochondria into cytosol liberates the DNA hybrid structures from the surface of GO and results in a “turn-on” fluorescence signal. This nanosensor is shown to possess rapid response, high sensitivity, and selectivity for AIF that enables real-time imaging of AIF translocation in VSMCs. Using this novel nanosensor, a better assessment of the apoptotic level of VSMCs and a more accurate evaluation of the extent of atherosclerotic lesions can be obtained. More importantly, the abundant binding between DNA hybrid structures and AIF inhibits the translocation of AIF into the nucleus and subsequent apoptosis in VSMCs. This inhibition may help stabilize plaque and reduce the risk of heart attack and stroke