The rare actinomycete Actinoplanes missouriensis forms terminal sporangia containing a few hundred flagellated spores, which can swim in aquatic environments after release from sporangium. However, gene regulation for its characteristic morphological development is largely unknown. Here, we report the functional analysis of an orphan response regulator, TcrA, which is encoded next to the chemotaxis-flagellar gene cluster. The tcrA null (ΔtcrA) mutant formed sporangium, in which sporulation proceeded. However, many distorted spores were produced and some spores ectopically germinated in the mutant sporangia. In addition, spores were hardly released from the mutant sporangia. A comparative RNA-Seq analysis between the wild-type and ΔtcrA strains showed that TcrA upregulated the transcription of more than 263 genes, which were integrated into 185 transcriptional units. In silico searches identified a 21-bp direct repeat sequence, 5'-nnGCA(A/C)CCG-n -GCA(A/C)CCGn-3', as the TcrA box, which was confirmed by electrophoretic mobility shift assays. Finally, we identified 34 transcriptional units as the TcrA regulon. TcrA seems to regulate a few hundred genes through the transcriptional activation of three FliA-family sigma factor genes besides its own regulon. We concluded that TcrA is a global transcriptional activator that controls many aspects of sporangium formation, including flagellar biogenesis, spore dormancy and sporangium dehiscence.
In this report, a redox- and pH-sensitive poly(β-amino ester)-grafted disulfide methylene oxide poly(ethylene glycol) (PAE-g-DSMPEG) was synthesized, and it showed not only a sharp pH-dependent assembly–disassembly transition but also a quick shell shading in a high concentration of reducing agent by Michael addition polymerization. 1H NMR, dynamic light scattering, and transition electron microscopy were combined to characterize the redox- and pH-responsiveness in various triggered conditions. The hydrophobic drug doxorubicin (DOX) was used as the model drug to investigate the encapsulation and delivery ability of polymeric micelles, in both in vitro and in vivo experiments. Notably, antitumor experiments in tumor-bearing mice showed that DOX-loaded polymeric micelles effectively enhanced the therapeutic efficacy in comparison to free-DOX. These results were further confirmed by histopathological examinations. Taken together, the results suggested that PAE-g-DSMPEG could be a potential hydrophobic drug delivery vehicle.
A novel type of pH-responsive biodegradable copolymer was developed based on methyloxy-poly(ethylene glycol)-block-poly[dopamine-2-(dibutylamino) ethylamine-l-glutamate] (mPEG-b-P(DPA-DE)LG) and applied to act as an intelligent nanocarrier system for magnetic resonance imaging (MRI). The mPEG-b-P(DPA-DE)LG copolymer was synthesized by a typical ring opening polymerization of N-carboxyanhydrides (NCAs-ROP) using mPEG-NH2 as a macroinitiator, and two types of amine-terminated dopamine groups and pH-sensitive ligands were grafted onto a side chain by a sequential aminolysis reaction. This design greatly benefits from the addition of the dopamine groups to facilitate self-assembly, as these groups can act as high-affinity anchors for iron oxide nanoparticles, thereby increasing long-term stability at physiological pH. The mPEG moiety in the copolymers helped the nanoparticles to remain well-dispersed in an aqueous solution, and pH-responsive groups could control the release of hydrophobic Fe3O4 nanoparticles in an acidic environment. The particle size of the Fe3O4-loaded mPEG-b-P(DPA-DE)LG micelles was measured by dynamic light scattering (DLS) and cryo-TEM. The superparamagnetic properties of the Fe3O4-loaded mPEG-b-P(DPA-DE)LG micelles were confirmed by a superconducting quantum interference device (SQUID). T2-weighted magnetic resonance imaging (MRI) of Fe3O4-loaded mPEG-b-P(DPA-DE)LG phantoms exhibited enhanced negative contrast with an r2 relaxivity of approximately 106.7 mM(-1) s(-1). To assess the ability of the Fe3O4-loaded mPEG-P(DE-DPA)LG micelles to act as MRI probes, we utilized a cerebral ischemia disease rat model with acidic tissue. We found that a gradual change in contrast in the cerebral ischemic area could be visualized by MRI after 1 h, and maximal signal loss was detected after 24 h post-injection. These results demonstrated that the Fe3O4-loaded mPEG-b-P(DPA-DE)LG micelles can act as pH-triggered MRI probes for diagnostic imaging of acidic pathological tissues.
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