Heterologous Synthesis and Characterization of Thiocillin IV

Micrococcin P1 and P2 are thiopeptides with a wide range of biological functions including antibacterial and antimalarial activities. We previously demonstrated optimized enzymatic sequences for the exclusive and scalable biosynthesis of micrococcin P2. Thiocillin IV is predicted to be the congener of O-methylated micrococcin P2, but the exact structure has not been elucidated. In this study, we report the first scalable biosynthesis and full structural characterization of thiocillin IV, a 26-membered thiopeptide. This was achieved by generating a recombinant plasmid by inserting tclO, a gene encoding an O-methyltransferase, and genes responsible for micrococcin P2 production and incorporating them into a Bacillus strain. With the incorporation of precursor peptide genes and optimal culture conditions, production reached 2.4 mg/L of culture. The purified thiocillin IV structure was identified as O-methylated micrococcin P2 at the 8-Thr position, and its promising biological activity toward various Gram-positive pathogens was observed. This study provides tclO-mediated site-selective methylation and opens a biotechnological opportunity to produce selective thiopeptides

Image_1_Co-targeting B-RAF and PTEN Enables Sensory Axons to Regenerate Across and Beyond the Spinal Cord Injury.TIF

Primary sensory axons in adult mammals fail to regenerate after spinal cord injury (SCI), in part due to insufficient intrinsic growth potential. Robustly boosting their growth potential continues to be a challenge. Previously, we showed that constitutive activation of B-RAF (rapidly accelerated fibrosarcoma kinase) markedly promotes axon regeneration after dorsal root and optic nerve injuries. The regrowth is further augmented by supplemental deletion of PTEN (phosphatase and tensin homolog). Here, we examined whether concurrent B-RAF activation and PTEN deletion promotes dorsal column axon regeneration after SCI. Remarkably, genetically targeting B-RAF and PTEN selectively in DRG neurons of adult mice enables many DC axons to enter, cross, and grow beyond the lesion site after SCI; some axons reach ∼2 mm rostral to the lesion by 3 weeks post-injury. Co-targeting B-RAF and PTEN promotes more robust DC regeneration than a pre-conditioning lesion, which additively enhances the regeneration triggered by B-RAF/PTEN. We also found that post-injury targeting of B-RAF and PTEN enhances DC axon regeneration. These results demonstrate that co-targeting B-RAF and PTEN effectively enhances the intrinsic growth potential of DC axons after SCI and therefore may help to develop a novel strategy to promote robust long-distance regeneration of primary sensory axons.</p

Image_2_Co-targeting B-RAF and PTEN Enables Sensory Axons to Regenerate Across and Beyond the Spinal Cord Injury.TIF

Primary sensory axons in adult mammals fail to regenerate after spinal cord injury (SCI), in part due to insufficient intrinsic growth potential. Robustly boosting their growth potential continues to be a challenge. Previously, we showed that constitutive activation of B-RAF (rapidly accelerated fibrosarcoma kinase) markedly promotes axon regeneration after dorsal root and optic nerve injuries. The regrowth is further augmented by supplemental deletion of PTEN (phosphatase and tensin homolog). Here, we examined whether concurrent B-RAF activation and PTEN deletion promotes dorsal column axon regeneration after SCI. Remarkably, genetically targeting B-RAF and PTEN selectively in DRG neurons of adult mice enables many DC axons to enter, cross, and grow beyond the lesion site after SCI; some axons reach ∼2 mm rostral to the lesion by 3 weeks post-injury. Co-targeting B-RAF and PTEN promotes more robust DC regeneration than a pre-conditioning lesion, which additively enhances the regeneration triggered by B-RAF/PTEN. We also found that post-injury targeting of B-RAF and PTEN enhances DC axon regeneration. These results demonstrate that co-targeting B-RAF and PTEN effectively enhances the intrinsic growth potential of DC axons after SCI and therefore may help to develop a novel strategy to promote robust long-distance regeneration of primary sensory axons.</p

Praeruptorin A Inhibits <i>in Vitro</i> Migration of Preosteoclasts and <i>in Vivo</i> Bone Erosion, Possibly Due to Its Potential To Target Calmodulin

Excessive activity and/or increased number of osteoclasts lead to bone resorption-related disorders. Here, we investigated the potential of praeruptorin A to inhibit migration/fusion of preosteoclasts <i>in vitro</i> and bone erosion <i>in vivo</i>. Praeruptorin A inhibited the RANKL-induced migration/fusion of preosteoclasts accompanied by the nuclear translocation of NFATc1, a master regulator of osteoclast differentiation. Antimigration/fusion activity of praeruptorin A was also confirmed by evaluating the mRNA expression of fusion-mediating molecules. <i>In silico</i> binding studies and several biochemical assays further revealed the potential of praeruptorin A to bind with Ca²⁺/calmodulin and inhibit its downstream signaling pathways, including the Ca²⁺/calmodulin-CaMKIV-CREB and Ca²⁺/calmodulin-calcineurin signaling axis responsible for controlling NFATc1. <i>In vivo</i> application of praeruptorin A significantly reduced lipopolysaccharide-induced bone erosion, indicating its possible use to treat bone resorption-related disorders. In conclusion, praeruptorin A has the potential to inhibit migration/fusion of preosteoclasts <i>in vitro</i> and bone erosion <i>in vivo</i> by targeting calmodulin and inhibiting the Ca²⁺/calmodulin-CaMKIV-CREB-NFATc1 and/or Ca²⁺/calmodulin-calcineurin-NFATc1 signaling axis

Acredinone C and the Effect of Acredinones on Osteoclastogenic and Osteoblastogenic Activity

A new inhibitor, acredinone C (<b>1</b>), of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation was isolated from the culture broth of the fungus <i>Acremonium</i> sp. (F9A015) along with acredinones A (<b>2</b>) and B (<b>3</b>). The structure of acredinone C (<b>1</b>), which incorporates benzophenone and xanthone moieties, was established by the analyses of combined spectroscopic data including 1D and 2D NMR and MS. All of the acredinones studied efficiently inhibited the RANKL-induced formation of TRAP⁺-MNCs in a dose-dependent manner without any cytotoxicity up to 10 μM. Acredinone A showed dual activity in both osteoclast and osteoblast differentiation <i>in vitro</i> and good efficacy in an animal disease model of bone formation

Effect of praeruptorin A on RANKL-induced osteoclast differentiation.

<p>(A) Chemical structure of praeruptorin A. (B) BMMs were pretreated with vehicle (0.1% DMSO) or praeruptorin A for 2 h and then incubated with RANKL (10 ng/ml) and M-CSF (30 ng/ml) for 4 days. Multinucleated cells were fixed, permeabilized, and stained with TRAP solution. Mature TRAP-positive multinucleated osteoclasts (MNCs) were photographed under a light microscope. TRAP-positive MNCs (nuclear number >3) were counted (C), and TRAP activity of osteoclasts was measured (D). (E) The effect of praeruptorin A on the viability of BMMs was evaluated by CCK-8 assay. *, <i>P</i><0.05; **, <i>P</i><0.01; ***<i>P</i><0.001.</p

Nanotherapeutics of PTEN Inhibitor with Mesoporous Silica Nanocarrier Effective for Axonal Outgrowth of Adult Neurons

Development of therapeutic strategies such as effective drug delivery is an urgent and yet unmet need for repair of damaged nervous systems. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) regulates axonal regrowth of central and peripheral nervous systems; its inhibition, meanwhile, facilitates axonal outgrowth of injured neurons. Here we show that nanotherapeutics based on mesoporous silica nanoparticles loading PTEN-inhibitor bisperoxovanadium (BpV) are effective for delivery of drug molecules and consequent improvement of axonal outgrowth. Mesoporous nanocarriers loaded BpV drug at large amount (27 μg per 1 mg of carrier), and released sustainably over 10 d. Nanocarrier-BpV treatment of primary neurons from the dorsal root ganglions (DRGs) of rats and mice at various concentrations induced them to actively take up the nanocomplexes with an uptake efficiency as high as 85%. The nanocomplex-administered neurons exhibited significantly enhanced axonal outgrowth compared with those treated with free-BpV drug. The expression of a series of proteins involved in PTEN inhibition and downstream signaling was substantially up-/down-regulated by the nanocarrier-BpV system. Injection of the nanocarriers into neural tissues (DRG, brain cortex, and spinal cord), moreover, demonstrated successful integration into neurons, glial cells, oligodendrocytes, and macrophages, suggesting the possible nanotherapeutics applications in vivo. Together, PTEN-inhibitor delivery via mesoporous nanocarriers can be considered a promising strategy for stimulating axonal regeneration in central and peripheral nervous systems

Anti-Osteoclastogenic Activity of Praeruptorin A via Inhibition of p38/Akt-c-Fos-NFATc1 Signaling and PLCγ-Independent Ca²⁺ Oscillation

<div><p>Background</p><p>A decrease of bone mass is a major risk factor for fracture. Several natural products have traditionally been used as herbal medicines to prevent and/or treat bone disorders including osteoporosis. Praeruptorin A is isolated from the dry root extract of <i>Peucedanum praeruptorum</i> Dunn and has several biological activities, but its anti-osteoporotic activity has not been studied yet.</p><p>Materials and Methods</p><p>The effect of praeruptorin A on the differentiation of bone marrow–derived macrophages into osteoclasts was examined by phenotype assay and confirmed by real-time PCR and immunoblotting. The involvement of NFATc1 in the anti-osteoclastogenic action of praeruptorin A was evaluated by its lentiviral ectopic expression. Intracellular Ca²⁺ levels were also measured.</p><p>Results</p><p>Praeruptorin A inhibited the RANKL-stimulated osteoclast differentiation accompanied by inhibition of p38 and Akt signaling, which could be the reason for praeruptorin A-downregulated expression levels of c-Fos and NFATc1, transcription factors that regulate osteoclast-specific genes, as well as osteoclast fusion-related molecules. The anti-osteoclastogenic effect of praeruptorin A was rescued by overexpression of NFATc1. Praeruptorin A strongly prevented the RANKL-induced Ca²⁺ oscillation without any changes in the phosphorylation of PLCγ.</p><p>Conclusion</p><p>Praeruptorin A could exhibit its anti-osteoclastogenic activity by inhibiting p38/Akt-c-Fos-NFATc1 signaling and PLCγ-independent Ca²⁺ oscillation.</p></div

Effect of NFATc1 on anti-osteoclastogenic action of praeruptorin A.

<p>(A) BMMs were infected with retroviruses harboring the control GFP or Ca-NFATc1-GFP vectors. Transduced BMMs were cultured with RANKL (10 ng/ml) and M-CSF (30 ng/ml) in the presence of praeruptorin A (10 µM) or vehicle (DMSO). After incubation for 2 days, GFP expression was visualized under a fluorescence microscope. After 2 additional days, mature TRAP-positive multinucleated osteoclasts were visualized by TRAP staining. (B) TRAP-positive cells (nuclear number >3) were counted as osteoclasts, and TRAP activity was measured at 405 nm. On the differentiation day 2, the mRNA and protein expression levels of osteoclastogenesis-related molecules were analyzed by real-time PCR (C) and Western blot analysis, respectively (D). Densitometric analysis was performed using ImageJ software and the relative, normalized ratios of NFATc1/actin, p-Akt/Akt or p-p38/p38 were presented. *, <i>P</i><0.05; **, <i>P</i><0.01; ***<i>P</i><0.001.</p