Iron-Catalyzed <i>N</i>‑Arylsulfonamide Formation through Directly Using Nitroarenes as Nitrogen Sources

One-step, catalytic synthesis of <i>N</i>-arylsulfonamides via the construction of N–S bonds from the direct coupling of sodium arylsulfinates with nitroarenes was realized in the presence of FeCl₂ and NaHSO₃ under mild conditions. In this process, stable and readily available nitroarenes were used as nitrogen sources, and NaHSO₃ acted as a reductant to provide <i>N</i>-arylsulfonamides in good to excellent yields. A broad range of functional groups were very well-tolerated in this reaction system. In addition, mechanistic studies indicated that the N–S bond might be generated through direct coupling of nitroarene with sodium arylsulfinate prior to the reduction of nitroarenes by NaHSO₃. Accordingly, a reaction mechanism involving <i>N</i>-aryl-<i>N</i>-arenesulfonylhydroxylamine as an intermediate was proposed

DEX inhibits myogenic differentiation via the glucocorticoid receptor (GR).

(A and B) Differentiating C2C12 myoblasts were treated with various concentrations of DEX (0 to 10−4 M) for 48 h or 4 d. Western blot analysis showing the protein expression levels of myogenin at 48 h (A) and MyHC at 4 d (B). (*P−5 M DEX and harvested for cytotoxicity assays using a CCK-8 kit at 12 h, 24 h, and 48 h. (D) Application of a GR antagonist (RU-486) nearly restored the protein expression level of MyHC in a dose-dependent manner. (*PP<0.05.</p

GSK-3β knockdown attenuates DEX-induced impairment of myotube formation.

<p>(A) shRNA interference of GSK-3β was performed in C2C12 myoblasts, and GSK-3β abundance was assessed by Western blot analysis to detect the silencing efficiency after transfection for 24 h. (B) shNC myoblast cells and shGSK-3β myoblast cells were differentiated in the absence (CON, panel a, b) or presence of DEX (DEX, panel c, d) for 4 d. Immunofluorescence detection of MyHC (red) and DAPI (blue) were used to detect myotubes. The scale bar is 50 µm. (C) Quantitative analysis of the fusion index using data from (B). The data are shown as the means ± SEM of three independent experiments. *<i>P</i><0.05; ^&<i>P</i><0.05 versus shNC/CON; ^#<i>P</i><0.05 versus shNC/DEX.</p

Pharmacological inhibition of GSK-3β rescues the impaired myogenic differentiation caused by dexamethasone.

<p>C2C12 myoblasts were treated with DEX (10⁻⁵ M) or a combination of DEX and various concentrations of LiCl (2.5, 5, 7.5 mM) after switching to DM. (A and B) Western blot analysis of MyHC protein expression when induced for 4 d (A) and 6 d (B). (C and D) Western blot analysis of MyoD and myogenin expression at the early stage of myogenic differentiation, <i>i.e.</i>, induced for 24 h (C) and 48 h (D). (E) C2C12 myoblasts were treated with DEX (10⁻⁵ M) or a combination of DEX and LiCl (5 mM) and induced to differentiate for 4 d. Immunofluorescence detection of MyHC (red) and DAPI counterstaining of nuclei (blue) were used to label myotubes. The scale bar is 50 µm. (F) Quantitative analysis of the fusion index from (E). (G) C2C12 myoblasts were induced to differentiate for 3 d in the presence of DEX (10⁻⁵ M) or a combination of DEX and LiCl (5 mM), and MCK mRNA levels were measured by real-time RT-PCR and compared to control myoblasts (without DEX or LiCl in DM). The data are shown as the means ± SEM of three independent experiments. *<i>P</i><0.05 versus the control group (without DEX or LiCl in DM).</p

GSK-3β inhibition attenuates dexamethasone-mediated repression of myogenic differentiation of primary satellite cells.

<p>(A) Immunofluorescence analysis of PAX-7 (green) and DAPI (blue) expression in primary satellite cells. (B) Differentiating primary satellite cells were treated with 10⁻⁵ M DEX and harvested for cytotoxicity assays using a CCK-8 kit at 12 h, 24 h, and 48 h. (C) Myogenic differentiation assay to determine the GR specificity of DEX using RU-486. Immunofluorescence detection of MyHC (red) and DAPI (blue) was used to detect myotubes (left panel). The fusion index is shown in right panel. The scale bar is 50 µm. (*<i>P</i><0.05 versus RU-486(−)/CON; ^&<i>P</i><0.05 versus RU-486(−)/DEX. n = 3 independent experiments). (D) Differentiating primary satellite cells were incubated with DEX and RU-486 (10 µM), alone or in combination, or control DM (CON). Phosphorylation of GSK-3β at serine 9 at the indicated time points was determined using Western blot analysis. (E and F) Primary satellite cells were treated with DEX (10⁻⁵ M) or a combination of DEX and 5 mM LiCl after switching to DM. Western blot analysis of MyoD and myogenin at 24 h and 48 h (E) and MyHC at 4 d and 6 d (F). (G) The GSK-3β abundance was assessed by Western blot analysis to detect the silencing efficiency after shRNA transfection for 24 h. (H) shNC and shGSK-3β satellite cells were differentiated in the absence (CON) or presence of DEX (DEX) for 4 d. Immunofluorescence detection of MyHC (red) and DAPI (blue) were used to detect myotubes (left panel). The fusion index is shown in right panel. The scale bar is 50 µm. The data are shown as the means ± SEM of three independent experiments. *<i>P</i><0.05; ^&<i>P</i><0.05 versus shNC/CON; ^#<i>P</i><0.05 versus shNC/DEX.</p

Table_5_Endophytic Communities of Transgenic Poplar Were Determined by the Environment and Niche Rather Than by Transgenic Events.xlsx

Microbial communities associated with plants represent key determinants of plant health, survival, and growth. However, a good understanding of the structural composition of the bacterial and fungal microbiome present in different plant tissues and growing environments, especially in transgenic woody plants, is required. In the present study, we hypothesized that environmental conditions, ecological niches, and transgenic events could influence the community structure of plant-associated microorganisms (bacterial and fungal endophytes). We sampled the root and stem endospheres of field-grown transgenic and non-transgenic poplar trees (Populus alba × P. berolinensis) and applied 16S rRNA and internal transcribed spacer amplicon Illumina MiSeq sequencing to determine the bacterial and fungal communities associated with the different plant habitats and tissues. We found that actinobacteria, proteobacteria, bacteroidetes, and firmicutes were the dominant endophytic bacteria, and the fungal community was dominated by dothideomycetes, agaricomycetes, leotiomycetes, and sordariomycetes. In conclusion, transgenic events did not affect the endophytic bacterial and fungal diversity of poplar trees. The bacterial and fungal community structure depends on the pH and the soil organic matter content. Each plant tissue represents a unique ecological niche for the microbial communities. Finally, we identified the indicator operational taxonomic units (OTUs) and core microbiome associated with the different plant tissues of Populus and different environmental conditions. The results provide a basis for further study of host-microbial interactions with the identified abundant OTUs of Populus.</p

Inhibition of Glycogen Synthase Kinase-3β Attenuates Glucocorticoid-Induced Suppression of Myogenic Differentiation <i>In Vitro</i>

<div><p>Glucocorticoids are the only therapy that has been demonstrated to alter the progress of Duchenne muscular dystrophy (DMD), the most common muscular dystrophy in children. However, glucocorticoids disturb skeletal muscle metabolism and hamper myogenesis and muscle regeneration. The mechanisms involved in the glucocorticoid-mediated suppression of myogenic differentiation are not fully understood. Glycogen synthase kinase-3β (GSK-3β) is considered to play a central role as a negative regulator in myogenic differentiation. Here, we showed that glucocorticoid treatment during the first 48 h in differentiation medium decreased the level of phosphorylated Ser9-GSK-3β, an inactive form of GSK-3β, suggesting that glucocorticoids affect GSK-3β activity. We then investigated whether GSK-3β inhibition could regulate glucocorticoid-mediated suppression of myogenic differentiation <i>in vitro</i>. Two methods were employed to inhibit GSK-3β: pharmacological inhibition with LiCl and GSK-3β gene knockdown. We found that both methods resulted in enhanced myotube formation and increased levels of muscle regulatory factors and muscle-specific protein expression. Importantly, GSK-3β inhibition attenuated glucocorticoid-induced suppression of myogenic differentiation. Collectively, these data suggest the involvement of GSK-3β in the glucocorticoid-mediated impairment of myogenic differentiation. Therefore, the inhibition of GSK-3β may be a strategy for preventing glucocorticoid-induced muscle degeneration.</p></div

Real-time PCR primer sequences used in this study.

<p>Real-time PCR primer sequences used in this study.</p

GSK-3β knockdown stimulates myogenic differentiation and confers resistance to DEX-induced inhibition of myogenic markers of differentiation.

<p>shGSK-3β and shNC myoblasts cells were induced to differentiate in the absence of DEX (CON), and Western blot analysis was used to assess the expression of MyoD and myogenin after induction for 24 h or 48 h (A) and the expression of MyHC after differentiation for 4 d or 6 d (B). shGSK-3β and shNC myoblast cells were induced to differentiate in the presence of DEX. Western blot analysis showing the expression of MyoD and myogenin after induction for 24 h or 48 h (C) and the expression of MyHC after induction for 4 d or 6 d (D). (E) MCK mRNA levels were measured in shGSK-3β and shNC myoblast cells after differentiation induction for 3 d in the absence (CON) or presence of DEX. The relative mRNA levels were assessed by real-time RT-PCR and compared with shNC myoblasts induced in control DM (without DEX). The data are shown as the means ± SEM of three independent experiments. *<i>P</i><0.05; ^&<i>P</i><0.05 versus shNC/CON; ^#<i>P</i><0.05 versus shNC/DEX.</p

Image_1_Endophytic Communities of Transgenic Poplar Were Determined by the Environment and Niche Rather Than by Transgenic Events.TIF

Microbial communities associated with plants represent key determinants of plant health, survival, and growth. However, a good understanding of the structural composition of the bacterial and fungal microbiome present in different plant tissues and growing environments, especially in transgenic woody plants, is required. In the present study, we hypothesized that environmental conditions, ecological niches, and transgenic events could influence the community structure of plant-associated microorganisms (bacterial and fungal endophytes). We sampled the root and stem endospheres of field-grown transgenic and non-transgenic poplar trees (Populus alba × P. berolinensis) and applied 16S rRNA and internal transcribed spacer amplicon Illumina MiSeq sequencing to determine the bacterial and fungal communities associated with the different plant habitats and tissues. We found that actinobacteria, proteobacteria, bacteroidetes, and firmicutes were the dominant endophytic bacteria, and the fungal community was dominated by dothideomycetes, agaricomycetes, leotiomycetes, and sordariomycetes. In conclusion, transgenic events did not affect the endophytic bacterial and fungal diversity of poplar trees. The bacterial and fungal community structure depends on the pH and the soil organic matter content. Each plant tissue represents a unique ecological niche for the microbial communities. Finally, we identified the indicator operational taxonomic units (OTUs) and core microbiome associated with the different plant tissues of Populus and different environmental conditions. The results provide a basis for further study of host-microbial interactions with the identified abundant OTUs of Populus.</p