Heterologous Expression of the Oceanobacillus iheyensis SigW and Its Anti-Protein RsiW in Bacillus subtilis

Pairs of the ECF sigma factor and its anti-sigma factor, SigW and RsiW, of Bacillus-related species that inhabit extreme environments were heterologously expressed in B. subtilis. All the RsiWs, membrane proteins, failed to fill their function of repressing cognate SigW activity, despite their close structural similarities. Particularly, uncontrolled expression of Oceanobacillus iheyensis OISigW due to abortive OIRsiW was harmful to B. subtilis. Analysis of revertants of this growth defect and site-directed mutagenesis indicated that the insertion of six and a minimum of three hydrophobic amino acid residues occurring in the transmembrane region allowed OIRsiW to function as anti-OISigW. Subcellular localization of OIRsiW was detected by immunoblot analysis, suggesting that both the wild-type and the mutant form of OIRsiW were localized to the membrane. An appropriate length of a transmembrane region required for proper integration into the membrane after translocation might vary among these Bacillus-related species.     

Assessing glycolytic flux alterations resulting from genetic perturbations in E. coli using a biosensor

We describe the development of an optimized glycolytic flux biosensor and its application in detecting altered flux in a production strain and in a mutant library. The glycolytic flux biosensor is based on the Cra-regulated ppsA promoter of E. coli controlling fluorescent protein synthesis. We validated the glycolytic flux dependency of the biosensor in a range of different carbon sources in six different E. coli strains and during mevalonate production. Furthermore, we studied the flux-altering effects of genome-wide single gene knock-outs in E. coli in a multiplex FlowSeq experiment. From a library consisting of 2126 knock-out mutants, we identified 3 mutants with high-flux and 95 mutants with low-flux phenotypes that did not have severe growth defects. This approach can improve our understanding of glycolytic flux regulation improving metabolic models and engineering efforts.     

Wetland drying indirectly influences plant community and seed bank diversity through soil pH

High altitude wetlands on the Tibetan Plateau have been shrinking due to anthropogenic disturbances and global climate change. However, the few studies that have been conducted on wetlands are inconclusive about the effect of soil moisture on seed banks and potential of seed banks in wetlands with different levels of soil moisture for regeneration of dried wetlands. We investigated seed banks and plant communities along a soil moisture gradient. A structural equation model was used to analyze the direct and indirect effects of soil moisture on seed banks, as well as the relationship between plant communities and seed banks. Although soil moisture had no direct effects on seed bank richness and density and indirect effects on seed banks through plant community, it had indirect effects on the seed bank through soil pH. Soil moisture also did not have direct effects on plant community richness, but it had indirect effects through soil pH. Plant community composition changed with soil moisture, but aboveground plant abundance and seed banks composition did not change. Low similarity exists between plant community and seed banks for all wetlands, and similarity decreased along the moisture gradient. The key factor determining plant community diversity was soil pH, while seed bank diversity was mainly affected by soil pH and plant community diversity with wetland drying. Although potential for regenerating the plant community from the seed bank decreased with an increase in soil moisture, drained wetlands still have enough residual seeds for successful restoration of species-rich alpine meadows.     

Identification of ‘cystic fibrosis protein’ as a complex of two calcium-binding proteins present in human cells of myeloid origin

Cystic fibrosis protein is a serum protein characterized by a pI close to 8.4 and present with a higher concentration in serum and plasma of cystic fibrosis carriers than in controls. This protein was found immunologically indistinguishable from the cystic fibrosis antigen isolated from granulocytes and presenting a sequence analogous to that of MRP-8, a calcium-binding protein expressed in the myeloid cell lineage. Using antibodies directed against MRP-8 and its closely associated calcium-binding protein, MRP-14, we demonstrate here that cystic fibrosis protein purified from serum is a complex of the two proteins MRP-8 and MRP-14.     

1,3-Diphenylpropanes from Daphne giraldii induced apoptosis in hepatocellular carcinoma cells through nuclear factor kappa-B inhibition

One new 1,3-diphenylpropane (1) together with six known analogues (2–7) were firstly isolated from the stem and root bark of Daphne giraldii. Their structures were determined by comprehensive NMR and HRESIMS spectroscopic data analyses. All the isolates were evaluated for their cytotoxicity against two hepatocellular carcinoma cell lines (HepG2 and Hep3B). Among them, compound 5 showed the most significant cytotoxicity against Hep3B cells, with an IC₅₀ value of 17.21 μM. A further study demonstrated that 5 obviously induced apoptotic cell death as well as the inactivation of nuclear factor kappa B p65 (NF-κB p65) in Hep3B cells. In addition, BAY 11-7082 (BAY), a NF-кB inhibitor, was used to determine the role of NF-кB signaling in 5-treated Hep3B cells. The results suggested that BAY could enhance 5-induced apoptosis of Hep3B cells. In conclusion, the data provided that 5 might be a potential candidate for the treatment of hepatocellular carcinoma through NF-κB inhibition.     

Single‐molecule force spectroscopy applied to heparin‐induced thrombocytopenia

Heparin‐induced thrombocytopenia (HIT), occurring up to approximately 1% to 5% of patients receiving the antithrombotic drug heparins, has a complex pathogenesis involving multiple partners ranging from small molecules to cells/platelets. Recently, insights into the mechanism of HIT have been achieved by using single‐molecule force spectroscopy (SMFS), a methodology that allows direct measurements of interactions among HIT partners. Here, the potential of SMFS in unraveling the mechanism of the initial steps in the pathogenesis of HIT at single‐molecule resolution is highlighted. The new findings ranging from the molecular binding strengths and kinetics to the determination of the boundary between risk and non‐risk heparin drugs or platelet‐surface and platelet‐platelet interactions will be reviewed. These novel results together have contributed to elucidate the mechanisms underlying HIT and demonstrate how SMFS can be applied to develop safer drugs with a reduced risk profile.     

Resistance training-induced muscle hypertrophy is mediated by TGF-β1-Smad signaling pathway in male Wistar rats

The TGF-β1-Smad pathway is a well-known negative regulator of muscle growth; however, its potential role in resistance training-induced muscle hypertrophy is not clear. The present study proposed to determine whether and how this pathway may be involved in resistance training-induced muscle hypertrophy. Skeletal muscle samples were collected from the control, trained (RT), control + SB431542 (CI_TGF), and trained + SB431542 (RTI_TGF) animals following 3, 5, and 8 weeks of resistance training. Inhibition of the TGF-β1-Smad pathway by SB431542 augmented muscle satellite cells activation, upregulated Akt/mTOR/S6K1 pathway, and attenuated FOXO1 and FOXO3a expression in the CI_TGF group (all p < .01), thereby causing significant muscle hypertrophy in animals from the CI_TGF. Resistance training significantly decreased muscle TGF-β1 expression and Smad3 (P-Smad3^S423/425) phosphorylation at COOH-terminal residues, augmented Smad2 (P-Smad2-L^S245/250/255) and Smad3 (P-Smad3-L^Ser208) phosphorylation levels at linker sites (all p < .01), and led to a muscle hypertrophy which was unaffected by SB431542, suggesting that the TGF-β1-Smad signaling pathway is involved in resistance training-induced muscle hypertrophy. The effects of inhibiting the TGF-β1-Smad signaling pathway were not additive to the resistance training effects on FOXO1 and FOXO3a expression, muscle satellite cells activation, and the Akt/mTOR/S6K1 pathway. Resistance training effect of satellite cell differentiation was independent of the TGF-β1-Smad signaling pathway. These results suggested that the effect of the TGF-β1-Smad signaling pathway on resistance training-induced muscle hypertrophy can be attributed mainly to its diminished inhibitory effects on satellite cell activation and protein synthesis. Suppressed P-Smad3^S423/425 and enhanced P-Smad2-L^S245/250/255 and P-Smad3-L^Ser208 are the molecular mechanisms that link the TGF-β1-Smad signaling pathway to resistance training-induced muscle hypertrophy.