(E)-1-(3,4-dihydroxyphenethyl)-3-styrylurea inhibits proliferation of MCF-7 cells through G1 cell cycle arrest and mitochondria-mediated apoptosis

Growing interest in the beneficial effects of antioxidants has inspired the synthesis of new phenolic acid phenethyl ureas (PAPUs) with enhanced antioxidant potential. We have previously shown the capacity of one PAPU compound, (E)-1-(3,4-dihydroxyphenethyl)-3-styrylurea (PAPU1), to induce caspase-dependent apoptosis in melanoma cells. In the present study, we examined the anti-proliferative effects of PAPU compounds on MCF-7 human breast cancer cells and determined the molecular mechanisms involved. Treatment with PAPU compounds inhibited predominantly proliferation in these cells, where the PAPU1 was the most efficient form. Flow cytometric analysis showed that PAPU1 blocked cell cycle progression in the G₀/G₁ phase, and reduced the proportion of cells in G₂/M phase. This was related to the inhibition of cell cycle regulatory factors, including cyclin D/E and cyclin-dependent kinase (CDK) 2/4, through induction of p21^Cip1. PAPU1 also induced the mitochondrial-mediated and caspase-dependent apoptosis in MCF-7 cells. This was evidenced by cellular changes in the levels of Bcl-2 and Bax, loss of the mitochondrial membrane potential, release of cytochrome c into the cytosol, and caspase-9 activation. Collectively, our results suggest that G₁ cell cycle regulatory proteins and mitochondrial pathways are the crucial targets of PAPU1 in the chemoprevention of breast cancer cells.     

Overexpression of Newcastle disease virus (NDV) V protein enhances NDV production kinetics in chicken embryo fibroblasts

Newcastle disease virus (NDV) is not only one of the most economically important pathogen of poultry but also has a potential as anticancer virotherapy. The role of NDV V protein in virus-production kinetics was investigated using DF-1 cell-based production system. The presence of an anti-interferon (IFN)-alpha antibody resulted in enhanced NDV production kinetics in a dose-dependent manner by blocking binding of NDV-induced IFN to its receptor. To prepare DF-1 cell whose cellular IFN signaling is blocked efficiently, stable cell lines expressing either lentogenic or velogenic NDV V protein known as an IFN antagonist were established. The overexpression of NDV V protein enhanced NDV production kinetics and expedited the rate of NDV production, while it had no effect on Japanese encephalitis virus production. NDV V protein functions as an IFN antagonist by inhibiting the increase in type I IFNs by NDV infection. The IFN signals in cells expressing NDV V protein were weakened by decreased activation or expression of the dsRNA-activated enzymes. These IFN antagonist activities enhance rapid virus replication and spread in the early phase of viral infection and will be useful in improving the production of viral vaccine strains.     

Efficient constitutive expression of thermostable 4-α-glucanotransferase in Bacillus subtilis using dual promoters

4-α-Glucanotransferases possess strong transglycosylation activity which has been used in various carbohydrate chemistry fields. Due to safety issues of the recombinant enzymes we chose Bacillus subtilis as an expression host to produce a thermostable 4-α-glucanotransferase from Thermus scotoductus (TSαGT). The HpaII promoter in the Gram-positive bacterial vector pUB110 was used first to express TSαGT gene in B. subtilis. However, the activity of TSαGT in B. subtilis was only 4% of that in our previous Escherichia coli system. Two expression systems constructed by sequential alignment of another constitutive promoter for either α-amylase from B. subtilis NA64 or maltogenic amylase from Bacillus licheniformis downstream of the HpaII promoter elevated the TSαGT productivity by 11- and 12-fold, respectively, compared to the single HpaII promoter system. In conclusion, the dual promoter systems in this study were much better than the single promoter system to express the TSαGT gene in B. subtilis.     

The PDZ-binding motif of the avian NS1 protein affects transmission of the 2009 influenza A(H1N1) virus

By nature of their segmented RNA genome, influenza A viruses (IAVs) have the potential to generate variants through a reassortment process. The influenza nonstructural (NS) gene is critical for a virus to counteract the antiviral responses of the host. Therefore, a newly acquired NS segment potentially determines the replication efficiency of the reassortant virus in a range of different hosts. In addition, the C-terminal PDZ-binding motif (PBM) has been suggested as a pathogenic determinant of IAVs. To gauge the pandemic potential from human and avian IAV reassortment, we assessed the replication properties of NS-reassorted viruses in cultured cells and in the lungs of mice and determined their transmissibility in guinea pigs. Compared with the recombinant A/Korea/01/2009 virus (rK09; 2009 pandemic H1N1 strain), the rK09/VN:NS virus, in which the NS gene was adopted from the A/Vietnam/1203/2004 virus (a human isolate of the highly pathogenic avian influenza H5N1 virus strains), exhibited attenuated virulence and reduced transmissibility. However, the rK09/VN:NS-PBM virus, harboring the PBM in the C-terminus of the NS1 protein, recovered the attenuated virulence of the rK09/VN:NS virus. In a guinea pig model, the rK09/VN:NS-PBM virus showed even greater transmission efficiency than the rK/09 virus. These results suggest that the PBM in the NS1 protein may determine viral persistence in the human and avian IAV interface.     

1D Stretchable Block Copolymer Yarn‐Based Energy Harvesters via BaTiO3/Polydimethylsiloxane Composite‐Carbon Conductive Ink

Highly stretchable self‐powered energy sources are promising options for powering diverse wearable smart electronics. However, commercially existing energy sources are disadvantaged by tensile strain limitations and constrained deformability. Here, 1D thread‐based highly stretchable triboelectric nanogenerators (HS‐TENGs), a crucial step toward overcoming these obstacles, are developed based on a highly stretchable coaxial‐type poly[styrene‐b‐isoprene‐b‐styrene] (SIS) elastomer tube. Carbon conductive ink is injected into the SIS tube as a core 1D electrode that remains almost unaffected even under 250% stretching because of its low Young's modulus. To further facilitate power generation by the HS‐TENG, a composite of barium titanate nanoparticles (BaTiO₃ NPs) and polydimethylsiloxane (PDMS) is coated on the initial SIS tube to modulate the dielectric permittivity based on variations in the BaTiO₃ NPs volume ratio. The 1D PDMS/BaTiO₃ NP composite‐coated SIS and a nylon 6‐coated 2D Ni–Cu conductive fabric are selected as triboelectric bottom and top layers, respectively. Woven HS‐TENGs textiles yield consistent power output under various extreme and harsh conditions, including folded, twisted, and washed states. These experimental findings indicate that the approach may become useful for realizing stretchable multifunctional power sources for various wearable electronics.     

Anaerobic bio-hydrogen production from ethanol fermentation: the role of pH

Hydrogen was produced by an ethanol-acetate fermentation at pH of 5.0 +/- 0.2 and HRT of 3 days. The yield of hydrogen was 100-200 ml g Glu(-1) with a hydrogen content of 25-40%. This fluctuation in the hydrogen yield was attributed to the formation of propionate and the activity of hydrogen utilizing methanogens. The change in the operational pH for the inhibition of this methanogenic activity induced a change in the main fermentation pathway. In this study, the main products were butyrate, ethanol and propionate, in the pH ranges 4.0-4.5, 4.5-5.0 and 5.0-6.0, respectively. However, the activity of all the microorganisms was inhibited below pH 4.0. Therefore, pH 4.0 was regarded as the operational limit for the anaerobic bio-hydrogen production process. These results indicate that the pH plays an important role in determining the type of anaerobic fermentation pathway in anaerobic bio-hydrogen processes.     

Structural and biochemical implications of single amino acid substitutions in the nucleotide-dependent switch regions of the nitrogenase Fe protein from<Emphasis Type="Italic"> Azotobacter vinelandii</Emphasis>

The structures of nitrogenase Fe proteins with defined amino acid substitutions in the previously implicated nucleotide-dependent signal transduction pathways termed switch I and switch II have been determined by X-ray diffraction methods. In the Fe protein of nitrogenase the nucleotide-dependent switch regions are responsible for communication between the sites responsible for nucleotide binding and hydrolysis and the [4Fe-4S] cluster of the Fe protein and the docking interface that interacts with the MoFe protein upon macromolecular complex formation. In this study the structural characterization of the Azotobacter vinelandii nitrogenase Fe protein with Asp at position 39 substituted by Asn in MgADP-bound and nucleotide-free states provides an explanation for the experimental observation that the altered Fe proteins form a trapped complex subsequent to a single electron transfer event. The structures reveal that the substitution allows the formation of a hydrogen bond between the switch I Asn39 and the switch II Asp125. In the structure of the native enzyme the analogous interaction between the side chains of Asp39 and Asp125 is precluded due to electrostatic repulsion. These results suggest that the electrostatic repulsion between Asp39 and Asp125 is important for dissociation of the Fe protein:MoFe protein complex during catalysis. In a separate study, the structural characterization of the Fe protein with Asp129 substituted by Glu provides the structural basis for the observation that the Glu129-substituted variant in the absence of bound nucleotides has biochemical properties in common with the native Fe protein with bound MgADP. Interactions of the longer Glu side chain with the phosphate binding loop (P-loop) results in a similar conformation of the switch II region as the conformation that results from the binding of the phosphate of ADP to the P-loop.     

A novel splice acceptor mutation in the DSPP gene causing dentinogenesis imperfecta type II

The dentin sialophosphoprotein (DSPP) gene (4q21.3) encodes two major noncollagenous dentin matrix proteins: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Defects in the human gene encoding DSPP cause inherited dentin defects, and these defects can be associated with bilateral progressive high-frequency sensorineural hearing loss. Clinically, five different patterns of inherited dentin defects are distinguished and are classified as dentinogenesis imperfecta (DGI) types I, II, and III, and dentin dysplasia types I and II. The genetic basis for this clinical heterogeneity is unknown. Among the 11 members recruited from the studied kindred, five were affected with autosomal dominant DGI type II. The mutation (g.1188CG, IVS2-3CG) lay in the third from the last nucleotide of intron 2 and changed its sequence from CAG to GAG. The mutation was correlated with the affection status and was absent in 104 unaffected individuals (208 alleles) with the same ethnic and geological background. The proband was in the primary dentition stage and presented with multiple pulp exposures. The occlusal surface of his dental enamel was generally abraded, and the dentin was heavily worn and uniformly shaded brown. The dental pulp chambers appeared originally to be within normal limits without any sign of obliteration, but over time (by age 4), the pulp chambers became partially or completely obliterated. The oldest affected member (age 59) showed mild hearing loss at high-frequency (8 kHz). Permanent dentition was severely affected in the adults, who had advanced dental attrition, premature loss of teeth, and extensive dental reconstruction.     

Chaperonin CCT-Mediated AIB1 Folding Promotes the Growth of ERα-Positive Breast Cancer Cells on Hard Substrates

Clinical observations have revealed a strong association between estrogen receptor alpha (ERα)-positive tumors and the development of bone metastases, however, the mechanism underlying this association remains unknown. We cultured MCF-7 (ERα-positive) on different rigidity substrates. Compared with cells grown on more rigid substrates (100 kPa), cells grown on soft substrates (10 kPa) exhibited reduced spreading ability, a lower ratio of cells in the S and G2/M cell cycle phases, and a decreased proliferation rate. Using stable isotope labeling by amino acids (SILAC), we further compared the whole proteome of MCF-7 cells grown on substrates of different rigidity (10 and 100 kPa), and found that the expression of eight members of chaperonin CCT increased by at least 2-fold in the harder substrate. CCT folding activity was increased in the hard substrate compared with the soft substrates. Amplified in breast cancer 1 (AIB1), was identified in CCT immunoprecipitates. CCT folding ability of AIB1 increased on 100-kPa substrate compared with 10- and 30-kPa substrates. Moreover, using mammalian two-hybrid protein-protein interaction assays, we found that the polyglutamine repeat sequence of the AIB1 protein was essential for interaction between CCTζ and AIB1. CCTζ-mediated AIB1 folding affects the cell area spreading, growth rate, and cell cycle. The expressions of the c-myc, cyclin D1, and PgR genes were higher on hard substrates than on soft substrate in both MCF-7 and T47D cells. ERα and AIB1 could up-regulate the mRNA and protein expression levels of the c-myc, cyclin D1, and PgR genes, and that 17 β-estradiol could enhance this effects. Conversely, 4-hydroxytamoxifen, could inhibit these effects. Taken together, our studies demonstrate that some ERα-positive breast cancer cells preferentially grow on more rigid substrates. CCT-mediated AIB1 folding appears to be involved in the rigidity response of breast cancer cells, which provides novel insight into the mechanisms of bone metastasis.     

Complete genome sequence of Celeribacter bacteriophage P12053L

The Roseobacter clade has been recognized as one of the abundant bacterial lineages in marine environments, which makes the characterization of bacteriophages infecting members of the clade important. Here we report the complete genome sequence of bacteriophage P12053L, which infects Celeribacter sp. strain IMCC12053, a member of the Roseobacter clade.