Synthesis and Electro‐Optical Properties of a Novel DNA–Lipid Complex Carrying Carbazole Moieties

A novel DNA–lipid complex carrying carbazole (Cz) moieties was prepared by substituting the sodium counter cation with cationic lipid, namely lipid(2Cz), in which the actual mole ratio of phosphate to lipid was 1:1.05. The DNA–lipid(2Cz) complex was soluble in common organic solvents including CHCl₃, CH₂Cl₂, methanol, and ethanol, while insoluble in THF, toluene, and water. CD spectroscopy revealed that the DNA–lipid complex took a predominantly double helical structure in methanol and that the helical structure was fairly stable against heating. A solution of the DNA–lipid(2Cz) complex emitted fluorescence in 40.0% quantum yield, which was lower than that of the corresponding lipid(2Cz) (76%). The cyclic voltammograms of the complex indicated that the oxidation potential of DNA–lipid(2Cz) was 0.65 V. The onset temperature of weight loss of the DNA–lipid complex is 230 °C according to TGA in air.

     

Effect of O6‐chloroethylguanine DNA lesions on the kinetics and mechanism of micronucleus induction in vivo

The aim of this work was to determine the kinetics of micronucleus production because of an increase in O⁶‐chloroethyl guanine (O6‐ChlEt‐G) DNA lesions in murine bone marrow cells in vivo. We increased the frequency of O6‐ChlEt‐G lesions by pretreatment with an inhibitor of O⁶‐methylguanine‐DNA methyltransferase (MGMT), O⁶‐benzylguanine (O6BG), and subsequent treatment with bis‐chloroethylnitrosourea (BCNU). The kinetics of micronucleated‐polychromatic erythrocyte (MN‐PCE) induction was established by scoring the frequency of MN‐PCEs per 2000 PCEs in peripheral blood at 8‐hr intervals from immediately prior to treatment to 72‐hr post‐treatment. We examined groups of five mice treated with (i) dimethylsulfoxide (DMSO), (ii) O6BG in DMSO, (iii) BCNU, or (iv) O6BG in DMSO plus BCNU. The data indicate that O6BG pretreatment causes: (i) ían increase in MN‐PCEs induced by BCNU, (ii) a delay in the time of maximal MN‐PCE induction produced by the different BCNU doses, and (iii) an increase in cytotoxicity. These data confirm that O6‐ChlEt‐G is a lesion involved in DNA break induction and in the subsequent production of micronuclei, and also that these lesions seem to be stoichiometrically reduced by MGMT. These data also show that induction of MN‐PCEs by BCNU is delayed by pretreatment with O6BG for more than 6 hr, perhaps due to the time required for repair of crosslinks derived from O6‐ChlEt‐G and/or for DNA duplication, which is required for adduct transformation into crosslinks. Environ. Mol. Mutagen. 2010. © 2009 Wiley‐Liss, Inc.     

Solution‐Processable Donor‐Acceptor‐Donor Oligomers with Cross‐Linkable Functionality

Electron‐acceptor units, combined with bithiophene substituted with flexible chains end‐functionalized with cross‐linkable moieties, provide soluble donor‐acceptor‐donor (DAD) π‐conjugated oligomer‐type molecules with cross‐linking ability and broad absorption in the visible spectrum. A study on the cross‐linking conditions of the new oligomers to yield insoluble polymer networks is presented, including conditions for obtaining polymer films over poly(3,4‐ethylenedioxythiophene):polystyrene sulfonate‐covered substrates. The combination of the DAD molecular design and cross‐linking functionality opens prospects for applications in solution‐processed small‐molecule solar cells with morphologically‐stable organic layers.

     

Reorganization of Lamellar Diblock Copolymer Poly(ε‐caprolactone)‐block‐poly(4‐vinylpyridine) in the Melting Temperature Range

The reorganization kinetics of the “original” lamellar diblock copolymer poly(ε‐caprolactone)‐block‐poly(4‐vinylpyridine) crystals formed at 260 K is studied in the melting region from 270 K (10 K below the onset of the melting peak of original crystals) to 310 K (the melting peak temperature) on the time scale starting from 10^?4 to 10² s by ultrafast differential scanning calorimetry. Different reorganization pathways are observed in this temperature range. Annealing at temperatures below 295 K leads to further stabilization of original crystals by secondary crystallization. At annealing temperatures higher than 295 K, crystals partially melt and the reorganization occurs via the melting–recrystallization. For even higher temperature, such as 310 K, the melting is completed within a few milliseconds and recrystallization starts from the nuclei formation. The sigmoidal recrystallization kinetics is analyzed by the Avrami equation. It is found that the copolymer experiences about one order of magnitude slower recrystallization rate and has higher melting peak temperatures of crystals formed after recrystallization than the homopolymer. The slower recrystallization kinetics in the copolymer is discussed from the viewpoint of the nanoscale spatial constraint and the intermediate state prior to the recrystallization.

     

The Influence of Magnesium Acetate on the Structure Formation of Polystyrene‐block‐poly(4‐vinylpyridine)‐Based Integral‐Asymmetric Membranes

The influence of magnesium acetate on the non‐solvent‐induced phase separation (NIPS) process of amphiphilic polystyrene‐block‐poly(4‐vinylpyridine)s to gain integral‐asymmetric membranes is investigated. Highly uniform pores over the large areas of the membrane can be achieved, and the average pore diameter is adjusted by varying the total molar mass of the block copolymers used. These stimuli‐responsive membranes, which are solution cast in the absence or the presence of small amounts of magnesium acetate, are directly compared, showing a remarkable effect on the pore structures and their openness. Minor salt addition is considered to influence the polarity of the solvents used in a positive manner such that the NIPS process can be improved significantly.

     

Contribution of microRNA 24–3p and Erk1/2 to interleukin‐6‐mediated plasma cell survival

Plasma cells can survive for long periods and continuously secrete protective antibodies, but plasma cell production of autoantibodies or transformation to tumor cells is detrimental. Plasma cell survival depends on exogenous factors from the surrounding microenvironment, and largely unknown intracellular mediators that regulate cell homeostasis. Here we investigated the contribution of the microRNA 24–3p (miR‐24–3p) to the survival of human plasma cells under the influence of IL‐6 and SDF‐1α (stromal cell derived factor 1), both of which are bone marrow survival niche mediators. Deep sequencing revealed a strong expression of miR‐24–3p in primary B cells, plasma blasts, plasma cells, and in plasmacytoma cells. In vitro studies using primary cells and the plasmacytoma cell line RPMI‐8226 revealed that (i) expression of miR‐24–3p mediates plasma cell survival, (ii) miR‐24–3p is upregulated by IL‐6 and SDF‐1α, (iii) IL‐6 mediates cell survival under ER stress conditions via miR‐24–3p expression, and (iv) IL‐6‐induced miR‐24–3p expression depends on the activity of the MAP kinase Erk1/2. These results suggest a direct connection between an external survival signal and an intracellular microRNA in regulating plasma cell survival. miR‐24–3p could therefore be a promising target for new therapeutic strategies for autoimmune and allergic diseases and for multiple myeloma.     

Synthesis of 12,12‐Ammonium Ionenes with Functionality for Chain Extension and Cross‐Linking via UV Irradiation

A novel synthetic methodology for preparing chain‐extended and cross‐linked ionenes using UV irradiation is described. 12,12‐Ammonium ionenes were functionalized with either cinnamate or styrenic functionality at the chain ends and were subsequently exposed to UV irradiation. In addition, a novel copolymer (poly(cinDMPC‐co‐bocDMPC)) was mixed with the cinnamate‐functionalized 12,12‐ammonium ionene to prepare photo‐cross‐linked networks. The UV irradiation process was monitored via UV–Vis spectroscopy after each exposure. The chain‐extended 12,12‐ammonium ionene possessed superior tensile properties compared to the original 12,12‐ammonium ionene. Successful cross‐linking was confirmed with solubility testing of the thin films in methanol. The results indicated that photo‐cross‐linked 12,12‐ammonium ionenes offer potential in membrane technology applications.

     

A Novel Poly(N‐Isopropylacrylamide‐co‐acryloylamidobenzo‐12‐crown‐4) Microgel with Rapid Stimuli‐Responsiveness for Molecule‐Specific Adsorption of γ‐Cyclodextrin

A novel poly(N‐isopropylacrylamide‐co‐acryloylamidobenzo‐12‐crown‐4) (PNB) microgel with rapid γ‐cyclodextrin (CD)‐responsive characteristics and adsorption property is developed. The microgel is composed of benzo‐12‐crown‐4 (B₁₂C₄) units as molecule‐recognition receptors and poly(N‐isopropylacrylamide) networks as phase‐transition actuators as well as adsorbent backbone chains. The PNB microgels significantly increase their volumes induced by γ‐CD, and adsorb γ‐CD molecules within PNB networks because of formation of γ‐CD/B₁₂C₄ inclusion complexes. Detection of γ‐CD‐concentration and the molecule‐specific adsorption processes of the microgels are investigated systematically. The PNB microgels provide a new tool for rapid concentration measurement and effective separation of γ‐CD.     

The human lung cell line A549 does not develop adaptive protection against the DNA‐damaging action of formaldehyde

The alkaline comet assay was used to further characterize the induction of DNA‐protein crosslinks (DPX) by formaldehyde (FA) and their removal in the human lung cell line A549. DPX were indirectly measured as the reduction of gamma ray‐induced DNA migration. Repeated treatments of A549 cells with low FA concentrations (up to 100 μM) did not lead to significant differences in the induction of DPX in comparison with a single treatment. Pretreatment with higher FA‐concentrations (200 μM and above) enhanced the crosslinking effect. There was no indication for an adaptive protection against the induction of DPX by FA. These findings are in agreement with RT‐PCR measurements of the expression of genes that encode the main enzymes involved in FA detoxification. A549 cells exposed to FA (50–300 μM) for 1, 4, or 24 hr did not reveal altered expression of the GSH‐dependent formaldehyde dehydrogenase (FDH, which is identical to alcohol dehydrogenase 3; ADH3), the cytosolic aldehyde dehydrogenase 1 (ALDH1A1) and the mitochondrial ALDH2. Pretreatment of A549 cells with a low FA concentration (50 μM) also did not enhance the removal of DPX induced by higher FA concentrations. Taken together, these results suggest that A549 cells do not develop adaptive protection against the genotoxic action of FA. Neither metabolic inactivation of FA nor the repair of FA‐induced DPX seems to be enhanced in cells pretreated with FA. Environ. Mol. Mutagen., 2010. © 2009 Wiley‐Liss, Inc.     

Coincident diabetes mellitus modulates Th1‐, Th2‐, and Th17‐cell responses in latent tuberculosis in an IL‐10‐ and TGF‐β‐dependent manner

Type 2 diabetes mellitus (DM) is a risk factor for the development of active tuberculosis (TB), although its role in the TB‐induced responses in latent TB (LTB) is not well understood. Since Th1, Th2, and Th17 responses are important in immunity to LTB, we postulated that coincident DM could alter the function of these CD4⁺ T‐cell subsets. To this end, we examined mycobacteria‐induced immune responses in the whole blood of individuals with LTB‐DM and compared them with responses of individuals without DM (LTB‐NDM). T‐cell responses from LTB‐DM are characterized by diminished frequencies of mono‐ and dual‐functional CD4⁺ Th1, Th2, and Th17 cells at baseline and following stimulation with mycobacterial antigens‐purified protein derivative, early secreted antigen‐6, and culture filtrate protein‐10. This modulation was at least partially dependent on IL‐10 and TGF‐β, since neutralization of either cytokine resulted in significantly increased frequencies of Th1 and Th2 cells but not Th17 cells in LTB‐DM but not LTB individuals. LTB‐DM is therefore characterized by diminished frequencies of Th1, Th2, and Th17 cells, indicating that DM alters the immune response in latent TB leading to a suboptimal induction of protective CD4⁺ T‐cell responses, thereby providing a potential mechanism for increased susceptibility to active disease.     

Activating the AKT2–nuclear factor‐κB–lipocalin‐2 axis elicits an inflammatory response in age‐related macular degeneration

Age‐related macular degeneration (AMD) is a complex and progressive degenerative eye disease resulting in severe loss of central vision. Recent evidence indicates that immune system dysregulation could contribute to the development of AMD. We hypothesize that defective lysosome‐mediated clearance causes accumulation of waste products in the retinal pigmented epithelium (RPE), activating the immune system and leading to retinal tissue injury and AMD. We have generated unique genetically engineered mice in which lysosome‐mediated clearance (both by phagocytosis and autophagy) in RPE cells is compromised, causing the development of features of early AMD. Our recent data indicate a link between lipocalin‐2 (LCN‐2) and the inflammatory responses induced in this mouse model. We show that nuclear factor‐κB (NF‐κB) and STAT‐1 may function as a complex in our animal model system, together controlling the upregulation of LCN‐2 expression in the retina and stimulating an inflammatory response. This study revealed increased infiltration of LCN‐2‐positive neutrophils in the choroid and retina of early AMD patients as compared with age‐matched controls. Our results demonstrate that, both in our animal model and in human AMD, the AKT2–NF‐κB–LCN‐2 signalling axis is involved in activating the inflammatory response, making this pathway a potential target for AMD treatment. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

DHEA improves impaired activation of Akt and PKC ζ/λ‐GLUT4 pathway in skeletal muscle and improves hyperglycaemia in streptozotocin‐induced diabetes rats

Aim: Addition of dehydroepiandrosterone (DHEA) to a cultured skeletal muscle locally synthesizes 5α‐dihydrotestosterone (DHT). It induced activation of glucose metabolism‐related signalling pathway via protein kinase B (Akt) and protein kinase C zeta/lambda (PKC ζ/λ)‐glucose transporter‐4 (GLUT4) proteins. However, such an effect of DHEA in vivo remains unclear. Methods: Using streptozotocin (STZ)‐induced rats with type 1 diabetes mellitus, we tested the hypothesis that a single bout of DHEA injection in the rats improves hyperglycaemia and muscle GLUT4‐regulated signalling pathway. After 1 week of STZ injection (55 mg kg^?1) with male Wistar rats, fasting glucose concentrations were determined in a blood sample taken from the tail vein. Blood glucose levels were then monitored for 180 min after DHEA or sesame oil (control) was injected (n = 10 for each group). Results: Blood glucose levels decreased significantly for 30–150 min after 2 mg DHEA injection in the STZ rats. In the skeletal muscle, expression and translocation of GLUT4 protein, phosphorylation of Akt and PKC ζ/λ, and phosphofructokinase and hexokinase enzyme activities increased significantly by DHEA injection. However, DHEA‐induced improvements in Akt and PKC ζ/λ‐GLUT4 pathways were blocked by a DHT inhibitor. Conclusion: These results suggest that a single bout of DHEA injection can improve hyperglycaemia and activate the glucose metabolism‐related signalling pathway via Akt and PKC ζ/λ‐GLUT4 proteins of skeletal muscles in rats. Moreover, these results show that a DHEA‐induced increase in muscle glucose uptake and utilization might contribute to improvement in hyperglycaemia in type 1 diabetes mellitus.     

Cross‐Linked Poly(ε‐caprolactone/D,L‐lactide) Copolymers with Elastic Properties

Cross‐linked ε‐caprolactone (CL) and D ,L ‐lactide (DLLA) copolymers with elastic properties were synthesized in three steps. First, the monomers were copolymerized in ring‐opening polymerization to obtain telechelic star‐shaped oligomers with almost completely random monomer distribution. The oligomers were methacrylated with methacrylic anhydride in the second step and cured in a third. Molar CL/DLLA compositions of 30/70, 50/50, 70/30, 90/10, and 100/0 were used to obtain elastic structures with a wide range of properties. The effect of the average length of the copolymer block on the properties of the networks was evaluated with three different co‐initiator contents (0.5, 1.0, and 2.0/100) in the oligomer synthesis. The oligomers were characterized by ¹³C NMR spectroscopy, size‐exclusion chromatography (SEC), and differential‐scanning calorimetry (DSC). The formation of elastic networks was confirmed by the absence of a flow region in dynamic mechanical analysis (DMA), the increase in T_g in DSC, and the full recovery of the sample dimensions after tensile testing. In addition, gel contents were high and the samples swelled in CH₂Cl₂. The networks possessed break stresses from 0.7–9.7 MPa with elongations from 80–350%. Networks with 100 or 90% of ε‐caprolactone retained their form in vitro for 12 weeks, but an increase in lactide content made the networks more vulnerable to hydrolysis.

Water absorption of the polymers during hydrolysis.     

Assessment of 5‐fluorouracil and 4‐nitroquinoline‐1‐oxide in vivo genotoxicity with Pig‐a mutation and micronucleus endpoints

Genotoxicity assessments were conducted on male Sprague Dawley rats treated with 5‐fluorouracil (5‐FU) and 4‐nitroquinoline‐1–oxide (4NQO) as part of an international validation trial of the Pig‐a mutant phenotype assay. Rats were orally exposed to 0, 11.5, 23, or 46 mg/kg/day 5‐FU for three consecutive days (Days 1–3); blood was sampled on Days ?1, 4, 15, 29, and 45. Pig‐a mutant phenotype reticulocyte (RET^CD59?) and mutant phenotype erythrocyte (RBC^CD59?) frequencies were determined on Days ?1, 15, 29, and 45, and percent micronucleated reticulocytes (%MN‐RET) were measured on Day 4. Rats were treated with 4NQO for 28 consecutive days by oral gavage, at doses of 1.5, 3, or 6 mg/kg/day. RBC^CD59? and RET^CD59? frequencies were determined on Days ?1, 15, and 29, and MN‐RET were quantified on Day 29. Whereas 5‐FU was found to increase %MN‐RET, no significant increases were observed for RBC^CD59? or RET^CD59? at any of the time points studied. The high dose of 4NQO (6 mg/kg/day) was observed to markedly increase RBC^CD59? and RET^CD59? frequencies, and this same dose level caused a weak but significantly elevated increase in MN‐RET (approximately twofold). Collectively, the results provide additional support for the combination of Pig‐a mutation and MN‐RET into acute and 28‐day repeat‐dose studies. Environ. Mol. Mutagen. 55:735–740, 2014. © 2014 Wiley Periodicals, Inc.     

Poly[2,7‐(9,9‐dihexylfluorene)]‐block‐Poly(2‐vinylpyridine) Rod–Coil Star‐block Copolymers: Synthesis,Micellar Structures,and Photophysical Properties

The first successful synthesis of conjugated rod–coil star block copolymer, (PF‐b‐P2VP)_n, containing conjugated poly[2,7‐(9,9‐dihexylfluorene)] (PF), and coil‐like poly(2‐vinylpyridine) (P2VP) by combining a Suzuki coupling reaction and living anionic polymerization is reported. With increasing methanol content in THF/methanol mixtures (PF‐b‐P2VP)_n symmetric star‐block copolymers maintain spherical micelles, but PF‐b‐P2VP asymmetric diblock copolymers vary from spherical micelles to vesicles. Both the absorption and emission spectra of PF‐b‐P2VP blue shift with increasing methanol content, suggesting an “H‐type” aggregation. However, (PF‐b‐P2VP)_n star‐block exhibits no shift in absorption but a red shift in the emission spectra, indicating a different type of aggregation. These results suggest the significance of polymer architectures on microphase‐separated morphologies and photophysical properties.