The Zn2+ Complex of 1,4,7,10-Tetraazacyclododecane as an Artificial Nucleobase

{2-Deoxy-3-O-[2-cyanoethoxy(diisopropylamino)phosphino]-5-O-(4,4′-dimethoxytrityl)-α-D- erythro-pentofuranosyl}-N-{2-[4,7,10-tris(2,2,2-trifluoroacetyl)-1,4,7,10-tetraazacyclododecan-1- yl]ethyl}acetamide (1) was prepared and incorporated into a 2′-O-methyl oligoribonucleotide. The hybridization of this oligonucleotide with complementary 2′-O-methyl oligoribonucleotides incorporating one to five uracil bases opposite to the azacrown structure was studied in the absence and presence of Zn²⁺. Introduction of Zn²⁺ moderately stabilized the duplex with U-bulged targets.     

A simple heterogeneous one-step assay for screening estrogenic compounds

Estrogen receptor (ER) modulators are a serious health issue but estrogenic compounds, especially antagonists of ER function, are widely screened for in search of novel therapeutics against hormonal diseases such as the breast cancer. Here we report a novel and a simple bioassay for estrogenic and anti-estrogenic compounds based on ligand-dependent recruitment of ER co-activator steroid receptor co-activator 1 (SRC-1) to purified Renilla luciferase-tagged ERα. In this assay, in vivo-biotinylated (E. coli) SRC-1, purified Renilla luciferase-ERα, and the analyte sample are mixed and incubated for 2 h in a streptavidin-coated microtiter wells, and after one washing step, luminescence is measured with a simple instrument. The assay does not require chemical labeling of the components and shows good sensitivity (25 pM E₂) and wide dynamic range of more than four orders of magnitude.     

Analysis of the length distribution of amyloid fibrils by centrifugal sedimentation

The aggregation of normally soluble peptides and proteins into amyloid fibrils is a process associated with a wide range of pathological conditions, including Alzheimer's and Parkinson's diseases. It has become apparent that aggregates of different sizes possess markedly different biological effects, with aggregates of lower relative molecular weight being associated with stronger neurotoxicity. Yet, although many approaches exist to measure the total mass concentration of aggregates, the ability to probe the length distribution of growing aggregates in solution has remained more elusive. In this work, we applied a differential centrifugation technique to measure the sedimentation coefficients of amyloid fibrils produced during the aggregation process of the amyloid β (M1–42) peptide (Aβ42). The centrifugal method has the advantage of providing structural information on the fibril distribution directly in solution and affording a short analysis time with respect to alternative imaging and analytical centrifugation approaches. We show that under quiescent conditions interactions between Aβ42 fibrils lead to lateral association and to the formation of entangled clusters. By contrast, aggregation under shaking generates a population of filaments characterized by shorter lengths. The results, which have been validated by cryogenic transmission electron microscopy (cryo-TEM) analysis, highlight the important role that fibril–fibril assembly can play in the deposition of aggregation-prone peptides.     

Aminothiazoles inhibit osteoclastogenesis and PGE2 production in LPS‐stimulated co‐cultures of periodontal ligament and RAW 264.7 cells,and RANKL‐mediated osteoclastogenesis and bone resorption in PBMCs

Inflammatory mediator prostaglandin E₂ (PGE₂) contributes to bone resorption in several inflammatory conditions including periodontitis. The terminal enzyme, microsomal prostaglandin E synthase‐1 (mPGES‐1) regulating PGE₂ synthesis is a promising therapeutic target to reduce inflammatory bone loss. The aim of this study was to investigate effects of mPGES‐1 inhibitors, aminothiazoles TH‐848 and TH‐644, on PGE₂ production and osteoclastogenesis in co‐cultures of periodontal ligament (PDL) and osteoclast progenitor cells RAW 264.7, stimulated by lipopolysaccharide (LPS), and bone resorption in RANKL‐mediated peripheral blood mononuclear cells (PBMCs). PDL and RAW 264.7 cells were cultured separately or co‐cultured and treated with LPS alone or in combination with aminothiazoles. Multinucleated cells stained positively for tartrate‐resistant acid phosphatase (TRAP) were scored as osteoclast‐like cells. Levels of PGE₂, osteoprotegerin (OPG) and interleukin‐6, as well as mRNA expression of mPGES‐1, OPG and RANKL were analysed in PDL cells. PBMCs were treated with RANKL alone or in combination with aminothiazoles. TRAP‐positive multinucleated cells were analysed and bone resorption was measured by the CTX‐I assay. Aminothiazoles reduced LPS‐stimulated osteoclast‐like cell formation both in co‐cultures and in RAW 264.7 cells. Additionally, aminothiazoles inhibited PGE₂ production in LPS‐stimulated cultures, but did not affect LPS‐induced mPGES‐1, OPG or RANKL mRNA expression in PDL cells. In PBMCs, inhibitors decreased both osteoclast differentiation and bone resorption. In conclusion, aminothiazoles reduced the formation of osteoclast‐like cells and decreased the production of PGE₂ in co‐cultures as well as single‐cell cultures. Furthermore, these compounds inhibited RANKL‐induced bone resorption and differentiation of PBMCs, suggesting these inhibitors for future treatment of inflammatory bone loss such as periodontitis.     

A High Power‐Density,Mediator‐Free,Microfluidic Biophotovoltaic Device for Cyanobacterial Cells

Biophotovoltaics has emerged as a promising technology for generating renewable energy because it relies on living organisms as inexpensive, self‐repairing, and readily available catalysts to produce electricity from an abundant resource: sunlight. The efficiency of biophotovoltaic cells, however, has remained significantly lower than that achievable through synthetic materials. Here, a platform is devised to harness the large power densities afforded by miniaturized geometries. To this effect, a soft‐lithography approach is developed for the fabrication of microfluidic biophotovoltaic devices that do not require membranes or mediators. Synechocystis sp. PCC 6803 cells are injected and allowed to settle on the anode, permitting the physical proximity between cells and electrode required for mediator‐free operation. Power densities of above 100 mW m^‐2 are demonstrated for a chlorophyll concentration of 100 μM under white light, which is a high value for biophotovoltaic devices without extrinsic supply of additional energy.     

Cellulose structure and lignin distribution in normal and compression wood of the Maidenhair tree(Ginkgo biloba L.)

We studied in detail the mean micro fibril angle and the width of cellulose crystals from the pith to the bark of a 15-year-old Maidenhair tree(Ginkgo biloba L.). The orientation of cellulose micro fibrils with respect to the cell axis and the width and length of cellulose crystallites were determined using Xray diffraction. Raman microscopy was used to compare the lignin distribution in the cell wall of normal/opposite and compression wood, which was found near the pith. Ginkgo biloba showed a relatively large mean micro fibril angle,varying between 19° and 39° in the S2 layer, and the average width of cellulose crystallites was 3.1–3.2 nm. Mild compression wood without any intercellular spaces or helical cavities was observed near the pith. Slit-like bordered pit openings and a heavily lignified S2 L layer con firmed the presence of compression wood. Ginkgo biloba showed typical features present in the juvenile wood of conifers. The micro fibril angle remained large over the 14 annual rings. The entire stem disc,with a diameter of 18 cm, was considered to consist of juvenile wood. The properties of juvenile and compression wood as well as the cellulose orientation and crystalline width indicate that the wood formation of G. biloba is similar to that of modern conifers.     

BpMADS4 has a central role in inflorescence initiation in silver birch (Betula pendula)

Acceleration of flowering would be beneficial for breeding trees with a long juvenile phase; conversely, inhibition of flowering would prevent the spread of transgenes from the genetically modified trees. We have previously isolated and characterized several MADS genes from silver birch ( Betula pendula Roth). In this study, we investigated the more detailed function of one of them, BpMADS4 , a member of the APETALA1/FRUITFULL group of MADS genes. The expression of BpMADS4 starts at very early stage of the male and female inflorescence development and the activity is high in the apex of the developing inflorescence. Later, some expression is detected in the bracts and in the flower initials. Ectopic expression of BpMADS4 accelerates flowering dramatically in normally flowering clones and also in the early-flowering birch clone, in which the earliest line flowered about 11 days after rooting, when the saplings were only 3 cm high. The birches transformed with the BpMADS4 antisense construct showed remarkable delay in flowering and the number of flowering individuals was reduced. Two of the transformed lines did not show any signs of flower development during our 2-year study, whereas all the control plants formed inflorescences within 107 days. Our results show that BpMADS4 has a critical role in the initiation of birch inflorescence development and that BpMADS4 seems to be involved in the transition from vegetative to reproductive development. Therefore, BpMADS4 provides a promising tool for the genetic enhancement of forest trees.