Involvement of the leaf-specific multidrug and toxic compound extrusion (MATE) transporter Nt-JAT2 in vacuolar sequestration of nicotine in Nicotiana tabacum

Alkaloids play a key role in higher plant defense against pathogens and herbivores. Following its biosynthesis in root tissues, nicotine, the major alkaloid of Nicotiana species, is translocated via xylem transport toward the accumulation sites, leaf vacuoles. Our transcriptome analysis of methyl jasmonate-treated tobacco BY-2 cells identified several multidrug and toxic compound extrusion (MATE) transporter genes. In this study, we characterized a MATE gene, Nicotiana tabacum jasmonate-inducible alkaloid transporter 2 (Nt-JAT2), which encodes a protein that has 32% amino acid identity with Nt-JAT1. Nt-JAT2 mRNA is expressed at a very low steady state level in whole plants, but is rapidly upregulated by methyl jasmonate treatment in a leaf-specific manner. To characterize the function of Nt-JAT2, yeast cells were used as the host organism in a cellular transport assay. Nt-JAT2 was localized at the plasma membrane in yeast cells. When incubated in nicotine-containing medium, the nicotine content in Nt-JAT2-expressing cells was significantly lower than in control yeast. Nt-JAT2-expressing cells also showed lower content of other alkaloids like anabasine and anatabine, but not of flavonoids, suggesting that Nt-JAT2 transports various alkaloids including nicotine. Fluorescence assays in BY-2 cells showed that Nt-JAT2-GFP was localized to the tonoplast. These findings indicate that Nt-JAT2 is involved in nicotine sequestration in leaf vacuoles following the translocation of nicotine from root tissues

Fibroblasts Show More Potential as Target Cells than Keratinocytes in COL7A1 Gene Therapy of Dystrophic Epidermolysis Bullosa

Dystrophic epidermolysis bullosa (DEB) is an inherited blistering skin disorder caused by mutations in the type VII collagen gene (COL7A1). Therapeutic introduction of COL7A1 into skin cells holds significant promise for the treatment of DEB. The purpose of this study was to establish an efficient retroviral transfer method for COL7A1 into DEB epidermal keratinocytes and dermal fibroblasts, and to determine which gene-transferred cells can most efficiently express collagen VII in the skin. We demonstrated that gene transfer using a combination of G protein of vesicular stomatitis virus-pseudotyped retroviral vector and retronectin introduced COL7A1 into keratinocytes and fibroblasts from a DEB patient with the lack of COL7A1 expression. Real-time polymerase chain reaction analysis of the normal human skin demonstrated that the quantity of COL7A1 expression in the epidermis was significantly higher than that in the dermis. Subsequently, we have produced skin grafts with the gene-transferred or untreated DEB keratinocytes and fibroblasts, and have transplanted them into nude rats. Interestingly, the series of skin graft experiments showed that the gene-transferred fibroblasts supplied higher amount of collagen VII to the new dermal–epidermal junction than the gene-transferred keratinocytes. An ultrastructural study revealed that collagen VII from gene-transferred cells formed proper anchoring fibrils. These results suggest that fibroblasts may be a better gene therapy target of DEB treatment than keratinocytes

Numerical Study on Acoustic Oscillations of 2D and 3D Flue Organ Pipe Like Instruments with Compressible LES

Acoustic oscillations of flue instruments are investigated numerically using compressible Large Eddy Simulation (LES). Investigating 2D and 3D models of flue instruments, we reproduce acoustic oscillations excited in the resonators as well as an important characteristic feature of flue instruments – the relation between the acoustic frequency and the jet velocity described by the semi-empirical theory developed by Cremer & Ising, Coltman and Fletcher et al. based on experimental results. Both 2D and 3D models exhibit almost the same oscillation frequency for a given jet velocity, but the acoustic oscillation as well as the jet motion is more stable in the 3D model than in the 2D model, due to less stability in 3D fluid of the rolled up eddies created by the collision of the jet with the edge, which largely disturb the jet motion and acoustic field in the 2D model. We also investigate the ratio of the amplitude of the acoustic flow through the mouth opening to the jet velocity, comparing with the experimental results and semi-empirical theory given by Hirschberg et al.

Cefazolin-containing poly(ε-caprolactone) sponge pad to reduce pin tract infection rate in rabbits

AbstractIn our previous study, a fibroblast growth factor-2 (FGF-2)–apatite composite layer coated on titanium screws effectively prevented pin tract infection in rabbits because of enhanced wound healing; however, the FGF-2–apatite composite layers did not completely prevent pin tract infection. Thus, we recently developed a poly(ε-caprolactone) (PCL) sponge pad embedded with cefazolin sodium (+CEZ), which has a fast-acting bactericidal effect. The pad is placed on the skin around the screws. The purpose of this study was to determine the anti-infective efficacy of the +CEZ pad on the pin–skin interface of the FGF-2–apatite-coated titanium screws. The +CEZ pads were prepared by mixing PCL and CEZ in 1,4-dioxane, followed by freeze-drying and compaction. They were analyzed regarding their surface structure, in vitro CEZ release profile, and bactericidal activity. The FGF-2–apatite-coated screws were implanted percutaneously in bilateral rabbit proximal tibial metaphyses—with and without the +CEZ pad—for 4 weeks (n = 20). The + CEZ pads consisted of a porous matrix of PCL in which CEZ was embedded. The CEZ-release profile showed an initial burst on Day 1 and a sustained release lasting for 30 days. The +CEZ pad retained its bactericidal activity against Staphylococcus aureus after preincubation on an agar plate for 7 days. Based on visual inspection, the pin tract infection rate was successfully reduced from 72.2% to 15.0% with the +CEZ pad (p < 0.05), which reduced the bacterial count, especially S. aureus (p < 0.05). The histological inflammation rate of the soft tissues was also significantly lower with the +CEZ pad than without it (p < 0.05). The pin tract infection rate was reduced to one-fifth with the +CEZ pad. Using it as described improves infection resistance during percutaneous implantation

Dysplasia Epiphysealis Hemimelica of The Trochlear Epiphysis

Dysplasia epiphysealis hemimelica is an uncommon disorder primarily involving the epiphyes of the lower extremities. We report imaging findings of a case of dysplasia epiphysealis hemimelica involving the trochlear epiphysis of the humerus. Conventional radiography showed irregular ossification in the area of trochlear epiphysis, the finding thought to be diagnostic of this discorder. However, other imaging modalities, such as double contrast arthrography, computed tomography and MR imaging, provided valuable information about the nature and extent of the epiphyseal pathology

Directed Differentiation of Patient-Specific Induced Pluripotent Stem Cells Identifies the Transcriptional Repression and Epigenetic Modification of NKX2-5, HAND1, and NOTCH1 in Hypoplastic Left Heart Syndrome

The genetic basis of hypoplastic left heart syndrome (HLHS) remains unknown, and the lack of animal models to reconstitute the cardiac maldevelopment has hampered the study of this disease. This study investigated the altered control of transcriptional and epigenetic programs that may affect the development of HLHS by using disease-specific induced pluripotent stem (iPS) cells. Cardiac progenitor cells (CPCs) were isolated from patients with congenital heart diseases to generate patient-specific iPS cells. Comparative gene expression analysis of HLHS- and biventricle (BV) heart-derived iPS cells was performed to dissect the complex genetic circuits that may promote the disease phenotype. Both HLHS- and BV heart-derived CPCs were reprogrammed to generate disease-specific iPS cells, which showed characteristic human embryonic stem cell signatures, expressed pluripotency markers, and could give rise to cardiomyocytes. However, HLHS-iPS cells exhibited lower cardiomyogenic differentiation potential than BV-iPS cells. Quantitative gene expression analysis demonstrated that HLHS-derived iPS cells showed transcriptional repression of NKX2-5, reduced levels of TBX2 and NOTCH/HEY signaling, and inhibited HAND1/2 transcripts compared with control cells. Although both HLHS-derived CPCs and iPS cells showed reduced SRE and TNNT2 transcriptional activation compared with BV-derived cells, co-transfection of NKX2-5, HAND1, and NOTCH1 into HLHS-derived cells resulted in synergistic restoration of these promoters activation. Notably, gain- and loss-of-function studies revealed that NKX2-5 had a predominant impact on NPPA transcriptional activation. Moreover, differentiated HLHS-derived iPS cells showed reduced H3K4 dimethylation as well as histone H3 acetylation but increased H3K27 trimethylation to inhibit transcriptional activation on the NKX2-5 promoter. These findings suggest that patient-specific iPS cells may provide molecular insights into complex transcriptional and epigenetic mechanisms, at least in part, through combinatorial expression of NKX2-5, HAND1, and NOTCH1 that coordinately contribute to cardiac malformations in HLHS