A tyrosinase, mTyr-CNK, that is functionally available as a monophenol monooxygenase

Tyrosinase efficiently catalyzes the ortho-hydroxylation of monophenols and the oxidation of diphenols without any additional cofactors. Although it is of significant interest for the biosynthesis of catechol derivatives, the rapid catechol oxidase activity and inactivation of tyrosinase have hampered its practical utilization as a monophenol monooxygenase. Here, we prepared a functional tyrosinase that exhibited a distinguished monophenolase/diphenolase activity ratio (V max mono/ V max di = 3.83) and enhanced catalytic efficiency against L-tyrosine (k cat  = 3.33 ± 0.18 s−1, K m  = 2.12 ± 0.14 mM at 20 °C and pH 6.0). This enzyme was still highly active in ice water (>80%), and its activity was well conserved below 30 °C. In vitro DOPA modification, with a remarkably high yield as a monophenol monooxygenase, was achieved by the enzyme taking advantage of these biocatalytic properties. These results demonstrate the strong potential for this enzyme’s use as a monophenol monooxygenase in biomedical and industrial applications.113Nsciescopu

Toward Green Synthesis of Graphene Oxide Using Recycled Sulfuric Acid via Couette-Taylor Flow

Developing eco-friendly and cost-effective processes for the synthesis of graphene oxide (GO) is essential for its widespread industrial applications. In this work, we propose a green synthesis technique for GO production using recycled sulfuric acid and filter-processed oxidized natural graphite obtained from a Couette-Taylor flow reactor. The viscosity of reactant mixtures processed from Couette-Taylor flow was considerably lower (???200 cP at 25 ??C) than that of those from Hummers' method, which enabled the simple filtration process. The filtered sulfuric acid can be recycled and reused for the repetitive GO synthesis with negligible differences in the as-synthesized GO qualities. This removal of sulfuric acid has great potential in lowering the overall GO production cost as the amount of water required during the fabrication process, which takes a great portion of the total production cost, can be dramatically reduced after such acid filtration. The proposed eco-friendly GO fabrication process is expected to promote the commercial application of graphene materials into industry shortly

An isoform of the plastid RNA polymerase-associated protein FSD3 negatively regulates chloroplast development

Background Plastid-encoded RNA polymerase (PEP) plays an essential role in chloroplast development by governing the expression of genes involved in photosynthesis. At least 12 PEP-associated proteins (PAPs), including FSD3/PAP4, regulate PEP activity and chloroplast development by modulating formation of the PEP complex. Results In this study, we identified FSD3S, a splicing variant of FSD3; the FSD3 and FSD3S transcripts encode proteins with identical N-termini, but different C-termini. Characterization of FSD3 and FSD3S proteins showed that the C-terminal region of FSD3S contains a transmembrane domain, which promotes FSD3S localization to the chloroplast membrane but not to nucleoids, in contrast to FSD3, which localizes to the chloroplast nucleoid. We also found that overexpression of FSD3S negatively affects photosynthetic activity and chloroplast development by reducing expression of genes involved in photosynthesis. In addition, FSD3S failed to complement the chloroplast developmental defects in the fsd3 mutant. Conclusion These results suggest FSD3 and FSD3S, with their distinct localization patterns, have different functions in chloroplast development, and FSD3S negatively regulates expression of PEP-dependent chloroplast genes, and development of chloroplasts.This work was carried out with the support of the Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01323901 and PJ01364301) Rural Development Administration, Republic of Korea, and the National Research Foundation of Korea Grant funded by the Korean Government (MOE) [NRF-2019R1A2C1007103]

Toward reduction of artifacts in fused images

Most fusion satellite image methodologies at pixel-level introduce false spatial details, i.e.artifacts, in the resulting fusedimages. In many cases, these artifacts appears because image fusion methods do not consider the differences in roughness or textural characteristics between different land covers. They only consider the digital values associated with single pixels. This effect increases as the spatial resolution image increases. To minimize this problem, we propose a new paradigm based on local measurements of the fractal dimension (FD). Fractal dimension maps (FDMs) are generated for each of the source images (panchromatic and each band of the multi-spectral images) with the box-counting algorithm and by applying a windowing process. The average of source image FDMs, previously indexed between 0 and 1, has been used for discrimination of different land covers present in satellite images. This paradigm has been applied through the fusion methodology based on the discrete wavelet transform (DWT), using the à trous algorithm (WAT). Two different scenes registered by optical sensors on board FORMOSAT-2 and IKONOS satellites were used to study the behaviour of the proposed methodology. The implementation of this approach, using the WAT method, allows adapting the fusion process to the roughness and shape of the regions present in the image to be fused. This improves the quality of the fusedimages and their classification results when compared with the original WAT metho

Quadruple 9-mer-based protein binding microarray with DsRed fusion protein

<p>Abstract</p> <p>Background</p> <p>The interaction between a transcription factor and DNA motif (<it>cis</it>-acting element) is an important regulatory step in gene regulation. Comprehensive genome-wide methods have been developed to characterize protein-DNA interactions. Recently, the universal protein binding microarray (PBM) was introduced to determine if a DNA motif interacts with proteins in a genome-wide manner.</p> <p>Results</p> <p>We facilitated the PBM technology using a DsRed fluorescent protein and a concatenated sequence of oligonucleotides. The PBM was designed in such a way that target probes were synthesized as quadruples of all possible 9-mer combinations, permitting unequivocal interpretation of the <it>cis</it>-acting elements. The complimentary DNA strands of the features were synthesized with a primer and DNA polymerase on microarray slides. Proteins were labeled via N-terminal fusion with DsRed fluorescent protein, which circumvents the need for a multi-step incubation. The PBM presented herein confirmed the well-known DNA binding sequences of Cbf1 and CBF1/DREB1B, and it was also applied to elucidate the unidentified <it>cis</it>-acting element of the OsNAC6 rice transcription factor.</p> <p>Conclusion</p> <p>Our method demonstrated PBM can be conveniently performed by adopting: (1) quadruple 9-mers may increase protein-DNA binding interactions in the microarray, and (2) a one-step incubation shortens the wash and hybridization steps. This technology will facilitate greater understanding of genome-wide interactions between proteins and DNA.</p

Trib2 regulates the pluripotency of embryonic stem cells and enhances reprogramming efficiency

Embryonic stem (ES) cells are pluripotent cells characterized by self-renewability and differentiation potential. Induced pluripotent stem (iPS) cells are ES cell-equivalent cells derived from somatic cells by the introduction of core reprogramming factors. ES and iPS cells are important sources for understanding basic biology and for generating therapeutic cells for clinical applications. Tribbles homolog 2 (Trib2) functions as a scaffold in signaling pathways. However, the relevance of Trib2 to the pluripotency of ES and iPS cells is unknown. In the present study, we elucidated the importance of Trib2 in maintaining pluripotency in mouse ES cells and in generating iPS cells from somatic cells through the reprogramming process. Trib2 expression decreased as ES cells differentiated, and Trib2 knockdown in ES cells changed their colony morphology while reducing the activity of alkaline phosphatase and the expression of the pluripotency marker genes Oct4, Sox2, Nanog and Klf4. Trib2 directly interacted with Oct4 and elevated Oct4 promoter activity. During the generation of iPS cells, Trib2 knockdown decreased the reprogramming efficiency of mouse embryonic fibroblasts, whereas Trib2 overexpression significantly increased their reprogramming efficiency. In summary, our results suggest that Trib2 is important for maintaining self-renewal in ES cells and for pluripotency induction during the reprogramming process

Patient-Specific Orthotopic Glioblastoma Xenograft Models Recapitulate the Histopathology and Biology of Human Glioblastomas In Situ

SummaryFrequent discrepancies between preclinical and clinical results of anticancer agents demand a reliable translational platform that can precisely recapitulate the biology of human cancers. Another critical unmet need is the ability to predict therapeutic responses for individual patients. Toward this goal, we have established a library of orthotopic glioblastoma (GBM) xenograft models using surgical samples of GBM patients. These patient-specific GBM xenograft tumors recapitulate histopathological properties and maintain genomic characteristics of parental GBMs in situ. Furthermore, in vivo irradiation, chemotherapy, and targeted therapy of these xenograft tumors mimic the treatment response of parental GBMs. We also found that establishment of orthotopic xenograft models portends poor prognosis of GBM patients and identified the gene signatures and pathways signatures associated with the clinical aggressiveness of GBMs. Together, the patient-specific orthotopic GBM xenograft library represent the preclinically and clinically valuable “patient tumor’s phenocopy” that represents molecular and functional heterogeneity of GBMs