High Efficiency Secondary Somatic Embryogenesis in Hovenia dulcis

Embryogenic callus was obtained from mature seed explants on medium supplemented with 2,4-dichlorophenoxyacetic acid. Primary somatic embryos (SEs) can only develop into abnormal plants. Well-developed SEs could be obtained through secondary somatic embryogenesis both in solid and liquid cultures. Temperature strongly affected induction frequency of secondary embryogenesis. Relatively high temperature (30∘C) and germinated SEs explants were effective for induction of secondary somatic embryos, and low temperature (20∘C) was more suitable for further embryo development, plantlet conversion, and transplant survival. Somatic embryos formed on agar medium had larger cotyledons than those of embryos formed in liquid medium. Supplementing 0.1 mg L−1 6-benzyladenine (BA) was effective for plant conversion; the rate of plant conversion was 43.3% in somatic embryos from solid culture and 36.5% in embryos from liquid culture. In vitro plants were successfully acclimatized in the greenhouse. The protocol established in this study will be helpful for large-scale vegetative propagation of this medicinal tree

Nanotopography as a tool for the investigation of molecular mechanisms of osteogenesis of MSCs

Nanotopographical patterning of biomaterial substrates has great potential for biofunctionalisation of devices for clinical applications, such as in orthopaedics. Nanotopography comprising 120 nm diameter nanopits with a partially disordered arrangement of up to +/- 50 nm offset from a square lattice with 300 nm centre to centre spacing (NSQ50, fabricated by electron beam lithography) has been characterized as being osteogenic. Following the finding of osteogenesis of mesenchymal stromal cells derived from human bone marrow (MSCs) on the NSQ50 nanotopography, MSCs cultured on ε-polycaprolactone (PCL) embossed with the NSQ50 pattern was used for this study on molecular mechanisms underlying NSQ50 induced MSC osteogenesis: the functional coupling of gene expression and osteogenesis, the molecular regulatory events driving gene expression and osteogenesis, and the possible link of metabolomics with molecular signalling of MSCs on the NSQ50 surface. Temporal analysis of gene expression for MSCs on the NSQ50 surface revealed that MSC fate commitment and osteogenic differentiation was transcriptionally controlled. The cell cycle and growth regulating transcription factor C-MYC was found to be significantly repressed, whereas the osteogenic transcription factor RUNX2 was up-regulated at 5 days of cell culture, and this was followed upregulation of the osteoblast specific transcription factor osterix (OSX) at days 11 and 13. Following this transcription factor activation, osteoblast specific marker genes were induced with increased alkaline phosphatase (ALP) observed at day 16, increased osteopontin (OPN) at day 20 and increased osteocalcin (OCN) at day 28. These data suggested that transcription factors regulated MSC osteogenic commitment at the early stage, and induced osteogenic specific marker gene expression at the late stages of cell culture on the NSQ50 surface, resulting in osteogenesis of the MSCs. Signalling pathway analysis illustrated that bone morphogenetic protein 2 (BMP2) was the initial signalling molecule that triggered osteogenic differentiation of MSCs by inducing RUNX2 expression via the canonical SMAD pathway. BMP2 and its transmembrane receptor type 1A (BMPR1A) were stimulated by 3 nanotopographical cues by 3 days of cell culture on the NSQ50 surface, whereas the induction of other transmembrane receptors, including the low density lipoprotein-receptor related protein 5 (LRP5) and integrin subunits α3, α4, β1, and β3 were not observed. Inhibition of BMP2 signalling by the BMP2 antagonist noggin resulted in down-regulation of RUNX2 and ALP. Further analysis of BMP2 signalling revealed that BMP2 also modulated expression of the microRNA (miR)- 23b which targets RUNX2. The effect of BMP2 signalling on the expression of RUNX2 was enhanced by co-localizing with integrin αvβ5 (the vitronectin (VN) receptor) which was found to be up-regulated after 5 days cell culture. Metabolomics data for MSCs on the NSQ50 surface during early osteogenic differentiation was analysed. MSC cellular metabolite analysis revealed possible changes in bioenergetic balance with shifts towards more mitochondrial oxidative process, possibly indicating a switch in MSCs on the surface towards lineage-specific commitment. Further analysis of the metabolomics data illustrated PPARG ligands from the polyunsaturated fatty acid family was downregulated, suggesting the inhibition of adipocyte differentiation in MSCs on the surface. The down-regulation of unsaturated fatty acids could also be involved in the regulation of Ca2+ channels which positively regulate BMP2 expression. The metabolomics data, together with gene expression and signalling pathway analysis demonstrated that MSCs on the NSQ50 surface initiated osteogenic commitment after 3 days of cell culture, with BMP2 initiating osteogenic transcription factor stimulation of mature and functional osteoblasts on the surface

Converting EOS Data from HDF-EOS to netCDF

A C-language computer program accepts, as input, a set of scientific data and metadata from an Earth Observing System (EOS) satellite and converts the set from (1) the format in which it was created and delivered to (2) another format for processing and exchange of data on Earth

Converting from XML to HDF-EOS

A computer program recreates an HDF-EOS file from an Extensible Markup Language (XML) representation of the contents of that file. This program is one of two programs written to enable testing of the schemas described in the immediately preceding article to determine whether the schemas capture all details of HDF-EOS files

HDF-EOS 5 Validator

A computer program partly automates the task of determining whether an HDF-EOS 5 file is valid in that it conforms to specifications for such characteristics as attribute names, dimensionality of data products, and ranges of legal data values. ["HDF-EOS" and variants thereof are defined in "Converting EOS Data From HDF-EOS to netCDF" (GSC-15007-1), which is the first of several preceding articles in this issue of NASA Tech Briefs.] Previously, validity of a file was determined in a tedious and error-prone process in which a person examined human-readable dumps of data-file-format information. The present software helps a user to encode the specifications for an HDFEOS 5 file, and then inspects the file for conformity with the specifications: First, the user writes the specifications in Extensible Markup Language (XML) by use of a document type definition (DTD) that is part of the program. Next, the portion of the program (denoted the validator) that performs the inspection is executed, using, as inputs, the specifications in XML and the HDF-EOS 5 file to be validated. Finally, the user examines the output of the validator

HDF-EOS Web Server

A shell script has been written as a means of automatically making HDF-EOS-formatted data sets available via the World Wide Web. ("HDF-EOS" and variants thereof are defined in the first of the two immediately preceding articles.) The shell script chains together some software tools developed by the Data Usability Group at Goddard Space Flight Center to perform the following actions: Extract metadata in Object Definition Language (ODL) from an HDF-EOS file, Convert the metadata from ODL to Extensible Markup Language (XML), Reformat the XML metadata into human-readable Hypertext Markup Language (HTML), Publish the HTML metadata and the original HDF-EOS file to a Web server and an Open-source Project for a Network Data Access Protocol (OPeN-DAP) server computer, and Reformat the XML metadata and submit the resulting file to the EOS Clearinghouse, which is a Web-based metadata clearinghouse that facilitates searching for, and exchange of, Earth-Science data

Human amniotic fluid stem cells do not differentiate into dopamine neurons in vitro or after transplantation in vivo.

Although embryonic stem (ES) cells can generate dopamine (DA) neurons that are potentially useful as a cell replacement therapy in Parkinson\u27s disease (PD), associated ethical and practical concerns remain major stumbling blocks to their eventual use in humans. In this study, we examined human amniotic fluid stem (hAFS) cells derived from routine amniocenteses for their potential to give rise to DA neurons in vitro and following transplantation into the 6-hydroxydopamine-lesioned rat brain. We show that undifferentiated hAFS cells constitutively expressed mRNAs and proteins typical of stem cells but also cell derivatives of all three germ layers, including neural progenitors/neurons (nestin, beta-tubulin III, neurofilament). Additionally, these cells expressed mRNAs of an immature DA phenotype (Lmx1a, Pitx-3, Nurr1, Aldh1a1) but not the corresponding proteins. Importantly, treatment with DA differentiation factors using a variety of protocols did not further promote the development of fully differentiated DA neurons from hAFS cells. Thus, Lmx1a, Aldh1a1, AADC, TH, and DAT proteins were not detected in hAFS cells in culture or after transplantation into the PD rat brain. Moreover, by 3 weeks after implantation, there were no surviving AFS cells in the graft, likely as a result of an acute immunorejection response, as evidenced by the abundant presence of CD11+ macrophage/microglia and reactive GFAP+ astrocytes in the host brain. Taken together, these results suggest that further studies will be needed to improve differentiation procedures in culture and to prolong cell survival in vivo if hAFS cells are to be useful as replacement cells in PD

Novel insights into bacterial dimethylsulfoniopropionate catabolism in the East China Sea

The compatible solute Dimethylsulfoniopropionate (DMSP), made by many marine organisms, is one of Earth’s most abundant organosulfur molecules. Many marine bacteria import DMSP and can degrade it as a source of carbon and/or sulfur via DMSP cleavage or DMSP demethylation pathways, which can generate the climate active gases dimethyl sulfide (DMS) or methanthiol (MeSH), respectively. Here we used culture-dependent and -independent methods to study bacteria catabolising DMSP in East China Sea (ECS). Of bacterial isolates, 42.11% showed DMSP-dependent DMS (Ddd+) activity, and 12.28% produced detectable levels of MeSH. Interestingly, although most Ddd+ isolates were Alphaproteobacteria (mainly Roseobacters), many gram-positive Actinobacteria were also shown to cleave DMSP producing DMS. The mechanism by which these Actinobacteria cleave DMSP is unknown, since no known functional ddd genes have been identified in genome sequences of Ddd+ Microbacterium and Agrococcus isolates or in any other sequenced Actinobacteria genomes. Gene probes to the DMSP demethylation gene dmdA and the DMSP lyase gene dddP demonstrated that these DMSP-degrading genes are abundant and widely distributed in ECS seawaters. dmdA was present in relatively high proportions in both surface (19.53% ± 6.70%) and bottom seawater bacteria (16.00% ± 8.73%). In contrast, dddP abundance positively correlated with chlorophyll a, and gradually decreased with the distance from land, which implies that the bacterial DMSP lyase gene dddP might be from bacterial groups that closely associate with phytoplankton. Bacterial community analysis showed positive correlations between Rhodobacteraceae abundance and concentrations of DMS and DMSP, further confirming the link between this abundant bacterial class and the environmental DMSP cycling