Appropriately differentiated ARPE-19 cells regain phenotype and gene expression profiles similar to those of native RPE cells.

PurposeThe RPE cell line ARPE-19 provides a dependable and widely used alternative to native RPE. However, replication of the native RPE phenotype becomes more difficult because these cells lose their specialized phenotype after multiple passages. Compounding this problem is the widespread use of ARPE-19 cells in an undifferentiated state to attempt to model RPE functions. We wished to determine whether suitable culture conditions and differentiation could restore the RPE-appropriate expression of genes and proteins to ARPE-19, along with a functional and morphological phenotype resembling native RPE. We compared the transcriptome of ARPE-19 cells kept in long-term culture with those of primary and other human RPE cells to assess the former's inherent plasticity relative to the latter.MethodsARPE-19 cells at passages 9 to 12 grown in DMEM containing high glucose and pyruvate with 1% fetal bovine serum were differentiated for up to 4 months. Immunocytochemistry was performed on ARPE-19 cells grown on filters. Total RNA extracted from ARPE-19 cells cultured for either 4 days or 4 months was used for RNA sequencing (RNA-Seq) analysis using a 2 × 50 bp paired end protocol. The RNA-Seq data were analyzed to identify the affected pathways and recognize shared ontological classification among differentially expressed genes. RPE-specific mRNAs and miRNAs were assessed with quantitative real-time (RT)-PCR, and proteins with western blotting.ResultsARPE-19 cells grown for 4 months developed the classic native RPE phenotype with heavy pigmentation. RPE-expressed genes, including RPE65, RDH5, and RDH10, as well as miR-204/211, were greatly increased in the ARPE-19 cells maintained at confluence for 4 months. The RNA-Seq analysis provided a comprehensive view of the relative abundance and differential expression of the genes in the differentiated ARPE-19 cells. Of the 16,757 genes with detectable signals, nearly 1,681 genes were upregulated, and 1,629 genes were downregulated with a fold change of 2.5 or more differences between 4 months and 4 days of culture. Gene Ontology analysis showed that the upregulated genes were associated with visual cycle, phagocytosis, pigment synthesis, cell differentiation, and RPE-related transcription factors. The majority of the downregulated genes play a role in cell cycle and proliferation.ConclusionsThe ARPE-19 cells cultured for 4 months developed a phenotype characteristic of native RPE and expressed proteins, mRNAs, and miRNAs characteristic of the RPE. Comparison of the ARPE-19 RNA-Seq data set with that of primary human fetal RPE, embryonic stem cell-derived RPE, and native RPE revealed an important overall similar expression ratio among all the models and native tissue. However, none of the cultured models reached the absolute values in the native tissue. The results of this study demonstrate that low-passage ARPE-19 cells can express genes specific to native human RPE cells when appropriately cultured and differentiated

Thermal Decomposition of Ba(II) & Sr(II) Bis(oxalato)-oxovanadates(IV)

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Soup to tree: the phylogeny of beetles inferred by mitochondrial metagenomics of a Bornean rainforest sample

In spite of the growth of molecular ecology, systematics and next-generation sequencing, the discovery and analysis of diversity is not currently integrated with building the tree-of-life. Tropical arthropod ecologists are well placed to accelerate this process if all specimens obtained via masstrapping, many of which will be new species, could be incorporated routinely in phylogeny reconstruction. Here we test a shotgun sequencing approach, whereby mitochondrial genomes are assembled from complex ecological mixtures via mitochondrial metagenomics, and demonstrate how the approach overcomes many of the taxonomic impediments to the study of biodiversity. DNA from ~500 beetle specimens, originating from a single rainforest canopy fogging sample from Borneo, was pooled and shotgun sequenced, followed by de novo assembly of complete and partial mitogenomes for 175 species. The phylogenetic tree obtained from this local sample was highly similar to that from existing mitogenomes selected for global coverage of major lineages of Coleoptera. When all sequences were combined, only minor topological changes are induced against this reference set, indicating an increasingly stable estimate of coleopteran phylogeny, whilst the ecological sample expands the tip-level representation of several lineages. Robust trees generated from ecological samples now enable an evolutionary framework for ecology. Meanwhile, the inclusion of uncharacterized samples in the tree-of-life rapidly expands taxon and biogeographic representation of lineages without morphological identification. Mitogenomes from shotgun sequencing of unsorted environmental samples and their associated metadata, placed robustly into the phylogenetic tree, constitute novel DNA ‘superbarcodes’ for testing hypotheses regarding global patterns of diversity

Coexisting aegirine and magnesioriebeckite from Bababudan Hills, Mysore State

This article does not have an abstract

Soup to tree: the phylogeny of beetles inferred by mitochondrial metagenomics of a Bornean rainforest sample

In spite of the growth of molecular ecology, systematics and next-generation sequencing, the discovery and analysis of diversity is not currently integrated with building the tree-of-life. Tropical arthropod ecologists are well placed to accelerate this process if all specimens obtained via masstrapping, many of which will be new species, could be incorporated routinely in phylogeny reconstruction. Here we test a shotgun sequencing approach, whereby mitochondrial genomes are assembled from complex ecological mixtures via mitochondrial metagenomics, and demonstrate how the approach overcomes many of the taxonomic impediments to the study of biodiversity. DNA from ~500 beetle specimens, originating from a single rainforest canopy fogging sample from Borneo, was pooled and shotgun sequenced, followed by de novo assembly of complete and partial mitogenomes for 175 species. The phylogenetic tree obtained from this local sample was highly similar to that from existing mitogenomes selected for global coverage of major lineages of Coleoptera. When all sequences were combined, only minor topological changes are induced against this reference set, indicating an increasingly stable estimate of coleopteran phylogeny, whilst the ecological sample expands the tip-level representation of several lineages. Robust trees generated from ecological samples now enable an evolutionary framework for ecology. Meanwhile, the inclusion of uncharacterized samples in the tree-of-life rapidly expands taxon and biogeographic representation of lineages without morphological identification. Mitogenomes from shotgun sequencing of unsorted environmental samples and their associated metadata, placed robustly into the phylogenetic tree, constitute novel DNA ‘superbarcodes’ for testing hypotheses regarding global patterns of diversity

Compressibility of CeMIn5Ce M In_5 and Ce2MIn8Ce_2 M In_8 (M = Rh, Ir and Co) Compounds

The lattice parameters of the tetragonal compounds Ce$M$In$_{5}$ and Ce$_{2}M$In$_{8}$($M=$Rh, Ir and Co) have been studied as a function of pressure up to 15 GPa using a diamond anvil cell under both hydrostatic and quasihydrostatic conditions at room temperature. The addition of $M$In$_{2}$ layers to the parent CeIn$_{3}$ compound is found to stiffen the lattice as the 2-layer systems (average of bulk modulus values $B_{0}$ is 70.4 GPa) have a larger $B_{0}$ than CeIn$_{3}$ (67 GPa), while the 1-layer systems with the are even stiffer (average of $B_{0}$ is 81.4 GPa). Estimating the hybridization using parameters from tight binding calculations shows that the dominant hybridization is $fp$ in nature between the Ce and In atoms. The values of $V_{pf}$ at the pressure where the superconducting transition temperature $T_{c}$ reaches a maximum is the same for all Ce$M$In$_{5}$ compounds. By plotting the maximum values of the superconducting transition temperature $T_{c}$ versus $c/a$ for the studied compounds and Pu-based superconductors, we find a universal $T_{c}$ versus $c/a$ behavior when these quantities are normalized appropriately. These results are consistent with magnetically mediated superconductivity.Comment: Updated version resubmitted to Phys. Rev.