James B. Cooper

Lab-Specific Gene Expression Signatures in Pluripotent Stem Cells

Pluripotent stem cells derived from both embryonic and reprogrammed somatic cells have significant potential for human regenerative medicine. Despite similarities in developmental potential, however, several groups have found fundamental differences between embryonic stem cell (ESC) and induced-pluripotent stem cell (iPSC) lines that may have important implications for iPSC-based medical therapies. Using an unsupervised clustering algorithm, we further studied the genetic homogeneity of iPSC and ESC lines by reanalyzing microarray gene expression data from seven different laboratories. Unexpectedly, this analysis revealed a strong correlation between gene expression signatures and specific laboratories in both ESC and iPSC lines. Nearly one-third of the genes with lab-specific expression signatures are also differentially expressed between ESCs and iPSCs. These data are consistent with the hypothesis that in vitro microenvironmental context differentially impacts the gene expression signatures of both iPSCs and ESCs.

Systems-level analysis of age-related macular degeneration reveals global biomarkers and phenotype-specific functional networks.

Please see related commentary: http://www.biomedcentral.com/1741-7015/10/21/abstractBackgroundAge-related macular degeneration (AMD) is a leading cause of blindness that affects the central region of the retinal pigmented epithelium (RPE), choroid, and neural retina. Initially characterized by an accumulation of sub-RPE deposits, AMD leads to progressive retinal degeneration, and in advanced cases, irreversible vision loss. Although genetic analysis, animal models, and cell culture systems have yielded important insights into AMD, the molecular pathways underlying AMDs onset and progression remain poorly delineated. We sought to better understand the molecular underpinnings of this devastating disease by performing the first comparative transcriptome analysis of AMD and normal human donor eyes.MethodsRPE-choroid and retina tissue samples were obtained from a common cohort of 31 normal, 26 AMD, and 11 potential pre-AMD human donor eyes. Transcriptome profiles were generated for macular and extramacular regions, and statistical and bioinformatic methods were employed to identify disease-associated gene signatures and functionally enriched protein association networks. Selected genes of high significance were validated using an independent donor cohort.ResultsWe identified over 50 annotated genes enriched in cell-mediated immune responses that are globally over-expressed in RPE-choroid AMD phenotypes. Using a machine learning model and a second donor cohort, we show that the top 20 global genes are predictive of AMD clinical diagnosis. We also discovered functionally enriched gene sets in the RPE-choroid that delineate the advanced AMD phenotypes, neovascular AMD and geographic atrophy. Moreover, we identified a graded increase of transcript levels in the retina related to wound response, complement cascade, and neurogenesis that strongly correlates with decreased levels of phototransduction transcripts and increased AMD severity. Based on our findings, we assembled protein-protein interactomes that highlight functional networks likely to be involved in AMD pathogenesis.ConclusionsWe discovered new global biomarkers and gene expression signatures of AMD. These results are consistent with a model whereby cell-based inflammatory responses represent a central feature of AMD etiology, and depending on genetics, environment, or stochastic factors, may give rise to the advanced AMD phenotypes characterized by angiogenesis and/or cell death. Genes regulating these immunological activities, along with numerous other genes identified here, represent promising new targets for AMD-directed therapeutics and diagnostics.

XSTREAM: A practical algorithm for identification and architecture modeling of tandem repeats in protein sequences

BackgroundBiological sequence repeats arranged in tandem patterns are widespread in DNA and proteins. While many software tools have been designed to detect DNA tandem repeats (TRs), useful algorithms for identifying protein TRs with varied levels of degeneracy are still needed.ResultsTo address limitations of current repeat identification methods, and to provide an efficient and flexible algorithm for the detection and analysis of TRs in protein sequences, we designed and implemented a new computational method called XSTREAM. Running time tests confirm the practicality of XSTREAM for analyses of multi-genome datasets. Each of the key capabilities of XSTREAM (e.g., merging, nesting, long-period detection, and TR architecture modeling) are demonstrated using anecdotal examples, and the utility of XSTREAM for identifying TR proteins was validated using data from a recently published paper.ConclusionWe show that XSTREAM is a practical and valuable tool for TR detection in protein and nucleotide sequences at the multi-genome scale, and an effective tool for modeling TR domains with diverse architectures and varied levels of degeneracy. Because of these useful features, XSTREAM has significant potential for the discovery of naturally-evolved modular proteins with applications for engineering novel biostructural and biomimetic materials, and identifying new vaccine and diagnostic targets.

Efficient selection of biomineralizing DNA aptamers using deep sequencing and population clustering.

DNA-based information systems drive the combinatorial optimization processes of natural evolution, including the evolution of biominerals. Advances in high-throughput DNA sequencing expand the power of DNA as a potential information platform for combinatorial engineering, but many applications remain to be developed due in part to the challenge of handling large amounts of sequence data. Here we employ high-throughput sequencing and a recently developed clustering method (AutoSOME) to identify single-stranded DNA sequence families that bind specifically to ZnO semiconductor mineral surfaces. These sequences were enriched from a diverse DNA library after a single round of screening, whereas previous screening approaches typically require 5-15 rounds of enrichment for effective sequence identification. The consensus sequence of the largest cluster was poly d(T)30. This consensus sequence exhibited clear aptamer behavior and was shown to promote the synthesis of crystalline ZnO from aqueous solution at near-neutral pH. This activity is significant, as the crystalline form of this wide-bandgap semiconductor is not typically amenable to solution synthesis in this pH range. High-resolution TEM revealed that this DNA synthesis route yields ZnO nanoparticles with an amorphous-crystalline core-shell structure, suggesting that the mechanism of mineralization involves nanoscale coacervation around the DNA template. We thus demonstrate that our new method, termed Single round Enrichment of Ligands by deep Sequencing (SEL-Seq), can facilitate biomimetic synthesis of technological nanomaterials by accelerating combinatorial selection of biomolecular-mineral interactions. Moreover, by enabling direct characterization of sequence family demographics, we anticipate that SEL-Seq will enhance aptamer discovery in applications employing additional rounds of screening.

NITZSCHIA OVALIS (BACILLARIOPHYCEAE) MONO LAKE STRAIN ACCUMULATES 1,4/2,5 CYCLOHEXANETETROL IN RESPONSE TO INCREASED SALINITY(1).

The growth of microalgae in hypersaline conditions requires that cells accumulate osmoprotectants. In many instances, these are polyols. We isolated the diatom Nitzschia ovalis H. J. Arn. from the saline and alkaline water body Mono Lake (CA, USA). This isolate can grow in salinities ranging from 5 to 120 parts per thousand (ppt) of salt but normally at 90 ppt salinity. In this report, we identified the major polyol osmoprotectant as 1,4/2,5 cyclohexanetetrol by electron ionization‐mass spectrometry (EI–MS), 1H, 13C nuclear magnetic resonance spectroscopy (NMR), and infrared (IR) and showed an increase in cellular concentration in response to rising salinity. This increase in the cyclitol concentration was evaluated by gas chromatography of the derived tetraacetylated cyclohexanetetrol obtaining an average of 0.7 fmol · cell−1 at 5 ppt and rising to 22.5 fmol · cell−1 at 120 ppt. The 1,4/2,5 cyclohexanetetrol was also detected in the red alga Porphyridium purpureum. Analysis of the free amino acid content in N. ovalis cultures exposed to changes in salinity showed that proline and lysine also accumulate with increased salinity, but the cellular concentration of these amino acids is about 10‐fold lower than the concentration of 1,4/2,5 cyclohexanetetrol. The comparison of amino acid concentration per cell with cyclitol suggests that this polyol is important in compensating the cellular osmotic pressure due to increased salinity, but other physiological functions could also be considered.

Global Analysis of Proline-Rich Tandem Repeat Proteins Reveals Broad Phylogenetic Diversity in Plant Secretomes

Cell walls, constructed by precisely choreographed changes in the plant secretome, play critical roles in plant cell physiology and development. Along with structural polysaccharides, secreted proline-rich Tandem Repeat Proteins (TRPs) are important for cell wall function, yet the evolutionary diversity of these structural TRPs remains virtually unexplored. Using a systems-level computational approach to analyze taxonomically diverse plant sequence data, we identified 31 distinct Pro-rich TRP classes targeted for secretion. This analysis expands upon the known phylogenetic diversity of extensins, the most widely studied class of wall structural proteins, and demonstrates that extensins evolved before plant vascularization. Our results also show that most Pro-rich TRP classes have unexpectedly restricted evolutionary distributions, revealing considerable differences in plant secretome signatures that define unexplored diversity.

A nodulin cDNA with homology to protochlorophyllide reductase.

Nodulins are defined as plant gene products expressed specifically in symbiotic root nodules, where they are thought to play roles in nodule development and physiology (Legocki and Verma, 1979). Nodulin clones provide important tools for dissecting the molecular genetic regulation of Rhizobiumlegume symbioses. During the isolation and characterization of Pro-rich nodulin cDNAs from Medicago truncatula, we isolated one cDNA clone that did not encode a Pro-rich protein (Table I). Sequence analysis of this 1329-bp clone, designated MtNOD712, revealed an open reading frame for a nove1 329-amino acid polypeptide with significant homology to pea PChR (Spano et al., 1992). Overall, MtNOD712 shares 31% identity and 53% similarity with pea PChR. The N-terminal half of MtNOD712 shows 41% identity and 59% similarity with the N-terminal portion of pea PChR, and one region of the deduced MtNOD712 protein, spanning amino acid residues 21 to 41, shows 57% identity and 85% similarity with the analogous amino acid sequences from pea PChR. This region of highest homology is included in the proposed dinucleotide-binding domain of PChR (Rossman et al., 1975; Darrah et al., 1990). In contrast, the C-terminal half of MtNOD712 shows only 24% identity and 48% similarity with the C-terminal half of pea PChR. Similar degrees of homology were found between MtNOD712 and PChR sequences from other plant species (oat, barley, and Arabidopsis). The N-terminal region of MtNOD712 also shows some homology with a small number of dehydrogenases/oxidoreductases in organisms ranging from Escherichia coli to human, including P-ketoacyl reductases, hydroxysteroid reductases, and oxoacyl-ACP reductases (e.g. the nodG gene product of R. meliloti). Transcripts corresponding to MtNOD712 were detected in poly(A)+ RNA isolated from mature symbiotic root nodules, but not in RNA isolated from roots, root nodules at early stages of development (before leghemoglobin expression and nitrogen fixation commences), or nodules formed in response to an infection-deficient Rhizobium mutant strain (exoB::Tn5). It is likely that the protein encoded by clone MtNOD712 functions during the latter stages of nodule development. Severa1 NAD(P)H-dependent reductases, thought to be flavoproteins or metalloflavoproteins, have been isolated from the cytosol of soybean root nodules (Puppo et al., 1980; Ji et

Identifying Stem Cell Gene Expression Patterns and Phenotypic Networks with AutoSOME

Stem cells have the unique property of differentiation and self-renewal and play critical roles in normal development, tissue repair, and disease. To promote systems-wide analysis of cells and tissues, we developed AutoSOME, a machine-learning method for identifying coordinated gene expression patterns and correlated cellular phenotypes in whole-transcriptome data, without prior knowledge of cluster number or structure. Here, we present a facile primer demonstrating the use of AutoSOME for identification and characterization of stem cell gene expression signatures and for visualization of transcriptome networks using Cytoscape. This protocol should serve as a general foundation for gene expression cluster analysis of stem cells, with applications for studying pluripotency, multi-lineage potential, and neoplastic disease.