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ItemGene expression profiling supports the hypothesis that human ovarian surface epithelia are multipotent and capable of serving as ovarian cancer initiating cells(Georgia Institute of Technology, 2009-12-29) Bowen, Nathan J. ; Walker, L. DeEtte ; Matyunina, Lilya V. ; Logani, Sanjay ; Totten, Kimberly A. ; Benigno, Benedict B. ; McDonald, John F.Background Accumulating evidence suggests that somatic stem cells undergo mutagenic transformation into cancer initiating cells. The serous subtype of ovarian adenocarcinoma in humans has been hypothesized to arise from at least two possible classes of progenitor cells: the ovarian surface epithelia (OSE) and/or an as yet undefined class of progenitor cells residing in the distal end of the fallopian tube. Methods Comparative gene expression profiling analyses were carried out on OSE removed from the surface of normal human ovaries and ovarian cancer epithelial cells (CEPI) isolated by laser capture micro-dissection (LCM) from human serous papillary ovarian adenocarcinomas. The results of the gene expression analyses were randomly confirmed in paraffin embedded tissues from ovarian adenocarcinoma of serous subtype and non-neoplastic ovarian tissues using immunohistochemistry. Differentially expressed genes were analyzed using gene ontology, molecular pathway, and gene set enrichment analysis algorithms. Results Consistent with multipotent capacity, genes in pathways previously associated with adult stem cell maintenance are highly expressed in ovarian surface epithelia and are not expressed or expressed at very low levels in serous ovarian adenocarcinoma. Among the over 2000 genes that are significantly differentially expressed, a number of pathways and novel pathway interactions are identified that may contribute to ovarian adenocarcinoma development.
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ItemA Threading-Based Method for the Prediction of DNABinding Proteins with Application to the Human GenomeProteins with Application to the Human Genome(Georgia Institute of Technology, 2009-11-13) Gao, Mu ; Skolnick, JeffreyDiverse mechanisms for DNA-protein recognition have been elucidated in numerous atomic complex structures from various protein families. These structural data provide an invaluable knowledge base not only for understanding DNA protein interactions, but also for developing specialized methods that predict the DNA-binding function from protein structure. While such methods are useful, a major limitation is that they require an experimental structure of the target as input. To overcome this obstacle, we develop a threading-based method, DNA-Binding-Domain-Threader (DBD-Threader, for the prediction of DNA-binding domains and associated DNA-binding protein residues. Our method, which uses a template library composed of DNA-protein complex structures, requires only the target protein’s sequence. In our approach,fold similarity and DNA-binding propensity are employed as two functional discriminating properties. In benchmark tests on 179 DNA-binding and 3,797 non-DNA-binding proteins, using templates whose sequence identity is less than 30% to the target, DBD-Threader achieves a sensitivity/precision of 56%/86%. This performance is considerably better than the standard sequence comparison method PSI-BLAST and is comparable to DBD-Hunter, which requires an experimental structure as input. Moreover, for over 70% of predicted DNA-binding domains, the backbone Root Mean Square Deviations (RMSDs) of the top-ranked structural models are within 6.5 A°of their experimental structures, with their associated DNA binding sites identified at satisfactory accuracy. Additionally, DBD-Threader correctly assigned the SCOP superfamily for most predicted domains. To demonstrate that DBD-Threader is useful for automatic function annotation on a large-scale, DBD-Threader was applied to 18,631 protein sequences from the human genome; 1,654 proteins are predicted to have DNA-binding function. Comparison with existing Gene Ontology (GO) annotations suggests that ,30% of our predictions are new. Finally, we present some interesting predictions in detail. In particular, it is estimated that 20% of classic zinc finger domains play a functional role not related to direct DNA-binding.
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ItemDifferent Effects of Species Diversity on Temporal Stability in Single‐Trophic and Multitrophic Communities(Georgia Institute of Technology, 2009-11) Jiang, Lin ; Pu, ZhichaoThe question of how species diversity affects ecological stability has long interested ecologists and yet remains largely unresolved. Historically, attempts to answer this question have been hampered by the presence of multiple potentially confounding stability concepts, confusion over responses at different levels of ecological organization, discrepancy between theoretical predictions, and, particularly, the paucity of empirical studies. Here we used meta‐analyses to synthesize results of empirical studies published primarily in the past 2 decades on the relationship between species diversity and temporal stability. We show that the overall effect of increasing diversity was positive for community‐level temporal stability but neutral for population‐level temporal stability. There were, however, striking differences in the diversity‐stability relationship between single‐ and multitrophic systems, with diversity stabilizing both population and community dynamics in multitrophic but not single‐trophic communities. These patterns were broadly equivalent across experimental and observational studies as well as across terrestrial and aquatic studies. We discuss possible mechanisms for population stability to increase with diversity in multitrophic systems and for diversity to influence community‐level stability in general. Overall, our results indicate that diversity can affect temporal stability, but the effects may critically depend on trophic complexity.
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ItemAAV recombineering with single strand oligonucleotides(Georgia Institute of Technology, 2009-11) Hirsch, Matthew L. ; Storici, Francesca ; Li, Chengwen ; Choi, Vivian W. ; Samulski, R. JudeAdeno-associated virus (AAV) transduction initiates a signaling cascade that culminates in a transient DNA damage response. During this time, host DNA repair proteins convert the linear single-strand AAV genomes to double-strand circular monomers and concatemers in processes stimulated by the AAV inverted terminal repeats (ITRs). As the orientation of AAV genome concatemerization appears unbiased, the likelihood of concatemerization in a desired orientation is low (less than 1 in 6). Using a novel recombineering method, Oligo-Assisted AAV Genome Recombination (OAGR), this work demonstrates the ability to direct concatemerization specifically to a desired orientation in human cells. This was achieved by a singlestrand DNA oligonucleotide (oligo) displaying homology to distinct AAV genomes capable of forming an intermolecular bridge for recombination. This DNA repair process results in concatemers with genomic junctions corresponding to the sequence of oligo homology. Furthermore, OAGR was restricted to single-strand, not duplexed, AAV genomes suggestive of replication-dependent recombination. Consistent with this process, OAGR demonstrated oligo polarity biases in all tested configurations except when a portion of the oligo targeted the ITR. This approach, in addition to being useful for the elucidation of intermolecular homologous recombination, may find eventual relevance for AAV mediated large gene therapy.
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ItemUnsupervised statistical clustering of environmental shotgun sequences(Georgia Institute of Technology, 2009-10-02) Kislyuk, Andrey ; Bhatnagar, Srijak ; Dushoff, Jonathan ; Weitz, Joshua S.Background: The development of effective environmental shotgun sequence binning methods remains an ongoing challenge in algorithmic analysis of metagenomic data. While previous methods have focused primarily on supervised learning involving extrinsic data, a first-principles statistical model combined with a self-training fitting method has not yet been developed. Results: We derive an unsupervised, maximum-likelihood formalism for clustering short sequences by their taxonomic origin on the basis of their k-mer distributions. The formalism is implemented using a Markov Chain Monte Carlo approach in a k-mer feature space. We introduce a space transformation that reduces the dimensionality of the feature space and a genomic fragment divergence measure that strongly correlates with the method's performance. Pairwise analysis of over 1000 completely sequenced genomes reveals that the vast majority of genomes have sufficient genomic fragment divergence to be amenable for binning using the present formalism. Using a highperformance implementation, the binner is able to classify fragments as short as 400 nt with accuracy over 90% in simulations of low-complexity communities of 2 to 10 species, given sufficient genomic fragment divergence. The method is available as an open source package called LikelyBin. Conclusion: An unsupervised binning method based on statistical signatures of short environmental sequences is a viable stand-alone binning method for low complexity samples. For medium and high complexity samples, we discuss the possibility of combining the current method with other methods as part of an iterative process to enhance the resolving power of sorting reads into taxonomic and/or functional bins.
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ItemGenetic determinants of mate recognition in Brachionus manjavacas (Rotifera)(Georgia Institute of Technology, 2009-09-09) Snell, Terry W. ; Shearer, Tonya L ; Smith, Hilary A. ; Kubanek, Julia ; Gribble, Kristin E. ; Welch , David B. MarkBackground: Mate choice is of central importance to most animals, influencing population structure, speciation, and ultimately the survival of a species. Mating behavior of male brachionid rotifers is triggered by the product of a chemosensory gene, a glycoprotein on the body surface of females called the mate recognition pheromone. The mate recognition pheromone has been biochemically characterized, but little was known about the gene(s). We describe the isolation and characterization of the mate recognition pheromone gene through protein purification, N-terminal amino acid sequence determination, identification of the mate recognition pheromone gene from a cDNA library, sequencing, and RNAi knockdown to confirm the functional role of the mate recognition pheromone gene in rotifer mating. Results: A 29 kD protein capable of eliciting rotifer male circling was isolated by high-performance liquid chromatography. Two transcript types containing the N-terminal sequence were identified in a cDNA library; further characterization by screening a genomic library and by polymerase chain reaction revealed two genes belonging to each type. Each gene begins with a signal peptide region followed by nearly perfect repeats of an 87 to 92 codon motif with no codons between repeats and the final motif prematurely terminated by the stop codon. The two Type A genes contain four and seven repeats and the two Type B genes contain three and five repeats, respectively. Only the Type B gene with three repeats encodes a peptide with a molecular weight of 29 kD. Each repeat of the Type B gene products contains three asparagines as potential sites for N-glycosylation; there are no asparagines in the Type A genes. RNAi with Type A double-stranded RNA did not result in less circling than in the phosphate-buffered saline control, but transfection with Type B double-stranded RNA significantly reduced male circling by 17%. The very low divergence between repeat units, even at synonymous positions, suggests that the repeats are kept nearly identical through a process of concerted evolution. Information-rich molecules like surface glycoproteins are well adapted for chemical communication and aquatic animals may have evolved signaling systems based on these compounds, whereas insects use cuticular hydrocarbons. Conclusion: Owing to its critical role in mating, the mate recognition pheromone gene will be a useful molecular marker for exploring the mechanisms and rates of selection and the evolution of reproductive isolation and speciation using rotifers as a model system. The phylogenetic variation in the mate recognition pheromone gene can now be studied in conjunction with the large amount of ecological and population genetic data being gathered for the Brachionus plicatilis species complex to understand better the evolutionary drivers of cryptic speciation.
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ItemOvarian Cancer Detection from Metabolomic Liquid Chromatography/Mass Spectrometry Data by Support Vector Machines(Georgia Institute of Technology, 2009-08-22) Guan, Wei ; Zhou, Manshui ; Hampton, Christina Young ; Benigno, Benedict B. ; Walker, L. DeEtte ; Gray, Alexander ; McDonald, John F. ; Fernández, Facundo M.Background: The majority of ovarian cancer biomarker discovery efforts focus on the identification of proteins that can improve the predictive power of presently available diagnostic tests. We here show that metabolomics, the study of metabolic changes in biological systems, can also provide characteristic small molecule fingerprints related to this disease. Results: In this work, new approaches to automatic classification of metabolomic data produced from sera of ovarian cancer patients and benign controls are investigated. The performance of support vector machines (SVM) for the classification of liquid chromatography/time-of-flight mass spectrometry (LC/TOF MS) metabolomic data focusing on recognizing combinations or "panels" of potential metabolic diagnostic biomarkers was evaluated. Utilizing LC/TOF MS, sera from 37 ovarian cancer patients and 35 benign controls were studied. Optimum panels of spectral features observed in positive or/and negative ion mode electrospray (ESI) MS with the ability to distinguish between control and ovarian cancer samples were selected using state-of-the-art feature selection methods such as recursive feature elimination and L1-norm SVM. Conclusion: Three evaluation processes (leave-one-out-cross-validation, 12-fold-cross-validation, 52-20-split-validation) were used to examine the SVM models based on the selected panels in terms of their ability for differentiating control vs. disease serum samples. The statistical significance for these feature selection results were comprehensively investigated. Classification of the serum sample test set was over 90% accurate indicating promise that the above approach may lead to the development of an accurate and reliable metabolomic-based approach for detecting ovarian cancer.
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ItemCharacterization of DNA conformation inside bacterial viruses(Georgia Institute of Technology, 2009-08) Petrov, Anton S. ; Locker, C. Rebecca ; Harvey, Stephen C.In this study we develop a formalism to describe the organization of DNA inside bacteriophage capsids during genome packaging. We have previously shown that DNA inside bacteriophage phi29 (ϕ29) is organized into folded toroids [A. S. Petrov and S. C. Harvey, Structure 15, 21 (2007)], whereas epsilon15 (ε15) reveals the coaxial organization of the genetic material [A. S. Petrov, K. Lim-Hing, and S. C. Harvey, Structure 15, 807 (2007)]. We now show that each system undergoes two consecutive transitions. The first transition corresponds to the formation of global conformations and is analogous to a disorder-order conformational transition. The second transition is characterized by a significant loss of DNA mobility at the local level leading to glasslike dynamic behavior. Packing genetic material inside bacteriophages can be used as a general model to study the behavior of semiflexible chains inside confined spaces, and the proposed formalism developed here can be used to study other systems of linear polymer chains confined to closed spaces.
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ItemComparisons of Structural Iron Reduction in Smectites by Bacteria and Dithionite: II. A variable-temperature Mössbauer spectroscopic study of Garfield nontronite(Georgia Institute of Technology, 2009-07) Ribeiro, Fabiana R. ; Fabris, José D. ; Kostka, Joel E. ; Komadel, Peter ; Stucki, Joseph W.The reduction of structural Fe in smectite may be mediated either abiotically by reaction with chemical reducing agents or biotically by reaction with various bacterial species. The effects of abiotic reduction on clay surface chemistry are much better known than the effects of biotic reduction, and differences between them are still in need of investigation. The purpose of the present study was to compare the effects of dithionite (abiotic) and bacteria (biotic) reduction of structural Fe in nontronite on the clay structure as observed by variabletemperature Mössbauer spectroscopy. Biotic reduction was accomplished by incubating Na-saturated Garfield nontronite (sample API 33a) with Shewanella oneidensis strain MR-1 (FeII/total Fe achieved was ~17 %). Partial abiotic reduction (FeII/total Fe ~23 %) was achieved using pH-buffered sodium dithionite. The nontronite was also reduced abiotically to FeII/total Fe ~96 %. Parallel samples were reoxidized by bubbling O2 gas through the reduced suspensions at room temperature prior to Mössbauer analysis at 77 and 4 K. At 77 K, the reduction treatments all gave spectra composed of doublets for structural FeII and FeIII in the nontronite. The spectra for reoxidized samples were largely restored to that of the un - altered sample, except for the sample reduced to 96 %. At 4 K, the spectrum for the 96 % reduced sample was highly complex and clearly reflected magnetic order in the sample. When partially reduced, the spectrum also exhibited magnetic order, but the features were completely different depending on whether reduced biotically or abiotically. The biotically reduced sample appeared to contain distinctly separate domains of FeII and FeIII within the structure, whereas partial abiotic reduction produced a spectrum representative of FeII–FeIII pairs as the dominant domain type. The 4 K spectra of the partially reduced, fully reoxidized samples were virtually the same as at 77 K, whereas reoxidation of the 96 % reduced sample produced a spectrum consisting of a magnetically ordered sextet with a minor contribution from a FeII doublet, indicating significant structural alterations compared to the un - altered sample.
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ItemFINDSITE LHM: A Threading-Based Approach to Ligand Homology Modeling(Georgia Institute of Technology, 2009-06-05) Brylinski, Michal ; Skolnick, JeffreyLigand virtual screening is a widely used tool to assist in new pharmaceutical discovery. In practice, virtual screening approaches have a number of limitations, and the development of new methodologies is required. Previously, we showed that remotely related proteins identified by threading often share a common binding site occupied by chemically similar ligands. Here, we demonstrate that across an evolutionarily related, but distant family of proteins, the ligands that bind to the common binding site contain a set of strongly conserved anchor functional groups as well as a variable region that accounts for their binding specificity. Furthermore, the sequence and structure conservation of residues contacting the anchor functional groups is significantly higher than those contacting ligand variable regions. Exploiting these insights, we developed FINDSITELHM that employs structural information extracted from weakly related proteins to perform rapid ligand docking by homology modeling. In large scale benchmarking, using the predicted anchor-binding mode and the crystal structure of the receptor, FINDSITELHM outperforms classical docking approaches with an average ligand RMSD from native of ,2.5 A° . For weakly homologous receptor protein models, using FINDSITELHM, the fraction of recovered binding residues and specific contacts is 0.66 (0.55) and 0.49 (0.38) for highly confident (all) targets, respectively. Finally, in virtual screening for HIV-1 protease inhibitors, using similarity to the ligand anchor region yields significantly improved enrichment factors. Thus, the rather accurate, computationally inexpensive FINDSITELHM algorithm should be a useful approach to assist in the discovery of novel biopharmaceuticals.