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1.  Pharmacokinetics of Natural and Engineered Secreted Factors Delivered by Mesenchymal Stromal Cells 
PLoS ONE  2014;9(2):e89882.
Transient cell therapy is an emerging drug class that requires new approaches for pharmacological monitoring during use. Human mesenchymal stem cells (MSCs) are a clinically-tested transient cell therapeutic that naturally secrete anti-inflammatory factors to attenuate immune-mediated diseases. MSCs were used as a proof-of-concept with the hypothesis that measuring the release of secreted factors after cell transplantation, rather than the biodistribution of the cells alone, would be an alternative monitoring tool to understand the exposure of a subject to MSCs. By comparing cellular engraftment and the associated serum concentration of secreted factors released from the graft, we observed clear differences between the pharmacokinetics of MSCs and their secreted factors. Exploration of the effects of natural or engineered secreted proteins, active cellular secretion pathways, and clearance mechanisms revealed novel aspects that affect the systemic exposure of the host to secreted factors from a cellular therapeutic. We assert that a combined consideration of cell delivery strategies and molecular pharmacokinetics can provide a more predictive model for outcomes of MSC transplantation and potentially other transient cell therapeutics.
PMCID: PMC3931832  PMID: 24587097
2.  Endobiont Viruses Sensed by the Human Host – Beyond Conventional Antiparasitic Therapy 
PLoS ONE  2012;7(11):e48418.
Wide-spread protozoan parasites carry endosymbiotic dsRNA viruses with uncharted implications to the human host. Among them, Trichomonas vaginalis, a parasite adapted to the human genitourinary tract, infects globally ∼250 million each year rendering them more susceptible to devastating pregnancy complications (especially preterm birth), HIV infection and HPV-related cancer. While first-line antibiotic treatment (metronidazole) commonly kills the protozoan pathogen, it fails to improve reproductive outcome. We show that endosymbiotic Trichomonasvirus, highly prevalent in T. vaginalis clinical isolates, is sensed by the human epithelial cells via Toll-like receptor 3, triggering Interferon Regulating Factor -3, interferon type I and proinflammatory cascades previously implicated in preterm birth and HIV-1 susceptibility. Metronidazole treatment amplified these proinflammatory responses. Thus, a new paradigm targeting the protozoan viruses along with the protozoan host may prevent trichomoniasis-attributable inflammatory sequelae.
PMCID: PMC3492353  PMID: 23144878
3.  A public genome-scale lentiviral expression library of human ORFs 
Nature methods  2011;8(8):659-661.
Functional characterization of the human genome requires tools for systematically modulating gene expression in both loss- and gain-of-function experiments. We describe the production of a sequence-confirmed, clonal collection of over 16,100 human open-reading frames (ORFs) encoded in a versatile Gateway vector system. Utilizing this ORFeome resource, we created a genome-scale expression collection in a lentiviral vector, thereby enabling both targeted experiments and high-throughput screens in diverse cell types.
PMCID: PMC3234135  PMID: 21706014
4.  Evidence for Transcript Networks Composed of Chimeric RNAs in Human Cells 
PLoS ONE  2012;7(1):e28213.
The classic organization of a gene structure has followed the Jacob and Monod bacterial gene model proposed more than 50 years ago. Since then, empirical determinations of the complexity of the transcriptomes found in yeast to human has blurred the definition and physical boundaries of genes. Using multiple analysis approaches we have characterized individual gene boundaries mapping on human chromosomes 21 and 22. Analyses of the locations of the 5′ and 3′ transcriptional termini of 492 protein coding genes revealed that for 85% of these genes the boundaries extend beyond the current annotated termini, most often connecting with exons of transcripts from other well annotated genes. The biological and evolutionary importance of these chimeric transcripts is underscored by (1) the non-random interconnections of genes involved, (2) the greater phylogenetic depth of the genes involved in many chimeric interactions, (3) the coordination of the expression of connected genes and (4) the close in vivo and three dimensional proximity of the genomic regions being transcribed and contributing to parts of the chimeric RNAs. The non-random nature of the connection of the genes involved suggest that chimeric transcripts should not be studied in isolation, but together, as an RNA network.
PMCID: PMC3251577  PMID: 22238572
5.  Metabolic network analysis integrated with transcript verification for sequenced genomes 
Nature methods  2009;6(8):589-592.
With sequencing of thousands of organisms completed or in progress, there is a growing need to integrate gene prediction with metabolic network analysis. Using Chlamydomonas reinhardtii as a model, we describe a systems-level methodology bridging metabolic network reconstruction with experimental verification of enzyme encoding open reading frames. Our quantitative and predictive metabolic model and its associated cloned open reading frames provide useful resources for metabolic engineering.
PMCID: PMC3139173  PMID: 19597503
6.  COT/MAP3K8 drives resistance to RAF inhibition through MAP kinase pathway reactivation 
Nature  2010;468(7326):968-972.
Oncogenic mutations in the serine/threonine kinase B-RAF are found in 50–70% of malignant melanomas1. Pre-clinical studies have demonstrated that the B-RAFV600E mutation predicts a dependency on the mitogen activated protein kinase (MAPK) signaling cascade in melanoma1–5—an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials6–8. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance9–11. Identification of resistance mechanisms in a manner that elucidates alternative ‘druggable’ targets may inform effective long-term treatment strategies12. Here, we expressed ~600 kinase and kinase-related open reading frames (ORFs) in parallel to functionally interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (COT/TPL2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAFV600E cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signaling. Moreover, COT expression is associated with de novo resistance in B-RAFV600E cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibition. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.
PMCID: PMC3058384  PMID: 21107320
7.  An experimentally derived confidence score for binary protein-protein interactions 
Nature methods  2008;6(1):91-97.
Information on protein-protein interactions is of central importance for many areas of biomedical research. Currently no method exists to systematically and experimentally assess the quality of individual interactions reported in interaction mapping experiments. To provide a standardized confidence-scoring method that can be applied to tens of thousands of protein interactions we have developed an interaction tool-kit consisting of four complementary high-throughput (HT) protein interaction assays. These assays were benchmarked against positive and random reference sets (PRS and RRS) consisting of well documented human interaction pairs and randomly chosen protein pairs, respectively. A logistic regression model was trained using the PRS/RRS data to combine the assay outputs and calculate the probability that any novel interaction pair is a true biophysical interaction once it has been tested in the tool-kit. This general approach will allow a systematic and empirical assignment of confidence scores to all individual protein-protein interactions in interactome networks.
PMCID: PMC2976677  PMID: 19060903
8.  An empirical framework for binary interactome mapping 
Nature methods  2008;6(1):83-90.
Several attempts have been made at systematically mapping protein-protein interaction, or “interactome” networks. However, it remains difficult to assess the quality and coverage of existing datasets. We describe a framework that uses an empirically-based approach to rigorously dissect quality parameters of currently available human interactome maps. Our results indicate that high-throughput yeast two-hybrid (HT-Y2H) interactions for human are superior in precision to literature-curated interactions supported by only a single publication, suggesting that HT-Y2H is suitable to map a significant portion of the human interactome. We estimate that the human interactome contains ~130,000 binary interactions, most of which remain to be mapped. Similar to estimates of DNA sequence data quality and genome size early in the human genome project, estimates of protein interaction data quality and interactome size are critical to establish the magnitude of the task of comprehensive human interactome mapping and to illuminate a path towards this goal.
PMCID: PMC2872561  PMID: 19060904
9.  High Quality Binary Protein Interaction Map of the Yeast Interactome Network 
Science (New York, N.Y.)  2008;322(5898):104-110.
Current yeast interactome network maps contain several hundred molecular complexes with limited and somewhat controversial representation of direct binary interactions. We carried out a comparative quality assessment of current yeast interactome datasets, demonstrating that high-throughput yeast two-hybrid (Y2H) provides high-quality binary interaction information. As a large fraction of the yeast binary interactome remains to be mapped, we developed an empirically-controlled mapping framework to produce a “second-generation” high-quality high-throughput Y2H dataset covering ~20% of all yeast binary interactions. Both Y2H and affinity-purification followed by mass spectrometry (AP/MS) data are of equally high quality but of a fundamentally different and complementary nature resulting in networks with different topological and biological properties. Compared to co-complex interactome models, this binary map is enriched for transient signaling interactions and inter-complex connections with a highly significant clustering between essential proteins. Rather than correlating with essentiality, protein connectivity correlates with genetic pleiotropy.
PMCID: PMC2746753  PMID: 18719252
10.  Isoform discovery by targeted cloning, “deep well” pooling, and parallel sequencing 
Nature methods  2008;5(7):597-600.
Describing the “ORFeome” of an organism, including all major isoforms, is essential for a systems understanding of any species; however, conventional cloning and sequencing approaches are prohibitively costly and labor-intensive. We describe a potentially genome-wide methodology for efficiently capturing novel coding isoforms using RT-PCR recombinational cloning, “deep well” pooling, and a “next generation” sequencing platform. This ORFeome discovery pipeline will be applicable to any eukaryotic species with a sequenced genome.
PMCID: PMC2743938  PMID: 18552854
11.  Efficient targeted transcript discovery via array-based normalization of RACE libraries 
Nature methods  2008;5(7):629-635.
RACE (Rapid Amplification of cDNA Ends) is a widely used approach for transcript identification. Random clone selection from the RACE mixture, however, is an ineffective sampling strategy if the dynamic range of transcript abundances is large. Here, we describe a strategy that uses array hybridization to improve sampling efficiency of human transcripts. The products of the RACE reaction are hybridized onto tiling arrays, and the exons detected are used to delineate a series of RT-PCR reactions, through which the original RACE mixture is segregated into simpler RT-PCR reactions. These are independently cloned, and randomly selected clones are sequenced. This approach is superior to direct cloning and sequencing of RACE products: it specifically targets novel transcripts, and often results in overall normalization of transcript abundances. We show theoretically and experimentally that this strategy leads indeed to efficient sampling of novel transcripts, and we investigate multiplexing it by pooling RACE reactions from multiple interrogated loci prior to hybridization.
PMCID: PMC2713501  PMID: 18500348
Neuropsychologia  2007;45(11):2508-2518.
We examined the implicit acquisition and mental representation of a novel verb in patients with probable Alzheimer’s disease (AD). Patients were exposed to the new verb in a naturalistic manner as part of a simple picture story. We probed grammatical, semantic and thematic matrix knowledge of the verb soon after presentation and again one week later. We found partial verb acquisition that was retained over one week. AD patients did not differ from controls in their acquisition and retention of a new verb’s major grammatical subcategory, although they acquired little of its semantic properties and displayed minimal acquisition of the new word’s thematic matrix. Moreover, AD patients appeared to maintain their acquired grammatical knowledge over one week. We discuss the implications of these findings from several perspectives, including the modularity of the language processing system, the relationship between episodic memory and semantic memory, and the role of the preserved implicit memory system in AD patients’ partially successful lexical acquisition.
PMCID: PMC1986669  PMID: 17482652
Alzheimer’s; learning; semantic; grammar
13.  Clinical, Cognitive and Anatomical Evolution from Nonfluent Progressive Aphasia to Corticobasal Syndrome: A Case Report 
Neurocase  2004;10(6):426-436.
Recent clinical and pathological studies have suggested that frontotemporal lobar degeneration (FTLD) and corticobasal syndrome (CBS) show clinical and pathological overlap. We present four years of longitudinal clinical, cognitive and anatomical data in the case of a 56-year-old woman, AS, whose clinical picture evolved from FTLD to CBS. For the first three years, AS showed a progressive speech and language disorder compatible with a diagnosis of the nonfluent aphasia variant of FTLD. At year four, 10 years after her first symptom, AS developed the classical clinical signs of CBS, including alien limb phenomenon and dystonia. Voxel-based morphometry (VBM) applied to AS’s four annual scans showed progression of atrophy from the inferior posterior frontal gyrus, to the left insula and finally to the medial frontal lobe. This case demonstrates the clinical overlap between FTLD and CBS and shows that the two can appear in the same patient at different stages of the disease in relation to the progression of anatomical damage.
PMCID: PMC2365737  PMID: 15788282

Results 1-13 (13)