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1.  Ferredoxin reductase affects p53-dependent, 5-fluorouracil–induced apoptosis in colorectal cancer cells 
Nature medicine  2001;7(10):1111-1117.
Loss of p53 gene function, which occurs in most colon cancer cells, has been shown to abolish the apoptotic response to 5-fluorouracil (5-FU). To identify genes downstream of p53 that might mediate these effects, we assessed global patterns of gene expression following 5-FU treatment of isogenic cells differing only in their p53 status. The gene encoding mitochondrial ferredoxin reductase (protein, FR; gene, FDXR) was one of the few genes significantly induced by p53 after 5-FU treatment. The FR protein was localized to mitochondria and suppressed the growth of colon cancer cells when over-expressed. Targeted disruption of the FDXR gene in human colon cancer cells showed that it was essential for viability, and partial disruption of the gene resulted in decreased sensitivity to 5-FU-induced apoptosis. These data, coupled with the effects of pharmacologic inhibitors of reactive oxygen species, indicate that FR contributes to p53-mediated apoptosis through the generation of oxidative stress in mitochondria.
PMCID: PMC4086305  PMID: 11590433
2.  Cardioprotection by S-nitrosation of a cysteine switch on mitochondrial complex I 
Nature medicine  2013;19(6):753-759.
Oxidative damage from elevated production of reactive oxygen species (ROS) contributes to ischemia-reperfusion injury in myocardial infarction and stroke. The mechanism by which the increase in ROS occurs is not known, and it is unclear how this increase can be prevented. A wide variety of nitric oxide donors and S-nitrosating agents protect the ischemic myocardium from infarction, but the responsible mechanisms are unclear1–6. Here we used a mitochondria-selective S-nitrosating agent, MitoSNO, to determine how mitochondrial S-nitrosation at the reperfusion phase of myocardial infarction is cardioprotective in vivo in mice. We found that protection is due to the S-nitrosation of mitochondrial complex I, which is the entry point for electrons from NADH into the respiratory chain. Reversible S-nitrosation of complex I slows the reactivation of mitochondria during the crucial first minutes of the reperfusion of ischemic tissue, thereby decreasing ROS production, oxidative damage and tissue necrosis. Inhibition of complex I is afforded by the selective S-nitrosation of Cys39 on the ND3 subunit, which becomes susceptible to modification only after ischemia. Our results identify rapid complex I reactivation as a central pathological feature of ischemia-reperfusion injury and show that preventing this reactivation by modification of a cysteine switch is a robust cardioprotective mechanism and hence a rational therapeutic strategy.
PMCID: PMC4019998  PMID: 23708290
3.  Resolution of Mitochondrial Oxidative Stress Rescues Coronary Collateral Growth in Zucker Obese Fatty Rats 
We have previously found abrogated ischemia-induced coronary collateral growth in Zucker obese fatty rats (ZOF) compared to Zucker lean rats (ZLN). Because ZOF have structural abnormalities in their mitochondria suggesting dysfunction, and also show increased production of O2ׄ−, we hypothesized that mitochondrial dysfunction, caused by oxidative stress impairs coronary collateral growth in ZOF.
Methods and Results
Increased levels of ROS were observed in aortic endothelium and smooth muscle cells in ZOF compared to ZLN. ROS levels were decreased by the mitochondria-targeted antioxidants MitoQuinone (MQ) and MitoTempol (MT) as assessed by MitoSox Red and DHE staining. Lipid peroxides (a marker of oxidized lipids) were increased in ZOF by ∼47 % compared to ZLN. The elevation in oxidative stress was accompanied by increased antioxidant enzymes, except GPx-1, and by increased uncoupling protein-2 in ZOF vs ZLN. In addition, elevated respiration rates were also observed in the obese compared to leans. Administration of MQ significantly normalized the metabolic profiles and reduced lipid peroxides in ZOF to the same level observed in leans. The protective effect of MQ also suppressed the induction of UCP-2 in the obese rats. Resolution of mitochondrial oxidative stress by MQ or MT restored coronary collateral growth to the same magnitude observed in ZLN in response to repetitive ischemia.
We conclude that mitochondrial oxidative stress and dysfunction play a key role in disrupting coronary collateral growth in obesity and the metabolic syndrome, and elimination of the mitochondrial oxidative stress with MQ or MT rescues collateral growth.
PMCID: PMC4013346  PMID: 22155454
MitoQuinone; MitoTempol; metabolic syndrome; arteriogenesis; uncoupling protein-2; lipid peroxidation
4.  Monitoring Fibrous Scaffold Guidance of Three-Dimensional Collagen Organisation Using Minimally-Invasive Second Harmonic Generation 
PLoS ONE  2014;9(2):e89761.
The biological and mechanical function of connective tissues is largely determined by controlled cellular alignment and therefore it seems appropriate that tissue-engineered constructs should be architecturally similar to the in vivo tissue targeted for repair or replacement. Collagen organisation dictates the tensile properties of most tissues and so monitoring the deposition of cell-secreted collagen as the construct develops is essential for understanding tissue formation. In this study, electrospun fibres with a random or high degree of orientation, mimicking two types of tissue architecture found in the body, were used to culture human fibroblasts for controlling cell alignment. The minimally-invasive technique of second harmonic generation was used with the aim of monitoring and profiling the deposition and organisation of collagen at different construct depths over time while construct mechanical properties were also determined over the culture period. It was seen that scaffold fibre organisation affected cell migration and orientation up to 21 days which in turn had an effect on collagen organisation. Collagen in random fibrous constructs was deposited in alternating configurations at different depths however a high degree of organisation was observed throughout aligned fibrous constructs orientated in the scaffold fibre direction. Three-dimensional second harmonic generation images showed that deposited collagen was more uniformly distributed in random constructs but aligned constructs were more organised and had higher intensities. The tensile properties of all constructs increased with increasing collagen deposition and were ultimately dictated by collagen organisation. This study highlights the importance of scaffold architecture for controlling the development of well-organised tissue engineered constructs and the usefulness of second harmonic generation imaging for monitoring collagen maturation in a minimally invasive manner.
PMCID: PMC3938545  PMID: 24587017
5.  A mitochondria-targeted mass spectrometry probe to detect glyoxals: implications for diabetes☆ 
Free Radical Biology & Medicine  2014;67(100):437-450.
The glycation of protein and nucleic acids that occurs as a consequence of hyperglycemia disrupts cell function and contributes to many pathologies, including those associated with diabetes and aging. Intracellular glycation occurs after the generation of the reactive 1,2-dicarbonyls methylglyoxal and glyoxal, and disruption of mitochondrial function is associated with hyperglycemia. However, the contribution of these reactive dicarbonyls to mitochondrial damage in pathology is unclear owing to uncertainties about their levels within mitochondria in cells and in vivo. To address this we have developed a mitochondria-targeted reagent (MitoG) designed to assess the levels of mitochondrial dicarbonyls within cells. MitoG comprises a lipophilic triphenylphosphonium cationic function, which directs the molecules to mitochondria within cells, and an o-phenylenediamine moiety that reacts with dicarbonyls to give distinctive and stable products. The extent of accumulation of these diagnostic heterocyclic products can be readily and sensitively quantified by liquid chromatography–tandem mass spectrometry, enabling changes to be determined. Using the MitoG-based analysis we assessed the formation of methylglyoxal and glyoxal in response to hyperglycemia in cells in culture and in the Akita mouse model of diabetes in vivo. These findings indicated that the levels of methylglyoxal and glyoxal within mitochondria increase during hyperglycemia both in cells and in vivo, suggesting that they can contribute to the pathological mitochondrial dysfunction that occurs in diabetes and aging.
•A mitochondria-targeted mass spectrometric probe, MitoG, has been developed to measure glyoxal and methylglyoxal.•Using MitoG we show that mitochondrial glyoxal and methylglyoxal can be measured in hyperglycemic cells.•MitoG can also be used in vivo to infer mitochondrial glyoxal and methylglyoxal production in a mouse model of type I diabetes.•These findings suggest that the accumulation of glyoxal and methylglyoxal within mitochondria may contribute to mitochondrial dysfunction in diabetes.
PMCID: PMC3978666  PMID: 24316194
Mitochondria; Exomarker; Methylglyoxal; Glyoxal; Hyperglycemia; MitoG; Free radicals
6.  Primary cilia respond to fluid shear stress and mediate flow-induced calcium deposition in osteoblasts 
The FASEB Journal  2014;28(1):430-439.
Bone turnover in vivo is regulated by mechanical forces such as shear stress originating from interstitial oscillatory fluid flow (OFF), and bone cells in vitro respond to mechanical loading. However, the mechanisms by which bone cells sense mechanical forces, resulting in increased mineral deposition, are not well understood. The aim of this study was to investigate the role of the primary cilium in mechanosensing by osteoblasts. MLO-A5 murine osteoblasts were cultured in monolayer and subjected to two different OFF regimens: 5 short (2 h daily) bouts of OFF followed by morphological analysis of primary cilia; or exposure to chloral hydrate to damage or remove primary cilia and 2 short bouts (2 h on consecutive days) of OFF. Primary cilia were shorter and there were fewer cilia per cell after exposure to periods of OFF compared with static controls. Damage or removal of primary cilia inhibited OFF-induced PGE2 release into the medium and mineral deposition, assayed by Alizarin red staining. We conclude that primary cilia are important mediators of OFF-induced mineral deposition, which has relevance for the design of bone tissue engineering strategies and may inform clinical treatments of bone disorders causes by load-deficiency.—Delaine-Smith, R. M., Sittichokechaiwut, A., Reilly, G. C. Primary cilia respond to fluid shear stress and mediate flow-induced calcium deposition in osteoblasts.
PMCID: PMC4012163  PMID: 24097311
mechanotransduction; oscillatory fluid flow; osteogenesis; extracellular matrix
7.  Sequence signatures extracted from proximal promoters can be used to predict distal enhancers 
Genome Biology  2013;14(10):R117.
Gene expression is controlled by proximal promoters and distal regulatory elements such as enhancers. While the activity of some promoters can be invariant across tissues, enhancers tend to be highly tissue-specific.
We compiled sets of tissue-specific promoters based on gene expression profiles of 79 human tissues and cell types. Putative transcription factor binding sites within each set of sequences were used to train a support vector machine classifier capable of distinguishing tissue-specific promoters from control sequences. We obtained reliable classifiers for 92% of the tissues, with an area under the receiver operating characteristic curve between 60% (for subthalamic nucleus promoters) and 98% (for heart promoters). We next used these classifiers to identify tissue-specific enhancers, scanning distal non-coding sequences in the loci of the 200 most highly and lowly expressed genes. Thirty percent of reliable classifiers produced consistent enhancer predictions, with significantly higher densities in the loci of the most highly expressed compared to lowly expressed genes. Liver enhancer predictions were assessed in vivo using the hydrodynamic tail vein injection assay. Fifty-eight percent of the predictions yielded significant enhancer activity in the mouse liver, whereas a control set of five sequences was completely negative.
We conclude that promoters of tissue-specific genes often contain unambiguous tissue-specific signatures that can be learned and used for the de novo prediction of enhancers.
PMCID: PMC3983659  PMID: 24156763
8.  Massively parallel decoding of mammalian regulatory sequences supports a flexible organizational model 
Nature genetics  2013;45(9):1021-1028.
Despite continual progress in the cataloging of vertebrate regulatory elements, little is known about their organization and regulatory architecture. Here we describe a massively parallel experiment to systematically test the impact of copy number, spacing, combination and order of transcription factor binding sites on gene expression. A complex library of ~5,000 synthetic regulatory elements containing patterns from 1 2 liver-specific transcription factor binding sites was assayed in mice and in HepG2 cells. We find that certain transcription factors act as direct drivers of gene expression in homotypic clusters of binding sites, independent of spacing between sites, whereas others function only synergistically. Heterotypic enhancers are stronger than their homotypic analogs and favor specific transcription factor binding site combinations, mimicking putative native enhancers. Exhaustive testing of binding site permutations suggests that there is flexibility in binding site order. Our findings provide quantitative support for a flexible model of regulatory element activity and suggest a framework for the design of synthetic tissue-specific enhancers.
PMCID: PMC3775494  PMID: 23892608
9.  A compact, in vivo screen of all 6-mers reveals drivers of tissue-specific expression and guides synthetic regulatory element design 
Genome Biology  2013;14(7):R72.
Large-scale annotation efforts have improved our ability to coarsely predict regulatory elements throughout vertebrate genomes. However, it is unclear how complex spatiotemporal patterns of gene expression driven by these elements emerge from the activity of short, transcription factor binding sequences.
We describe a comprehensive promoter extension assay in which the regulatory potential of all 6 base-pair (bp) sequences was tested in the context of a minimal promoter. To enable this large-scale screen, we developed algorithms that use a reverse-complement aware decomposition of the de Bruijn graph to design a library of DNA oligomers incorporating every 6-bp sequence exactly once. Our library multiplexes all 4,096 unique 6-mers into 184 double-stranded 15-bp oligomers, which is sufficiently compact for in vivo testing. We injected each multiplexed construct into zebrafish embryos and scored GFP expression in 15 tissues at two developmental time points. Twenty-seven constructs produced consistent expression patterns, with the majority doing so in only one tissue. Functional sequences are enriched near biologically relevant genes, match motifs for developmental transcription factors, and are required for enhancer activity. By concatenating tissue-specific functional sequences, we generated completely synthetic enhancers for the notochord, epidermis, spinal cord, forebrain and otic lateral line, and show that short regulatory sequences do not always function modularly.
This work introduces a unique in vivo catalog of short, functional regulatory sequences and demonstrates several important principles of regulatory element organization. Furthermore, we provide resources for designing compact, reverse-complement aware k-mer libraries.
PMCID: PMC4054837  PMID: 23867016
10.  P-glycoprotein (Mdr1a/1b) and breast cancer resistance protein (Bcrp) decrease the uptake of hydrophobic alkyl triphenylphosphonium cations by the brain 
Biochimica et Biophysica Acta  2013;1830(6):3458-3465.
Mitochondrial dysfunction contributes to degenerative neurological disorders, consequently there is a need for mitochondria-targeted therapies that are effective within the brain. One approach to deliver pharmacophores is by conjugation to the lipophilic triphenylphosphonium (TPP) cation that accumulates in mitochondria driven by the membrane potential. While this approach has delivered TPP-conjugated compounds to the brain, the amounts taken up are lower than by other organs.
To discover why uptake of hydrophobic TPP compounds by the brain is relatively poor, we assessed the role of the P-glycoprotein (Mdr1a/b) and breast cancer resistance protein (Bcrp) ATP binding cassette (ABC) transporters, which drive the efflux of lipophilic compounds from the brain thereby restricting the uptake of lipophilic drugs. We used a triple transgenic mouse model lacking two isoforms of P-glycoprotein (Mdr1a/1b) and the Bcrp.
There was a significant increase in the uptake into the brain of two hydrophobic TPP compounds, MitoQ and MitoF, in the triple transgenics following intra venous (IV) administration compared to control mice. Greater amounts of the hydrophobic TPP compounds were also retained in the liver of transgenic mice compared to controls. The uptake into the heart, white fat, muscle and kidneys was comparable between the transgenic mice and controls.
Efflux of hydrophobic TPP compounds by ABC transporters contributes to their lowered uptake into the brain and liver.
General significance
These findings suggest that strategies to bypass ABC transporters in the BBB will enhance delivery of mitochondria-targeted antioxidants, probes and pharmacophores to the brain.
► Brain accumulation of triphenylphosphonium cations is decreased by ABC transporters. ► ABC-transporter inactivation increases brain uptake of triphenylphosphonium cations. ► Bypassing ABC transporters may increase the effectiveness of mitochondrial therapies.
PMCID: PMC3898886  PMID: 23454352
ABC proteins, ATP binding cassette proteins; BBB, blood–brain barrier; Bcrp, breast cancer resistance protein; CsA, cyclosporin A; IP, intra peritoneal; IV, intra venous; Mdr1, multi drug resistance 1; MitoF, 11-fluoroundecyltriphenylphosphonium mesylate; MitoQ, [10-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)decyl]triphenylphosphonium mesylate; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; TPB, tetraphenylborate; TPP, triphenylphosphonium cation; ROS, reactive oxygen species; TPMP, methyltriphenylphosphonium; Mitochondria; Lipophilic cation; Blood–brain barrier; ABC transporters; MitoQ
11.  Pharmacogene regulatory elements: from discovery to applications 
Genome Medicine  2012;4(5):45.
Regulatory elements play an important role in the variability of individual responses to drug treatment. This has been established through studies on three classes of elements that regulate RNA and protein abundance: promoters, enhancers and microRNAs. Each of these elements, and genetic variants within them, are being characterized at an exponential pace by next-generation sequencing (NGS) technologies. In this review, we outline examples of how each class of element affects drug response via regulation of drug targets, transporters and enzymes. We also discuss the impact of NGS technologies such as chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq), and the ramifications of new techniques such as high-throughput chromosome capture (Hi-C), chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) and massively parallel reporter assays (MPRA). NGS approaches are generating data faster than they can be analyzed, and new methods will be required to prioritize laboratory results before they are ready for the clinic. However, there is no doubt that these approaches will bring about a systems-level understanding of the interplay between genetic variants and drug response. An understanding of the importance of regulatory variants in pharmacogenomics will facilitate the identification of responders versus non-responders, the prevention of adverse effects and the optimization of therapies for individual patients.
PMCID: PMC3506911  PMID: 22630332
ChIP-Seq; enhancers; miRNA; next-generation sequencing; pharmacogenomics; promoters; RNA-Seq
12.  Re-Directing an Alkylating Agent to Mitochondria Alters Drug Target and Cell Death Mechanism 
PLoS ONE  2013;8(4):e60253.
We have successfully delivered a reactive alkylating agent, chlorambucil (Cbl), to the mitochondria of mammalian cells. Here, we characterize the mechanism of cell death for mitochondria-targeted chlorambucil (mt-Cbl) in vitro and assess its efficacy in a xenograft mouse model of leukemia. Using a ρ° cell model, we show that mt-Cbl toxicity is not dependent on mitochondrial DNA damage. We also illustrate that re-targeting Cbl to mitochondria results in a shift in the cell death mechanism from apoptosis to necrosis, and that this behavior is a general feature of mitochondria-targeted Cbl. Despite the change in cell death mechanisms, we show that mt-Cbl is still effective in vivo and has an improved pharmacokinetic profile compared to the parent drug. These findings illustrate that mitochondrial rerouting changes the site of action of Cbl and also alters the cell death mechanism drastically without compromising in vivo efficacy. Thus, mitochondrial delivery allows the exploitation of Cbl as a promiscuous mitochondrial protein inhibitor with promising therapeutic potential.
PMCID: PMC3621862  PMID: 23585833
13.  Science Incubators: Synthesis Centers and Their Role in the Research Ecosystem 
PLoS Biology  2013;11(1):e1001468.
How should funding agencies enable researchers to explore high-risk but potentially high-reward science? One model that appears to work is the NSF-funded synthesis center, an incubator for community-led, innovative science.
PMCID: PMC3545866  PMID: 23335860
14.  Transcriptional Response of Two Core Photosystem Genes in Symbiodinium spp. Exposed to Thermal Stress 
PLoS ONE  2012;7(12):e50439.
Mutualistic symbioses between scleractinian corals and endosymbiotic dinoflagellates (Symbiodinium spp.) are the foundation of coral reef ecosystems. For many coral-algal symbioses, prolonged episodes of thermal stress damage the symbiont's photosynthetic capability, resulting in its expulsion from the host. Despite the link between photosynthetic competency and symbiont expulsion, little is known about the effect of thermal stress on the expression of photosystem genes in Symbiodinium. This study used real-time PCR to monitor the transcript abundance of two important photosynthetic reaction center genes, psbA (encoding the D1 protein of photosystem II) and psaA (encoding the P700 protein of photosystem I), in four cultured isolates (representing ITS2-types A13, A20, B1, and F2) and two in hospite Symbiodinium spp. within the coral Pocillopora spp. (ITS2-types C1b-c and D1). Both cultured and in hospite Symbiodinium samples were exposed to elevated temperatures (32°C) over a 7-day period and examined for changes in photochemistry and transcript abundance. Symbiodinium A13 and C1b-c (both thermally sensitive) demonstrated significant declines in both psbA and psaA during the thermal stress treatment, whereas the transcript levels of the other Symbiodinium types remained stable. The downregulation of both core photosystem genes could be the result of several different physiological mechanisms, but may ultimately limit repair rates of photosynthetic proteins, rendering some Symbiodinium spp. especially susceptible to thermal stress.
PMCID: PMC3517614  PMID: 23236373
15.  Mesenchymal stem cell responses to mechanical stimuli 
Mesenchymal stem cells (MSCs) have the potential to replace or restore the function of damaged tissues and offer much promise in the successful application of tissue engineering and regenerative medicine strategies. Optimising culture conditions for the pre-differentiation of MSCs is a key goal for the research community, and this has included a number of different approaches, one of which is the use of mechanical stimuli. Mesenchymal tissues are subjected to mechanical stimuli in vivo and terminally differentiated cells from the mesenchymal lineage respond to mechanical stimulation in vivo and in vitro. MSCs have also been shown to be highly mechanosensitive and this may present an ideal method for controlling MSC differentiation. Here we present an overview of the response of MSCs to various mechanical stimuli, focusing on their differentiation towards the mesenchymal tissue lineages including bone, cartilage, tendon/ligament, muscle and adipose tissue. More research is needed to elucidate the complex interactions between biochemically and mechanically stimulated differentiation pathways.
PMCID: PMC3666521  PMID: 23738294
mechanical stimuli; mesenchymal stem cell; osteogenesis; tenogenesis
16.  Massively parallel functional dissection of mammalian enhancers in vivo 
Nature biotechnology  2012;30(3):265-270.
The functional consequences of genetic variation in mammalian regulatory elements are poorly understood. We report the in vivo dissection of three mammalian liver enhancers at single nucleotide resolution via a massively parallelized reporter assay. For each enhancer, we synthesized a library of >100,000 mutant haplotypes with 2–3% divergence from wild-type. Each haplotype was linked to a unique sequence tag embedded within a transcriptional cassette. We introduced each enhancer library into mouse liver and measured the relative activities of individual haplotypes en masse by sequencing of the transcribed tags. Linear regression yielded highly reproducible estimates of the impact of every possible single nucleotide change on enhancer activity. The functional impact of most mutations was modest, with ~22% impacting activity by >1.2-fold, and only ~3% by >2-fold. These results suggest that mammalian enhancers are relatively robust to single nucleotide changes. Several, but not all positions with higher impact showed evidence for purifying selection, or co-localized with known liver-associated transcription factor binding sites, demonstrating the value of empirical high-resolution functional analysis.
PMCID: PMC3402344  PMID: 22371081
17.  Surgical residency training and international volunteerism: a national survey of residents from 2 surgical specialties 
Canadian Journal of Surgery  2012;55(4 Suppl 2):S191-S199.
Many low- and middle-income countries (LMICs) lack basic surgical resources, resulting in avoidable disability and mortality. Recently, residents in surgical training programs have shown increasing interest in overseas elective experiences to assist surgical programs in LMICs. The purpose of this study was to survey Canadian surgical residents about their interest in international volunteerism.
We sent a web-based survey to all general and orthopedic surgery residents enrolled in surgical training programs in Canada. The survey assessed residents’ interests, attitudes and motivations, and perceived barriers and aids with respect to international volunteerism.
In all, 361 residents completed the survey for a response rate of 38.0%. Half of the respondents indicated that the availability of an international surgery elective would have positively influenced their selection of a residency program. Excluding the 18 residents who had volunteered during residency, 63.8% of the remaining residents confirmed an interest in international volunteering with “contributing to an important cause,” “teaching” and “tourism/cultural enhancement” as the leading reasons for their interest. Perceived barriers included “lack of financial support” and “lack of available organized opportunities.” All (100%) respondents who had done an international elective during residency confirmed that they would pursue such work in the future.
Administrators of Canadian surgical programs should be aware of strong resident interest in global health care and accordingly develop opportunities by encouraging faculty mentorships and resources for global health teaching.
PMCID: PMC3432249  PMID: 22854155
18.  Peripheral Nervous System Genes Expressed in Central Neurons Induce Growth on Inhibitory Substrates 
PLoS ONE  2012;7(6):e38101.
Trauma to the spinal cord and brain can result in irreparable loss of function. This failure of recovery is in part due to inhibition of axon regeneration by myelin and chondroitin sulfate proteoglycans (CSPGs). Peripheral nervous system (PNS) neurons exhibit increased regenerative ability compared to central nervous system neurons, even in the presence of inhibitory environments. Previously, we identified over a thousand genes differentially expressed in PNS neurons relative to CNS neurons. These genes represent intrinsic differences that may account for the PNS’s enhanced regenerative ability. Cerebellar neurons were transfected with cDNAs for each of these PNS genes to assess their ability to enhance neurite growth on inhibitory (CSPG) or permissive (laminin) substrates. Using high content analysis, we evaluated the phenotypic profile of each neuron to extract meaningful data for over 1100 genes. Several known growth associated proteins potentiated neurite growth on laminin. Most interestingly, novel genes were identified that promoted neurite growth on CSPGs (GPX3, EIF2B5, RBMX). Bioinformatic approaches also uncovered a number of novel gene families that altered neurite growth of CNS neurons.
PMCID: PMC3368946  PMID: 22701605
19.  Successful treatment of Leuconostoc bacteremia in a neutropenic patient with tigecycline 
Infectious Disease Reports  2012;4(2):e31.
Leuconostoc lactis is a recognised cause of infection in immunocompromised hosts. It is intrinsically resistant to multiple antibiotics and treatment options may be limited. We report a case of safe and effective use of tigecycline in the treatment of Leuconostoc catheter-related line sepsis in a neutropenic patient. To our knowledge, this is the first reported case of successful use of tigecycline for Leuconostoc bacteremia.
PMCID: PMC3892628  PMID: 24470945
Leuconostoc; tigecycline; bacteremia; immunocompromise; neutropenia.
20.  BioAssay Ontology Annotations Facilitate Cross-Analysis of Diverse High-throughput Screening Data Sets 
Journal of biomolecular screening  2011;16(4):415-426.
High-throughput screening data repositories, such as PubChem, represent valuable resources for the development of small molecule chemical probes and can serve as entry points for drug discovery programs. While the loose data format offered by PubChem allows for great flexibility, important annotations, such as the assay format and technologies employed, are not explicitly indexed. We have previously developed a BioAssay Ontology (BAO) and curated over 350 assays with standardized BAO terms. Here we describe the use of BAO annotations to analyze a large set of assays that employ luciferase- and β-lactamase-based technologies. We identified promiscuous chemotypes pertaining to different sub-categories of assays and specific mechanisms by which these chemotypes interfere in reporter gene assays. Our results show that the data in PubChem can be used to identify promiscuous compounds that interfere non-specifically with particular technologies. Furthermore, we show that BAO is a valuable toolset for the identification of related assays and for the systematic generation of insights that are beyond the scope of individual assays or screening campaigns.
PMCID: PMC3167204  PMID: 21471461
compound promiscuity; assay ontology; reporter gene assays; high-throughput screening data analysis; cheminformatics
21.  De novo sequencing of circulating miRNAs identifies novel markers predicting clinical outcome of locally advanced breast cancer 
MicroRNAs (miRNAs) have been recently detected in the circulation of cancer patients, where they are associated with clinical parameters. Discovery profiling of circulating small RNAs has not been reported in breast cancer (BC), and was carried out in this study to identify blood-based small RNA markers of BC clinical outcome.
The pre-treatment sera of 42 stage II-III locally advanced and inflammatory BC patients who received neoadjuvant chemotherapy (NCT) followed by surgical tumor resection were analyzed for marker identification by deep sequencing all circulating small RNAs. An independent validation cohort of 26 stage II-III BC patients was used to assess the power of identified miRNA markers.
More than 800 miRNA species were detected in the circulation, and observed patterns showed association with histopathological profiles of BC. Groups of circulating miRNAs differentially associated with ER/PR/HER2 status and inflammatory BC were identified. The relative levels of selected miRNAs measured by PCR showed consistency with their abundance determined by deep sequencing. Two circulating miRNAs, miR-375 and miR-122, exhibited strong correlations with clinical outcomes, including NCT response and relapse with metastatic disease. In the validation cohort, higher levels of circulating miR-122 specifically predicted metastatic recurrence in stage II-III BC patients.
Our study indicates that certain miRNAs can serve as potential blood-based biomarkers for NCT response, and that miR-122 prevalence in the circulation predicts BC metastasis in early-stage patients. These results may allow optimized chemotherapy treatments and preventive anti-metastasis interventions in future clinical applications.
PMCID: PMC3342150  PMID: 22400902
Breast cancer; miRNA; Biomarker; Neoadjuvant chemotherapy; Metastasis
22.  Transcriptional Profiling of Intrinsic PNS Factors in the Postnatal Mouse 
Neurons in the peripheral nervous system (PNS) display a higher capacity to regenerate after injury than those in the central nervous system, suggesting cell specific transcriptional modules underlying axon growth and inhibition. We report a systems biology based search for PNS specific transcription factors (TFs). Messenger RNAs enriched in dorsal root ganglion (DRG) neurons compared to cerebellar granule neurons (CGNs) were identified using subtractive hybridization and DNA microarray approaches. Network and transcription factor binding site enrichment analyses were used to further identify TFs that may be differentially active. Combining these techniques, we identified 32 TFs likely to be enriched and/or active in the PNS. Twenty-five of these TFs were then tested for an ability to promote CNS neurite outgrowth in an overexpression screen. Real-time PCR and immunohistochemical studies confirmed that one representative TF, STAT3, is intrinsic to PNS neurons, and that constitutively active STAT3 is sufficient to promote CGN neurite outgrowth.
PMCID: PMC3003944  PMID: 20696251
Dorsal Root Ganglion; Transcription Factor; High Content Analysis; Screen; Systems Biology; Cerebellar Granule Neuron; STAT3
23.  Host–symbiont recombination versus natural selection in the response of coral–dinoflagellate symbioses to environmental disturbance 
Mutualisms between reef-building corals and endosymbiotic dinoflagellates are particularly sensitive to environmental stress, yet the ecosystems they construct have endured major oscillations in global climate. During the winter of 2008, an extreme cold-water event occurred in the Gulf of California that bleached corals in the genus Pocillopora harbouring a thermally ‘sensitive’ symbiont, designated Symbiodinium C1b-c, while colonies possessing Symbiodinium D1 were mostly unaffected. Certain bleached colonies recovered quickly while others suffered partial or complete mortality. In most colonies, no appreciable change was observed in the identity of the original symbiont, indicating that these partnerships are stable. During the initial phases of recovery, a third species of symbiont B1Aiptasia, genetically identical to that harboured by the invasive anemone, Aiptasia sp., grew opportunistically and was visible as light-yellow patches on the branch tips of several colonies. However, this symbiont did not persist and was displaced in all cases by C1b-c several months later. Colonies with D1 were abundant at inshore habitats along the continental eastern Pacific, where seasonal turbidity is high relative to offshore islands. Environmental conditions of the central and southern coasts of Mexico were not sufficient to explain the exclusivity of D1 Pocillopora in these regions. It is possible that mass mortalities associated with major thermal disturbances during the 1997–1998 El Niño Southern Oscillation eliminated C1b-c holobionts from these locations. The differential loss of Pocillopora holobionts in response to thermal stress suggests that natural selection on existing variation can cause rapid and significant shifts in the frequency of particular coral–algal partnerships. However, coral populations may take decades to recover following episodes of severe selection, thereby raising considerable uncertainty about the long-term viability of these communities.
PMCID: PMC2982020  PMID: 20444713
climate change; coral bleaching; eastern Pacific; holobiont; natural selection; Symbiodinium
24.  Pain and other symptom severity in women with fibromyalgia and a previous hysterectomy 
Journal of Pain Research  2011;4:325-329.
Fibromyalgia is a troubling disease characterized by chronic pain. This study explored whether pain and other fibromyalgia symptoms are worse among women who had undergone a hysterectomy with or without an oophorectomy versus those who had not.
Consecutive women who were seen at the Fibromyalgia Treatment Program at a tertiary medical center between 2001 and 2004 and who completed the Fibromyalgia Impact Questionnaire (FIQ) and Short Form-36 Health Survey (SF-36) at initial evaluation were included in this study.
A total of 813 women were included; 328 had had a hysterectomy. Total FIQ scores from women who had had a hysterectomy were higher (worse symptoms) than those who had not (58.1 vs 56.4, P = 0.002). FIQ subscale scores of pain (P = 0.003), fatigue (P = 0.030), stiffness (P = 0.035), and depression (P = 0.008) were also worse in women who had had a hysterectomy. Similar to the FIQ, SF-36 physical component scores were worse in women who had had a hysterectomy (P = 0.045).
Pain and other fibromyalgia symptom severity was worse in women who had had a hysterectomy with or without an oophorectomy.
PMCID: PMC3191932  PMID: 22003306
fibromyalgia; hysterectomy; oophorectomy; symptom severity; surgical menopause
25.  BioAssay Ontology (BAO): a semantic description of bioassays and high-throughput screening results 
BMC Bioinformatics  2011;12:257.
High-throughput screening (HTS) is one of the main strategies to identify novel entry points for the development of small molecule chemical probes and drugs and is now commonly accessible to public sector research. Large amounts of data generated in HTS campaigns are submitted to public repositories such as PubChem, which is growing at an exponential rate. The diversity and quantity of available HTS assays and screening results pose enormous challenges to organizing, standardizing, integrating, and analyzing the datasets and thus to maximize the scientific and ultimately the public health impact of the huge investments made to implement public sector HTS capabilities. Novel approaches to organize, standardize and access HTS data are required to address these challenges.
We developed the first ontology to describe HTS experiments and screening results using expressive description logic. The BioAssay Ontology (BAO) serves as a foundation for the standardization of HTS assays and data and as a semantic knowledge model. In this paper we show important examples of formalizing HTS domain knowledge and we point out the advantages of this approach. The ontology is available online at the NCBO bioportal
After a large manual curation effort, we loaded BAO-mapped data triples into a RDF database store and used a reasoner in several case studies to demonstrate the benefits of formalized domain knowledge representation in BAO. The examples illustrate semantic querying capabilities where BAO enables the retrieval of inferred search results that are relevant to a given query, but are not explicitly defined. BAO thus opens new functionality for annotating, querying, and analyzing HTS datasets and the potential for discovering new knowledge by means of inference.
PMCID: PMC3149580  PMID: 21702939

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