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1.  PET Regional Cerebral Blood Flow Change During Working and Declarative Memory: Relationship With Task Performance 
Neuropsychology  1997;11(2):222-231.
Functional and anatomical relationships between working and declarative memory were investigated by contrasting regional cerebral blood flow (rCBF) change during standard working (Wisconsin Card Sorting Test, WCST) and declarative memory (Paired Associate Recognition Test, PART) tasks using identical stimulus–response modalities. The tasks and a resting baseline were administered to 30 participants (16 men, 14 women) during successive 10-min positron emission tomography 15O-water measures of rCBF. For both tasks, rCBF increased over baseline in inferior frontal and occipitotemporal regions, with more consistent dorsolateral prefrontal activation for WCST than PART. Additional orbitofrontal increases and dorsomedial decreases were seen for the PART. Activation patterns diverged when performance was considered. For the WCST, high performers activated dorsolateral and inferior frontal regions, whereas top PART performers activated only the occipitotemporal region. These results suggest operation of a frontotemporal network subserving both types of memory function that becomes more focal as performance increases.
PMCID: PMC4332579  PMID: 9110329
2.  Effect of Schizophrenia on Frontotemporal Activity During Word Encoding and Recognition: A PET Cerebral Blood Flow Study 
The American journal of psychiatry  2001;158(7):1114-1125.
Neuropsychological studies have shown that deficits in verbal episodic memory in schizophrenia occur primarily during encoding and retrieval stages of information processing. The current study used positron emission tomography to examine the effect of schizophrenia on change in cerebral blood flow (CBF) during these memory stages.
CBF was measured in 23 healthy comparison subjects and 23 patients with schizophrenia during four conditions: resting baseline, motor baseline, word encoding, and word recognition. The motor baseline was used as a reference that was subtracted from encoding and recognition conditions by using statistical parametric mapping.
Patients’ performance was similar to that of healthy comparison subjects. During word encoding, patients showed reduced activation of left prefrontal and superior temporal regions. Reduced left prefrontal activation in patients was also seen during word recognition, and additional differences were found in the left anterior cingulate, left mesial temporal lobe, and right thalamus. Although patients’ performance was similar to that of healthy comparison subjects, left inferior prefrontal activation was associated with better performance only in the comparison subjects.
Left frontotemporal activation during episodic encoding and retrieval, which is associated with better recognition in healthy people, is disrupted in schizophrenia despite relatively intact recognition performance and right prefrontal function. This may reflect impaired strategic use of semantic information to organize encoding and facilitate retrieval.
PMCID: PMC4332582  PMID: 11431234
3.  Genome-wide identification of signaling center enhancers in the developing limb 
Development (Cambridge, England)  2014;141(21):4194-4198.
The limb is widely used as a model developmental system and changes to gene expression patterns in its signaling centers, notably the zone of polarizing activity (ZPA) and the apical ectodermal ridge (AER), are known to cause limb malformations and evolutionary differences in limb morphology. Although several genes that define these limb signaling centers have been described, the identification of regulatory elements that are active within these centers has been limited. By dissecting mouse E11.5 limbs that fluorescently mark the ZPA or AER, followed by fluorescence-activated cell sorting and low-cell H3K27ac ChIP-seq, we identified thousands of specific signaling-center enhancers. Our ChIP-seq datasets show strong correlation with ZPA- and AER-expressed genes, previously characterized functional ZPA and AER enhancers and enrichment for relevant biological terms related to limb development and malformation for the neighboring genes. Using transgenic assays, we show that several of these sequences function as ZPA and AER enhancers. Our results identify novel ZPA and AER enhancers that could be important regulators of genes involved in the establishment of these specialized regions and the patterning of tetrapod limbs.
PMCID: PMC4302890  PMID: 25273087
Enhancer; AER; ZPA; Limb; Mouse
4.  Systematic Dissection of Coding Exons at Single Nucleotide Resolution Supports an Additional Role in Cell-Specific Transcriptional Regulation 
PLoS Genetics  2014;10(10):e1004592.
In addition to their protein coding function, exons can also serve as transcriptional enhancers. Mutations in these exonic-enhancers (eExons) could alter both protein function and transcription. However, the functional consequence of eExon mutations is not well known. Here, using massively parallel reporter assays, we dissect the enhancer activity of three liver eExons (SORL1 exon 17, TRAF3IP2 exon 2, PPARG exon 6) at single nucleotide resolution in the mouse liver. We find that both synonymous and non-synonymous mutations have similar effects on enhancer activity and many of the deleterious mutation clusters overlap known liver-associated transcription factor binding sites. Carrying a similar massively parallel reporter assay in HeLa cells with these three eExons found differences in their mutation profiles compared to the liver, suggesting that enhancers could have distinct operating profiles in different tissues. Our results demonstrate that eExon mutations could lead to multiple phenotypes by disrupting both the protein sequence and enhancer activity and that enhancers can have distinct mutation profiles in different cell types.
Author Summary
Exons that code for protein can also have additional functions, such as regulating gene transcription through enhancer activity. Here, we changed every nucleotide in three different exons that also function as enhancers, and examined their enhancer activity to test whether nucleotide changes in these exons can affect both the protein sequence and enhancer function. We found that mutations with a significant effect on enhancer function can reside both in regions that change the protein sequence (non-synonymous) and regions that do not change it (synonymous). When we conducted a similar analysis in a different cell type, we observed a difference in the nucleotide changes that cause a significant effect on enhancer activity, suggesting that the enhancer functional units can differ between tissues.
PMCID: PMC4207465  PMID: 25340400
5.  Characteristics of Memory B Cells Elicited by a Highly Efficacious HPV Vaccine in Subjects with No Pre-existing Immunity 
PLoS Pathogens  2014;10(10):e1004461.
Licensed human papillomavirus (HPV) vaccines provide near complete protection against the types of HPV that most commonly cause anogenital and oropharyngeal cancers (HPV 16 and 18) when administered to individuals naive to these types. These vaccines, like most other prophylactic vaccines, appear to protect by generating antibodies. However, almost nothing is known about the immunological memory that forms following HPV vaccination, which is required for long-term immunity. Here, we have identified and isolated HPV 16-specific memory B cells from female adolescents and young women who received the quadrivalent HPV vaccine in the absence of pre-existing immunity, using fluorescently conjugated HPV 16 pseudoviruses to label antigen receptors on the surface of memory B cells. Antibodies cloned and expressed from these singly sorted HPV 16-pseudovirus labeled memory B cells were predominantly IgG (>IgA>IgM), utilized diverse variable genes, and potently neutralized HPV 16 pseudoviruses in vitro despite possessing only average levels of somatic mutation. These findings suggest that the quadrivalent HPV vaccine provides an excellent model for studying the development of B cell memory; and, in the context of what is known about memory B cells elicited by influenza vaccination/infection, HIV-1 infection, or tetanus toxoid vaccination, indicates that extensive somatic hypermutation is not required to achieve potent vaccine-specific neutralizing antibody responses.
Author Summary
There is an urgent need to better understand how to reliably generate effective vaccines, particularly subunit vaccines, as certain pathogens are considered to pose too great of a safety risk to be developed as live, attenuated or killed vaccines (e.g., HIV-1). The human papillomavirus (HPV) vaccines are two of the most effective subunit vaccines ever developed and have continued to show protection against HPV associated disease up to and beyond five years post-vaccination. Moreover, the target population for these vaccines have essentially no pre-existing immunity to the HPV types covered by the vaccine; therefore, these vaccines provide an excellent model for studying the immunity elicited by a highly effective subunit vaccine. As the HPV vaccines, like most vaccines, protect by generating antibodies, we are interested in characterizing the memory B cells elicited by the HPV vaccine. Memory B cells help to sustain antibody levels over time by rapidly differentiating into antibody secreting cells upon pathogen re-exposure. Although previous studies have provided evidence that the HPV vaccines elicit memory B cells, they did not characterize these cells. Here, we have isolated HPV-specific memory B cells from adolescent females and women who received the quadrivalent HPV vaccine and have cloned antibodies from these cells. Importantly, we find that these antibodies potently inhibit HPV and that the memory B cells from which they derive exhibit hallmarks of long-lived memory B cells.
PMCID: PMC4199765  PMID: 25330199
6.  Genome-Wide Discovery of Drug-Dependent Human Liver Regulatory Elements 
PLoS Genetics  2014;10(10):e1004648.
Inter-individual variation in gene regulatory elements is hypothesized to play a causative role in adverse drug reactions and reduced drug activity. However, relatively little is known about the location and function of drug-dependent elements. To uncover drug-associated elements in a genome-wide manner, we performed RNA-seq and ChIP-seq using antibodies against the pregnane X receptor (PXR) and three active regulatory marks (p300, H3K4me1, H3K27ac) on primary human hepatocytes treated with rifampin or vehicle control. Rifampin and PXR were chosen since they are part of the CYP3A4 pathway, which is known to account for the metabolism of more than 50% of all prescribed drugs. We selected 227 proximal promoters for genes with rifampin-dependent expression or nearby PXR/p300 occupancy sites and assayed their ability to induce luciferase in rifampin-treated HepG2 cells, finding only 10 (4.4%) that exhibited drug-dependent activity. As this result suggested a role for distal enhancer modules, we searched more broadly to identify 1,297 genomic regions bearing a conditional PXR occupancy as well as all three active regulatory marks. These regions are enriched near genes that function in the metabolism of xenobiotics, specifically members of the cytochrome P450 family. We performed enhancer assays in rifampin-treated HepG2 cells for 42 of these sequences as well as 7 sequences that overlap linkage-disequilibrium blocks defined by lead SNPs from pharmacogenomic GWAS studies, revealing 15/42 and 4/7 to be functional enhancers, respectively. A common African haplotype in one of these enhancers in the GSTA locus was found to exhibit potential rifampin hypersensitivity. Combined, our results further suggest that enhancers are the predominant targets of rifampin-induced PXR activation, provide a genome-wide catalog of PXR targets and serve as a model for the identification of drug-responsive regulatory elements.
Author Summary
Drug response varies between individuals and can be caused by genetic factors. Nucleotide variation in gene regulatory elements can have a significant effect on drug response, but due to the difficulty in identifying these elements, they remain understudied. Here, we used various genomic assays to analyze human liver cells treated with or without the antibiotic rifampin and identified drug-induced regulatory elements genome-wide. The testing of numerous active promoters in human liver cells showed only a few to be induced by rifampin treatment. A similar analysis of enhancers found several of them to be induced by the drug. Nucleotide variants in one of these enhancers were found to alter its activity. Combined, this work identifies numerous novel gene regulatory elements that can be activated due to drug response and thus provides candidate sequences in the human genome where nucleotide variation can lead to differences in drug response. It also provides a universally applicable method to detect these elements for other drugs.
PMCID: PMC4183418  PMID: 25275310
7.  NMDA Receptor Blockade by Ketamine Abrogates Lipopolysaccharide-Induced Depressive-Like Behavior in C57BL/6J Mice 
Neuropsychopharmacology  2013;38(9):1609-1616.
We have previously demonstrated that lipopolysaccharide (LPS) induces depressive-like behavior by activating indoleamine 2,3 dioxygenase (IDO; O'Connor et al, 2009c). IDO degrades tryptophan along the kynurenine pathway. Using mass-spectrometry (LC-MS) analysis of kynurenine metabolites in the brain of mice injected at the periphery with 1 mg/kg LPS, we show that LPS activates the kynurenine 3-monooxygenase pathway that ultimately degrades kynurenine into quinolinic acid. As quinolinic acid acts as an N-methyl-𝒟-aspartate (NMDA) receptor agonist, we used the NMDA receptor antagonist ketamine to assess the role of NMDA receptor activation in LPS-induced depressive-like behavior. Here, we report that a low dose of ketamine (6 mg/kg, intraperitoneally) immediately before administration of LPS (0.83 mg/kg, intraperitoneally) in C57Bl/6 J mice abrogated the development of LPS-induced depressive-like behavior, without altering LPS-induced sickness measured by body weight loss, decreased motor activity, and reduced food intake. Depressive-like behavior was measured 24 h after LPS by decreased sucrose preference and increased immobility in the forced swim test (FST). Ketamine had no effect on LPS-induced cytokine expression in the liver and brain, IDO activation, and brain-derived neurotrophic factor (BDNF) transcripts. The ability of ketamine to abrogate LPS-induced depressive-like behavior independently of a possible interference with LPS-induced inflammatory signaling was confirmed when ketamine was administered 10 h after LPS instead of immediately before LPS. In contrast, ketamine had no effect when administered 24 h before LPS. To confirm that NMDA receptor antagonism by ketamine mediates the antidepressant-like activity of this compound in LPS-treated mice, mice were pretreated with the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline-2,3-dione (NBQX) to block enhanced AMPA receptor glutamatergic neurotransmission after NMDA receptor antagonism by ketamine. NBQX administered at the dose of 10 mg/kg intraperitoneally 15 min before ketamine in mice treated with LPS 24 h earlier restored LPS-induced decreased sucrose preference. These findings indicate that LPS-induced depressive-like behavior is mediated by NMDA receptor activation, probably as a consequence of formation of quinolinic acid.
PMCID: PMC3717543  PMID: 23511700
AMPA receptor; animal models; behavioral science; biological psychiatry; depression; depression; unipolar/bipolar; inflammation; ketamine; NMDA receptor; ketamine; depression; NMDA receptor; AMPA receptor; lipopolysaccharide; inflammation
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  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
18.  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
19.  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
20.  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
21.  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
22.  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
23.  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
24.  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
25.  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

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