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1.  Aneurysmal ‘pepper-pot’ atrial septal defect in an older gentleman with multiple cerebrovascular attacks 
BMJ Case Reports  2012;2012:bcr1220115402.
Our patient presented to a large university teaching hospital with a history of light-headedness, falls and multiple cerebrovascular ischaemic events. This caused a right sided hemiplegia and the patient experienced significant functional limitation. Extensive investigations were carried out to exclude any causative factors such as carotid artery disease and the patient had all identifiable cardiovascular risk factors identified and modified. No significant pathology was found and a referral was made to the cardiology service. Transthoracic echocardiography revealed a complex type 3 perforate ‘pepper pot’ atrial septal aneurysm with associated thrombus. The patient was commenced on warfarin and appropriate rate limiting medication. After discussion of all interventional modalities, the patient opted for conservative management.
PMCID: PMC3351657  PMID: 22605008
2.  Are chest compressions safe for the patient reconstructed with sternal plates? Evaluating the safety of cardiopulmonary resuscitation using a human cadaveric model 
Plate and screw fixation is a recent addition to the sternal wound treatment armamentarium. Patients undergoing cardiac and major vascular surgery have a higher risk of postoperative arrest than other elective patients. Those who undergo sternotomy for either cardiac or major vascular procedures are at a higher risk of postoperative arrest. Sternal plate design allows quick access to the mediastinum facilitating open cardiac massage, but chest compressions are the mainstay of re-establishing cardiac output in the event of arrest. The response of sternal plates and the chest wall to compressions when plated has not been studied. The safety of performing this maneuver is unknown. This study intends to demonstrate compressions are safe after sternal plating.
We investigated the effect of chest compressions on the plated sternum using a human cadaveric model. Cadavers were plated, an arrest was simulated, and an experienced physician performed a simulated resuscitation. Intrathoracic pressure was monitored throughout to ensure the plates encountered an appropriate degree of force. The hardware and viscera were evaluated for failure and trauma respectively.
No hardware failure or obvious visceral trauma was observed. Rib fractures beyond the boundaries of the plates were noted but the incidence was comparable to control and to the fracture incidence after resuscitation previously cited in the literature.
From this work we believe chest compressions are safe for the patient with sternal plates when proper plating technique is used. We advocate the use of this life-saving maneuver as part of an ACLS resuscitation in the event of an arrest for rapidly re-establishing circulation.
PMCID: PMC2933603  PMID: 20718981
3.  Pharmacological and Immunological Identification of Native α7 Nicotinic Receptors: Evidence for Homomeric and Heteromeric α7 Receptors 
Life sciences  2007;81(16):1317-1322.
Controversy surrounds the expression of α7 nicotinic acetylcholine receptors (nAChRs) in adrenal chromaffin cells. In these studies, α7 nAChRs expressed in bovine adrenal chromaffin cells are investigated. Using radiolabeled ligand binding techniques, [125I]α-bungarotoxin (αBGT) binding reaches equilibrium within 4 hours and is saturable with a Kd value of 4.2 nM. Using homologous competition experiments, the Ki for binding of αBGT was 1.9 nM. These data are consistent with the expression of homomeric α7 nAChRs. Methyllycaconatine (MLA), which binds α7 nAChRs with high affinity, inhibits [125I]αBGT binding in a concentration-dependent manner with a Ki of 30.6 nM; this value is ∼10 fold higher than the reported affinity of MLA for α7 nAChRs. We also document the ability of bromoacetylcholine (brACh) to alkylate α7 nAChRs, as has been previous demonstrated for bovine adrenal α3β4 nAChRs. When adrenal nAChRs are immunoprecipated with mAb 319, an antibody which recognizes α7 nAChR protein, and then probed with mAb 319 using Western blot analysis, a single band of ∼53 kD is identified. When adrenal nAChRs are immunoprecipated with mAb35, an antibody which recognizes α3 and α5 nAChR proteins, and then probed with mAb319 using Western blot analysis, a single band of ∼53 kD is identified. Together, these results support the expression of α7 nAChRs in bovine adrenal chromaffin cells. However, these data suggest that the subunit composition of some of these receptors may include heteromeric α7 nAChRs.
PMCID: PMC2083560  PMID: 17928008
Nicotinic acetylcholine receptor; binding; adrenal medulla; methyllycaconitine; α-bungarotoxin; α7
4.  Integration of untargeted metabolomics with transcriptomics reveals active metabolic pathways 
Metabolomics : Official journal of the Metabolomic Society  2014;2014(August):
While recent advances in metabolomic measurement technologies have been dramatic, extracting biological insight from complex metabolite profiles remains a challenge. We present an analytical strategy that uses data obtained from high resolution liquid chromatography–mass spectrometry and a bioinformatics toolset for detecting actively changing metabolic pathways upon external perturbation. We begin with untargeted metabolite profiling to nominate altered metabolites and identify pathway candidates, followed by validation of those pathways with transcriptomics. Using the model organisms Rhodospirillum rubrum and Bacillus subtilis, our results reveal metabolic pathways that are interconnected with methionine salvage. The rubrum-type methionine salvage pathway is interconnected with the active methyl cycle in which re-methylation, a key reaction for recycling methionine from homocysteine, is unexpectedly suppressed; instead, homocysteine is catabolized by the transsulfuration pathway. Notably, the non-mevalonate pathway is repressed, whereas the rubrum-type methionine salvage pathway contributes to isoprenoid biosynthesis upon 5’-methylthioadenosine feeding. In this process, glutathione functions as a coenzyme in vivo when 1-methylthio-d-xylulose 5-phosphate (MTXu 5-P) methylsulfurylase catalyzes dethiomethylation of MTXu 5-P. These results clearly show that our analytical approach enables unexpected metabolic pathways to be uncovered.
PMCID: PMC4334135
Active pathway detection; Isoprenoid biosynthesis; Liquid chromatography–mass spectrometry; Metabolomics; Methionine salvage; Quantitative real time polymerase chain reaction; Transcriptomics
5.  FGF receptor genes and breast cancer susceptibility: results from the Breast Cancer Association Consortium 
Agarwal, D | Pineda, S | Michailidou, K | Herranz, J | Pita, G | Moreno, L T | Alonso, M R | Dennis, J | Wang, Q | Bolla, M K | Meyer, K B | Menéndez-Rodríguez, P | Hardisson, D | Mendiola, M | González-Neira, A | Lindblom, A | Margolin, S | Swerdlow, A | Ashworth, A | Orr, N | Jones, M | Matsuo, K | Ito, H | Iwata, H | Kondo, N | Hartman, M | Hui, M | Lim, W Y | T-C Iau, P | Sawyer, E | Tomlinson, I | Kerin, M | Miller, N | Kang, D | Choi, J-Y | Park, S K | Noh, D-Y | Hopper, J L | Schmidt, D F | Makalic, E | Southey, M C | Teo, S H | Yip, C H | Sivanandan, K | Tay, W-T | Brauch, H | Brüning, T | Hamann, U | Dunning, A M | Shah, M | Andrulis, I L | Knight, J A | Glendon, G | Tchatchou, S | Schmidt, M K | Broeks, A | Rosenberg, E H | van't Veer, L J | Fasching, P A | Renner, S P | Ekici, A B | Beckmann, M W | Shen, C-Y | Hsiung, C-N | Yu, J-C | Hou, M-F | Blot, W | Cai, Q | Wu, A H | Tseng, C-C | Van Den Berg, D | Stram, D O | Cox, A | Brock, I W | Reed, M W R | Muir, K | Lophatananon, A | Stewart-Brown, S | Siriwanarangsan, P | Zheng, W | Deming-Halverson, S | Shrubsole, M J | Long, J | Shu, X-O | Lu, W | Gao, Y-T | Zhang, B | Radice, P | Peterlongo, P | Manoukian, S | Mariette, F | Sangrajrang, S | McKay, J | Couch, F J | Toland, A E | Yannoukakos, D | Fletcher, O | Johnson, N | Silva, I dos Santos | Peto, J | Marme, F | Burwinkel, B | Guénel, P | Truong, T | Sanchez, M | Mulot, C | Bojesen, S E | Nordestgaard, B G | Flyer, H | Brenner, H | Dieffenbach, A K | Arndt, V | Stegmaier, C | Mannermaa, A | Kataja, V | Kosma, V-M | Hartikainen, J M | Lambrechts, D | Yesilyurt, B T | Floris, G | Leunen, K | Chang-Claude, J | Rudolph, A | Seibold, P | Flesch-Janys, D | Wang, X | Olson, J E | Vachon, C | Purrington, K | Giles, G G | Severi, G | Baglietto, L | Haiman, C A | Henderson, B E | Schumacher, F | Le Marchand, L | Simard, J | Dumont, M | Goldberg, M S | Labrèche, F | Winqvist, R | Pylkäs, K | Jukkola-Vuorinen, A | Grip, M | Devilee, P | Tollenaar, R A E M | Seynaeve, C | García-Closas, M | Chanock, S J | Lissowska, J | Figueroa, J D | Czene, K | Eriksson, M | Humphreys, K | Darabi, H | Hooning, M J | Kriege, M | Collée, J M | Tilanus-Linthorst, M | Li, J | Jakubowska, A | Lubinski, J | Jaworska-Bieniek, K | Durda, K | Nevanlinna, H | Muranen, T A | Aittomäki, K | Blomqvist, C | Bogdanova, N | Dörk, T | Hall, P | Chenevix-Trench, G | Easton, D F | Pharoah, P D P | Arias-Perez, J I | Zamora, P | Benítez, J | Milne, R L
British Journal of Cancer  2014;110(4):1088-1100.
Breast cancer is one of the most common malignancies in women. Genome-wide association studies have identified FGFR2 as a breast cancer susceptibility gene. Common variation in other fibroblast growth factor (FGF) receptors might also modify risk. We tested this hypothesis by studying genotyped single-nucleotide polymorphisms (SNPs) and imputed SNPs in FGFR1, FGFR3, FGFR4 and FGFRL1 in the Breast Cancer Association Consortium.
Data were combined from 49 studies, including 53 835 cases and 50 156 controls, of which 89 050 (46 450 cases and 42 600 controls) were of European ancestry, 12 893 (6269 cases and 6624 controls) of Asian and 2048 (1116 cases and 932 controls) of African ancestry. Associations with risk of breast cancer, overall and by disease sub-type, were assessed using unconditional logistic regression.
Little evidence of association with breast cancer risk was observed for SNPs in the FGF receptor genes. The strongest evidence in European women was for rs743682 in FGFR3; the estimated per-allele odds ratio was 1.05 (95% confidence interval=1.02–1.09, P=0.0020), which is substantially lower than that observed for SNPs in FGFR2.
Our results suggest that common variants in the other FGF receptors are not associated with risk of breast cancer to the degree observed for FGFR2.
PMCID: PMC3929867  PMID: 24548884
breast cancer; SNP; FGF receptors; susceptibility; disease subtypes
6.  Physician-led, hospital-linked, birth care centers can decrease Cesarean section rates without increasing rates of adverse events 
Birth (Berkeley, Calif.)  2013;40(3):155-163.
This study compares outcomes at a hospital-linked, physician-led, birthing center to a traditional hospital labor and delivery service.
Using de-identified electronic medical records, a retrospective cohort design was employed to evaluate 32,174 singleton births during 1998–2005.
Compared to hospital service, birth care center delivery was associated with a lower rate of cesarean sections (adjusted Relative Risk =0.73, 95 percent confidence interval 0.59–0.91; p<0.001) without an increased rate of operative vaginal delivery (adjusted Relative Risk=1.04, 95 percent confidence interval 0.97–1.13; p=0.25) and a higher initiation of breast feeding (adjusted Relative Risk = 1.28, 95 percent confidence interval 1.25–1.30 (p=<0.001). A maternal length of stay greater than 72 hours occurred less frequently in the birth care center (adjusted Relative Risk =0.60, 95 percent confidence interval 0.55–0.66; p<0.001). Comparing only women without major obstetrical risk factors, the differences in outcomes were reduced but not eliminated. Adverse maternal and infant outcomes were not increased at the birth care center.
A hospital-linked, physician-led, birth care center has the potential to lower rates of cesarean sections without increasing rates of operative vaginal delivery or other adverse maternal and infant outcomes.
PMCID: PMC4321785  PMID: 24635500
hospital-linked; birth care center; delivery; obstetric; pregnancy outcome; physician
7.  Salud Mesoamérica 2015 Initiative: design, implementation, and baseline findings 
Health has improved markedly in Mesoamerica, the region consisting of southern Mexico and Central America, over the past decade. Despite this progress, there remain substantial inequalities in health outcomes, access, and quality of medical care between and within countries. Poor, indigenous, and rural populations have considerably worse health indicators than national or regional averages. In an effort to address these health inequalities, the Salud Mesoamérica 2015 Initiative (SM2015), a results-based financing initiative, was established.
For each of the eight participating countries, health targets were set to measure the progress of improvements in maternal and child health produced by the Initiative. To establish a baseline, we conducted censuses of 90,000 households, completed 20,225 household interviews, and surveyed 479 health facilities in the poorest areas of Mesoamerica. Pairing health facility and household surveys allows us to link barriers to care and health outcomes with health system infrastructure components and quality of health services.
Indicators varied significantly within and between countries. Anemia was most prevalent in Panama and least prevalent in Honduras. Anemia varied by age, with the highest levels observed among children aged 0 to 11 months in all settings. Belize had the highest proportion of institutional deliveries (99%), while Guatemala had the lowest (24%). The proportion of women with four antenatal care visits with a skilled attendant was highest in El Salvador (90%) and the lowest in Guatemala (20%). Availability of contraceptives also varied. The availability of condoms ranged from 83% in Nicaragua to 97% in Honduras. Oral contraceptive pills and injectable contraceptives were available in just 75% of facilities in Panama. IUDs were observed in only 21.5% of facilities surveyed in El Salvador.
These data provide a baseline of much-needed information for evidence-based action on health throughout Mesoamerica. Our baseline estimates reflect large disparities in health indicators within and between countries and will facilitate the evaluation of interventions and investments deployed in the region over the next three to five years. SM2015’s innovative monitoring and evaluation framework will allow health officials with limited resources to identify and target areas of greatest need.
Electronic supplementary material
The online version of this article (doi:10.1186/s12963-015-0034-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4327957
Results-based financing; Salud Mesoamerica 2015; Vaccination; Contraceptives; Skilled birth attendance; Antenatal care; Anemia; Wasting; Health facilities
8.  Age-related changes in afferent pathways and urothelial function in the male mouse bladder 
The Journal of Physiology  2014;592(3):537-549.
The prevalence of lower urinary tract storage disorders such as overactive bladder syndrome and urinary incontinence significantly increase with age. Previous studies have demonstrated age-related changes in detrusor function and urothelial transmitter release but few studies have investigated how the urothelium and sensory pathways are affected. The aim of this study was to investigate the effect of ageing on urothelial-afferent signalling in the mouse bladder. Three-month-old control and 24-month-old aged male mice were used. In vivo natural voiding behaviour, sensory nerve activity, urothelial cell function, muscle contractility, transmitter release and gene and protein expression were measured to identify how all three components of the bladder (neural, contractile and urothelial) are affected by ageing. In aged mice, increased voiding frequency and enhanced low threshold afferent nerve activity was observed, suggesting that ageing induces overactivity and hypersensitivity of the bladder. These changes were concurrent with altered ATP and acetylcholine bioavailability, measured as transmitter overflow into the lumen, increased purinergic receptor sensitivity and raised P2X3 receptor expression in the urothelium. Taken together, these data suggest that ageing results in aberrant urothelial function, increased afferent mechanosensitivity, increased smooth muscle contractility, and changes in gene and protein expression (including of P2X3). These data are consistent with the hypothesis that ageing evokes changes in purinergic signalling from the bladder, and further studies are now required to fully validate this idea.
PMCID: PMC3930438  PMID: 24297847
9.  Age-related changes in afferent pathways and urothelial function in the male mouse bladder 
The Journal of Physiology  2014;592(Pt 3):537-549.
The prevalence of lower urinary tract storage disorders such as overactive bladder syndrome and urinary incontinence significantly increase with age. Previous studies have demonstrated age-related changes in detrusor function and urothelial transmitter release but few studies have investigated how the urothelium and sensory pathways are affected. The aim of this study was to investigate the effect of ageing on urothelial-afferent signalling in the mouse bladder. Three-month-old control and 24-month-old aged male mice were used. In vivo natural voiding behaviour, sensory nerve activity, urothelial cell function, muscle contractility, transmitter release and gene and protein expression were measured to identify how all three components of the bladder (neural, contractile and urothelial) are affected by ageing. In aged mice, increased voiding frequency and enhanced low threshold afferent nerve activity was observed, suggesting that ageing induces overactivity and hypersensitivity of the bladder. These changes were concurrent with altered ATP and acetylcholine bioavailability, measured as transmitter overflow into the lumen, increased purinergic receptor sensitivity and raised P2X3 receptor expression in the urothelium. Taken together, these data suggest that ageing results in aberrant urothelial function, increased afferent mechanosensitivity, increased smooth muscle contractility, and changes in gene and protein expression (including of P2X3). These data are consistent with the hypothesis that ageing evokes changes in purinergic signalling from the bladder, and further studies are now required to fully validate this idea.
PMCID: PMC3930438  PMID: 24297847
10.  A genome-wide association study of marginal zone lymphoma shows association to the HLA region 
Vijai, Joseph | Wang, Zhaoming | Berndt, Sonja I. | Skibola, Christine F. | Slager, Susan L. | de Sanjose, Silvia | Melbye, Mads | Glimelius, Bengt | Bracci, Paige M. | Conde, Lucia | Birmann, Brenda M. | Wang, Sophia S. | Brooks-Wilson, Angela R. | Lan, Qing | de Bakker, Paul I. W. | Vermeulen, Roel C. H. | Portlock, Carol | Ansell, Stephen M. | Link, Brian K. | Riby, Jacques | North, Kari E. | Gu, Jian | Hjalgrim, Henrik | Cozen, Wendy | Becker, Nikolaus | Teras, Lauren R. | Spinelli, John J. | Turner, Jenny | Zhang, Yawei | Purdue, Mark P. | Giles, Graham G. | Kelly, Rachel S. | Zeleniuch-Jacquotte, Anne | Ennas, Maria Grazia | Monnereau, Alain | Bertrand, Kimberly A. | Albanes, Demetrius | Lightfoot, Tracy | Yeager, Meredith | Chung, Charles C. | Burdett, Laurie | Hutchinson, Amy | Lawrence, Charles | Montalvan, Rebecca | Liang, Liming | Huang, Jinyan | Ma, Baoshan | Villano, Danylo J. | Maria, Ann | Corines, Marina | Thomas, Tinu | Novak, Anne J. | Dogan, Ahmet | Liebow, Mark | Thompson, Carrie A. | Witzig, Thomas E. | Habermann, Thomas M. | Weiner, George J. | Smith, Martyn T. | Holly, Elizabeth A. | Jackson, Rebecca D. | Tinker, Lesley F. | Ye, Yuanqing | Adami, Hans-Olov | Smedby, Karin E. | De Roos, Anneclaire J. | Hartge, Patricia | Morton, Lindsay M. | Severson, Richard K. | Benavente, Yolanda | Boffetta, Paolo | Brennan, Paul | Foretova, Lenka | Maynadie, Marc | McKay, James | Staines, Anthony | Diver, W. Ryan | Vajdic, Claire M. | Armstrong, Bruce K. | Kricker, Anne | Zheng, Tongzhang | Holford, Theodore R. | Severi, Gianluca | Vineis, Paolo | Ferri, Giovanni M. | Ricco, Rosalia | Miligi, Lucia | Clavel, Jacqueline | Giovannucci, Edward | Kraft, Peter | Virtamo, Jarmo | Smith, Alex | Kane, Eleanor | Roman, Eve | Chiu, Brian C. H. | Fraumeni, Joseph F. | Wu, Xifeng | Cerhan, James R. | Offit, Kenneth | Chanock, Stephen J. | Rothman, Nathaniel | Nieters, Alexandra
Nature Communications  2015;6:5751.
Marginal zone lymphoma (MZL) is the third most common subtype of B-cell non-Hodgkin lymphoma. Here we perform a two-stage GWAS of 1,281 MZL cases and 7,127 controls of European ancestry and identify two independent loci near BTNL2 (rs9461741, P=3.95 × 10−15) and HLA-B (rs2922994, P=2.43 × 10−9) in the HLA region significantly associated with MZL risk. This is the first evidence that genetic variation in the major histocompatibility complex influences MZL susceptibility.
Marginal zone lymphoma (MZL) is a common subtype of B-cell non-Hodgkin lymphoma. Here the authors carry out a two-stage genome-wide association study in over 8,000 Europeans and identify two new MZL risk loci at chromosome 6p, implicating the major histocompatibility complex in the disease for the first time.
PMCID: PMC4287989  PMID: 25569183
11.  Age dependent changes in the LPS induced transcriptome of bovine dermal fibroblasts occurs without major changes in the methylome 
BMC Genomics  2015;16(1):30.
By comparing fibroblasts collected from animals at 5-months or 16-months of age we have previously found that the cultures from older animals produce much more IL-8 in response to lipopolysaccharide (LPS) stimulation. We now expand this finding by examining whole transcriptome differences in the LPS response between cultures from the same animals at different ages, and also investigate the contribution of DNA methylation to the epigenetic basis for the age-dependent increases in responsiveness.
Age-dependent differences in IL-8 production by fibroblasts in response to LPS exposure for 24 h were abolished by pretreatment of cultures with a DNA demethylation agent, 5-aza-2′deoxycytidine (AZA). RNA-Seq analysis of fibroblasts collected from the same individuals at either 5 or 16 months of age and exposed in parallel to LPS for 0, 2, and 8 h revealed a robust response to LPS that was much greater in the cultures from older animals. Pro-inflammatory genes including IL-8, IL-6, TNF-α, and CCL20 (among many other immune associated genes), were more highly expressed (FDR < 0.05) in the 16-month old cultures following LPS exposure. Methylated CpG island recovery assay sequencing (MIRA-Seq) revealed numerous methylation peaks spread across the genome, combined with an overall hypomethylation of gene promoter regions, and a remarkable similarity, except for 20 regions along the genome, between the fibroblasts collected at the two ages from the same animals.
The fibroblast pro-inflammatory response to LPS increases dramatically from 5 to 16 months of age within individual animals. A better understanding of the mechanisms underlying this process could illuminate the physiological processes by which the innate immune response develops and possibly individual variation in innate immune response arises. In addition, although relatively unchanged by age, our data presents a general overview of the bovine fibroblast methylome as a guide for future studies in cattle epigenetics utilizing this cell type.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1223-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4312471  PMID: 25623529
Epigenetics; MIRA-Seq; RNA-Seq; LPS; TLR4
12.  Astrocytes Increase ATP Exocytosis Mediated Calcium Signaling in Response to Microgroove Structures 
Scientific Reports  2015;5:7847.
Following central nervous system (CNS) injury, activated astrocytes form glial scars, which inhibit axonal regeneration, leading to long-term functional deficits. Engineered nanoscale scaffolds guide cell growth and enhance regeneration within models of spinal cord injury. However, the effects of micro-/nanosize scaffolds on astrocyte function are not well characterized. In this study, a high throughput (HTP) microscale platform was developed to study astrocyte cell behavior on micropatterned surfaces containing 1 μm spacing grooves with a depth of 250 or 500 nm. Significant changes in cell and nuclear elongation and alignment on patterned surfaces were observed, compared to on flat surfaces. The cytoskeleton components (particularly actin filaments and focal adhesions) and nucleus-centrosome axis were aligned along the grooved direction as well. More interestingly, astrocytes on micropatterned surfaces showed enhanced mitochondrial activity with lysosomes localized at the lamellipodia of the cells, accompanied by enhanced adenosine triphosphate (ATP) release and calcium activities. These data indicate that the lysosome-mediated ATP exocytosis and calcium signaling may play an important role in astrocytic responses to substrate topology. These new findings have furthered our understanding of the biomechanical regulation of astrocyte cell–substrate interactions, and may benefit the optimization of scaffold design for CNS healing.
PMCID: PMC4297955  PMID: 25597401
13.  Preclinical therapeutic efficacy of a novel pharmacological inducer of apoptosis in malignant peripheral nerve sheath tumors 
Cancer research  2013;74(2):586-597.
Neurofibromatosis Type 1 (NF1) is an autosomal disorder that affects neural crest-derived tissues, leading to a wide spectrum of clinical presentations. Patients commonly present with plexiform neurofibromas, benign but debilitating growths that can transform into malignant peripheral nerve sheath tumors (MPNSTs), a main cause of mortality. Currently, surgery is the primary course of treatment for MPNST, but with the limitation that these tumors are highly invasive. Radiation therapy is another treatment option, but is undesirable because it can induce additional mutations. MPNST patients may also receive doxorubicin as therapy, but this DNA-intercalating agent has relatively low tumor specificity and limited efficacy. In this study, we exploited a robust genetically-engineered mouse model of MPNST that recapitulates human NF1 associated MPNST to identify a novel small chemical compound that inhibits tumor cell growth. Compound 21 (Cpd21) inhibits growth of all available in vitro models of MPNST and human MPNST cell lines, while remaining non-toxic to normally-dividing Schwann cells or mouse embryonic fibroblasts. We show that this compound delays the cell cycle and leads to cellular apoptosis. Moreover, Cpd21 can reduce MPNST burden in a mouse allograft model, underscoring the compound’s potential as a novel chemotherapeutic agent.
PMCID: PMC3947005  PMID: 24285727
Neurofibromatosis Type1; NF1; Malignant Peripheral Nerve Sheath Tumor; MPNST; small molecule; apoptosis
14.  An Injectable, Calcium Responsive Composite Hydrogel for the Treatment of Acute Spinal Cord Injury 
ACS Applied Materials & Interfaces  2014;6(3):1424-1438.
Immediately following spinal cord injury, further injury can occur through several secondary injury cascades. As a consequence of cell lysis, an increase in extracellular Ca2+ results in additional neuronal loss by inducing apoptosis. Thus, hydrogels that reduce extracellular Ca2+ concentration may reduce secondary injury severity. The goal of this study was to develop composite hydrogels consisting of alginate, chitosan, and genipin that interact with extracellular Ca2+ to enable in situ gelation while maintaining an elastic modulus similar to native spinal cord (∼1000 Pa). It was hypothesized that incorporation of genipin and chitosan would regulate hydrogel electrostatic characteristics and influence hydrogel porosity, degradation, and astrocyte behavior. Hydrogel composition was varied to create hydrogels with statistically similar mechanical properties (∼1000 Pa) that demonstrated tunable charge characteristics (6-fold range in free amine concentration) and degradation rate (complete degradation between 7 and 28 days; some blends persist after 28 days). Hydrogels demonstrate high sensitivity to Ca2+ concentration, as a 1 mM change during fabrication induced a significant change in elastic modulus. Additionally, hydrogels incubated in a Ca2+-containing solution exhibited an increased linear viscoelastic limit (LVE) and an increased elastic modulus above the LVE limit in a time dependent manner. An extension of the LVE limit implies a change in hydrogel cross-linking structure. Attachment assays demonstrated that addition of chitosan/genipin to alginate hydrogels induced up to a 4-fold increase in the number of attached astrocytes and facilitated astrocyte clustering on the hydrogel surface in a composition dependent manner. Furthermore, Western blots demonstrated tunable glial fibrillary acid protein (GFAP) expression in astrocytes cultured on hydrogel blends, with some hydrogel compositions demonstrating no significant increase in GFAP expression compared to astrocytes cultured on glass. Thus, alginate/chitosan/genipin hydrogel composites show promise as scaffolds that regulate astrocyte behavior and for the prevention of Ca2+-related secondary neuron damage during acute SCI.
PMCID: PMC3982972  PMID: 24397537
alginate; chitosan; hydrogel; astrocytes; spinal cord injury; glial fibrillary acidic protein
15.  Human Pluripotent Stem Cell Culture: Considerations for Maintenance, Expansion, and Therapeutics 
Cell stem cell  2014;14(1):13-26.
Human pluripotent stem cells (hPSCs) provide powerful resources for application in regenerative medicine and pharmaceutical development. In the past decade, various methods have been developed for large-scale hPSC culture that rely on combined use of multiple growth components, including media containing various growth factors, extracellular matrices, three-dimensional environmental (3D) cues and modes of multicellular association. In this review, we dissect these growth components by comparing cell culture methods and identifying the benefits and pitfalls associated with each one. We further provide criteria, considerations, and suggestions to achieve optimal cell growth for hPSC expansion, differentiation, and use in future therapeutic applications.
PMCID: PMC3915741  PMID: 24388173
Human embryonic stem cells; induced pluripotent stem cells; cell culture; expansion; differentiation; regenerative medicine
16.  Statistical Linkage Analysis of Substitutions in Patient-Derived Sequences of Genotype 1a Hepatitis C Virus Nonstructural Protein 3 Exposes Targets for Immunogen Design 
Journal of Virology  2014;88(13):7628-7644.
Chronic hepatitis C virus (HCV) infection is one of the leading causes of liver failure and liver cancer, affecting around 3% of the world's population. The extreme sequence variability of the virus resulting from error-prone replication has thwarted the discovery of a universal prophylactic vaccine. It is known that vigorous and multispecific cellular immune responses, involving both helper CD4+ and cytotoxic CD8+ T cells, are associated with the spontaneous clearance of acute HCV infection. Escape mutations in viral epitopes can, however, abrogate protective T-cell responses, leading to viral persistence and associated pathologies. Despite the propensity of the virus to mutate, there might still exist substitutions that incur a fitness cost. In this paper, we identify groups of coevolving residues within HCV nonstructural protein 3 (NS3) by analyzing diverse sequences of this protein using ideas from random matrix theory and associated methods. Our analyses indicate that one of these groups comprises a large percentage of residues for which HCV appears to resist multiple simultaneous substitutions. Targeting multiple residues in this group through vaccine-induced immune responses should either lead to viral recognition or elicit escape substitutions that compromise viral fitness. Our predictions are supported by published clinical data, which suggested that immune genotypes associated with spontaneous clearance of HCV preferentially recognized and targeted this vulnerable group of residues. Moreover, mapping the sites of this group onto the available protein structure provided insight into its functional significance. An epitope-based immunogen is proposed as an alternative to the NS3 epitopes in the peptide-based vaccine IC41.
IMPORTANCE Despite much experimental work on HCV, a thorough statistical study of the HCV sequences for the purpose of immunogen design was missing in the literature. Such a study is vital to identify epistatic couplings among residues that can provide useful insights for designing a potent vaccine. In this work, ideas from random matrix theory were applied to characterize the statistics of substitutions within the diverse publicly available sequences of the genotype 1a HCV NS3 protein, leading to a group of sites for which HCV appears to resist simultaneous substitutions possibly due to deleterious effect on viral fitness. Our analysis leads to completely novel immunogen designs for HCV. In addition, the NS3 epitopes used in the recently proposed peptide-based vaccine IC41 were analyzed in the context of our framework. Our analysis predicts that alternative NS3 epitopes may be worth exploring as they might be more efficacious.
PMCID: PMC4054436  PMID: 24760894
17.  The VUKA Family Program: Piloting a family-based psychosocial intervention to promote health and mental health among HIV infected early adolescents in South Africa 
AIDS care  2013;26(1):10.1080/09540121.2013.806770.
An increasing number of adolescents born with HIV in South Africa are on antiretroviral treatment and have to confront complex issues related to coping with a chronic, stigmatizing and transmittable illness. Very few evidence-based mental health and health promotion programs for this population exist in South Africa. This study builds on a previous collaboratively designed and developmentally-timed family-based intervention for early adolescents (CHAMP). The study uses community-based participatory approach as part of formative research to evaluate a pilot randomized control trial at two hospitals. The paper reports on the development, feasibility and acceptability of the VUKA family-based program and its short-term impact on a range of psychosocial variables for HIV+ pre-adolescents and their caregivers. A ten session intervention of approximately 3 months duration was delivered to 65 pre-adolescents aged 10-13 years and their families. VUKA participants were noted to improve on all dimensions, including mental health, youth behaviour, HIV treatment knowledge, stigma, communication and adherence to medication. VUKA shows promise as a family-based mental and HIV prevention program for HIV+ pre-adolescents and which could be delivered by trained lay staff.
PMCID: PMC3838445  PMID: 23767772
Family-based; Psychosocial intervention; Mental health; HIV+ adolescents
18.  Fucosylation with Fucosyltransferase (FT)-VI or FT-VII Improves Cord Blood Engraftment 
Cytotherapy  2013;16(1):10.1016/j.jcyt.2013.07.003.
Advantages associated with the use of cord blood (CB) transplantation include the availability of cryopreserved units, ethnic diversity and lower incidence of graft-versus-host disease when compared to bone marrow or mobilized peripheral blood. However, poor engraftment remains a major obstacle. We and others have found that ex vivo fucosylation can enhance engraftment in murine models and thus ex vivo treatment of CB with fucosyltransferase (FT)-VI prior to transplantation is under clinical evaluation (NCT01471067). However, FT-VII appears to be more relevant to hematopoietic cells and may alter acceptor substrate diversity. In this study, we compare the ability of FT-VI and FT-VII to improve the rapidity, magnitude, multi-lineage and multi-tissue engraftment of human CB hematopoietic stem and progenitor cells (HSPC) in vivo.
CD34-selected CB HSPC were treated with recombinant FT-VI, FT-VI or mock control, then injected into immunodeficient mice and monitored for multi-lineage and multi-tissue engraftment.
Both FT-VI and FT-VII fucosylated CB CD34+ cells in vitro, and both led to enhanced rates and magnitudes of engraftment when compared to untreated CB CD34+ cells in vivo. Engraftment following treatment with either fucosyltransferase was robust at multiple timepoints, and in multiple tissues, with similar multi-lineage potential. In contrast, only FT-VII was able to fucosylate T- and B-lymphocytes.
While we found that FT-VI and FT-VII were similarly able to fucosylate and enhance the engraftment of CB CD34+ cells, differences in their ability to fucosylate lymphocytes cells may modulate graft-versus-tumor and/or graft-versus-host effects and may allow further optimization of CB transplantation.
PMCID: PMC3883688  PMID: 24094497
19.  A Randomized Clinical Trial of Alcohol Care Management Delivered in Department of Veterans Affairs Primary Care Clinics Versus Specialty Addiction Treatment 
Alcohol use disorder is one of the leading causes of disability worldwide. Despite the availability of efficacious treatments, few individuals with an alcohol use disorder are actively engaged in treatment. Available evidence suggests that primary care may play a crucial role in the identification of patients with an alcohol use disorder, delivery of interventions, and the success of treatment.
The principal aims of this study were to test the effectiveness of a primary care-based Alcohol Care Management (ACM) program for alcohol use disorder and treatment engagement in veterans.
The design of the study was a 26-week single-blind randomized clinical trial. The study was conducted in the primary care practices at three VA medical centers. Participants were randomly assigned to treatment in ACM or standard treatment in a specialty outpatient addiction treatment program.
One hundred and sixty-three alcohol-dependent veterans were randomized.
ACM focused on the use of pharmacotherapy and psychosocial support. ACM was delivered in-person or by telephone within the primary care clinic.
Engagement in treatment and heavy alcohol consumption.
The ACM condition had a significantly higher proportion of participants engaged in treatment over the 26 weeks [OR = 5.36, 95 % CI = (2.99, 9.59)]. The percentage of heavy drinking days were significantly lower in the ACM condition [OR = 2.16, 95 % CI = (1.27, 3.66)], while overall abstinence did not differ between groups.
Results demonstrate that treatment for an alcohol use disorder can be delivered effectively within primary care, leading to greater rates of engagement in treatment and greater reductions in heavy drinking.
PMCID: PMC3889933  PMID: 24052453
addiction; primary care; treatment; randomized clinical trial
20.  Directed Differentiation of Human Induced Pluripotent Stem Cells Toward Bone and Cartilage: In Vitro Versus In Vivo Assays 
The ability to generate large numbers of bone- and cartilage-forming cells from induced pluripotent stem cells (iPSCs) would mark a major advance in tissue engineering. A number of protocols exist, but the overall quality and consistency of this type of differentiation are under-reported. In this study, the authors analyzed differentiated iPSCs in vitro and in vivo by stringent criteria, and found that in vitro analysis does not predict in vivo differentiation.
The ability to differentiate induced pluripotent stem cells (iPSCs) into committed skeletal progenitors could allow for an unlimited autologous supply of such cells for therapeutic uses; therefore, we attempted to create novel bone-forming cells from human iPSCs using lines from two distinct tissue sources and methods of differentiation that we previously devised for osteogenic differentiation of human embryonic stem cells, and as suggested by other publications. The resulting cells were assayed using in vitro methods, and the results were compared with those obtained from in vivo transplantation assays. Our results show that true bone was formed in vivo by derivatives of several iPSC lines, but that the successful cell lines and differentiation methodologies were not predicted by the results of the in vitro assays. In addition, bone was formed equally well from iPSCs originating from skin or bone marrow stromal cells (also known as bone marrow-derived mesenchymal stem cells), suggesting that the iPSCs did not retain a “memory” of their previous life. Furthermore, one of the iPSC-derived cell lines formed verifiable cartilage in vivo, which likewise was not predicted by in vitro assays.
PMCID: PMC4073820  PMID: 24855277
Induced pluripotent stem cells; Bone; Osteoblast; Chondrogenesis; Transplantation
21.  Rare variants of large effect in BRCA2 and CHEK2 affect risk of lung cancer 
Wang, Yufei | McKay, James D. | Rafnar, Thorunn | Wang, Zhaoming | Timofeeva, Maria | Broderick, Peter | Zong, Xuchen | Laplana, Marina | Wei, Yongyue | Han, Younghun | Lloyd, Amy | Delahaye-Sourdeix, Manon | Chubb, Daniel | Gaborieau, Valerie | Wheeler, William | Chatterjee, Nilanjan | Thorleifsson, Gudmar | Sulem, Patrick | Liu, Geoffrey | Kaaks, Rudolf | Henrion, Marc | Kinnersley, Ben | Vallée, Maxime | LeCalvez-Kelm, Florence | Stevens, Victoria L. | Gapstur, Susan M. | Chen, Wei V. | Zaridze, David | Szeszenia-Dabrowska, Neonilia | Lissowska, Jolanta | Rudnai, Peter | Fabianova, Eleonora | Mates, Dana | Bencko, Vladimir | Foretova, Lenka | Janout, Vladimir | Krokan, Hans E. | Gabrielsen, Maiken Elvestad | Skorpen, Frank | Vatten, Lars | Njølstad, Inger | Chen, Chu | Goodman, Gary | Benhamou, Simone | Vooder, Tonu | Valk, Kristjan | Nelis, Mari | Metspalu, Andres | Lener, Marcin | Lubiński, Jan | Johansson, Mattias | Vineis, Paolo | Agudo, Antonio | Clavel-Chapelon, Francoise | Bueno-de-Mesquita, H.Bas | Trichopoulos, Dimitrios | Khaw, Kay-Tee | Johansson, Mikael | Weiderpass, Elisabete | Tjønneland, Anne | Riboli, Elio | Lathrop, Mark | Scelo, Ghislaine | Albanes, Demetrius | Caporaso, Neil E. | Ye, Yuanqing | Gu, Jian | Wu, Xifeng | Spitz, Margaret R. | Dienemann, Hendrik | Rosenberger, Albert | Su, Li | Matakidou, Athena | Eisen, Timothy | Stefansson, Kari | Risch, Angela | Chanock, Stephen J. | Christiani, David C. | Hung, Rayjean J. | Brennan, Paul | Landi, Maria Teresa | Houlston, Richard S. | Amos, Christopher I.
Nature genetics  2014;46(7):736-741.
We conducted imputation to the 1000 Genomes Project of four genome-wide association studies of lung cancer in populations of European ancestry (11,348 cases and 15,861 controls) and genotyped an additional 10,246 cases and 38,295 controls for follow-up. We identified large-effect genome-wide associations for squamous lung cancer with the rare variants of BRCA2-K3326X (rs11571833; odds ratio [OR]=2.47, P=4.74×10−20) and of CHEK2-I157T (rs17879961; OR=0.38 P=1.27×10−13). We also showed an association between common variation at 3q28 (TP63; rs13314271; OR=1.13, P=7.22×10−10) and lung adenocarcinoma previously only reported in Asians. These findings provide further evidence for inherited genetic susceptibility to lung cancer and its biological basis. Additionally, our analysis demonstrates that imputation can identify rare disease-causing variants having substantive effects on cancer risk from pre-existing GWAS data.
PMCID: PMC4074058  PMID: 24880342
22.  Genome-Wide Significant Localization for Working and Spatial Memory: Identifying Genes for Psychosis Using Models of Cognition 
It is well established that risk for developing psychosis is largely mediated by the influence of genes, but identifying precisely which genes underlie that risk has been problematic. Focusing on endophenotypes, rather than illness risk, is one solution to this problem. Impaired cognition is a well-established endophenotype of psychosis. Here we aimed to characterize the genetic architecture of cognition using phenotypically detailed models as opposed to relying on general IQ or individual neuropsychological measures. In so doing we hoped to identify genes that mediate cognitive ability which might also contribute to psychosis risk. Hierarchical factor models of genetically clustered cognitive traits were subjected to linkage analysis followed by QTL region-specific association analyses in a sample of 1,269 Mexican American individuals from extended pedigrees. We identified four genome wide significant QTLs, two for working and two for spatial memory, and a number of plausible and interesting candidate genes. The creation of detailed models of cognition seemingly enhanced the power to detect genetic effects on cognition and provided a number of possible candidate genes for psychosis.
PMCID: PMC4106137  PMID: 24243780
schizophrenia; genetics; cognition; GWAS; linkage
23.  Birthweight and risk markers for type 2 diabetes and cardiovascular disease in childhood: the Child Heart and Health Study in England (CHASE) 
Diabetologia  2014;58:474-484.
Lower birthweight (a marker of fetal undernutrition) is associated with higher risks of type 2 diabetes and cardiovascular disease (CVD) and could explain ethnic differences in these diseases. We examined associations between birthweight and risk markers for diabetes and CVD in UK-resident white European, South Asian and black African-Caribbean children.
In a cross-sectional study of risk markers for diabetes and CVD in 9- to 10-year-old children of different ethnic origins, birthweight was obtained from health records and/or parental recall. Associations between birthweight and risk markers were estimated using multilevel linear regression to account for clustering in children from the same school.
Key data were available for 3,744 (66%) singleton study participants. In analyses adjusted for age, sex and ethnicity, birthweight was inversely associated with serum urate and positively associated with systolic BP. After additional height adjustment, lower birthweight (per 100 g) was associated with higher serum urate (0.52%; 95% CI 0.38, 0.66), fasting serum insulin (0.41%; 95% CI 0.08, 0.74), HbA1c (0.04%; 95% CI 0.00, 0.08), plasma glucose (0.06%; 95% CI 0.02, 0.10) and serum triacylglycerol (0.30%; 95% CI 0.09, 0.51) but not with BP or blood cholesterol. Birthweight was lower among children of South Asian (231 g lower; 95% CI 183, 280) and black African-Caribbean origin (81 g lower; 95% CI 30, 132). However, adjustment for birthweight had no effect on ethnic differences in risk markers.
Birthweight was inversely associated with urate and with insulin and glycaemia after adjustment for current height. Lower birthweight does not appear to explain emerging ethnic difference in risk markers for diabetes.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-014-3474-7) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
PMCID: PMC4320299  PMID: 25520157
Birthweight; Cardiovascular disease; Childhood; Ethnicity; Type 2 diabetes
24.  Patient safety skills in primary care: a national survey of GP educators 
BMC Family Practice  2014;15(1):206.
Clinicians have a vital role in promoting patient safety that goes beyond their technical competence. The qualities and attributes of the safe hospital doctor have been explored but similar work within primary care is lacking. Exploring the skills and attributes of a safe GP may help to inform the development of training programmes to promote patient safety within primary care.
This study aimed to determine the views of General Practice Educational Supervisors (GPES) regarding the qualities and attributes of a safe General Practitioner (GP) and the perceived trainability of these ‘safety skills’ and to compare selected results with those generated by a previous study of hospital doctors.
This was a two-stage study comprising content validation of a safety skills questionnaire (originally developed for hospital doctors) (Stage 1) and a prospective survey of all GPES in Scotland (n = 691) (Stage 2).
Stage 1: The content-validated questionnaire comprised 66 safety skills/attributes across 17 broad categories with an overall content validation index of 0.92.
Stage 2: 348 (50%) GPES completed the survey. GPES felt the skills/attributes most important to being a safe GP were honesty (93%), technical clinical skills (89%) and conscientiousness (89%). That deemed least important/relevant to being a safe GP was leadership (36%). This contrasts sharply with the views of hospital doctors in the previous study. GPES felt the most trainable safety skills/attributes were technical skills (93%), situation awareness (75%) and anticipation/preparedness (71%). The least trainable were honesty (35%), humility (33%) and patient awareness/empathy (30%). Additional safety skills identified as relevant to primary care included patient advocacy, negotiation skills, accountability/ownership and clinical intuition (‘listening to that worrying little inner voice’).
GPES believe a broad range of skills and attributes contribute to being a safe GP. Important but subtle differences exist between what primary care and secondary care doctors perceive as core safety attributes. Educationalists, GPs and patient safety experts should collaborate to develop and implement training in these skills to ensure that current and future GPs possess the necessary competencies to engage and lead in safety improvement efforts.
Electronic supplementary material
The online version of this article (doi:10.1186/s12875-014-0206-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4275946  PMID: 25515429
General practice; Patient safety; Medical education; Skills
25.  Biomarkers in diabetic nephropathy: Present and future 
World Journal of Diabetes  2014;5(6):763-776.
Diabetic nephropathy (DN) is the leading cause of end stage renal disease in the Western world. Microalbuminuria (MA) is the earliest and most commonly used clinical index of DN and is independently associated with cardiovascular risk in diabetic patients. Although MA remains an essential tool for risk stratification and monitoring disease progression in DN, a number of factors have called into question its predictive power. Originally thought to be predictive of future overt DN in 80% of patients, we now know that only around 30% of microalbuminuric patients progress to overt nephropathy after 10 years of follow up. In addition, advanced structural alterations in the glomerular basement membrane may already have occurred by the time MA is clinically detectable.Evidence in recent years suggests that a significant proportion of patients with MA can revert to normoalbuminuria and the concept of nonalbuminuric DN is well-documented, reflecting the fact that patients with diabetes can demonstrate a reduction in glomerular filtration rate without progressing from normo-to MA. There is an unmet clinical need to identify biomarkers with potential for earlier diagnosis and risk stratification in DN and recent developments in this field will be the focus of this review article.
PMCID: PMC4265863  PMID: 25512779
Diabetes; Nephropathy; Microalbuminuria; Proteinuria; Biomarkers

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