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1.  Hyperinsulinemia Potentiates Airway Responsiveness to Parasympathetic Nerve Stimulation in Obese Rats 
Obesity is a substantial risk factor for developing asthma, but the molecular mechanisms underlying this relationship are unclear. We tested the role of insulin in airway responsiveness to nerve stimulation using rats genetically prone or resistant to diet-induced obesity. Airway response to vagus nerve stimulation and airway M2 and M3 muscarinic receptor function were measured in obese-prone and -resistant rats with high or low circulating insulin. The effects of insulin on nerve-mediated human airway smooth muscle contraction and human M2 muscarinic receptor function were tested in vitro. Our data show that increased vagally mediated bronchoconstriction in obesity is associated with hyperinsulinemia and loss of inhibitory M2 muscarinic receptor function on parasympathetic nerves. Obesity did not induce airway inflammation or increase airway wall thickness. Smooth muscle contraction to acetylcholine was not increased, indicating that hyperresponsiveness is mediated at the level of airway nerves. Reducing serum insulin with streptozotocin protected neuronal M2 receptor function and prevented airway hyperresponsiveness to vagus nerve stimulation in obese rats. Replacing insulin restored dysfunction of neuronal M2 receptors and airway hyperresponsiveness to vagus nerve stimulation in streptozotocin-treated obese rats. Treatment with insulin caused loss of M2 receptor function, resulting in airway hyperresponsiveness to vagus nerve stimulation in obese-resistant rats, and inhibited human neuronal M2 receptor function in vitro. This study shows that it is not obesity per se but hyperinsulinemia accompanying obesity that potentiates vagally induced bronchoconstriction by inhibiting neuronal M2 muscarinic receptors and increasing acetylcholine release from airway parasympathetic nerves.
PMCID: PMC4148040  PMID: 24605871
hyperinsulinemia; obesity; asthma; airway responsiveness; neural M2 muscarinic receptor
2.  A phase I dose escalation study of oral bexarotene in combination with intravenous decitabine in patients with AML 
American journal of hematology  2014;89(8):E103-E108.
The response rate of non-M3 AML to all trans retinoic acid (ATRA) has been limited. Using Affymetrix expression arrays, we found that in diverse AML blasts RXRA was expressed at higher levels than RARA and that mouse Ctsg-PML-RARA leukemia responded to bexarotene, a ligand for RXRA. We therefore performed a phase I study of combination bexarotene and decitabine in elderly and relapsed AML patients. We found that this combination was well tolerated, although outcomes were modest (1 CRi, and 3 PR among 19 patients). Correlative studies found that patients with clinical response had increased differentiation to bexarotene both in vivo and ex vivo, suggesting that pre-treatment analysis might identify a more susceptible subgroup of patients.
PMCID: PMC4108244  PMID: 24723466
3.  QnrS1 structure–activity relationships 
Loop B is important for low-level quinolone resistance conferred by Qnr proteins. The role of individual amino acids within QnrS1 loop B in quinolone resistance and gyrase protection was assessed.
qnrS1 and 11 qnrS1 alleles with site-directed Ala mutations in loop B were expressed in Escherichia coli BL21(DE3) and proteins were purified by affinity chromatography. Ciprofloxacin MICs were determined with and without IPTG. Gyrase DNA supercoiling was measured with and without ciprofloxacin IC50 and with various concentrations of QnrS1 proteins.
Wild-type QnrS1 and QnrS1 with Asn-110→Ala and Arg-111→Ala substitutions increased the ciprofloxacin MIC 12-fold in BL21(DE3), although QnrS1 with Gln-107→Ala replacement increased it 2-fold more than wild-type did. However, QnrS1 with Ala substitutions at His-106, Val-108, Ser-109, Met-112, Tyr-113, Phe-114, Cys-115 and Ser-116 increased ciprofloxacin MIC 1.4- to 8-fold less than wild-type QnrS1. Induction by 10–1000 μM IPTG increased ciprofloxacin MICs for all mutants, reaching values similar to those for wild-type. Purified wild-type and mutated proteins differed in protection of gyrase from ciprofloxacin action. Wild-type QnrS1 produced complete protection of gyrase supercoiling from ciprofloxacin (1.8 μM) action at 0.05 nM and half protection at 0.5 pM, whereas QnrS1 with Ala replacements that conferred the least increase in ciprofloxacin MICs also required the highest QnrS1 concentrations for protection.
Key individual residues in QnrS1 loop B affect ciprofloxacin resistance and gyrase protection from ciprofloxacin action, supporting the concept that loop B is key for interaction with gyrase necessary for quinolone resistance.
PMCID: PMC4100707  PMID: 24729602
pentapeptide repeat proteins; QnrS; quinolone resistance
4.  Tissue Optical Clearing, Three-Dimensional Imaging, and Computer Morphometry in Whole Mouse Lungs and Human Airways 
In whole adult mouse lung, full identification of airway nerves (or other cellular/subcellular objects) has not been possible due to patchy distribution and micron-scale size. Here we describe a method using tissue clearing to acquire the first complete image of three-dimensional (3D) innervation in the lung. We then created a method to pair analysis of nerve (or any other colabeled epitope) images with identification of 3D tissue compartments and airway morphometry by using fluorescent casting and morphometry software (which we designed and are making available as open-source). We then tested our method to quantify a sparse heterogeneous nerve population by examining visceral pleural nerves. Finally, we demonstrate the utility of our method in human tissue to image full thickness innervation in irregular 3D tissue compartments and to quantify sparse objects (intrinsic airway ganglia). Overall, this method can uniquely pair the advantages of whole tissue imaging and cellular/subcellular fluorescence microscopy.
PMCID: PMC4091855  PMID: 24471696
nerve; clearing; modeling; visceral pleura; morphometry
5.  Phase I Study of Oral Clofarabine Consolidation in Adults Aged 60 and Older with Acute Myeloid Leukemia 
American journal of hematology  2014;89(5):487-492.
Clofarabine has shown activity and tolerability in older patients with acute myeloid leukemia (AML). We investigated the safety and tolerability of an oral formulation of clofarabine for consolidation therapy of patients aged 60 and older with AML. In this phase I study, twenty-two patients older than 60 years with AML in first complete remission were treated once daily with oral clofarabine for 14 or 21 days of a 28 day cycle, for up to 5 cycles. Dose escalation from 1 mg to 6 mg daily using a 3 + 3 design was used to determine dose-limiting toxicities (DLT), the maximum tolerated dose (MTD), and tolerability of oral clofarabine. No DLTs or Grade 3-4 nonhematologic toxicities were observed. The primary toxicities were hematologic, including uncomplicated grade 3-4 neutropenia (50%) and thrombocytopenia (50%). Given that myelosuppression necessitating dose delays/reductions was observed more commonly at higher doses, the recommended phase II dose is 2 mg daily for 21 of 28 days. At doses equal to or greater than 2 mg, the median relapse-free survival was 28.35 months. Oral clofarabine was well-tolerated with encouraging activity in patients older than 60 years. Further investigation of oral clofarabine as a consolidation and/or maintenance therapy in AML for older individuals is warranted. (
PMCID: PMC4378713  PMID: 24415560
6.  Metastability in lipid based particles exhibits temporally deterministic and controllable behavior 
Scientific Reports  2015;5:9481.
The metastable-to-stable phase-transition is commonly observed in many fields of science, as an uncontrolled independent process, highly sensitive to microscopic fluctuations. In particular, self-assembled lipid suspensions exhibit phase-transitions, where the underlying driving mechanisms and dynamics are not well understood. Here we describe a study of the phase-transition dynamics of lipid-based particles, consisting of mixtures of dilauroylphosphatidylethanolamine (DLPE) and dilauroylphosphatidylglycerol (DLPG), exhibiting a metastable liquid crystalline-to-stable crystalline phase transition upon cooling from 60°C to 37°C. Surprisingly, unlike classically described metastable-to-stable phase transitions, the manner in which recrystallization is delayed by tens of hours is robust, predetermined and controllable. Our results show that the delay time can be manipulated by changing lipid stoichiometry, changing solvent salinity, adding an ionophore, or performing consecutive phase-transitions. Moreover, the delay time distribution indicates a deterministic nature. We suggest that the non-stochastic physical mechanism responsible for the delayed recrystallization involves several rate-affecting processes, resulting in a controllable, non-independent metastability. A qualitative model is proposed to describe the structural reorganization during the phase transition.
PMCID: PMC4377625  PMID: 25820650
7.  Epigenetic Therapy for Friedreich Ataxia 
Annals of neurology  2014;76(4):489-508.
To investigate whether a histone deacetylase inhibitor (HDACi) would be effective in an in vitro model for the neurodegenerative disease Friedreich ataxia (FRDA) and to evaluate safety and surrogate markers of efficacy in a phase I clinical trial in patients.
We used a human FRDA neuronal cell model, derived from patient induced pluripotent stem cells, to determine the efficacy of a 2-aminobenzamide HDACi (109) as a modulator of FXN gene expression and chromatin histone modifications. FRDA patients were dosed in 4 cohorts, ranging from 30mg/day to 240mg/day of the formulated drug product of HDACi 109, RG2833. Patients were monitored for adverse effects as well as for increases in FXN mRNA, frataxin protein, and chromatin modification in blood cells.
In the neuronal cell model, HDACi 109/RG2833 increases FXN mRNA levels and frataxin protein, with concomitant changes in the epigenetic state of the gene. Chromatin signatures indicate that histone H3 lysine 9 is a key residue for gene silencing through methylation and reactivation through acetylation, mediated by the HDACi. Drug treatment in FRDA patients demonstrated increased FXN mRNA and H3 lysine 9 acetylation in peripheral blood mononuclear cells. No safety issues were encountered.
Drug exposure inducing epigenetic changes in neurons in vitro is comparable to the exposure required in patients to see epigenetic changes in circulating lymphoid cells and increases in gene expression. These findings provide a proof of concept for the development of an epigenetic therapy for this fatal neurological disease.
PMCID: PMC4361037  PMID: 25159818
8.  Pedigreed Primate Embryonic Stem Cells Express Homogeneous Familial Gene Profiles 
Stem cells (Dayton, Ohio)  2007;25(11):2695-2704.
Human embryonic stem cells (hESCs) hold great biomedical promise, but experiments comparing them produce heterogeneous results, raising concerns regarding their reliability and utility, although these variations may result from their disparate and anonymous origins. To determine whether primate ESCs have intrinsic biological limitations compared with mouse ESCs, we examined expression profiles and pluripotency of newly established nonhuman primate ESC (nhpESCs). Ten pedigreed nhpESC lines, seven full siblings (fraternal quadruplets and fraternal triplets), and nine half siblings were derived from 41 rhesus embryos; derivation success correlated with embryo quality. Each line has been growing continuously for ~1 year with stable diploid karyo-type (except for one stable trisomy) and expresses in vitro pluripotency markers, and eight have already formed teratomas. Unlike the heterogeneous gene expression profiles found among hESCs, these nhpESCs display remarkably homogeneous profiles (>97%), with full-sibling lines nearly identical (>98.2%). Female nhpESCs express genes distinct from their brother lines; these sensitive analyses are enabled because of the very low background differences. Experimental comparisons among these primate ESCs may prove more reliable than currently available hESCs, since they are akin to inbred mouse strains in which genetic variables are also nearly eliminated. Finally, contrasting the biological similarities among these lines with the heterogeneous hESCs might suggest that additional, more uniform hESC lines are justified. Taken together, pedigreed primate ESCs display homogeneous and reliable expression profiles. These similarities to mouse ESCs suggest that heterogeneities found among hESCs likely result from their disparate origins rather than intrinsic biological limitations with primate embryonic stem cells.
PMCID: PMC4357318  PMID: 17641389
Embryonic stem cells; Pluripotency; Differentiation; Gene expression; Primates
9.  Disaster Preparedness of Poison Control Centers in the USA: A 15-year Follow-up Study 
Journal of Medical Toxicology  2013;10(1):19-25.
There is limited published literature on the extent to which United States (US) Poison Control Centers (PCCs) are prepared for responding to disasters. We describe PCCs' disaster preparedness activities and compare and contrast these results to those previously reported in the medical literature. We also describe the extent to which PCCs are engaged in disaster and terrorism preparedness planning and other public health roles such as surveillance. An electronic questionnaire was sent via email to the managing directors of the 57 member PCCs of the American Association of Poison Control Centers. Collected data included the population served and number of calls received, extent of disaster preparedness including the presence of a written disaster plan and elements included in that plan, the presence and nature of regular disaster drills, experience with disaster including periods of inability to operate, involvement in terrorism and disaster preparedness/response policy development, and public health surveillance of US PCCs. Descriptive statistics were performed on collected data. Comparisons with the results from a previously published survey were performed. A response was obtained from 40/57 (70 %) PCCs. Each PCC serves a larger population (p < 0.0001) and receives more calls per year (p = 0.0009) than the previous descriptions of PCC preparedness. More centers report the presence of a written disaster plan (p < 0.0001), backup by another center (p < 0.0001), regular disaster drills (p < 0.0001), and comfort with ability to operate in a disaster (p < 0.0001) than previously described. PCCs are involved in disaster (34/40, 85 %) and terrorism (29/40, 73 %) preparedness at the local, state, or federal levels. PCCs (36/40, 90 %) are also involved in public health functions (illness surveillance or answering “after hours” public health calls). Despite an increase in calls received and population served per center as compared to previous descriptions, more PCCs report the presence of a written disaster plan, backup by another center, regular disaster drills, and comfort in ability to operate in a disaster. PCCs are actively involved in terrorism and disaster preparedness and response planning and traditional public health responsibilities such as surveillance.
PMCID: PMC3951635  PMID: 23842905
Poison Control Center; Disasters; Disaster planning; Terrorism; Mass casualty incidents
10.  Myocardial T2 mapping reveals age- and sex-related differences in volunteers 
T2 mapping indicates to be a sensitive method for detection of tissue oedema hidden beyond the detection limits of T2-weighted Cardiovascular Magnetic Resonance (CMR). However, due to variability of baseline T2 values in volunteers, reference values need to be defined. Therefore, the aim of the study was to investigate the effects of age and sex on quantitative T2 mapping with a turbo gradient-spin-echo (GRASE) sequence at 1.5 T. For that reason, we studied sensitivity issues as well as technical and biological effects on GRASE-derived myocardial T2 maps. Furthermore, intra- and interobserver variability were calculated using data from a large volunteer group.
GRASE-derived multiecho images were analysed using dedicated software. After sequence optimization, validation and sensitivity measurements were performed in muscle phantoms ex vivo and in vivo. The optimized parameters were used to analyse CMR images of 74 volunteers of mixed sex and a wide range of age with typical prevalence of hypertension and diabetes. Myocardial T2 values were analysed globally and according to the 17 segment model. Strain-encoded (SENC) imaging was additionally performed to investigate possible effects of myocardial strain on global or segmental T2 values.
Ex vivo studies in muscle phantoms showed, that GRASE-derived T2 values were comparable to those acquired by a standard multiecho spinecho sequence but faster by a factor of 6. Besides that, T2 values reflected tissue water content. The in vivo measurements in volunteers revealed intra- and interobserver correlations with R2=0.91 and R2=0.94 as well as a coefficients of variation of 2.4% and 2.2%, respectively. While global T2 time significantly decreased towards the heart basis, female volunteers had significant higher T2 time irrespective of myocardial region. We found no correlation of segmental T2 values with maximal systolic, diastolic strain or heart rate. Interestingly, volunteers´ age was significantly correlated to T2 time while that was not the case for other coincident cardiovascular risk factors.
GRASE-derived T2 maps are highly reproducible. However, female sex and aging with typical prevalence of hypertension and diabetes were accompanied by increased myocardial T2 values. Thus, sex and age must be considered as influence factors when using GRASE in a diagnostic manner.
Electronic supplementary material
The online version of this article (doi:10.1186/s12968-015-0118-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4318191  PMID: 25656484
Cardiovascular magnetic resonance; T2 mapping; Volunteer study; SENC
14.  The natural history of conducting and reporting clinical trials: interviews with trialists 
Trials  2015;16:16.
To investigate the nature of the research process as a whole, factors that might influence the way in which research is carried out, and how researchers ultimately report their findings.
Semi-structured qualitative telephone interviews with authors of trials, identified from two sources: trials published since 2002 included in Cochrane systematic reviews selected for the ORBIT project; and trial reports randomly sampled from 14,758 indexed on PubMed over the 12-month period from August 2007 to July 2008.
A total of 268 trials were identified for inclusion, 183 published since 2002 and included in the Cochrane systematic reviews selected for the ORBIT project and 85 randomly selected published trials indexed on PubMed. The response rate from researchers in the former group was 21% (38/183) and in the latter group was 25% (21/85). Overall, 59 trialists were interviewed from the two different sources. A number of major but related themes emerged regarding the conduct and reporting of trials: establishment of the research question; identification of outcome variables; use of and adherence to the study protocol; conduct of the research; reporting and publishing of findings. Our results reveal that, although a substantial proportion of trialists identify outcome variables based on their clinical experience and knowing experts in the field, there can be insufficient reference to previous research in the planning of a new trial. We have revealed problems with trial recruitment: not reaching the target sample size, over-estimation of recruitment potential and recruiting clinicians not being in equipoise. We found a wide variation in the completeness of protocols, in terms of detailing study rationale, outlining the proposed methods, trial organisation and ethical considerations.
Our results confirm that the conduct and reporting of some trials can be inadequate. Interviews with researchers identified aspects of clinical research that can be especially challenging: establishing appropriate and relevant outcome variables to measure, use of and adherence to the study protocol, recruiting of study participants and reporting and publishing the study findings. Our trialists considered the prestige and impact factors of academic journals to be the most important criteria for selecting those to which they would submit manuscripts.
Electronic supplementary material
The online version of this article (doi:10.1186/s13063-014-0536-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4322554  PMID: 25619208
Qualitative; Interviews; Trialists; Research reporting; Recruitment; Trial protocols; Equipoise
15.  Plasmid-mediated quinolone resistance 
Microbiology spectrum  2014;2(2):10.1128/microbiolspec.PLAS-0006-2013.
Three mechanisms for plasmid-mediated quinolone resistance (PMQR) have been discovered since 1998. Plasmid genes qnrA, qnrB, qnrC, qnrD, qnrS, and qnrVC code for proteins of the pentapeptide repeat family that protects DNA gyrase and topoisomerase IV from quinolone inhibition. The qnr genes appear to have been acquired from chromosomal genes in aquatic bacteria, are usually associated with mobilizing or transposable elements on plasmids, and are often incorporated into sul1-type integrons. The second plasmid-mediated mechanism involves acetylation of quinolones with an appropriate amino nitrogen target by a variant of the common aminoglycoside acetyltransferase AAC(6′)-Ib. The third mechanism is enhanced efflux produced by plasmid genes for pumps QepAB and OqxAB. PMQR has been found in clinical and environmental isolates around the world and appears to be spreading. The plasmid-mediated mechanisms provide only low-level resistance that by itself does not exceed the clinical breakpoint for susceptibility but nonetheless facilitates selection of higher-level resistance and makes infection by pathogens containing PMQR harder to treat.
PMCID: PMC4288778  PMID: 25584197
16.  The Role of Reminding in the Effects of Spaced Repetitions on Cued Recall: Sufficient but not Necessary 
Three experiments examined the role of study-phase retrieval (reminding) in the effects of spaced repetitions on cued recall. Remindings were brought under task control to evaluate their effects. Participants studied two lists of word pairs containing three item types: single items that appeared once in List 2, within-list repetitions that appeared twice in List 2, and between-list repetitions that appeared once in List 1 and once in List 2. Our primary interest was in performance on between-list repetitions. Detection of between-list repetitions was encouraged in an n-back condition by instructing participants to indicate when a presented item was a repetition of any preceding item, including items presented in List 1. In contrast, detection of between-list repetitions was discouraged in a within-list back condition by instructing participants only to indicate repetitions occurring in List 2. Cued recall of between-list repetitions was enhanced when instructions encouraged detection of List 1 presentations. These results accord with those from prior experiments showing a role of study-phase retrieval in effects of spacing repetitions. Past experiments have relied on conditionalized data to draw conclusions, producing the possibility that performance benefits merely reflected effects of item selection. By bringing effects under task control, we avoided that problem. Our results provide evidence that reminding resulting from retrieval of earlier presentations plays a role in the effects of spaced repetitions on cued recall. However, our results also reveal that such retrievals are not necessary to produce an effect of spacing repetitions.
PMCID: PMC4032790  PMID: 23937236
cognitive control; reminding; repetition effects; spacing effects; study-phase retrieval
17.  In Memoriam: Robert C. Moellering, Jr. 
PMCID: PMC4068561  PMID: 24798286
18.  Protective effect of CMV reactivation on relapse after allogeneic hematopoietic cell transplantation in AML patients is influenced by their conditioning regimen 
Cytomegalovirus (CMV) reactivation after allogeneic hematopoietic cell transplant (allo-HCT) has been associated with reduced risk of relapse in patients with acute myeloid leukemia (AML). However the influence of the conditioning regimen on this protective effect of CMV reactivation after allo-HCT is relatively unexplored. To address this, we evaluated the risk of relapse in 264 AML patients who received T cell replete, 6/6 HLA matched sibling or 10/10 HLA matched unrelated donor transplantation at a single institution between 2006 and 2011. Out of these 264 patients, 206 received myeloablative (MA) and 58 received reduced intensity conditioning (RIC) regimens. CMV reactivation was observed in 88 patients with MA conditioning and 37 patients with RIC. At a median follow up of 299 days, CMV reactivation was associated with significantly lower risk of relapse in patients who received MA conditioning both in univariate (P= .01) and multivariate analyses (hazard ratio of 0.5246, P= .006), however CMV reactivation did not significantly affect the risk of relapse in our RIC cohort. These results confirm the protective effect of CMV reactivation on relapse in AML patients after allo-HCT reported by previous studies, however they suggest that this protective effect of CMV reactivation on relapse is influenced by the conditioning regimen used with the transplant.
PMCID: PMC4029772  PMID: 24120526
19.  Early Mortality and Primary Causes of Death in Mothers of Children with Intellectual Disability or Autism Spectrum Disorder: A Retrospective Cohort Study 
PLoS ONE  2014;9(12):e113430.
Mothers of children with intellectual disability or autism spectrum disorder (ASD) have poorer health than other mothers. Yet no research has explored whether this poorer health is reflected in mortality rates or whether certain causes of death are more likely. We aimed to calculate the hazard ratios for death and for the primary causes of death in mothers of children with intellectual disability or ASD compared to other mothers.
The study population comprised all mothers of live-born children in Western Australia from 1983–2005. We accessed state-wide databases which enabled us to link socio-demographic details, birth dates, diagnoses of intellectual disability or ASD in the children and dates and causes of death for all mothers who had died prior to 2011. Using Cox Regression with death by any cause and death by each of the three primary causes as the event of interest, we calculated hazard ratios for death for mothers of children intellectual disability or ASD compared to other mothers.
Results and Discussion
During the study period, mothers of children with intellectual disability or ASD had more than twice the risk of death. Mothers of children with intellectual disability were 40% more likely to die of cancer; 150% more likely to die of cardiovascular disease and nearly 200% more likely to die from misadventure than other mothers. Due to small numbers, only hazard ratios for cancer were calculated for mothers of children with ASD. These mothers were about 50% more likely to die from cancer than other mothers. Possible causes and implications of our results are discussed.
Similar studies, pooling data from registries elsewhere, would improve our understanding of factors increasing the mortality of mothers of children with intellectual disability or ASD. This would allow the implementation of informed services and interventions to improve these mothers' longevity.
PMCID: PMC4275172  PMID: 25535971
20.  The Application of the Open Pharmacological Concepts Triple Store (Open PHACTS) to Support Drug Discovery Research 
PLoS ONE  2014;9(12):e115460.
Integration of open access, curated, high-quality information from multiple disciplines in the Life and Biomedical Sciences provides a holistic understanding of the domain. Additionally, the effective linking of diverse data sources can unearth hidden relationships and guide potential research strategies. However, given the lack of consistency between descriptors and identifiers used in different resources and the absence of a simple mechanism to link them, gathering and combining relevant, comprehensive information from diverse databases remains a challenge. The Open Pharmacological Concepts Triple Store (Open PHACTS) is an Innovative Medicines Initiative project that uses semantic web technology approaches to enable scientists to easily access and process data from multiple sources to solve real-world drug discovery problems. The project draws together sources of publicly-available pharmacological, physicochemical and biomolecular data, represents it in a stable infrastructure and provides well-defined information exploration and retrieval methods. Here, we highlight the utility of this platform in conjunction with workflow tools to solve pharmacological research questions that require interoperability between target, compound, and pathway data. Use cases presented herein cover 1) the comprehensive identification of chemical matter for a dopamine receptor drug discovery program 2) the identification of compounds active against all targets in the Epidermal growth factor receptor (ErbB) signaling pathway that have a relevance to disease and 3) the evaluation of established targets in the Vitamin D metabolism pathway to aid novel Vitamin D analogue design. The example workflows presented illustrate how the Open PHACTS Discovery Platform can be used to exploit existing knowledge and generate new hypotheses in the process of drug discovery.
PMCID: PMC4270790  PMID: 25522365
21.  Growth and metastasis of B16-F10 melanoma cells is not critically dependent on host CD73 expression in mice 
BMC Cancer  2014;14:898.
Recent studies have suggested that adenosine generated by ecto-5′-nucleotidase (CD73) in the tumor microenvironment plays a major role in promoting tumor growth by suppressing the immune response and stimulating angiogenesis via A2A and A2B receptors. However, adenosine has also been reported to inhibit tumor growth acting via A1 and A3 receptors. Therefore the aim of this study was to clarify the role of host CD73, which catalyzes the extracellular hydrolysis of AMP to adenosine, on tumor growth and metastasis of B16-F10 melanoma cells.
CD73 and alkaline phosphatase (AP) activity of B16-F10 melanoma cells were measured by HPLC. Tumor cells were injected either subcutaneously or intradermally in WT and CD73−/− mice and tumor growth was monitored by MRI at 9.4 T. Immune cell subpopulations within tumors were assessed by FACS after enzymatic digestion. An endothelium specific CD73−/− was created using Tie2-Cre+ mice and CD73flox/flox (loxP) mice. Chimeric mice lacking CD73−/− on hematopoietic cells was generated by bone marrow transplantation. Lung metastatic spread was measured after intravenous B16-F10 application.
B16-F10 cells showed very little CD73 and negligible AP activity. Neither complete loss of host CD73 nor specific knockout of CD73 on endothelial cells or hematopoietic cells affected tumor growth after subcutaneous or intradermal tumor cell application. Only peritumoral edema formation was significantly attenuated in global CD73−/− mice in the intradermal model. Immune cell composition revealed no differences in the different transgenic mice models. Also lung metastasis after intravenous B16-F10 injection was not altered in CD73−/− mice.
CD73 expression on host cells, particularly on endothelial and hematopoietic cells, does not modulate tumor growth and metastatic spread of B16-F10 melanoma cells most likely because of insufficient adenosine formation by the tumor itself.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-898) contains supplementary material, which is available to authorized users.
PMCID: PMC4265456  PMID: 25465225
CD73; B16-F10 melanoma; Adenosine; Immune system; Tumor; Mice
22.  Risk factors and clinical characteristics of patients with qnr-positive Klebsiella pneumoniae bacteraemia 
Journal of Antimicrobial Chemotherapy  2013;68(12):2907-2914.
Plasmid-mediated quinolone resistance (PMQR) caused by qnr genes has been known for 15 years. Information about global distribution and prevalence of qnr genes is abundant, but clinical information concerning infections produced by these isolates and risk factors for their acquisition is limited.
Klebsiella pneumoniae blood isolates (n = 227) from a 1 year prospective cohort of patients in Taiwan were studied. MICs of quinolones were determined for all isolates, and multiplex PCR for the presence of PMQR genes and DNA gyrase mutations was applied to all 24 isolates with ciprofloxacin MICs ≥0.12 mg/L and a control group of 72 isolates with MICs ≤0.06 mg/L.
All qnr isolates were in the group with ciprofloxacin MICs ≥0.12 mg/L, constituting 9.4% of tested isolates and 3.9% (qnrB 2.6% and qnrS 1.3%) of total isolates. aac(6′)-Ib-cr and qepA were not found. Risk factors for qnr included nosocomial infection, bedridden status, surgery within 3 months, non-K1/K2 serotypes and prior antimicrobial use. Ciprofloxacin MIC ≥0.12 mg/L was associated with prior quinolone use; in contrast, prior cephalosporin use was more closely linked to the presence of qnr. Fourteen-day mortality was similar in patients infected with qnr-positive versus qnr-negative isolates, but there was a trend for increased in-hospital mortality in patients infected with qnr-positive isolates.
In K. pneumoniae blood isolates collected at a hospital in Taiwan, the overall prevalence of qnr genes was 3.9%. Prior quinolone use was linked to increased ciprofloxacin MIC, but not with the prevalence of qnr, which was most strongly linked to exposure to other antimicrobials, especially cephalosporins.
PMCID: PMC3820109  PMID: 23956373
K. pneumoniae; PMQR; resistance
23.  One-bone forearm procedure for Hajdu–Cheney syndrome: a case report 
Hand (New York, N.Y.)  2013;8(4):479-482.
One-bone forearm surgery is generally regarded as one of the last available salvage procedures that could be used to treat patients with longitudinal forearm instability secondary to a congenital, oncologic, or a post-traumatic etiology. We performed this procedure on a 23-year-old patient with longitudinal forearm instability secondary to Hajdu–Cheney syndrome, which is a rare genetic disorder characterized by generalized ligamentous laxity, skeletal dysplasia, acro-osteolysis, and generalized osteoporosis. The patient developed shoulder pain secondary to overuse 28 months following treatment, and was managed conservatively. Eight years after surgery, the patient had not undergone any additional procedures, had no pain, reported a Quick Disabilities of the Arm Shoulder and Hand score of 21, and was completely satisfied with treatment. Although OBF procedure is a radical first-line salvage option, in unique circumstances and appropriate patient selection, it may provide acceptable, durable, and predictable results.
PMCID: PMC3840764  PMID: 24426971
24.  The DNA Double-Strand Break Response Is Abnormal in Myeloblasts From Patients With Therapy-Related Acute Myeloid Leukemia 
Leukemia  2013;28(6):1242-1251.
The complex chromosomal aberrations found in therapy related acute myeloid leukemia (t-AML) suggest that the DNA double strand break (DSB) response may be altered. In this study we examined the DNA DSB response of primary bone marrow cells from t-AML patients and performed next-generation sequencing of 37 canonical homologous recombination (HR) and non-homologous end-joining (NHEJ) DNA repair genes, and a subset of DNA damage response genes using tumor and paired normal DNA obtained from t-AML patients. Our results suggest that the majority of t-AML patients (11 of 15) have tumor cell-intrinsic, functional dysregulation of their DSB response. Distinct patterns of abnormal DNA damage response in myeloblasts correlated with acquired genetic alterations in TP53 and the presence of inferred chromothripsis. Furthermore, the presence of trisomy 8 in tumor cells was associated with persistently elevated levels of DSBs. Although tumor-acquired point mutations or small indels in canonical HR and NHEJ genes do not appear to be a dominant means by which t-AML leukemogenesis occurs, our functional studies suggest that an abnormal response to DNA damage is a common finding in t-AML.
PMCID: PMC4047198  PMID: 24304937
therapy-related AML; DNA damage; DNA repair; Trisomy 8

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