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1.  Ribosome Synthesis and MAPK Activity Modulate Ionizing Radiation-Induced Germ Cell Apoptosis in Caenorhabditis elegans 
PLoS Genetics  2013;9(11):e1003943.
Synthesis of ribosomal RNA by RNA polymerase I (RNA pol I) is an elemental biological process and is key for cellular homeostasis. In a forward genetic screen in C. elegans designed to identify DNA damage-response factors, we isolated a point mutation of RNA pol I, rpoa-2(op259), that leads to altered rRNA synthesis and a concomitant resistance to ionizing radiation (IR)-induced germ cell apoptosis. This weak apoptotic IR response could be phenocopied when interfering with other factors of ribosome synthesis. Surprisingly, despite their resistance to DNA damage, rpoa-2(op259) mutants present a normal CEP-1/p53 response to IR and increased basal CEP-1 activity under normal growth conditions. In parallel, rpoa-2(op259) leads to reduced Ras/MAPK pathway activity, which is required for germ cell progression and physiological germ cell death. Ras/MAPK gain-of-function conditions could rescue the IR response defect in rpoa-2(op259), pointing to a function for Ras/MAPK in modulating DNA damage-induced apoptosis downstream of CEP-1. Our data demonstrate that a single point mutation in an RNA pol I subunit can interfere with multiple key signalling pathways. Ribosome synthesis and growth-factor signalling are perturbed in many cancer cells; such an interplay between basic cellular processes and signalling might be critical for how tumours evolve or respond to treatment.
Author Summary
Maintenance of genome integrity and tissue homeostasis is critically important for organisms to survive. For adequate responses to DNA damage or other types of stress, cells have evolved intricate surveillance mechanisms such as repair, cell cycle arrest and apoptosis. Disruption of these response pathways leads to an enhanced probability of cancer. The nematode C. elegans has proven a valuable model to study the genetic basis of apoptosis as an adaptive response to genotoxic or cytotoxic insults. Here, we have used C. elegans in a non-biased genetic screen to identify further regulators of DNA damage-induced apoptosis and find that ribosome synthesis factors critically determine cell death levels. We show that the newly isolated mutant rpoa-2(op259), which has a ribosomal RNA synthesis defect, is resistant to apoptosis as a result of concurrent alterations in p53 and Ras/MAP kinase signalling. Changes in ribosome synthesis and altered Ras/MAPK activity can be observed in many tumor types, and perturbed p53 activity is among the most frequently detected abnormities in human cancer. The interaction between ribosome synthesis and major signalling pathways is potentially of high relevance for tumor biology.
doi:10.1371/journal.pgen.1003943
PMCID: PMC3836707  PMID: 24278030
2.  K-RAS Mutant Pancreatic Tumors Show Higher Sensitivity to MEK than to PI3K Inhibition In Vivo 
PLoS ONE  2012;7(8):e44146.
Activating K-RAS mutations occur at a frequency of 90% in pancreatic cancer, and to date no therapies exist targeting this oncogene. K-RAS signals via downstream effector pathways such as the MAPK and the PI3K signaling pathways, and much effort has been focused on developing drugs targeting components of these pathways. To better understand the requirements for K-RAS and its downstream signaling pathways MAPK and PI3K in pancreatic tumor maintenance, we established an inducible K-RAS knock down system that allowed us to ablate K-RAS in established tumors. Knock down of K-RAS resulted in impaired tumor growth in all pancreatic xenograft models tested, demonstrating that K-RAS expression is indeed required for tumor maintenance of K-RAS mutant pancreatic tumors. We further examined signaling downstream of K-RAS, and detected a robust reduction of pERK levels upon K-RAS knock down. In contrast, no effect on pAKT levels could be observed due to almost undetectable basal expression levels. To investigate the requirement of the MAPK and the PI3K pathways on tumor maintenance, three selected pancreatic xenograft models were tested for their response to MEK or PI3K inhibition. Tumors of all three models regressed upon MEK inhibition, but showed less pronounced response to PI3K inhibition. The effect of MEK inhibition on pancreatic xenografts could be enhanced further by combined application of a PI3K inhibitor. These data provide further rationale for testing combinations of MEK and PI3K inhibitors in clinical trials comprising a patient population with pancreatic cancer harboring mutations in K-RAS.
doi:10.1371/journal.pone.0044146
PMCID: PMC3432074  PMID: 22952903
3.  Towards crystal structure prediction of complex organic compounds – a report on the fifth blind test 
Following on from the success of the previous crystal structure prediction blind tests (CSP1999, CSP2001, CSP2004 and CSP2007), a fifth such collaborative project (CSP2010) was organized at the Cambridge Crystallographic Data Centre. A range of methodologies was used by the participating groups in order to evaluate the ability of the current computational methods to predict the crystal structures of the six organic molecules chosen as targets for this blind test. The first four targets, two rigid molecules, one semi-flexible molecule and a 1:1 salt, matched the criteria for the targets from CSP2007, while the last two targets belonged to two new challenging categories – a larger, much more flexible molecule and a hydrate with more than one polymorph. Each group submitted three predictions for each target it attempted. There was at least one successful prediction for each target, and two groups were able to successfully predict the structure of the large flexible molecule as their first place submission. The results show that while not as many groups successfully predicted the structures of the three smallest molecules as in CSP2007, there is now evidence that methodologies such as dispersion-corrected density functional theory (DFT-D) are able to reliably do so. The results also highlight the many challenges posed by more complex systems and show that there are still issues to be overcome.
doi:10.1107/S0108768111042868
PMCID: PMC3222142  PMID: 22101543
4.  Towards crystal structure prediction of complex organic compounds – a report on the fifth blind test 
The results of the fifth blind test of crystal structure prediction, which show important success with more challenging large and flexible molecules, are presented and discussed.
Following on from the success of the previous crystal structure prediction blind tests (CSP1999, CSP2001, CSP2004 and CSP2007), a fifth such collaborative project (CSP2010) was organized at the Cambridge Crystallographic Data Centre. A range of methodologies was used by the participating groups in order to evaluate the ability of the current computational methods to predict the crystal structures of the six organic molecules chosen as targets for this blind test. The first four targets, two rigid molecules, one semi-flexible molecule and a 1:1 salt, matched the criteria for the targets from CSP2007, while the last two targets belonged to two new challenging categories – a larger, much more flexible molecule and a hydrate with more than one polymorph. Each group submitted three predictions for each target it attempted. There was at least one successful prediction for each target, and two groups were able to successfully predict the structure of the large flexible molecule as their first place submission. The results show that while not as many groups successfully predicted the structures of the three smallest molecules as in CSP2007, there is now evidence that methodologies such as dispersion-corrected density functional theory (DFT-D) are able to reliably do so. The results also highlight the many challenges posed by more complex systems and show that there are still issues to be overcome.
doi:10.1107/S0108768111042868
PMCID: PMC3222142  PMID: 22101543
prediction; blind test; polymorph; crystal structure prediction
5.  Impact of ETIF Deletion on Safety and Immunogenicity of Equine Herpesvirus Type 1-Vectored Vaccines ▿ ‡  
Journal of Virology  2010;84(22):11602-11613.
Heterologous gene transfer by viral vector systems is often limited by factors such as preexisting immunity, toxicity, low packaging capacity, or weak immunogenic potential. A novel viral vector system derived from equine herpesvirus type 1 (EHV-1) not only overcomes some of these obstacles but also promotes the robust expression of a delivered transgene and the induction of antigen-specific immune responses. Regarding an enhanced safety profile, we assessed the impact of the gene encoding the sole essential tegument protein, ETIF, on the replication and immunogenicity of recombinant EHVs. The deletion of ETIF severely attenuates replication in permissive RK13 cells and a human lung epithelial cell line but without influencing transgene expression. Whereas the intranasal administration of a recombinant luciferase EHV in BALB/c mice resulted in transgene expression in nasal cavities and lungs for 5 to 6 days, the ETIF deletion limited expression to 2 days and resulted in 30-fold-less luminescence. Attenuated replication was accompanied by a decreased capacity to induce CD8+ T cells against a delivered HIV Gag transgene in BALB/c mice following repeated intranasal application. However, a single subcutaneous immunization with a gag DNA vaccine primed specific T cells for substantial expansion by two subsequent intranasal booster immunizations with either the gag recombinant ETIF mutant or the parental virus. In addition to inducing Gag-specific serum antibodies, this prime-boost strategy clearly outperformed three sequential immunizations with the parental or EHV-ΔETIF virus or repeated DNA vaccination by inducing substantial specific secretory IgA (sIgA) titers.
doi:10.1128/JVI.00677-10
PMCID: PMC2977905  PMID: 20826695
6.  Enhancement of the efficiency of non-viral gene delivery by application of pulsed magnetic field 
Nucleic Acids Research  2006;34(5):e40.
New approaches to increase the efficiency of non-viral gene delivery are still required. Here we report a simple approach that enhances gene delivery using permanent and pulsating magnetic fields. DNA plasmids and novel DNA fragments (PCR products) containing sequence encoding for green fluorescent protein were coupled to polyethylenimine coated superparamagnetic nanoparticles (SPIONs). The complexes were added to cells that were subsequently exposed to permanent and pulsating magnetic fields. Presence of these magnetic fields significantly increased the transfection efficiency 40 times more than in cells not exposed to the magnetic field. The transfection efficiency was highest when the nanoparticles were sedimented on the permanent magnet before the application of the pulsating field, both for small (50 nm) and large (200–250 nm) nanoparticles. The highly efficient gene transfer already within 5 min shows that this technique is a powerful tool for future in vivo studies, where rapid gene delivery is required before systemic clearance or filtration of the gene vectors occurs.
doi:10.1093/nar/gkl035
PMCID: PMC1408310  PMID: 16540591
7.  Cellular uptake of imatinib into leukemic cells is independent of human organic cation transporter 1 (OCT1) 
Purpose
In addition to mutated BCR-ABL1 kinase, the organic cation transporter 1 (OCT1, encoded by SLC22A1) has been considered to contribute to imatinib resistance in patients with chronic myeloid leukemia (CML). Since data are conflicting as to whether OCT1 transports imatinib and may serve as clinical biomarker we used a combination of different approaches including animal experiments to elucidate comprehensively the impact of OCT1 on cellular imatinib uptake.
Experimental Design
Transport of imatinib was studied using OCT1-expressing Xenopus oocytes, mammalian cell lines (HEK293, MDCK, V79) stably expressing OCT1, human leukemic cells, and Oct1-knockout mice. OCT1 mRNA and protein expression were analyzed in leukemic cells from imatinib naïve CML patients as well as in cell lines.
Results
Transport and inhibition studies showed that overexpression of functional OCT1 protein in Xenopus oocytes or mammalian cell lines did not lead to an increased cellular accumulation of imatinib. The CML cell lines (K562, Meg-01, LAMA84) and leukemic cells from patients expressed neither OCT1 mRNA nor protein as demonstrated by immunoblotting and immunofluorescence microscopy, yet they showed a considerable imatinib uptake. Oct1 deficiency in mice had no influence on plasma and hepatic imatinib concentrations.
Conclusions
These data clearly demonstrate that cellular uptake of imatinib is independent of OCT1 and therefore OCT1 is apparently not a valid biomarker for imatinib resistance.
doi:10.1158/1078-0432.CCR-13-1999
PMCID: PMC3932302  PMID: 24352644
imatinib resistance; organic cation transporter 1; CML; drug transport; SLC22A1
8.  Constitutive mitochondrial DNA copy number in peripheral blood of melanoma families with and without CDKN2A mutations 
Journal of carcinogenesis & mutagenesis  2014;2014(Suppl 4):006.
Quantitative changes in mitochondrial DNA (mtDNA) have been associated with the risk of a number of human cancers; however, the relationship between constitutive mtDNA copy number in blood and the risk of familial cutaneous malignant melanoma (CMM) has not been reported. We measured mtDNA copy number using quantitative PCR in blood-derived DNA from 136 CMM cases and 302 controls in 53 melanoma-prone families (23 segregating CDKN2A germline mutations). MtDNA copy number did not vary by age, sex, pigmentation characteristics, or CMM status. However, germline CDKN2A mutation carriers had significantly higher mean mtDNA copy number compared to non-carriers, particularly among CMM cases (geometric mean mtDNA copy number of 144 and 111 for carrier versus non-carrier, respectively; P= 0.02). When adjusting for age, sex, and familial correlation, having increasing mtDNA copy number was significantly associated with CDKN2A mutation status among CMM cases (OR=1.47, Ptrend=0.024). In particular, individuals with specific CDKN2A mutations with the potential to inactivate or reduce the level of the p16-INK4 reactive oxygen species (ROS) protective function had significantly increased mtDNA copy number levels (P=0.035). Future research in prospective studies is required to validate these findings and to further investigate mtDNA copy number in both blood and melanoma tissues in relation to CMM risk and CDKN2A mutation status.
doi:10.4172/2157-2518.S4-006
PMCID: PMC4326067
Familial melanoma; CDKN2A; mtDNA copy number; peripheral blood
9.  Comprehensive MALDI-TOF Biotyping of the Non-Redundant Harvard Pseudomonas aeruginosa PA14 Transposon Insertion Mutant Library 
PLoS ONE  2015;10(2):e0117144.
Background
Pseudomonas aeruginosa is a gram-negative bacterium that is ubiquitously present in the aerobic biosphere. As an antibiotic-resistant facultative pathogen, it is a major cause of hospital-acquired infections. Its rapid and accurate identification is crucial in clinical and therapeutic environments.
Methods
In a large-scale MALDI-TOF mass spectrometry-based screen of the Harvard transposon insertion mutant library of P. aeruginosa strain PA14, intact-cell proteome profile spectra of 5547 PA14 transposon mutants exhibiting a plethora of different phenotypes were acquired and analyzed.
Results
Of all P. aeruginosa PA14 mutant profiles 99.7% were correctly identified as P. aeruginosa with the Biotyper software on the species level. On the strain level, 99.99% of the profiles were mapped to five different individual P. aeruginosa Biotyper database entries. A principal component analysis-based approach was used to determine the most important discriminatory mass features between these Biotyper groups. Although technical replicas were consistently categorized to specific Biotyper groups in 94.2% of the mutant profiles, biological replicas were not, indicating that the distinct proteotypes are affected by growth conditions.
Conclusions
The PA14 mutant profile collection presented here constitutes the largest coherent P. aeruginosa MALDI-TOF spectral dataset publicly available today. Transposon insertions in thousands of different P. aeruginosa genes did not affect species identification from MALDI-TOF mass spectra, clearly demonstrating the robustness of the approach. However, the assignment of the individual spectra to sub-groups proved to be non-consistent in biological replicas, indicating that the differentiation between biotyper groups in this nosocomial pathogen is unassured.
doi:10.1371/journal.pone.0117144
PMCID: PMC4321832  PMID: 25665154
10.  Knowledge Retrieval from PubMed Abstracts and Electronic Medical Records with the Multiple Sclerosis Ontology 
PLoS ONE  2015;10(2):e0116718.
Background
In order to retrieve useful information from scientific literature and electronic medical records (EMR) we developed an ontology specific for Multiple Sclerosis (MS).
Methods
The MS Ontology was created using scientific literature and expert review under the Protégé OWL environment. We developed a dictionary with semantic synonyms and translations to different languages for mining EMR. The MS Ontology was integrated with other ontologies and dictionaries (diseases/comorbidities, gene/protein, pathways, drug) into the text-mining tool SCAIView. We analyzed the EMRs from 624 patients with MS using the MS ontology dictionary in order to identify drug usage and comorbidities in MS. Testing competency questions and functional evaluation using F statistics further validated the usefulness of MS ontology.
Results
Validation of the lexicalized ontology by means of named entity recognition-based methods showed an adequate performance (F score = 0.73). The MS Ontology retrieved 80% of the genes associated with MS from scientific abstracts and identified additional pathways targeted by approved disease-modifying drugs (e.g. apoptosis pathways associated with mitoxantrone, rituximab and fingolimod). The analysis of the EMR from patients with MS identified current usage of disease modifying drugs and symptomatic therapy as well as comorbidities, which are in agreement with recent reports.
Conclusion
The MS Ontology provides a semantic framework that is able to automatically extract information from both scientific literature and EMR from patients with MS, revealing new pathogenesis insights as well as new clinical information.
doi:10.1371/journal.pone.0116718
PMCID: PMC4321837  PMID: 25665127
11.  Cognitions as mediators in the relationship between self-compassion and affect 
Previous studies suggest that self-compassion is related to numerous facets of mental health, but the role of cognitions in this relationship remains unknown. To examine the mediating role of cognitions in the relationship between self-compassion and anxiety, depression, and life satisfaction when controlling for self-esteem in Japanese people, we conducted two studies. Study 1 (N = 231) examined the relationship between self-compassion and affect by modeling negative automatic thoughts as a mediator; Study 2 (N = 233) tested whether positive and negative automatic thoughts meditate this relationship. Results suggested that both self-compassion and self-esteem increased positive automatic thoughts and decreased trait anxiety, whereas only self-esteem increased life satisfaction and decreased depression directly. Positive automatic thoughts increased life satisfaction and decreased depression and trait anxiety, and positive automatic thoughts mediated the relationship between self-compassion and negative affect. These findings suggest that both positive and negative automatic thoughts mediate the relationship between self-compassion and affect in Japanese people.
doi:10.1016/j.paid.2014.10.008
PMCID: PMC4225636  PMID: 25395717
Self-compassion; Self-esteem; Automatic thoughts; Anxiety; Depression; Life satisfaction
12.  Aortic emergencies—diagnosis and treatment: a pictorial review 
Insights into Imaging  2015;6(1):17-32.
Objectives
To demonstrate the various presentations of acute aortic pathology and to present diagnostic and therapeutic approaches.
Methods
Diagnostic imaging is the key to the reliable diagnosis of acute aortic pathology with multi-slice computed tomography angiography (CTA) as the fastest and most robust modality. Endovascular aortic repair (EVAR) with stent grafts and open surgical repair are therapeutic approaches for aortic pathology.
Results
CTA is reliable in diagnosing and grading aortic trauma, measuring aortic diameter in aortic aneurysms and detecting vascular wall pathology in acute aortic syndrome and aortic inflammation. CTA enables planning the optimal therapeutic approach. Stent graft implantation and/or an open surgical approach can address vascular wall pathology and exclude aortic aneurysms.
Conclusion
Aortic emergencies have to be detected quickly. CTA is the imaging method of choice and helps to decide whether elective, urgent or emergent treatment is necessary with EVAR and open surgical repair as the main treatment approaches.
Teaching Points
• To present aortic pathology caused by trauma
• To present acute aortic syndrome (aortic dissection, intramural haematoma and penetrating ulcers)
• To present symptomatic and ruptured aortic aneurysm
• To present infection (mycotic aneurysms/aorto-duodenal fistulae) or iatrogenic injury of the aorta
• To understand different presentations for treatment planning (EVAR and open surgery)
doi:10.1007/s13244-014-0380-y
PMCID: PMC4330229  PMID: 25638646
Aortic pathology; Aortic Dissection; Aortic Aneurysm; Diagnostic imaging; Endovascular aortic repair (EVAR)
13.  A Co-Culture System with an Organotypic Lung Slice and an Immortal Alveolar Macrophage Cell Line to Quantify Silica-Induced Inflammation 
PLoS ONE  2015;10(1):e0117056.
There is growing evidence that amorphous silica nanoparticles cause toxic effects on lung cells in vivo as well as in vitro and induce inflammatory processes. The phagocytosis of silica by alveolar macrophages potentiates these effects. To understand the underlying molecular mechanisms of silica toxicity, we applied a co-culture system including the immortal alveolar epithelial mouse cell line E10 and the macrophage cell line AMJ2-C11. In parallel we exposed precision-cut lung slices (lacking any blood cells as well as residual alveolar macrophages) of wild type and P2rx7−/− mice with or without AMJ2-C11 cells to silica nanoparticles. Exposure of E10 cells as well as slices of wild type mice resulted in an increase of typical alveolar epithelial type 1 cell proteins like T1α, caveolin-1 and -2 and PKC-β1, whereas the co-culture with AMJ2-C11 showed mostly a slightly lesser increase of these proteins. In P2rx7−/− mice most of these proteins were slightly decreased. ELISA analysis of the supernatant of wild type and P2rx7−/− mice precision-cut lung slices showed decreased amounts of IL-6 and TNF-α when incubated with nano-silica. Our findings indicate that alveolar macrophages influence the early inflammation of the lung and also that cell damaging reagents e.g. silica have a smaller impact on P2rx7−/− mice than on wild type mice. The co-culture system with an organotypic lung slice is a useful tool to study the role of alveolar macrophages during lung injury at the organoid level.
doi:10.1371/journal.pone.0117056
PMCID: PMC4312074  PMID: 25635824
14.  Correlation of circular RNA abundance with proliferation – exemplified with colorectal and ovarian cancer, idiopathic lung fibrosis, and normal human tissues 
Scientific Reports  2015;5:8057.
Circular RNAs are a recently (re-)discovered abundant RNA species with presumed function as miRNA sponges, thus part of the competing endogenous RNA network. We analysed the expression of circular and linear RNAs and proliferation in matched normal colon mucosa and tumour tissues. We predicted >1,800 circular RNAs and proved the existence of five randomly chosen examples using RT-qPCR. Interestingly, the ratio of circular to linear RNA isoforms was always lower in tumour compared to normal colon samples and even lower in colorectal cancer cell lines. Furthermore, this ratio correlated negatively with the proliferation index. The correlation of global circular RNA abundance (the circRNA index) and proliferation was validated in a non-cancerous proliferative disease, idiopathic pulmonary fibrosis, ovarian cancer cells compared to cultured normal ovarian epithelial cells, and 13 normal human tissues. We are the first to report a global reduction of circular RNA abundance in colorectal cancer cell lines and cancer compared to normal tissues and discovered a negative correlation of global circular RNA abundance and proliferation. This negative correlation seems to be a general principle in human tissues as validated with three different settings. Finally, we present a simple model how circular RNAs could accumulate in non-proliferating cells.
doi:10.1038/srep08057
PMCID: PMC4306919  PMID: 25624062
15.  Determining the predictors of innovation implementation in healthcare: a quantitative analysis of implementation effectiveness 
Background
The failure rates for implementing complex innovations in healthcare organizations are high. Estimates range from 30% to 90% depending on the scope of the organizational change involved, the definition of failure, and the criteria to judge it. The innovation implementation framework offers a promising approach to examine the organizational factors that determine effective implementation. To date, the utility of this framework in a healthcare setting has been limited to qualitative studies and/or group level analyses. Therefore, the goal of this study was to quantitatively examine this framework among individual participants in the National Cancer Institute’s Community Clinical Oncology Program using structural equation modeling.
Methods
We examined the innovation implementation framework using structural equation modeling (SEM) among 481 physician participants in the National Cancer Institute’s Community Clinical Oncology Program (CCOP). The data sources included the CCOP Annual Progress Reports, surveys of CCOP physician participants and administrators, and the American Medical Association Physician Masterfile.
Results
Overall the final model fit well. Our results demonstrated that not only did perceptions of implementation climate have a statistically significant direct effect on implementation effectiveness, but physicians’ perceptions of implementation climate also mediated the relationship between organizational implementation policies and practices (IPP) and enrollment (p <0.05). In addition, physician factors such as CCOP PI status, age, radiological oncologists, and non-oncologist specialists significantly influenced enrollment as well as CCOP organizational size and structure, which had indirect effects on implementation effectiveness through IPP and implementation climate.
Conclusions
Overall, our results quantitatively confirmed the main relationship postulated in the innovation implementation framework between IPP, implementation climate, and implementation effectiveness among individual physicians. This finding is important, as although the model has been discussed within healthcare organizations before, the studies have been predominately qualitative in nature and/or at the organizational level. In addition, our findings have practical applications. Managers looking to increase implementation effectiveness of an innovation should focus on creating an environment that physicians perceive as encouraging implementation. In addition, managers should consider instituting specific organizational IPP aimed at increasing positive perceptions of implementation climate. For example, IPP should include specific expectations, support, and rewards for innovation use.
doi:10.1186/s12913-014-0657-3
PMCID: PMC4307151  PMID: 25608564
Innovation; Implementation effectiveness; Implementation climate; Innovation implementation framework; Community clinical oncology program
16.  Synanthropic rodents and their ectoparasites as carriers of a novel haemoplasma and vector-borne, zoonotic pathogens indoors 
Parasites & Vectors  2015;8:27.
Background
Despite their close association with human dwellings, the role of synanthropic rodents in the epidemiology of vector-borne infections is seldom studied. The aim of the present study was to compensate for this lack of information, by the molecular investigation of vector-borne bacteria in peridomestic rodents and their ectoparasites.
Findings
Fifty-two rodents (mainly house mice and brown rats) were caught alive in buildings and checked for blood-sucking ectoparasites; followed by molecular analysis of these, together with spleen samples, for the presence of vector-borne agents. Haemoplasma infection was significantly more prevalent among brown rats, than among house mice. A novel haemoplasma genotype (with only 92-93% similarity to Candidatus Mycoplasma turicensis and M. coccoides in its 16S rRNA gene) was detected in a harvest mouse and a brown rat. Sporadic occurrence of Rickettsia helvetica, Anaplasma phagocytophilum, Borrelia burgdorferi s.l. and Bartonella sp. was also noted in rodents and/or their ectoparasites.
Conclusions
These results indicate that synanthropic rodents, although with low prevalence, may carry zoonotic and vector-borne pathogens indoors.
doi:10.1186/s13071-014-0630-3
PMCID: PMC4299477  PMID: 25589174
Mouse; Rat; Rickettsia; Anaplasma; Borrelia; Bartonella; Haemoplasma
17.  An Intracellular Nanotrap Redirects Proteins and Organelles in Live Bacteria 
mBio  2015;6(1):e02117-14.
ABSTRACT 
Owing to their small size and enhanced stability, nanobodies derived from camelids have previously been used for the construction of intracellular “nanotraps,” which enable redirection and manipulation of green fluorescent protein (GFP)-tagged targets within living plant and animal cells. By taking advantage of intracellular compartmentalization in the magnetic bacterium Magnetospirillum gryphiswaldense, we demonstrate that proteins and even entire organelles can be retargeted also within prokaryotic cells by versatile nanotrap technology. Expression of multivalent GFP-binding nanobodies on magnetosomes ectopically recruited the chemotaxis protein CheW1-GFP from polar chemoreceptor clusters to the midcell, resulting in a gradual knockdown of aerotaxis. Conversely, entire magnetosome chains could be redirected from the midcell and tethered to one of the cell poles. Similar approaches could potentially be used for building synthetic cellular structures and targeted protein knockdowns in other bacteria.
Importance   Intrabodies are commonly used in eukaryotic systems for intracellular analysis and manipulation of proteins within distinct subcellular compartments. In particular, so-called nanobodies have great potential for synthetic biology approaches because they can be expressed easily in heterologous hosts and actively interact with intracellular targets, for instance, by the construction of intracellular “nanotraps” in living animal and plant cells. Although prokaryotic cells also exhibit a considerable degree of intracellular organization, there are few tools available equivalent to the well-established methods used in eukaryotes. Here, we demonstrate the ectopic retargeting and depletion of polar membrane proteins and entire organelles to distinct compartments in a magnetotactic bacterium, resulting in a gradual knockdown of magneto-aerotaxis. This intracellular nanotrap approach has the potential to be applied in other bacteria for building synthetic cellular structures, manipulating protein function, and creating gradual targeted knockdowns. Our findings provide a proof of principle for the universal use of fluorescently tagged proteins as targets for nanotraps to fulfill these tasks.
Importance  
Intrabodies are commonly used in eukaryotic systems for intracellular analysis and manipulation of proteins within distinct subcellular compartments. In particular, so-called nanobodies have great potential for synthetic biology approaches because they can be expressed easily in heterologous hosts and actively interact with intracellular targets, for instance, by the construction of intracellular “nanotraps” in living animal and plant cells. Although prokaryotic cells also exhibit a considerable degree of intracellular organization, there are few tools available equivalent to the well-established methods used in eukaryotes. Here, we demonstrate the ectopic retargeting and depletion of polar membrane proteins and entire organelles to distinct compartments in a magnetotactic bacterium, resulting in a gradual knockdown of magneto-aerotaxis. This intracellular nanotrap approach has the potential to be applied in other bacteria for building synthetic cellular structures, manipulating protein function, and creating gradual targeted knockdowns. Our findings provide a proof of principle for the universal use of fluorescently tagged proteins as targets for nanotraps to fulfill these tasks.
doi:10.1128/mBio.02117-14
PMCID: PMC4313912  PMID: 25587011
18.  Patterns of Breast Magnetic Resonance Imaging Use in Community Practice 
JAMA internal medicine  2014;174(1):125-132.
Importance
Breast magnetic resonance imaging (MRI) is increasingly used for breast cancer screening, diagnostic evaluation, and surveillance However, we lack data on national patterns of breast MRI use in community practice.
Objective
To describe 2005–2009 patterns of breast magnetic resonance imaging (MRI) use in U.S. community practice.
Design
Observational cohort study
Setting
Data collected from 2005–2009 on breast MRI and mammography from five national Breast Cancer Surveillance Consortium registries.
Participants
Data included 8931 breast MRI examinations and 1,288,924 screening mammograms from women aged 18–79 years.
Main measures
We calculated the rate of breast MRI examinations per 1000 women with breast imaging within the same year and described the clinical indications for the breast MRI examinations by year and age. We compared women screened with breast MRI to women screened with mammography alone for patient characteristics and lifetime breast cancer risk.
Results
The overall rate of breast MRI from 2005 through 2009 nearly tripled from 4.2 to 11.5 examinations per 1000 women with the most rapid rise from 2005–2007 (p=0.02). The most common clinical indication was diagnostic evaluation (40.3%), followed by screening (31.7%). Compared to women who received screening mammography alone, women who underwent screening breast MRI were more likely to be <50 years, white non-Hispanic, nulliparous, and have extremely dense breast tissue, a family history of breast cancer, and a personal history of breast cancer. The proportion of women screened by breast MRI at high lifetime risk for breast cancer (>20%) increased during the study period from 9% in 2005 to 29% in 2009.
Conclusions and relevance
Use of breast MRI for screening in high-risk women is increasing. However, our findings suggest there is a need to improve appropriate utilization, including among women who may benefit from screening breast MRI.
doi:10.1001/jamainternmed.2013.11963
PMCID: PMC3905972  PMID: 24247555
19.  Src kinase modulates the apoptotic p53 pathway by altering HIPK2 localization 
Cell Cycle  2013;13(1):115-125.
Non-receptor tyrosine kinase Src is a master regulator of cell proliferation. Hyperactive Src is a potent oncogene and a driver of cellular transformation and carcinogenesis. Homeodomain-interacting protein kinase 2 (HIPK2) is a tumor suppressor mediating growth suppression and apoptosis upon genotoxic stress through phosphorylation of p53 at Ser46. Here we show that Src phosphorylates HIPK2 and changes its subcellular localization. Using mass spectrometry we identified 9 Src-mediated Tyr-phosphorylation sites within HIPK2, 5 of them positioned in the kinase domain. By means of a phosphorylation-specific antibody we confirm that Src mediates phosphorylation of HIPK2 at Tyr354. We demonstrate that ectopic expression of Src increases the half-life of HIPK2 by interfering with Siah-1-mediated HIPK2 degradation. Moreover, we find that hyperactive Src binds HIPK2 and redistributes HIPK2 from the cell nucleus to the cytoplasm, where both kinases partially colocalize. Accordingly, we find that hyperactive Src decreases chemotherapeutic drug-induced p53 Ser46 phosphorylation and apoptosis activation. Together, our results suggest that Src kinase suppresses the apoptotic p53 pathway by phosphorylating HIPK2 and relocalizing the kinase to the cytoplasm.
doi:10.4161/cc.26857
PMCID: PMC3925721  PMID: 24196445
Src; HIPK2; p53; chemotherapeutic drug; apoptosis
20.  Genome and transcriptome of the porcine whipworm Trichuris suis 
Nature genetics  2014;46(7):701-706.
Trichuris (whipworm) infects 1 billion people worldwide, and causes a disease (trichuriasis) that results in major socioeconomic losses in both humans and pigs. Trichuriasis relates to an inflammation of the large intestine manifested in bloody diarrhoea, and chronic disease can cause malnourishment and stunting in children. Paradoxically, Trichuris of pigs has shown substantial promise as a treatment for human autoimmune disorders, including inflammatory bowel disease (IBD) and multiple sclerosis (MS). Here, we report ~80 megabase (Mb) draft assemblies of the genomes of adult male and female T. suis, and explore stage-, sex- and tissue-specific transcription of messenger and small non-coding RNAs.
doi:10.1038/ng.3012
PMCID: PMC4105696  PMID: 24929829
21.  The impact of free or standardized lifestyle and urine sampling protocol on metabolome recognition accuracy 
Genes & Nutrition  2014;10(1):441.
Urine contains a clear individual metabolic signature, although embedded within a large daily variability. Given the potential of metabolomics to monitor disease onset from deviations from the “healthy” metabolic state, we have evaluated the effectiveness of a standardized lifestyle in reducing the “metabolic” noise. Urine was collected from 24 (5 men and 19 women) healthy volunteers over a period of 10 days: phase I, days 1–7 in a real-life situation; phase II, days 8–10 in a standardized diet and day 10 plus exercise program. Data on dietary intake and physical activity have been analyzed by a nation-specific software and monitored by published protocols. Urine samples have been analyzed by 1H NMR followed by multivariate statistics. The individual fingerprint emerged and consolidated with increasing the number of samples and reaches ~100 % cross-validated accuracy for about 40 samples. Diet standardization reduced both the intra-individual and the interindividual variability; the effect was due to a reduction in the dispersion of the concentration values of several metabolites. Under standardized diet, however, the individual phenotype was still clearly visible, indicating that the individual’s signature was a strong feature of the metabolome. Consequently, cohort studies designed to investigate the relation of individual metabolic traits and nutrition require multiple samples from each participant even under highly standardized lifestyle conditions in order to exploit the analytical potential of metabolomics. We have established criteria to facilitate design of urine metabolomic studies aimed at monitoring the effects of drugs, lifestyle, dietary supplements, and for accurate determination of signatures of diseases.
doi:10.1007/s12263-014-0441-3
PMCID: PMC4235801  PMID: 25403096
Diet; Exercise; Metabolomics; Short term; Urine; NMR
22.  Mice Lacking the SLAM Family Member CD84 Display Unaltered Platelet Function in Hemostasis and Thrombosis 
PLoS ONE  2014;9(12):e115306.
Background
Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity by forming thrombi at sites of vascular injury. Although the early events of thrombus formation—platelet adhesion and aggregation—have been intensively studied, less is known about the mechanisms and receptors that stabilize platelet-platelet interactions once a thrombus has formed. One receptor that has been implicated in this process is the signaling lymphocyte activation molecule (SLAM) family member CD84, which can undergo homophilic interactions and becomes phosphorylated upon platelet aggregation.
Objective
The role of CD84 in platelet physiology and thrombus formation was investigated in CD84-deficient mice.
Methods and Results
We generated CD84-deficient mice and analyzed their platelets in vitro and in vivo. Cd84−/− platelets exhibited normal activation and aggregation responses to classical platelet agonists. Furthermore, CD84 deficiency did not affect integrin-mediated clot retraction and spreading of activated platelets on fibrinogen. Notably, also the formation of stable three-dimensional thrombi on collagen-coated surfaces under flow ex vivo was unaltered in the blood of Cd84−/− mice. In vivo, Cd84−/− mice exhibited unaltered hemostatic function and arterial thrombus formation.
Conclusion
These results show that CD84 is dispensable for thrombus formation and stabilization, indicating that its deficiency may be functionally compensated by other receptors or that it may be important for platelet functions different from platelet-platelet interactions.
doi:10.1371/journal.pone.0115306
PMCID: PMC4281120  PMID: 25551754
23.  β2 integrin mediates hantavirus-induced release of neutrophil extracellular traps 
The Journal of Experimental Medicine  2014;211(7):1485-1497.
β2 Integrin–mediated systemic release of neutrophil extracellular traps is a novel mechanism of immunopathology associated with hantavirus infection.
Rodent-borne hantaviruses are emerging human pathogens that cause severe human disease. The underlying mechanisms are not well understood, as hantaviruses replicate in endothelial and epithelial cells without causing any cytopathic effect. We demonstrate that hantaviruses strongly stimulated neutrophils to release neutrophil extracellular traps (NETs). Hantavirus infection induced high systemic levels of circulating NETs in patients and this systemic NET overflow was accompanied by production of autoantibodies to nuclear antigens. Analysis of the responsible mechanism using neutrophils from β2 null mice identified β2 integrin receptors as a master switch for NET induction. Further experiments suggested that β2 integrin receptors such as complement receptor 3 (CR3) and 4 (CR4) may act as novel hantavirus entry receptors. Using adenoviruses, we confirmed that viral interaction with β2 integrin induced strong NET formation. Collectively, β2 integrin–mediated systemic NET overflow is a novel viral mechanism of immunopathology that may be responsible for characteristic aspects of hantavirus-associated disease such as kidney and lung damage.
doi:10.1084/jem.20131092
PMCID: PMC4076588  PMID: 24889201
24.  Differences in Injury Pattern and Prevalence of Cartilage Lesions in Knee and Ankle Joints: A Retrospective Cohort Study 
Orthopedic Reviews  2014;6(4):5611.
Osteoarthritis (OA) is more common in the knee compared to the ankle joint. This can not be explained exclusively by anatomical and biomechanical differences. The aim of this study is to analyze and compare the injury pattern (clinically) and the cartilage lesions (arthroscopically) of knee and ankle joints in a cohort of patients from the same catchment area. A retrospective study of the clinical data of 3122 patients (2139 outpatients and 983 inpatients) was performed, who were treated due to an injury of the knee and ankle joint. Statistical analysis was performed using SigmaStat 3.0 (SPSS Inc, Chicago, USA). There is a higher prevalence of injuries in the ankle as compared to the knee joint in this population from the same catchment area. In contrast, high-grade cartilage lesions are more prevalent in the knee, whereas low grade cartilage lesions are equally distributed between knee and ankle. From this data it can be concluded that the frequency of injuries and the injury pattern of knee versus ankle joints do not correlate with the severity of cartilage lesions and may therefore have no direct influence on the differential incidence of OA in those two joints.
doi:10.4081/or.2014.5611
PMCID: PMC4274453  PMID: 25568732
osteoarthritis; knee; ankle; cartilage lesion; injury
25.  Exploring the Chemical Space of Multitarget Ligands Using Aligned Self-Organizing Maps 
ACS Medicinal Chemistry Letters  2013;4(12):1169-1172.
Design of multitarget drugs and polypharmacological compounds has become popular during the past decade. However, the main approach to design such compounds is to link two selective ligands via a flexible linker. Although such chimeric ligands often have reasonable potency in vitro, the in vivo efficacy is low due to high molecular weight, low ligand efficiency, and poor pharmacokinetic profile. We developed an unprecedented in silico approach for fragment-based design of multitarget ligands. It relies on superposition of the chemical spaces related to the affinity on single targets represented by self-organizing maps. We used this approach for screening of molecular fragments, which bind to the enzymes 5-lipoxygenase (5-LO) and soluble epoxide hydrolase (sEH). Using STD-NMR and activity-based assays, we were able to identify fragments binding to both targets. Furthermore, we were able to expand one of the fragments to a potent dual inhibitor bearing a reasonable molecular weight (MW = 446) and high affinity to both targets (IC50 of 0.03 μM toward 5-LO and 0.17 μM toward sEH).
doi:10.1021/ml4002562
PMCID: PMC4027374  PMID: 24900624
Aligned self-organizing maps; 5-lipoxygenase; soluble epoxide hydrolase; structure−activity relationships

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