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1.  The accurate assessment of small-angle X-ray scattering data 
A set of quantitative techniques is suggested for assessing SAXS data quality. These are applied in the form of a script, SAXStats, to a test set of 27 proteins, showing that these techniques are more sensitive than manual assessment of data quality.
Small-angle X-ray scattering (SAXS) has grown in popularity in recent times with the advent of bright synchrotron X-ray sources, powerful computational resources and algorithms enabling the calculation of increasingly complex models. However, the lack of standardized data-quality metrics presents difficulties for the growing user community in accurately assessing the quality of experimental SAXS data. Here, a series of metrics to quantitatively describe SAXS data in an objective manner using statistical evaluations are defined. These metrics are applied to identify the effects of radiation damage, concentration dependence and interparticle interactions on SAXS data from a set of 27 previously described targets for which high-resolution structures have been determined via X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. The studies show that these metrics are sufficient to characterize SAXS data quality on a small sample set with statistical rigor and sensitivity similar to or better than manual analysis. The development of data-quality analysis strategies such as these initial efforts is needed to enable the accurate and unbiased assessment of SAXS data quality.
PMCID: PMC4304685  PMID: 25615859
SAXS data quality; SAXStats
2.  A computational model unifies apparently contradictory findings concerning phantom pain 
Scientific Reports  2014;4:5298.
Amputation often leads to painful phantom sensations, whose pathogenesis is still unclear. Supported by experimental findings, an explanatory model has been proposed that identifies maladaptive reorganization of the primary somatosensory cortex (S1) as a cause of phantom pain. However, it was recently found that BOLD activity during voluntary movements of the phantom positively correlates with phantom pain rating, giving rise to a model of persistent representation. In the present study, we develop a physiologically realistic, computational model to resolve the conflicting findings. Simulations yielded that both the amount of reorganization and the level of cortical activity during phantom movements were enhanced in a scenario with strong phantom pain as compared to a scenario with weak phantom pain. These results suggest that phantom pain, maladaptive reorganization, and persistent representation may all be caused by the same underlying mechanism, which is driven by an abnormally enhanced spontaneous activity of deafferented nociceptive channels.
PMCID: PMC4058874  PMID: 24931344
3.  An increase of interleukin-33 serum levels after coronary stent implantation is associated with coronary in-stent restenosis 
Cytokine  2014;67(2):65-70.
•An association between IL-33 and restenosis in coronary artery disease exists.•IL-33 increase after stent implantation is associated with a higher rate of restenosis.•IL-33 estimation before and after PCI could determine patients at risk.
The study aim was to determine the predictive value of interleukin (IL)-33, a recently described member of the IL-1 family of cytokines, for the development of in-stent restenosis (ISR). IL-33 serum levels were measured in 387 consecutive patients undergoing percutaneous coronary intervention (PCI) of whom 193 had stable angina, 93 non-ST elevation myocardial infarction (NSTEMI), and 101 ST-elevation MI (STEMI), respectively. Blood was taken directly before and 24 h after stent implantation. The presence of ISR was initially evaluated by clinical means after six to eight months. When presence of myocardial ischemia was suspected, coronary angiography was performed to confirm the suspected diagnosis of ISR. Clinical ISR was present in total in 34 patients (8.8%). IL-33 was detectable in 185 patients and was below detection limit in 202 patients. In patients with decreased IL-33 (n = 95), unchanged or non-detectable levels (n = 210) or increased levels of IL-33 after PCI (n = 82), ISR-rate was 2.1%, 9.5% and 14.6%, respectively (p < 0.05). Accordingly, patients with ISR showed a significant increase of IL-33 upon PCI (p < 0.05). This association was independent from clinical presentation and risk factors as well as numbers and type of stents. In patients with both stable and unstable coronary artery disease, an increase of IL-33 serum levels after stent implantation is associated with a higher rate of in-stent restenosis.
PMCID: PMC3996548  PMID: 24725541
Interleukin-33; Restenosis; Coronary artery disease; Myocardial infarction; Percutaneous coronary intervention
4.  Adiponectin Isoforms Differentially Affect Gene Expression and the Lipidome of Primary Human Hepatocytes 
Metabolites  2014;4(2):394-407.
Adiponectin (APN) exerts multiple beneficial effects in obesity and protects from liver injury. Different APN isoforms circulate in serum, and here, the effect of low molecular weight (LMW) and higher molecular weight (HMW) APN on primary human hepatocytes (PHH) has been analyzed. APN is not detected in hepatocyte lysates; levels are strongly increased by HMW-APN, but not by LMW-APN, suggesting the distinct uptake/degradation of APN isoforms by PHH. Several genes with a role in fibrosis, glucose and lipid metabolism known to be regulated by HMW-APN are not affected by the LMW-isoform. Follistatin is reduced by HMW-APN and induced by LMW-APN in supernatants of PHH. Fibroblast growth factor 21 is repressed by both isoforms. Cellular triglycerides and cholesterol levels are not reduced by APN. Total phospholipids, including plasmalogens and sphingomyelins, are not changed upon APN incubation, while distinct species are either induced or repressed. Unexpectedly, total ceramide is increased by LMW-APN. Current data show that APN isoforms differentially affect hepatocyte gene expression, but do not grossly alter the hepatocyte lipidome.
PMCID: PMC4101512  PMID: 24957032
ceramide; phospholipids; gene expression; trimer; lipidomic
5.  Soluble ST2 and Interleukin-33 Levels in Coronary Artery Disease: Relation to Disease Activity and Adverse Outcome 
PLoS ONE  2014;9(4):e95055.
ST2 is a receptor for interleukin (IL)-33. We investigated an association of soluble ST2 (sST2) and IL-33 serum levels with different clinical stages of coronary artery disease. We assessed the predictive value of sST2 and IL-33 in patients with stable angina, non-ST elevation myocardial infarction (NSTEMI) and ST elevation myocardial infarction (STEMI).
We included 373 patients of whom 178 had stable angina, 97 had NSTEMI, and 98 had STEMI. Patients were followed for a mean of 43 months. The control group consisted of 65 individuals without significant stenosis on coronary angiography. Serum levels of sST2 and IL-33 were measured by ELISAs.
sST2 levels were significantly increased in patients with STEMI as compared to patients with NSTEMI and stable angina as well as with controls. IL-33 levels did not differ between the four groups. During follow-up, 37 (10%) patients died and the combined endpoint (all cause death, MI and rehospitalisation for cardiac causes) occurred in 66 (17.6%) patients. sST2 serum levels significantly predicted mortality in the total cohort. When patients were stratified according to their clinical presentation, the highest quintile of sST2 significantly predicted mortality in patients with STEMI, but not with NSTEMI or stable coronary artery disease. sST2 was a significant predictor for the combined endpoint in STEMI patients and in patients with stable angina. Serum levels of IL-33 were not associated with clinical outcome in the total cohort, but the highest quintile of IL-33 predicted mortality in patients with STEMI.
Serum levels of sST2 are increased in patients with acute coronary syndromes as compared to levels in patients with stable coronary artery disease and in individuals without coronary artery disease. sST2 and IL-33 were associated with mortality in patients with STEMI but not in patients with NSTEMI or stable angina.
PMCID: PMC3994012  PMID: 24751794
6.  Structure, Dynamics, Evolution, and Function of a Major Scaffold Component in the Nuclear Pore Complex 
The nuclear pore complex, composed of proteins termed nucleoporins (Nups), is responsible for nucleocytoplasmic transport in eukaryotes. Nuclear pore complexes (NPCs) form an annular structure composed of the nuclear ring, cytoplasmic ring, a membrane ring, and two inner rings. Nup192 is a major component of the NPC’s inner ring. We report the crystal structure of Saccharomyces cerevisiae Nup192 residues 2–960 [ScNup192(2–960)], which adopts an α-helical fold with three domains (i.e., D1, D2, and D3). Small angle X-ray scattering and electron microscopy (EM) studies reveal that ScNup192(2–960) could undergo long-range transition between “open” and “closed” conformations. We obtained a structural model of full-length ScNup192 based on EM, the structure of ScNup192(2–960), and homology modeling. Evolutionary analyses using the ScNup192(2–960) structure suggest that NPCs and vesicle-coating complexes are descended from a common membrane-coating ancestral complex. We show that suppression of Nup192 expression leads to compromised nuclear transport and hypothesize a role for Nup192 in modulating the permeability of the NPC central channel.
PMCID: PMC3755625  PMID: 23499021
7.  Attenuated and Protease-Profile Modified Sendai Virus Vectors as a New Tool for Virotherapy of Solid Tumors 
PLoS ONE  2014;9(3):e90508.
Multiple types of oncolytic viruses are currently under investigation in clinical trials. To optimize therapeutic outcomes it is believed that the plethora of different tumor types will require a diversity of different virus types. Sendai virus (SeV), a murine parainfluenza virus, displays a broad host range, enters cells within minutes and already has been applied safely as a gene transfer vector in gene therapy patients. However, SeV spreading naturally is abrogated in human cells due to a lack of virus activating proteases. To enable oncolytic applications of SeV we here engineered a set of novel recombinant vectors by a two-step approach: (i) introduction of an ubiquitously recognized cleavage-motive into SeV fusion protein now enabling continuous spreading in human tissues, and (ii) profound attenuation of these rSeV by the knockout of viral immune modulating accessory proteins. When employing human hepatoma cell lines, newly generated SeV variants now reached high titers and induced a profound tumor cell lysis. In contrast, virus release from untransformed human fibroblasts or primary human hepatocytes was found to be reduced by about three log steps in a time course experiment which enables the cumulation of kinetic differences of the distinct phases of viral replication such as primary target cell infection, target cell replication, and progeny virus particle release. In a hepatoma xenograft animal model we found a tumor-specific spreading of our novel recombinant SeV vectors without evidence of biodistribution into non-malignant tissues. In conclusion, we successfully developed novel tumor-selective oncolytic rSeV vectors, constituting a new tool for virotherapy of solid tumors being ready for further preclinical and clinical development to address distinct tumor types.
PMCID: PMC3944018  PMID: 24598703
8.  Plasma osmolality predicts clinical outcome in patients with acute coronary syndrome undergoing percutaneous coronary intervention 
The impact of plasma osmolality on clinical outcome in acute coronary syndrome (ACS) patients has not been investigated so far.
In a retrospective analysis, we included 985 patients with ACS undergoing percutaneous coronary intervention (PCI). Plasma osmolality was calculated using concentrations of sodium, plasma glucose, and blood urea nitrogen at admission. Patients were stratified by quartiles (Q) of admission osmolality, clinical outcome was compared between those groups. The primary endpoints were in-hospital, 30-day, and 1-year mortality.
Univariate analysis in the Cox proportional-hazards model revealed significantly higher rates of in-hospital death for patients with osmolality in Q4, as compared to patients with osmolality in Q1–3 (HR 5.4, 95% CI 3.3–9.0, p<0.01). After adjustment for confounding baseline variables, osmolality in Q4 was associated with 2.8-fold hazard of in-hospital death (HR 2.75, 95% CI 1.35–5.61, p=0.005). Upon multivariate analysis, admission osmolality in Q4 vs. Q1–3 was associated with higher mortality rates after 30 days (HR 2.53, 95% CI 1.23–5.21, p=0.012) and 1 year (HR 1.73, 95% CI 1.02–2.91, p=0.04). Moreover, we performed landmark analysis in order to exclude critically ill patients, which revealed similar adjusted rates of death beyond 30 days to 1 year (HR 1.21, 95% CI 0.55–2.66, p=0.642).
Using the 4th quartile of plasma osmolality at admission as a natural cut-off point, osmolality in Q4, as compared to Q1–3, was significantly predictive of short term but not long-term outcome in ACS patients undergoing coronary stenting. Our data suggest osmolality to be an independent, feasible, and cost-effective tool for rapid risk stratification in ACS patients.
PMCID: PMC3932780  PMID: 24337920
Acute coronary syndrome; osmolality; risk stratification
9.  Pain-related and negative semantic priming enhances perceived pain intensity 
‘Priming’ an individual by exposing them to pictures or words before a painful stimulus can significantly affect their subsequent pain experience. This study aimed to determine whether this priming effect is only associated with negative primes, or whether pain-related primes have the same, or increased, effects.
Negative affective and pain-related cues, such as pictures or words, have been shown to act as primes and enhance the perceived intensity of subsequent painful events. For pain-related semantic primes, it remains unclear whether this effect depends on negative valence itself or, specifically, on the pain-relatedness of the words.
To investigate the effect of pain-related, negative affective (pain-unrelated) and neutral semantic primes on the perception of subsequent noxious target stimuli.
Pain ratings in response to noxious electrical stimulation of light and moderate intensity were examined in 39 healthy subjects after subjects were exposed to semantic primes of different meaning and valence (pain-related, negative, positive and neutral adjectives) presented with different interstimulus intervals (0 ms, 500 ms and 1500 ms).
Increased pain ratings of noxious stimuli were observed following pain-related and negative compared with neutral primes.
The results support the motivational priming theory for semantic stimuli, indicating that affectively negative semantic primes increase subjective pain intensity. However, a specific pain-related priming effect was not reliably demonstrated. Additionally, it is shown that experimental parameters (ie, stimulus intensity and interstimulus interval) modify the extent of negative and pain-related semantic priming.
Verbal priming plays a role for the perception of noxious stimuli in a time-dependent manner.
PMCID: PMC4028655  PMID: 24716197
Affective words; Electrical stimulation; Pain ratings; Pain words; Semantic priming
10.  IL-6 signalling in patients with acute ST-elevation myocardial infarction 
Results in Immunology  2013;4:8-13.
Cytokines of the IL-6 family have been related to infarct size and prognosis in patients with myocardial infarction. The aims of the present study were to elucidate possible associations between myocardial necrosis and left ventricular impairment and members of the IL-6 transsignalling system including soluble (s) IL-6R and (s) glycoprotein 130 (sgp130) in patients with ST-elevation myocardial infarction (STEMI) treated with primary PCI.
In blood samples from 1028 STEMI patients, collected in-hosptial, we found significant correlations between peak TnT and IL-6 and CRP (p < 0.001, all) and between IL-6 and CRP and LV ejection fraction and NT-proBNP (p < 0.001, all). On the contrary, no significant associations were found between peak TnT and sgp130 or sIL-6R. Furthermore sgp130 was significantly elevated in diabetic patients and also associated with the glucometabolic state.
In conclusion, circulating levels of IL-6 and CRP, but not the soluble forms of the receptor (sIL-6R) or the receptor signalling subunit (sgp130) were associated with the extent of myocardial necrosis. The biological importance of the IL-6/gp130-mediated signalling pathways in patients with acute myocardial infarction and dysglycemia should be further elucidated.
•Circulating levels of IL-6 and CRP, but not sgp130 and sIL-6R were associated with the extent of myocardial necrosis.•Circulating levels of sgp130 were weakly associated with impaired LV function and glucometabolic state.•Circulating levels of sIL-6R were not associated with either impaired LV function or glucometabolic state.
PMCID: PMC3973821  PMID: 24707455
ST-elevation myocardial infarction; IL-6; sgp130; sIL-6R; Myocardial necrosis; sgp130, soluble glycoprotein 130; sIL-6R, soluble interleukin -6 receptor; PCI, percutaneous coronary intervention; STEMI, ST-elevation myocardial infarction; LVEF, left ventricular ejection fraction
12.  Resveratrol as a Pan-HDAC Inhibitor Alters the Acetylation Status of Jistone Proteins in Human-Derived Hepatoblastoma Cells 
PLoS ONE  2013;8(8):e73097.
The polyphenolic alcohol resveratrol has demonstrated promising activities for the prevention and treatment of cancer. Different modes of action have been described for resveratrol including the activation of sirtuins, which represent the class III histone deacetylases (HDACs). However, little is known about the activity of resveratrol on the classical HDACs of class I, II and IV, although these classes are involved in cancer development or progression and inhibitors of HDACs (HDACi) are currently under investigation as promising novel anticancer drugs. We could show by in silico docking studies that resveratrol has the chemical structure to inhibit the activity of different human HDAC enzymes. In vitro analyses of overall HDAC inhibition and a detailed HDAC profiling showed that resveratrol inhibited all eleven human HDACs of class I, II and IV in a dose-dependent manner. Transferring this molecular mechanism into cancer therapy strategies, resveratrol treatment was analyzed on solid tumor cell lines. Despite the fact that hepatocellular carcinoma (HCC) is known to be particularly resistant against conventional chemotherapeutics, treatment of HCC with established HDACi already has shown promising results. Testing of resveratrol on hepatoma cell lines HepG2, Hep3B and HuH7 revealed a dose-dependent antiproliferative effect on all cell lines. Interestingly, only for HepG2 cells a specific inhibition of HDACs and in turn a histone hyperacetylation caused by resveratrol was detected. Additional testing of human blood samples demonstrated a HDACi activity by resveratrol ex vivo. Concluding toxicity studies showed that primary human hepatocytes tolerated resveratrol, whereas in vivo chicken embryotoxicity assays demonstrated severe toxicity at high concentrations. Taken together, this novel pan-HDACi activity opens up a new perspective of resveratrol for cancer therapy alone or in combination with other chemotherapeutics. Moreover, resveratrol may serve as a lead structure for chemical optimization of bioavailability, pharmacology or HDAC inhibition.
PMCID: PMC3758278  PMID: 24023672
13.  Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME 
Godoy, Patricio | Hewitt, Nicola J. | Albrecht, Ute | Andersen, Melvin E. | Ansari, Nariman | Bhattacharya, Sudin | Bode, Johannes Georg | Bolleyn, Jennifer | Borner, Christoph | Böttger, Jan | Braeuning, Albert | Budinsky, Robert A. | Burkhardt, Britta | Cameron, Neil R. | Camussi, Giovanni | Cho, Chong-Su | Choi, Yun-Jaie | Craig Rowlands, J. | Dahmen, Uta | Damm, Georg | Dirsch, Olaf | Donato, María Teresa | Dong, Jian | Dooley, Steven | Drasdo, Dirk | Eakins, Rowena | Ferreira, Karine Sá | Fonsato, Valentina | Fraczek, Joanna | Gebhardt, Rolf | Gibson, Andrew | Glanemann, Matthias | Goldring, Chris E. P. | Gómez-Lechón, María José | Groothuis, Geny M. M. | Gustavsson, Lena | Guyot, Christelle | Hallifax, David | Hammad, Seddik | Hayward, Adam | Häussinger, Dieter | Hellerbrand, Claus | Hewitt, Philip | Hoehme, Stefan | Holzhütter, Hermann-Georg | Houston, J. Brian | Hrach, Jens | Ito, Kiyomi | Jaeschke, Hartmut | Keitel, Verena | Kelm, Jens M. | Kevin Park, B. | Kordes, Claus | Kullak-Ublick, Gerd A. | LeCluyse, Edward L. | Lu, Peng | Luebke-Wheeler, Jennifer | Lutz, Anna | Maltman, Daniel J. | Matz-Soja, Madlen | McMullen, Patrick | Merfort, Irmgard | Messner, Simon | Meyer, Christoph | Mwinyi, Jessica | Naisbitt, Dean J. | Nussler, Andreas K. | Olinga, Peter | Pampaloni, Francesco | Pi, Jingbo | Pluta, Linda | Przyborski, Stefan A. | Ramachandran, Anup | Rogiers, Vera | Rowe, Cliff | Schelcher, Celine | Schmich, Kathrin | Schwarz, Michael | Singh, Bijay | Stelzer, Ernst H. K. | Stieger, Bruno | Stöber, Regina | Sugiyama, Yuichi | Tetta, Ciro | Thasler, Wolfgang E. | Vanhaecke, Tamara | Vinken, Mathieu | Weiss, Thomas S. | Widera, Agata | Woods, Courtney G. | Xu, Jinghai James | Yarborough, Kathy M. | Hengstler, Jan G.
Archives of Toxicology  2013;87(8):1315-1530.
This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell–derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.
Electronic supplementary material
The online version of this article (doi:10.1007/s00204-013-1078-5) contains supplementary material, which is available to authorized users.
PMCID: PMC3753504  PMID: 23974980
Non-parenchymal cells; Mechanisms of gene regulation; DILI; 3D Models; Cryopreservation; Clearance; Mathematical modeling
14.  Extracting Labeled Topological Patterns from Samples of Networks 
PLoS ONE  2013;8(8):e70497.
An advanced graph theoretical approach is introduced that enables a higher level of functional interpretation of samples of directed networks with identical fixed pairwise different vertex labels that are drawn from a particular population. Compared to the analysis of single networks, their investigation promises to yield more detailed information about the represented system. Often patterns of directed edges in sample element networks are too intractable for a direct evaluation and interpretation. The new approach addresses the problem of simplifying topological information and characterizes such a sample of networks by finding its locatable characteristic topological patterns. These patterns, essentially sample-specific network motifs with vertex labeling, might represent the essence of the intricate topological information contained in all sample element networks and provides as well a means of differentiating network samples. Central to the accurateness of this approach is the null model and its properties, which is needed to assign significance to topological patterns. As a proof of principle the proposed approach has been applied to the analysis of networks that represent brain connectivity before and during painful stimulation in patients with major depression and in healthy subjects. The accomplished reduction of topological information enables a cautious functional interpretation of the altered neuronal processing of pain in both groups.
PMCID: PMC3741309  PMID: 23950945
15.  Nrf2 Activates Augmenter of Liver Regeneration (ALR) via Antioxidant Response Element and Links Oxidative Stress to Liver Regeneration 
Molecular Medicine  2013;19(1):237-244.
Liver regeneration can be impaired by permanent oxidative stress and activation of nuclear factor erythroid 2–related factor 2 (Nrf2), known to regulate the cellular antioxidant response, and has been shown to improve the process of liver regeneration. A variety of factors regulate hepatic tissue regeneration, among them augmenter of liver regeneration (ALR), attained great attention as being survival factors for the liver with proproliferative and antiapoptotic properties. Here we determined the Nrf2/antioxidant response element (ARE) regulated expression of ALR and show ALR as a target gene of Nrf2 in vitro and in vivo. The ALR promoter comprises an ARE binding site and, therefore, ALR expression can be induced by ARE-activator tertiary butylhydroquinone (tBHQ) in hepatoma cells and primary human hepatocytes (PHH). Promoter activity and expression of ALR were enhanced after cotransfection of Nrf2 compared with control and dominant negative mutant of Nrf2. Performing partial hepatectomy in livers from Nrf2+/+ mice compared with Nrf2−/− knock-out (KO) mice, we found increased expression of ALR in addition to known antioxidant ARE-regulated genes. Furthermore, we observed increased ALR expression in hepatitis B virus (HBV) compared with hepatitis C virus (HCV) positive hepatoma cells and PHH. Recently, it was demonstrated that HBV infection activates Nrf2 and, now, we add results showing increased ALR expression in liver samples from patients infected with HBV. ALR is regulated by Nrf2, acts as a liver regeneration and antioxidative protein and, therefore, links oxidative stress to hepatic regeneration to ensure survival of damaged cells.
PMCID: PMC3769531  PMID: 23887691
16.  Adiponectin upregulates hepatocyte CMKLR1 which is reduced in human fatty liver 
Chemokine-like receptor 1 (CMKLR1) ligands chemerin and resolvin E1 are suggested to have a role in non-alcoholic fatty liver disease (NAFLD). Here, expression of CMKLR1 in liver cells and NAFLD was studied. CMKLR1 was detected in primary human hepatocytes (PHH), Kupffer cells, bile-duct cells and hepatic stellate cells. In human and rodent fatty liver and in fibrotic liver of mice fed a methionine–choline deficient diet CMKLR1 was reduced. Hepatocytes are the major cells in the liver and effects of adipokines, cytokines and lipids on CMKLR1 in PHH were analyzed. Increased cellular triglyceride or cholesterol content, lipopolysaccharide, IL-6, TNF and leptin did not influence CMKLR1 levels in PHH whereas profibrotic TGFβ tended to reduce CMKLR1. Adiponectin strongly upregulated CMKLR1 mRNA and protein in PHH and hepatic CMKLR1 when injected into wild type mice. Further, CMKLR1 was suppressed in the liver of adiponectin deficient mice. These data indicate that low CMKLR1 in NAFLD may partly result from reduced adiponectin activity.
PMCID: PMC3670424  PMID: 22118966
Adipokine; Hepatic steatosis; Chemerin receptor; Liver
17.  An integrated high-throughput data acquisition system for biological solution X-ray scattering studies 
Journal of Synchrotron Radiation  2012;19(Pt 3):431-434.
A fully automated high-throughput solution X-ray scattering data collection system, developed for protein structure studies at beamline 4-2 of the Stanford Synchrotron Radiation Lightsource, is described.
A fully automated high-throughput solution X-ray scattering data collection system has been developed for protein structure studies at beamline 4-2 of the Stanford Synchrotron Radiation Lightsource. It is composed of a thin-wall quartz capillary cell, a syringe needle assembly on an XYZ positioning arm for sample delivery, a water-cooled sample rack and a computer-controlled fluid dispenser. It is controlled by a specifically developed software component built into the standard beamline control program Blu-Ice/DCS. The integrated system is intuitive and very simple to use, and enables experimenters to customize data collection strategy in a timely fashion in concert with an automated data processing program. The system also allows spectrophotometric determination of protein concentration for each sample aliquot in the beam via an in situ UV absorption spectrometer. A single set of solution scattering measurements requires a 20–30 µl sample aliquot and takes typically 3.5 min, including an extensive capillary cleaning cycle. Over 98.5% of measurements are valid and free from artefacts commonly caused by air-bubble contamination. The sample changer, which is compact and light, facilitates effortless switching with other sample-handling devices required for other types of non-crystalline X-ray scattering experiments.
PMCID: PMC3329959  PMID: 22514181
proteomics; structural genomics; X-ray scattering; laboratory automation; SAXS
18.  Network Redundancy Analysis of Effective Brain Networks; a Comparison of Healthy Controls and Patients with Major Depression 
PLoS ONE  2013;8(4):e60956.
This study investigated electroencephalographic correlates in chronically depressed patients compared to healthy controls using intracutaneously applied electrical pain stimulus, to better understand the interaction between pain processing and depression. A close interaction between pain and depression is generally recognized although the precise mechanisms are not yet fully understood. The present study focuses on the hypothesis that effective brain connectivity in major depression patients is altered. Multifunctional interactions between brain regions represent a robust index of effective interactions within the brain, and can be quantified by network redundancy. Thus, structural network differences between 18 normal controls and 18 major depression patients before as well as during the processing of moderately painful intracutaneous electrical stimuli were investigated on the basis of network redundancy differences. In our sample, both patients and control subjects exhibit comparable network redundancies before stimulus application. Caused by the stimulus, there is a global increase of network redundancy in both groups. This increase is diminished in the group of major depression patients. We found clear differences between patients and controls during the stimulus processing, where the network redundancy in normal controls is larger in comparison to patients. The differences might be explained by the fact that major depression patients are more restricted to the affective component of the processing. The well-established biasing to affective processing might suppress the somatosensory processing resulting in a lower number of connections within the considered network. This might then lead to a reduction in network redundancy during stimulus processing.
PMCID: PMC3630161  PMID: 23637778
19.  Somatosensory Abnormalities for Painful and Innocuous Stimuli at the Back and at a Site Distinct from the Region of Pain in Chronic Back Pain Patients 
PLoS ONE  2013;8(3):e58885.
Chronic low back pain (CLBP) was shown to be associated with pathophysiological changes at several levels of the sensorimotor system. Changes in sensory thresholds have been reported but complete profiles of Quantitative Sensory Testing (QST) were only rarely obtained in CLBP patients. The aim of the present study was to investigate comprehensive QST profiles in CLBP at the painful site (back) and at a site distinct from their painful region (hand) and to compare these data with similar data in healthy controls. We found increased detection thresholds in CLBP patients compared to healthy controls for all innocuous stimuli at the back and extraterritorial to the painful region at the hand. Additionally, CLBP patients showed decreased pain thresholds at both sites. Importantly, there was no interaction between the investigated site and group, i.e. thresholds were changed both at the affected body site and for the site distinct from the painful region (hand). Our results demonstrate severe, widespread changes in somatosensory sensitivity in CLBP patients. These widespread changes point to alterations at higher levels of the neuraxis or/and to a vulnerability to nociceptive plasticity in CLBP patients.
PMCID: PMC3598908  PMID: 23554950
20.  Plasticity in the Visual System is Associated with Prosthesis Use in Phantom Limb Pain 
The experience of strong phantom limb pain (PLP) in arm amputees was previously shown to be associated with structural neural plasticity in parts of the cortex that belong to dorsal and ventral visual streams. It has been speculated that this plasticity results from the extensive use of a functional prosthesis which is associated with increased visual feedback to control the artificial hand. To test this hypothesis, we reanalyzed data of cortical volumes of 21 upper limb amputees and tested the association between the amount of use of the hand prosthesis and cortical volume plasticity. On the behavioral level, we found no relation between PLP and the amount of prosthesis use for the whole patient group. However, by subdividing the patient group into patients with strong PLP and those with low to medium PLP, stronger pain was significantly associated with less prosthesis use whereas the group with low PLP did not show such an association. Most plasticity of cortical volume was identified within the dorsal stream. The more the patients that suffered from strong PLP used their prosthesis, the smaller was the volume of their posterior parietal cortex. Our data indicate a relationship between prosthesis use and cortical plasticity of the visual stream. This plasticity might present a brain adaptation process to new movement and coordination patterns needed to guide an artificial hand.
PMCID: PMC3690782  PMID: 23805096
phantom limb pain; magnetic resonance imaging; morphometry; use of myoelectric prostheses; visual stream
21.  Human Brain Stem Structures Respond Differentially to Noxious Heat 
Concerning the physiological correlates of pain, the brain stem is considered to be one core region that is activated by noxious input. In animal studies, different slopes of skin heating (SSH) with noxious heat led to activation in different columns of the midbrain periaqueductal gray (PAG). The present study aimed at finding a method for differentiating structures in PAG and other brain stem structures, which are associated with different qualities of pain in humans according to the structures that were associated with different behavioral significances to noxious thermal stimulation in animals. Brain activity was studied by functional MRI in healthy subjects in response to steep and shallow SSH with noxious heat. We found differential activation to different SSH in the PAG and the rostral ventromedial medulla (RVM). In a second experiment, we demonstrate that the different SSH were associated with different pain qualities. Our experiments provide evidence that brainstem structures, i.e., the PAG and the RVM, become differentially activated by different SSH. Therefore, different SSH can be utilized when brain stem structures are investigated and when it is aimed to activate these structures differentially. Moreover, percepts of first pain were elicited by shallow SSH whereas percepts of second pain were elicited by steep SSH. The stronger activation of these brain stem structures to SSH, eliciting percepts of second vs. first pain, might be of relevance for activating different coping strategies in response to the noxious input with the two types of SSH.
PMCID: PMC3764478  PMID: 24032012
A-delta fiber; C-fiber; second pain; pain descriptors; PAG; RVM; periaqueductal grey; rostral ventromedial medulla
22.  Potential impact of a nine-valent vaccine in human papillomavirus related cervical disease 
Information on human papillomavirus (HPV) type distribution is necessary to evaluate the potential impact of current and future HPV vaccines. We estimated the relative contribution (RC) to invasive cervical cancer (ICC) and precancerous cervical lesions of the nine HPV types (HPV 6/11/16/18/31/33/45/52/58) included in an HPV vaccine currently under development.
Estimations on ICC were based on an international study of 8,977 HPV positive cases and estimations on precancerous cervical lesions were extracted from a published meta-analysis including 115,789 HPV positive women. Globocan 2008 and 2010 World Population Prospects were used to estimate current and future projections of new ICC cases.
RC of the 9 HPV types in ICC was 89.4%, with 18.5% of cases positive for HPV 31/33/45/52/58. Regional variations were observed. RCs varied by histology, ranging between 89.1% in squamous cell carcinomas (SCC) and 95.5% in adenocarcinomas (ADC). HPV 16/18/45 were detected in 94.2% of ADC. RC of the 9 types altogether decreased with age (trend test p < 0.0001), driven by the decrease in older ages of HPV 16/18/45. In contrast, the RC of HPV 31/33/52/58 increased with age. Due to population growth alone, projected estimates of ICC cases attributable to the 9 types are expected to rise from 493,770 new cases in 2012 to 560,887 new cases in 2025.
The RCs of individual high risk HPV types varied by cytological and histological grades of HPV-positive precancerous cervical lesions, and there was an under representation of HPV 18 and 45 compared to ICC.
The addition of HPV 31/33/45/52/58 to HPV types included in current vaccines could prevent almost 90% of ICC cases worldwide. If the nine-valent vaccine achieves the same degree of efficacy than previous vaccines, world incidence rates could be substantially reduced.
PMCID: PMC3554470  PMID: 23273245
Human papillomavirus; Cervical cancer; Genotype; Epidemiology; Human papillomavirus vaccines
23.  Selective Protection of Human Liver Tissue in TNF-Targeting of Cancers of the Liver by Transient Depletion of Adenosine Triphosphate 
PLoS ONE  2012;7(12):e52496.
Tumor necrosis factor alpha (TNF) is able to kill cancer cells via receptor-mediated cell death requiring adenosine triphosphate (ATP). Clinical usage of TNF so far is largely limited by its profound hepatotoxicity. Recently, it was found in the murine system that specific protection of hepatocytes against TNF's detrimental effects can be achieved by fructose-mediated ATP depletion therein. Before employing this quite attractive selection principle in a first clinical trial, we here comprehensively investigated the interdependence between ATP depletion and TNF hepatotoxicity in both in vitro and ex vivo experiments based on usage of primary patient tissue materials.
Primary human hepatocytes, and both non-tumorous and tumorous patient-derived primary liver tissue slices were used to elucidate fructose-induced ATP depletion and TNF-induced cytotoxicity.
PHH as well as tissue slices prepared from non-malignant human liver specimen undergoing a fructose-mediated ATP depletion were both demonstrated to be protected against TNF-induced cell death. In contrast, due to tumor-specific overexpression of hexokinase II, which imposes a profound bypass on hepatocytic-specific fructose catabolism, this was not the case for human tumorous liver tissues.
Normal human liver tissues can be protected transiently against TNF-induced cell death by systemic pretreatment with fructose used in non-toxic/physiologic concentrations. Selective TNF-targeting of primary and secondary tumors of the liver by transient and specific depletion of hepatocytic ATP opens up a new clinical avenue for the TNF-based treatment of liver cancers.
PMCID: PMC3525543  PMID: 23272249
24.  Characterization of Lipid Matrices for Membrane Protein Crystallization by High-Throughput Small Angle X-ray Scattering 
Methods (San Diego, Calif.)  2011;55(4):342-349.
The lipidic cubic phase (LCP) has repeatedly proven to serve as a successful membrane-mimetic matrix for a variety of difficult-to-crystallize membrane proteins. While monoolein has been the predominant lipid of choice, there is a growing need for the characterization and use of other LCP host lipids, allowing exploration of a range of structural parameters such as bilayer thickness and curvature for optimal insertion, stability and crystallogenesis of membrane proteins. Here, we describe the development of a high-throughput (HT) pipeline to employ small angle X-ray scattering (SAXS) – the most direct technique to identify lipid mesophases and measure their structural parameters – to interrogate rapidly a large number of lipid samples under a variety of conditions, similar to those encountered during crystallization. Leveraging the identical setup format for LCP crystallization trials, this method allows the quickly assessment of lipid matrices for their utility in membrane protein crystallization, and could inform the tailoring of lipid and precipitant conditions to overcome specific crystallization challenges. As proof of concept, we present HT LCP-SAXS analysis of lipid samples made of monoolein with and without cholesterol, and of monovaccenin, equilibrated with solutions used for crystallization trials and LCP fluorescence recovery after photobleaching (FRAP) experiments.
PMCID: PMC3243793  PMID: 21903166
lipidic cubic phase; monoolein; phase behavior; SAXS
25.  Enhanced sensitivity to punctate painful stimuli in female patients with chronic low back pain 
BMC Neurology  2012;12:98.
Chronic low back pain (CLBP) has been shown to be associated with various pathophysiological changes at several level of the sensorimotor system, pointing to a general hypersensitivity in CLBP patients. The aim of the present study was to investigate signs of generalized mechanical pain hypersensitivity in CLBP patients on the hand and on the painful site of the back.
Pinprick stimulation according to a validated standardized quantitative sensory testing protocol was used in 14 female CLBP patients and 14 healthy controls (HC) matched for sex and age. Stimulus response functions to pinprick stimulation on the skin were examined at the affected back and reference sites (hand palmar and hand dorsum). Data from CLBP patients were compared with HC and with reference data from the German Research Network on Neuropathic Pain.
We found significant differences in the stimulus response functions between CLBP patients and HC. Pain ratings to the pinpricks were increased for low and moderate pinprick stimuli in CLBP patients. Importantly, this kind of specific pinprick hyperalgesia was found not only for the affected body site (back), but also for the remote reference sites (hand dorsum and hand palmar).
We interpret our results as pointing to changes in the nociceptive processing in CLBP at higher levels of the neuraxis, possibly thalamus and/or attentional control, rather than changes of spinal processing. Alternatively, there might be a higher vulnerability to noxious stimulation in CLBP patients.
PMCID: PMC3488472  PMID: 22998460
Chronic Low Back Pain (CLBP); Mechanical pain thresholds; Pinprick hyperalgesia; Allodynia

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