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1.  Redox State of Pentraxin 3 as a Novel Biomarker for Resolution of Inflammation and Survival in Sepsis* 
Molecular & Cellular Proteomics : MCP  2014;13(10):2545-2557.
In an endotoxaemic mouse model of sepsis, a tissue-based proteomics approach for biomarker discovery identified long pentraxin 3 (PTX3) as the lead candidate for inflamed myocardium. When the redox-sensitive oligomerization state of PTX3 was further investigated, PTX3 accumulated as an octamer as a result of disulfide-bond formation in heart, kidney, and lung—common organ dysfunctions seen in patients with sepsis. Oligomeric moieties of PTX3 were also detectable in circulation. The oligomerization state of PTX3 was quantified over the first 11 days in critically ill adult patients with sepsis. On admission day, there was no difference in the oligomerization state of PTX3 between survivors and non-survivors. From day 2 onward, the conversion of octameric to monomeric PTX3 was consistently associated with a greater survival after 28 days of follow-up. For example, by day 2 post-admission, octameric PTX3 was barely detectable in survivors, but it still constituted more than half of the total PTX3 in non-survivors (p < 0.001). Monomeric PTX3 was inversely associated with cardiac damage markers NT-proBNP and high-sensitivity troponin I and T. Relative to the conventional measurements of total PTX3 or NT-proBNP, the oligomerization of PTX3 was a superior predictor of disease outcome.
PMCID: PMC4188985  PMID: 24958171
2.  Effects of perhexiline-induced fuel switch on the cardiac proteome and metabolome 
Perhexiline is a potent anti-anginal drug used for treatment of refractory angina and other forms of heart disease. It provides an oxygen sparing effect in the myocardium by creating a switch from fatty acid to glucose metabolism through partial inhibition of carnitine palmitoyltransferase 1 and 2. However, the precise molecular mechanisms underlying the cardioprotective effects elicited by perhexiline are not fully understood. The present study employed a combined proteomics, metabolomics and computational approach to characterise changes in murine hearts upon treatment with perhexiline. According to results based on difference in-gel electrophoresis, the most profound change in the cardiac proteome related to the activation of the pyruvate dehydrogenase complex. Metabolomic analysis by high-resolution nuclear magnetic resonance spectroscopy showed lower levels of total creatine and taurine in hearts of perhexiline-treated mice. Creatine and taurine levels were also significantly correlated in a cross-correlation analysis of all metabolites. Computational modelling suggested that far from inducing a simple shift from fatty acid to glucose oxidation, perhexiline may cause complex rebalancing of carbon and nucleotide phosphate fluxes, fuelled by increased lactate and amino acid uptake, to increase metabolic flexibility and to maintain cardiac output. This article is part of a Special Issue entitled "Focus on Cardiac Metabolism".
Graphical abstract
► Mice were fed perhexiline to achieve steady state concentrations. ► Hearts were analysed using a combined proteomic and metabolomic approach. ► Computer modelling was used to cross-validate the findings. ► Perhexiline has more wide-ranging and complex metabolic effects than previously thought.
PMCID: PMC3573230  PMID: 23277191
CPT, carnitine palmitoyltransferase; DIGE, difference in-gel electrophoresis; FCS, foetal calf serum; FDR, false discovery rate; GO, Gene ontology; 1H NMR, proton nuclear magnetic resonance spectroscopy; LC-MS/MS, liquid chromatography tandem mass spectrometry; TCA, tricarboxylic acid; Metabolomics; Proteomics; Cardioprotection; Metabolism; Heart failure
3.  Novel Role of ADAMTS-5 Protein in Proteoglycan Turnover and Lipoprotein Retention in Atherosclerosis* 
The Journal of Biological Chemistry  2012;287(23):19341-19345.
Background: In atherosclerosis, proteoglycan accumulation results in increased lipoprotein retention.
Results: ADAMTS-5 is reduced in aortas of apolipoprotein E-null mice. ADAMTS-5 deficiency impairs processing of vascular proteoglycans, and ADAMTS-5 activity affects proteoglycan-mediated lipoprotein retention.
Conclusion: ADAMTS-5 regulates vascular proteoglycan catabolism and alters lipoprotein retention.
Significance: This is the first study implicating ADAMTS-5 proteolytic activity in atherosclerosis.
Atherosclerosis is initiated by the retention of lipoproteins on proteoglycans in the arterial intima. However, the mechanisms leading to proteoglycan accumulation and lipoprotein retention are poorly understood. In this study, we set out to investigate the role of ADAMTS-5 (a disintegrin and metalloprotease with thrombospondin motifs-5) in the vasculature. ADAMTS-5 was markedly reduced in atherosclerotic aortas of apolipoprotein E-null (apoE−/−) mice. The reduction of ADAMTS-5 was accompanied by accumulation of biglycan and versican, the major lipoprotein-binding proteoglycans, in atherosclerosis. ADAMTS-5 activity induced the release of ADAMTS-specific versican (DPEAAE441) and aggrecan (374ALGS) fragments as well as biglycan and link protein from the aortic wall. Fibroblast growth factor 2 (FGF-2) inhibited ADAMTS-5 expression in isolated aortic smooth muscle cells and blocked the spontaneous release of ADAMTS-generated versican and aggrecan fragments from aortic explants. In aortas of ADAMTS-5-deficient mice, DPEAAE441 versican neoepitopes were not detectable. Instead, biglycan levels were increased, highlighting the role of ADAMTS-5 in the catabolism of vascular proteoglycans. Importantly, ADAMTS-5 proteolytic activity reduced the LDL binding ability of biglycan and released LDL from human aortic lesions. This study provides the first evidence implicating ADAMTS-5 in the regulation of proteoglycan turnover and lipoprotein retention in atherosclerosis.
PMCID: PMC3365970  PMID: 22493487
Atherosclerosis; Cardiovascular Disease; Extracellular Matrix; Lipoprotein; Protease; Proteoglycan; ADAMTS-5
4.  Proteomics Analysis of the Cardiac Myofilament Subproteome Reveals Dynamic Alterations in Phosphatase Subunit Distribution* 
Myofilament proteins are responsible for cardiac contraction. The myofilament subproteome, however, has not been comprehensively analyzed thus far. In the present study, cardiomyocytes were isolated from rodent hearts and stimulated with endothelin-1 and isoproterenol, potent inducers of myofilament protein phosphorylation. Subsequently, cardiomyocytes were “skinned,” and the myofilament subproteome was analyzed using a high mass accuracy ion trap tandem mass spectrometer (LTQ Orbitrap XL) equipped with electron transfer dissociation. As expected, a small number of myofilament proteins constituted the majority of the total protein mass with several known phosphorylation sites confirmed by electron transfer dissociation. More than 600 additional proteins were identified in the cardiac myofilament subproteome, including kinases and phosphatase subunits. The proteomic comparison of myofilaments from control and treated cardiomyocytes suggested that isoproterenol treatment altered the subcellular localization of protein phosphatase 2A regulatory subunit B56α. Immunoblot analysis of myocyte fractions confirmed that β-adrenergic stimulation by isoproterenol decreased the B56α content of the myofilament fraction in the absence of significant changes for the myosin phosphatase target subunit isoforms 1 and 2 (MYPT1 and MYPT2). Furthermore, immunolabeling and confocal microscopy revealed the spatial redistribution of these proteins with a loss of B56α from Z-disc and M-band regions but increased association of MYPT1/2 with A-band regions of the sarcomere following β-adrenergic stimulation. In summary, we present the first comprehensive proteomics data set of skinned cardiomyocytes and demonstrate the potential of proteomics to unravel dynamic changes in protein composition that may contribute to the neurohormonal regulation of myofilament contraction.
PMCID: PMC2849712  PMID: 20037178
5.  Antifactor Xa activity in critically ill patients receiving antithrombotic prophylaxis with standard dosages of certoparin: a prospective, clinical study 
Critical Care  2005;9(5):R541-R548.
Deep venous thrombosis with subsequent pulmonary embolism or post-thrombotic syndrome is a feared complication in the intensive care unit. Therefore, routine prophylactic anticoagulation is widely recommended. Aside from unfractionated heparin, low molecular weight heparins, such as certoparin, have become increasingly used for prophylactic anticoagulation in critically ill patients. In this prospective study, we evaluated the potency of 3,000 IU certoparin administered once daily to reach antithrombotic antifactor Xa (aFXa) levels of 0.1 to 0.3 IU/ml in 62 critically ill patients.
AFXa levels were determined 4, 12 and 24 h after injection of certoparin. Prothrombin time, activated partial thromboplastin time, antithrombin, fibrinogen, hemoglobin, platelet count, serum urea and creatinine concentrations were documented before and 12 and 24 h after injection of certoparin.
Four hours after certoparin injection (n = 32), 28% of patients were within the antithrombotic aFXa range. After 12 and 24 h, 6% achieved antithrombotic aFXa levels. Because of a severe pulmonary embolism in one study patient, an interim analysis was performed, and the dosage of certoparin was increased to 3,000 IU twice daily. This regime attained recommended antithrombotic aFXa levels in 47%, 27%, 40% and 30% of patients at 4, 12, 16 and 24 h, respectively, after twice daily certoparin injection (n = 30). Antithrombin and fibrinogen concentrations slightly increased during the observation period. Low antithrombin concentrations before certoparin were independently correlated with underdosing of certoparin. Patients with aFXa levels <0.1 IU/ml 4 h after certoparin injection required vasopressors more often and had lower serum concentrations of creatinine and urea than patients with antithrombotic aFXa levels.
Standard dosages of certoparin of 3,000 IU given once or twice daily are ineffective for attaining the recommended aFXa levels of 0.1 to 0.3 IU/ml in critically ill patients. Low antithrombin levels before certoparin administration were independently associated with low aFXa levels. Renal function and vasopressor therapy may further influence the effectiveness of certoparin in ensuring adequate antithrombotic prophylaxis.
PMCID: PMC1297619  PMID: 16277716
6.  Exacerbated vein graft arteriosclerosis in protein kinase Cδ–null mice 
Journal of Clinical Investigation  2001;108(10):1505-1512.
Smooth muscle cell (SMC) accumulation is a key event in the development of atherosclerosis, including vein bypass graft arteriosclerosis. Because members of the protein kinase C (PKC) family signal cells to undergo proliferation, differentiation, or apoptosis, we generated PKCδ knockout mice and performed vein bypass grafts on these animals. PKCδ–/– mice developed normally and were fertile. Vein segments from PKCδ–/– mice isografted to carotid arteries of recipient mice of either genotype led to a more severe arteriosclerosis than was seen with PKCδ+/+ vein grafts. Arteriosclerotic lesions in PKCδ–/– mice showed a significantly higher number of SMCs than were found in wild-type animals; this was correlated with decreased SMC death in lesions of PKCδ–/– mice. SMCs derived from PKCδ–/– aortae were resistant to cell death induced by any of several stimuli, but they were similar to wild-type SMCs with respect to mitogen-stimulated cell proliferation in vitro. Furthermore, pro-apoptotic treatments led to diminished caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and cytochrome c release in PKCδ–/– relative to wild-type SMCs, suggesting that their apoptotic resistance involves the loss of free radical generation and mitochondrial dysfunction in response to stress stimuli. Our data indicate that PKCδ maintains SMC homeostasis and that its function in the vessel wall per se is crucial in the development of vein graft arteriosclerosis.
PMCID: PMC209416  PMID: 11714742
7.  Freebies 
PMCID: PMC2306017  PMID: 21289744
8.  Escherichia coli Nissle 1917 bacterial ghosts retain crucial surface properties and express chlamydial antigen: an imaging study of a delivery system for the ocular surface 
To target chronic inflammatory ocular surface diseases, a drug delivery platform is needed that is safe, possesses immunomodulatory properties, and can be used either for drug delivery, or as a foreign antigen carrier. A new therapeutic approach that we have previously proposed uses nonliving bacterial ghosts (BGs) as a carrier-delivery system which can be engineered to carry foreign antigens and/or be loaded with therapeutic drugs. The parent strain chosen for development of our BG delivery system is the probiotic Escherichia coli strain Nissle 1917 (EcN), whose intrinsic properties trigger the innate immune system with the flagella and fimbriae used to attach and stimulate epithelial cells. In previous studies, we have shown that EcN BGs are safe for the ocular surface route, but evidence that EcN BGs retain flagella and fimbriae after transformation, has never been visually confirmed. In this study, we used different visualization techniques to determine whether flagella and fimbriae are retained on EcN BGs engineered either for drug delivery or as a foreign antigen carrier. We have also shown by immunoelectron microscopy that EcN retains two foreign antigens after processing to become EcN BGs. Furthermore, we demonstrated that BGs derived from EcN and expressing a foreign antigen attachment to conjunctival epithelial cells in vitro without causing reduced cell viability. These results are an important step in constructing a delivery system based on a nonliving probiotic that is suitable for use in ocular surface diseases pairing immunomodulation and targeted delivery.
PMCID: PMC4516183
delivery system; flagella; fimbriae; foreign antigen; electron microscopy; epithelial cells
9.  Phosphoinositide and Inositol Phosphate Analysis in Lymphocyte Activation 
Lymphocyte antigen receptor engagement profoundly changes the cellular content of phosphoinositide lipids and soluble inositol phosphates. Among these, the phosphoinositides phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) play key signaling roles by acting as pleckstrin homology (PH) domain ligands that recruit signaling proteins to the plasma membrane. Moreover, PIP2 acts as a precursor for the second messenger molecules diacylglycerol and soluble inositol 1,4,5-trisphosphate (IP3), essential mediators of PKC, Ras/Erk, and Ca2+ signaling in lymphocytes. IP3 phosphorylation by IP3 3-kinases generates inositol 1,3,4,5-tetrakisphosphate (IP4), an essential soluble regulator of PH domain binding to PIP3 in developing T cells. Besides PIP2, PIP3, IP3, and IP4, lymphocytes produce multiple other phosphoinositides and soluble inositol phosphates that could have important physiological functions. To aid their analysis, detailed protocols that allow one to simultaneously measure the levels of multiple different phosphoinositide or inositol phosphate isomers in lymphocytes are provided here. They are based on thin layer, conventional and high-performance liquid chromatographic separation methods followed by radiolabeling or non-radioactive metal-dye detection. Finally, less broadly applicable nonchromatographic methods for detection of specific phosphoinositide or inositol phosphate isomers are discussed. Support protocols describe how to obtain pure unstimulated CD4+CD8+ thymocyte populations for analyses of inositol phosphate turnover during positive and negative selection, key steps in T cell development.
PMCID: PMC4500525  PMID: 19918943
lymphocyte; inositol; phosphoinositide; phospholipid; second messenger; T cell; thymocyte; signal transduction; IP3; IP4; IP5; IP6; PIP2; PIP3; HPLC; MDD-HPLC
10.  Rotational laxity and collateral ligament laxity following total knee arthroplasty with rotating platform 
International Orthopaedics  2014;38(7):1379-1386.
The aim of this study was to evaluate laxity in knees with pre-operative (preop) valgus alignment compared to knees with pre-operative varus alignment after total knee arthroplasty (TKA).
This was a retrospective study including 81 patients, with six years follow-up, for pre-operative valgus- or varus alignment of the leg. All patients had been supplied with the same cruciate retaining (CR) TKA with rotating platform. Clinical findings were assessed by KSS, OKS and IKDC 2000 score. Rotational knee laxity was evaluated by a validated instrument (Laxitester®) with 2 Nm torque in 30° flexion. Collateral ligament laxity was tested manually in 30° flexion with a bending moment of approximately 5 Nm. Biomechanical results were compared to the contralateral side.
Thirty-one patients had a preop valgus alignment of 8.96° and 50 patients a varus leg axis of 4.99° in the mean. In the preop valgus knees rotational analysis showed an increased laxity of 10.7° compared to preop varus knees (p = 0.001). There was no significant difference in medial (valgus 2.6 mm, varus 2.5 mm) and lateral (valgus 2.8 mm, varus 2.7 mm) laxity. KSS and OKS showed no significant differences in the follow-up results. In the IKDC 2000 objective score 50 % of the preop varus knees and 25.8 % of the preop valgus knees were classified as nearly normal. The difference in the IKDC objective was highly significant (p < 0.001).
Preop valgus knees show a significantly increased rotational laxity but no increased collateral ligament laxity compared to pre-operative varus knees six years after TKA with rotating platform. There is a significant difference in IKDC objective.
PMCID: PMC4071494  PMID: 24604621
Knee laxity; Total knee replacement; Rotational laxity knee; Rotating platform TKA; Cruciate retaining TKA; Laxitester
11.  Long-Term Memory and the Control of Attentional Control 
Cognitive psychology  2014;72:1-26.
Task-switch costs and in particular the switch-cost asymmetry (i.e., the larger costs of switching to a dominant than a non-dominant task) are usually explained in terms of trial-to-trial carry-over of task-specific control settings. Here we argue that task switches are just one example of situations that trigger a transition from working-memory maintenance to updating, thereby opening working memory to interference from long-term memory. We used a new paradigm that requires selecting a spatial location either on the basis of a central cue (i.e., endogenous control of attention) or a peripheral, sudden onset (i.e., exogenous control of attention). We found a strong cost asymmetry that occurred even after short interruptions of otherwise single-task blocks (Exp. 1-3), but that was much stronger when participants had experienced the competing task under conditions of conflict (Exp. 1-2). Experiment 3 showed that the asymmetric costs were due to interruptions per se, rather than to associative interference tied to specific interruption activities. Experiment 4 generalized the basic pattern across interruptions varying in length or control demands and Experiment 5 across primary tasks with response-selection conflict rather than attentional conflict. Combined, the results support a model in which costs of selecting control settings arise when (a) potentially interfering memory traces have been encoded in long-term memory and (b) working-memory is forced from a maintenance mode into an updating mode (e.g., through task interruptions), thereby allowing unwanted retrieval of the encoded memory traces.
PMCID: PMC4294623  PMID: 24650696
Attention; Executive Control; Task Switching
12.  Toward Clinically Compatible Phase-Contrast Mammography 
PLoS ONE  2015;10(6):e0130776.
Phase-contrast mammography using laboratory X-ray sources is a promising approach to overcome the relatively low sensitivity and specificity of clinical, absorption-based screening. Current research is mostly centered on identifying potential diagnostic benefits arising from phase-contrast and dark-field mammography and benchmarking the latter with conventional state-of-the-art imaging methods. So far, little effort has been made to adjust this novel imaging technique to clinical needs. In this article, we address the key points for a successful implementation to a clinical routine in the near future and present the very first dose-compatible and rapid scan-time phase-contrast mammograms of both a freshly dissected, cancer-bearing mastectomy specimen and a mammographic accreditation phantom.
PMCID: PMC4481352  PMID: 26110618
13.  The green tea catechin epigallocatechin gallate induces cell cycle arrest and shows potential synergism with cisplatin in biliary tract cancer cells 
The green tea catechin epigallocatechin gallate (EGCG) was shown to effectively inhibit tumor growth in various types of cancer including biliary tract cancer (BTC). For most BTC patients only palliative therapy is possible, leading to a median survival of about one year. Chemoresistance is a major problem that contributes to the high mortality rates of BTC. The aim of this study was to investigate the cytotoxic effect of EGCG alone or in combination with cisplatin on eight BTC cell lines and to investigate the cellular anti-cancer mechanisms of EGCG.
The effect of EGCG treatment alone or in combination with the standard chemotherapeutic cisplatin on cell viability was analyzed in eight BTC cell lines. Additionally, we analyzed the effects of EGCG on caspase activity, cell cycle distribution and gene expression in the BTC cell line TFK-1.
EGCG significantly reduced cell viability in all eight BTC cell lines (p < 0.05 or p < 0.01, respectively, for most cell lines and EGCG concentrations > 5 μM). Combined EGCG and cisplatin treatment showed a synergistic cytotoxic effect in five cell lines and an antagonistic effect in two cell lines. Furthermore, EGCG reduced the mRNA levels of various cell cycle-related genes, while increasing the expression of the cell cycle inhibitor p21 and the apoptosis-related death receptor 5 (p < 0.05). This observation was accompanied by an increase in caspase activity and cells in the sub-G1 phase of the cell cycle, indicating induction of apoptosis. EGCG also induced a down-regulation of expression of stem cell-related genes and genes that are associated with an aggressive clinical character of the tumor, such as cd133 and abcg2.
EGCG shows various anti-cancer effects in BTC cell lines and might therefore be a potential anticancer drug for future studies in BTC. Additionally, EGCG displays a synergistic cytotoxic effect with cisplatin in most tested BTC cell lines.
Graphical abstractSummary illustration
PMCID: PMC4477611  PMID: 26100134
EGCG; Green tea; Biliary tract cancer; Drug synergism; Cisplatin; Cell cycle arrest
14.  The elusive link between conflict and conflict adaptation 
Psychological research  2008;73(6):794-802.
A core tenet of the original conflict-monitoring model is that regulation is triggered automatically when conflict is present and that the same regulation mechanism explains both trial-to-trial adaptation effects as well as effects of block-wise conflict manipulations. We present here results from two experiments using the Stroop task that show (a) that adaptation effects in the absence of response repetitions may occur only at the beginning of testing and that (b) robust block-wise effects can be found even in the absence of trial-by-trial effects. Furthermore, we show that the failure to eliminate target-to-distractor repetitions can produce artificial trial-to-trial adaptation effects. Based on the evidence of a weak link between conflict and conflict adaptation, we argue that a wider range of possible reasons for conflict adaptation effects needs to be taken into consideration.
PMCID: PMC4476291  PMID: 19034501
15.  Tidal day organic and inorganic material flux of ponds in the Liberty Island freshwater tidal wetland 
SpringerPlus  2015;4:273.
The loss of inorganic and organic material export and habitat produced by freshwater tidal wetlands is hypothesized to be an important contributing factor to the long-term decline in fishery production in San Francisco Estuary. However, due to the absence of freshwater tidal wetlands in the estuary, there is little information on the export of inorganic and organic carbon, nutrient or phytoplankton community biomass and the associated mechanisms. A single-day study was conducted to assess the potential contribution of two small vegetated ponds and one large open-water pond to the inorganic and organic material flux within the freshwater tidal wetland Liberty Island in San Francisco Estuary. The study consisted of an intensive tidal day (25.5 h) sampling program that measured the flux of inorganic and organic material at three ponds using continuous monitoring of flow, chlorophyll a, turbidity and salt combined with discrete measurements of phytoplankton community carbon, total and dissolved organic carbon and nutrient concentration at 1.5 h intervals. Vegetated ponds had greater material concentrations than the open water pond and, despite their small area, contributed up to 81% of the organic and 61% of the inorganic material flux of the wetland. Exchange between ponds was important to wetland flux. The small vegetated pond in the interior of the wetland contributed as much as 72–87% of the total organic carbon and chlorophyll a and 10% of the diatom flux of the wetland. Export of inorganic and organic material from the small vegetated ponds was facilitated by small-scale topography and tidal asymmetry that produced a 40% greater material export on ebb tide. The small vegetated ponds contrasted with the large open water pond, which imported 29–96% of the inorganic and 4–81% of the organic material into the wetland from the adjacent river. This study identified small vegetated ponds as an important source of inorganic and organic material to the wetland and the importance of small scale physical processes within ponds to material flux of the wetland.
PMCID: PMC4469598  PMID: 26090320
Freshwater tidal wetland; Wetland material flux; Vegetated ponds; Carbon flux
16.  Inhibition of Neuroblastoma Tumor Growth by Ketogenic Diet and/or Calorie Restriction in a CD1-Nu Mouse Model 
PLoS ONE  2015;10(6):e0129802.
Neuroblastoma is a malignant pediatric cancer derived from neural crest cells. It is characterized by a generalized reduction of mitochondrial oxidative phosphorylation. The goal of the present study was to investigate the effects of calorie restriction and ketogenic diet on neuroblastoma tumor growth and monitor potential adaptive mechanisms of the cancer’s oxidative phosphorylation system.
Xenografts were established in CD-1 nude mice by subcutaneous injection of two neuroblastoma cell lines having distinct genetic characteristics and therapeutic sensitivity [SH-SY5Y and SK-N-BE(2)]. Mice were randomized to four treatment groups receiving standard diet, calorie-restricted standard diet, long chain fatty acid based ketogenic diet or calorie-restricted ketogenic diet. Tumor growth, survival, metabolic parameters and weight of the mice were monitored. Cancer tissue was evaluated for diet-induced changes of proliferation indices and multiple oxidative phosphorylation system parameters (respiratory chain enzyme activities, western blot analysis, immunohistochemistry and mitochondrial DNA content).
Ketogenic diet and/or calorie restriction significantly reduced tumor growth and prolonged survival in the xenograft model. Neuroblastoma growth reduction correlated with decreased blood glucose concentrations and was characterized by a significant decrease in Ki-67 and phospho-histone H3 levels in the diet groups with low tumor growth. As in human tumor tissue, neuroblastoma xenografts showed distinctly low mitochondrial complex II activity in combination with a generalized low level of mitochondrial oxidative phosphorylation, validating the tumor model. Neuroblastoma showed no ability to adapt its mitochondrial oxidative phosphorylation activity to the change in nutrient supply induced by dietary intervention.
Our data suggest that targeting the metabolic characteristics of neuroblastoma could open a new front in supporting standard therapy regimens. Therefore, we propose that a ketogenic diet and/or calorie restriction should be further evaluated as a possible adjuvant therapy for patients undergoing treatment for neuroblastoma.
PMCID: PMC4459995  PMID: 26053068
18.  A survey study on student preferences regarding pathology teaching in Germany: a call for curricular modernization 
BMC Medical Education  2015;15:94.
Pathology is a discipline that provides the basis of the understanding of disease in medicine. The past decades have seen a decline in the emphasis laid on pathology teaching in medical schools and outdated pathology curricula have worsened the situation. Student opinions and thoughts are central to the questions of whether and how such curricula should be modernized.
A survey was conducted among 1018 German medical students regarding their preferences in pathology teaching modalities and their satisfaction with lecture-based courses. A qualitative analysis was performed comparing a recently modernized pathology curriculum with a traditional lecture-based curriculum. The differences in modalities of teaching used were investigated.
Student satisfaction with the lecture-based curriculum positively correlated with student grades (spearman’s correlation coefficient 0.24). Additionally, students with lower grades supported changing the curriculum (spearman’s correlation coefficient 0.47). The majority supported virtual microscopy, autopsies, seminars and podcasts as preferred didactic methods.
The data supports the implementation of a pathology curriculum where tutorials, autopsies and supplementary computer-based learning tools play important roles.
PMCID: PMC4460630  PMID: 26032301
Pathology education; Curriculum development; Student survey; Teaching
19.  Thyroid Hormone Receptors Predict Prognosis in BRCA1 Associated Breast Cancer in Opposing Ways 
PLoS ONE  2015;10(6):e0127072.
Since BRCA1 associated breast cancers are frequently classified as hormone receptor negative or even triple negative, the application of endocrine therapies is rather limited in these patients. Like hormone receptors that bind to estrogen or progesterone, thyroid hormone receptors (TRs) are members of the nuclear hormone receptor superfamily. TRs might be interesting biomarkers - especially in the absence of classical hormone receptors. The current study aimed to investigate whether TRs may be specifically expressed in BRCA1 associated cancer cases and whether they are of prognostic significance in these patients as compared to sporadic breast cancer cases. This study analyzed TRα and TRβ immunopositivity in BRCA1 associated (n = 38) and sporadic breast cancer (n = 86). Further, TRs were studied in MCF7 (BRCA1 wildtype) and HCC3153 (BRCA1 mutated) cells. TRβ positivity rate was significantly higher in BRCA1 associated as compared to sporadic breast cancers (p = 0.001). The latter observation remained to be significant when cases that had been matched for clinicopathological criteria were compared (p = 0.037). Regarding BRCA1 associated breast cancer cases TRβ positivity turned out to be a positive prognostic factor for five-year (p = 0.007) and overall survival (p = 0.026) while TRα positivity predicted reduced five-year survival (p = 0.030). Activation of TRβ resulted in down-modulation of CTNNB1 while TRα inhibition reduced cell viability in HCC3153. However, only BRCA1 wildtype MCF7 cells were capable of rapidly degrading TRα1 in response to T3 stimulation. Significantly, this study identified TRβ to be up-regulated in BRCA1 associated breast cancer and revealed TRs to be associated with patients’ prognosis. TRs were also found to be expressed in triple negative BRCA1 associated breast cancer. Further studies need to be done in order to evaluate whether TRs may become interesting targets of endocrine therapeutic approaches, especially when tumors are triple-negative.
PMCID: PMC4451081  PMID: 26029931
20.  Immunoreactivity of the fully humanized therapeutic antibody PankoMab-GEX™ is an independent prognostic marker for breast cancer patients 
Mucin-1 (MUC1, CD227), more widely known as CA15-3, is an abundantly expressed epithelial cell surface antigen and has evolved to be the most predictive serum tumour marker in breast cancer. PankoMab-GEX™, which is currently being evaluated for its therapeutic efficacy in a phase IIb clinical trial, is a glyco-optimized anti-MUC1 antibody specifically recognizing a tumour-associated MUC1 epitope (TA-MUC1). The current study aimed to analyse the immunoreactivity of PankoMabGEX™ and its correlation with established clinico-pathological variables including 10-year and overall survival in a large cohort of breast cancer patients.
Breast cancer tissue sections (n = 227) underwent a standardized immunohistochemical staining protocol for TA-MUC1 by using PankoMab-GEX™ as a primary antibody. The staining was evaluated by two independent observers and quantified by applying the IR-score.
TA-MUC1 as detected by PankoMab-GEX™ was identified in 74.9% of breast cancer tissue sections. Patients were subdivided according to the subcellular localisation of TA-MUC1 and cases classified as mem-PankoMab-GEX™ (solely membranous) positive, cyt-PankoMab-GEX™ (solely cytoplasmic) positive, double positive or as completely negative were compared regarding their survival. Herein mem-PankoMab-GEX™-positive patients performed best, while double-negative ones presented with a significantly shortened survival. Positivity for mem-PankoMab-GEX™ as well as a double-negative immunophenotype turned out to be independent prognosticators for survival.
This is the first study to report on PankoMab-GEX™ in a large panel of breast cancer patients. The PankoMab-GEX™ epitope TA-MUC1 could be identified in the majority of cases and was found to be an independent prognosticator depending on its subcellular localisation. Since TA-MUC1 is known to be highly immunogenic cancers staining positive for PankoMab-GEX™ might be more compromised by host anti-tumour immune defence. Further, the observations reported here might be fundamental for selecting patients to undergo PankoMab-GEX™-containing chemotherapy protocols.
Electronic supplementary material
The online version of this article (doi:10.1186/s13046-015-0152-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4447018  PMID: 25986064
PankoMab-GEX™; Prognosis; Breast cancer; MUC1; TA-MUC1; Immunohistochemistry
21.  Understanding Patient Satisfaction Ratings for Radiology Services 
AJR. American journal of roentgenology  2013;201(6):1190-1196.
Under the Hospital Value-Based Purchasing Program of the Centers for Medicare & Medicaid Services patient satisfaction accounts for 30% of the measures of and payments for quality of care. Understanding what drives satisfaction data, how it is obtained, converted into scores, and formulated into rankings, is increasingly critical for imaging departments.
PMCID: PMC4427188  PMID: 24261356
22.  Dynamic Impedance Model of the Skin-Electrode Interface for Transcutaneous Electrical Stimulation 
PLoS ONE  2015;10(5):e0125609.
Transcutaneous electrical stimulation can depolarize nerve or muscle cells applying impulses through electrodes attached on the skin. For these applications, the electrode-skin impedance is an important factor which influences effectiveness. Various models describe the interface using constant or current-depending resistive-capacitive equivalent circuit. Here, we develop a dynamic impedance model valid for a wide range stimulation intensities. The model considers electroporation and charge-dependent effects to describe the impedance variation, which allows to describe high-charge pulses. The parameters were adjusted based on rectangular, biphasic stimulation pulses generated by a stimulator, providing optionally current or voltage-controlled impulses, and applied through electrodes of different sizes. Both control methods deliver a different electrical field to the tissue, which is constant throughout the impulse duration for current-controlled mode or have a very current peak for voltage-controlled. The results show a predominant dependence in the current intensity in the case of both stimulation techniques that allows to keep a simple model. A verification simulation using the proposed dynamic model shows coefficient of determination of around 0.99 in both stimulation types. The presented method for fitting electrode-skin impedance can be simple extended to other stimulation waveforms and electrode configuration. Therefore, it can be embedded in optimization algorithms for designing electrical stimulation applications even for pulses with high charges and high current spikes.
PMCID: PMC4420281  PMID: 25942010
23.  Eocene fossil is earliest evidence of flower-visiting by birds 
Biology Letters  2014;10(5):20140223.
Birds are important pollinators, but the evolutionary history of ornithophily (bird pollination) is poorly known. Here, we report a skeleton of the avian taxon Pumiliornis from the middle Eocene of Messel in Germany with preserved stomach contents containing numerous pollen grains of an eudicotyledonous angiosperm. The skeletal morphology of Pumiliornis is in agreement with this bird having been a, presumably nectarivorous, flower-visitor. It represents the earliest and first direct fossil evidence of flower-visiting by birds and indicates a minimum age of 47 million years for the origin of bird–flower interactions. As Pumiliornis does not belong to any of the modern groups of flower-visiting birds, the origin of ornithophily in some angiosperm lineages may have predated that of their extant avian pollinators.
PMCID: PMC4046380  PMID: 24872461
bird pollination; ornithophily; fossil birds; Messel; Eocene
24.  Risk of Cardiovascular Events in Survivors of Severe Sepsis 
Rationale: The risk of cardiovascular events after severe sepsis is not known, and these events may explain increased long-term mortality in survivors of severe sepsis.
Objectives: To determine whether survivors of severe sepsis hospitalization have high long-term risk of cardiovascular events. We examined whether higher risk is due to severe sepsis hospitalization or poor prehospitalization health status, and if the higher risk is also observed in patients hospitalized for infectious and noninfectious reasons, and in other critically ill patients.
Methods: Unmatched and matched-cohort analyses of Medicare beneficiaries. For unmatched analysis, we compared patients with severe sepsis admitted to the intensive care unit (ICU) and survived hospitalization (n = 4,179) to unmatched population control subjects (n = 819,283). For matched analysis, we propensity-score-matched each patient with severe sepsis to four control subjects (population, hospitalized, non–severe sepsis ICU control subjects, and infection hospitalization). Primary outcome was 1-year incidence rate of hospitalization for cardiovascular events.
Measurements and Main Results: Cardiovascular events were common among patients discharged alive after severe sepsis hospitalization (29.5%; 498.2 events/1,000 person-years). Survivors of severe sepsis had a 13-fold higher risk of cardiovascular events compared with unmatched control subjects (498.2 vs. 36 events/1,000 person-years; P < 0.0001), and a 1.9-fold higher risk compared with matched-population control subjects (P < 0.0001). Survivors of severe sepsis had 1.1-fold higher risk compared with matched hospitalized patients and infection hospitalizations (P = 0.002 and 0.001) and similar risk compared with matched-ICU control subjects.
Conclusions: Survivors of severe sepsis have high risk of cardiovascular events. The higher risk is mainly due to poor prehospitalization health status, and is also seen in a broader population of acutely ill patients.
PMCID: PMC4098105  PMID: 24456535
sepsis; severe sepsis; cardiovascular disease; mortality
25.  Distinct Mechanisms Regulate Exposure of Neutralizing Epitopes in the V2 and V3 Loops of HIV-1 Envelope 
Journal of Virology  2014;88(21):12853-12865.
Broadly neutralizing antibodies targeting the HIV-1 envelope (Env) are key components for protection against HIV-1. However, many cross-reactive epitopes are often occluded. This study investigates the mechanisms contributing to the masking of V2i (variable loop V2 integrin) epitopes compared to the accessibility of V3 epitopes. V2i are conformation-dependent epitopes encompassing the integrin α4β7-binding motif on the V1V2 loop of HIV-1 Env gp120. The V2i monoclonal antibodies (MAbs) display extensive cross-reactivity with gp120 monomers from many subtypes but neutralize only few viruses, indicating V2i's cryptic nature. First, we asked whether CD4-induced Env conformational changes affect V2i epitopes similarly to V3. CD4 treatment of BaL and JRFL pseudoviruses increased their neutralization sensitivity to V3 MAbs but not to the V2i MAbs. Second, the contribution of N-glycans in masking V2i versus V3 epitopes was evaluated by testing the neutralization of pseudoviruses produced in the presence of a glycosidase inhibitor, kifunensine. Viruses grown in kifunensine were more sensitive to neutralization by V3 but not V2i MAbs. Finally, we evaluated the time-dependent dynamics of the V2i and V3 epitopes. Extending the time of virus-MAb interaction to 18 h before adding target cells increased virus neutralization by some V2i MAbs and all V3 MAbs tested. Consistent with this, V2i MAb binding to Env on the surface of transfected cells also increased in a time-dependent manner. Hence, V2i and V3 epitopes are highly dynamic, but distinct factors modulate the antibody accessibility of these epitopes. The study reveals the importance of the structural dynamics of V2i and V3 epitopes in determining HIV-1 neutralization by antibodies targeting these sites.
IMPORTANCE Conserved neutralizing epitopes are present in the V1V2 and V3 regions of HIV-1 Env, but these epitopes are often occluded from Abs. This study reveals that distinct mechanisms contribute to the masking of V3 epitopes and V2i epitopes in the V1V2 domain. Importantly, V3 MAbs and some V2i MAbs display greater neutralization against relatively resistant HIV-1 isolates when the MAbs interact with the virus for a prolonged period of time. Given their highly immunogenic nature, V3 and V2i epitopes are valuable targets that would augment the efficacy of HIV vaccines.
PMCID: PMC4248937  PMID: 25165106

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