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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.  Ultra-early Predictive Assay for Treatment Failure Using Functional Magnetic Resonance Imaging and Clinical Prognostic Parameters in Cervical Cancer 
Cancer  2010;116(4):903-912.
We prospectively evaluated MRI-parameters quantifying heterogeneous perfusion pattern and residual tumor volume early during treatment in cervical cancer, and compared their predictive power for primary tumor recurrence and cancer death with the standard clinical prognostic factors (CPFs). A novel approach of augmenting the predictive power of CPFs with MRI-parameters was assessed.
Sixty-two cervical cancer patients underwent dynamic contrast-enhanced (DCE) MRI before and during early radiation/chemotherapy (2-2.5 weeks into treatment). Heterogeneous tumor perfusion was analyzed by signal intensity (SI) of each tumor voxel. Poorly-perfused tumor regions were quantitated as lower 10th percentile of SI (SI(10%)). DCE-MRI and three-dimensional (3D) tumor volumetry MRI-parameters were assessed as predictors of recurrence and cancer death (median follow-up, 4.1 years). Their discriminating capacity was compared with CPFs (stage, lymphnode status, histology) using sensitivity/specificity and Cox regression analysis.
SI(10%) and 3D-volume 2-2.5 weeks into therapy independently predicted recurrence (HR=2.6 [1.0-6.5], P=.04; HR=1.9 [1.1-3.5], P=.03, respectively) and death (HR=1.9 [1.0--3.5], P=.03; HR=1.9 [1.2-2.9], P=.01), and were superior to CPFs. The addition of MRI-parameters to CPFs increased sensitivity and specificity of CPFs from 71% and 51% to 100% and 71% for predicting recurrence; and from 79% and 54% to 93% and 60% for predicting death.
MRI-parameters reflecting heterogeneous tumor perfusion and subtle tumor volume change early during radiation/chemotherapy are independent and better predictors of tumor recurrence and death than CPFs. Combination of CPFs and MRI-parameters further improves early prediction of treatment failure and may enable a window of opportunity to alter treatment strategy.
PMCID: PMC4362726  PMID: 20052727
DCE magnetic resonance imaging; Functional tumor imaging; Cervical cancer; Outcome prediction; Tumor heterogeneity; Tumor perfusion
9.  The Evolution of HIV-1 group M Genetic Variability in Southern Cameroon is characterized by Several Emerging Recombinant Forms of CRF02_AG and viruses with drug resistance mutations 
Journal of medical virology  2013;86(3):385-393.
The HIV epidemic in Cameroon is marked by a broad genetic diversity dominated by Circulating Recombinant Forms (CRFs). Studies performed more than a decade ago in urban settings of Southern Cameroon revealed a dominance of the CRF02_AG and clade A variants in >90% of the infected subjects; however, little is known about the evolving viral variants circulating in this region. To document circulating HIV viral diversity, four regions of the viral genome (gag, PR, reverse transcriptase, env) in 116 HIV-1 positive individuals in Limbe, Southern Cameroon, were PCR-amplified. Sequences obtained at the RT and protease regions were analyzed for mutations that conferred drug resistance using the Stanford Drug Resistance Database. The present study reveals a broad genetic diversity characterized by several unique recombinant forms (URF) accounting for 36% of infections, 48.6% of patients infected with CRF02_AG, and the emergence of CRF22_01A1 in 7.2% of patients. Three out of 15 (20%) treated patients and 13 out of 93 (13.9%) drug naïve patients harbor drug resistance mutations to RT inhibitors, while 3.2% of drug naïve patients harbor drug resistance mutations associated with protease inhibitors. The high proportion (13.9%) of drug resistance mutations among the drug naïve patients reveals the ongoing transmission of these viruses in this region of Cameroon and highlights the need for drug resistance testing before starting treatment for patients infected with HIV-1.
PMCID: PMC4011137  PMID: 24248638
Cameroon; Genetic Diversity; Unique Recombinant Forms; Drug Resistance Mutations
10.  Modification of spasticity by transcutaneous spinal cord stimulation in individuals with incomplete spinal cord injury 
To examine the effects of transcutaneous spinal cord stimulation (tSCS) on lower-limb spasticity.
Interventional pilot study to produce preliminary data.
Department of Physical Medicine and Rehabilitation, Wilhelminenspital, Vienna, Austria.
Three subjects with chronic motor-incomplete spinal cord injury (SCI) who could walk ≥10 m.
Two interconnected stimulating skin electrodes (Ø 5 cm) were placed paraspinally at the T11/T12 vertebral levels, and two rectangular electrodes (8 × 13 cm) on the abdomen for the reference. Biphasic 2 ms-width pulses were delivered at 50 Hz for 30 minutes at intensities producing paraesthesias but no motor responses in the lower limbs.
Outcome measures
The Wartenberg pendulum test and neurological recordings of surface-electromyography (EMG) were used to assess effects on exaggerated reflex excitability. Non-functional co-activation during volitional movement was evaluated. The timed 10-m walk test provided measures of clinical function.
The index of spasticity derived from the pendulum test changed from 0.8 ± 0.4 pre- to 0.9 ± 0.3 post-stimulation, with an improvement in the subject with the lowest pre-stimulation index. Exaggerated reflex responsiveness was decreased after tSCS across all subjects, with the most profound effect on passive lower-limb movement (pre- to post-tSCS EMG ratio: 0.2 ± 0.1), as was non-functional co-activation during voluntary movement. Gait speed values increased in two subjects by 39%.
These preliminary results suggest that tSCS, similar to epidurally delivered stimulation, may be used for spasticity control, without negatively impacting residual motor control in incomplete SCI. Further study in a larger population is warranted.
PMCID: PMC4066429  PMID: 24090290
Humans; Movement; Muscle spasticity; Spinal cord injuries; Spinal cord stimulation; Paraplegia; Tetraplegia; Rehabilitation
11.  Control of Task Sequences: What is the Role of Language? 
It is almost a truism that language aids serial-order control through self-cuing of upcoming sequential elements. We measured speech onset latencies as subjects performed hierarchically organized task sequences while "thinking aloud" each task label. Surprisingly, speech onset latencies and response times (RTs) were highly synchronized, a pattern that is not consistent with the hypothesis that speaking aids proactive retrieval of upcoming sequential elements during serial-order control. We also found that when instructed to do so, participants were able to speak task labels prior to presentation of response-relevant stimuli and that this substantially reduced RT signatures of retrieval—however at the cost of more sequencing errors. Thus, while proactive retrieval is possible in principle, in natural situations it seems to be prevented through a strong, "gestalt-like" tendency to synchronize speech and action. We suggest that this tendency may support context updating rather than proactive control.
PMCID: PMC4301959  PMID: 24274386
15.  Cognitive Aging: Is There a Dark Side to Environmental Support? 
Trends in cognitive sciences  2013;18(1):7-15.
It has been known for some time that memory deficits among older adults increase when self-initiated processing is required, and decrease when the environment provides task-appropriate cues. We propose that this observation is not confined to memory but can be subsumed under a more general developmental trend. In perception, learning/memory, and action management, older adults often rely more on external information than younger adults, probably both as a direct reflection and indirect adaptation to difficulties in internally triggering and maintaining cognitive representations. This age-graded shift from internal towards environmental control is often associated with compromised performance. Cognitive aging research and the design of aging-friendly environments can benefit from paying closer attention to the developmental dynamics and implications of this shift.
PMCID: PMC3969029  PMID: 24210962
environmental support; self-initiated processing; cognitive aging; cognitive control
16.  Trans-Splicing Improvement by the Combined Application of Antisense Strategies 
Spliceosome-mediated RNA trans-splicing has become an emergent tool for the repair of mutated pre-mRNAs in the treatment of genetic diseases. RNA trans-splicing molecules (RTMs) are designed to induce a specific trans-splicing reaction via a binding domain for a respective target pre-mRNA region. A previously established reporter-based screening system allows us to analyze the impact of various factors on the RTM trans-splicing efficiency in vitro. Using this system, we are further able to investigate the potential of antisense RNAs (AS RNAs), presuming to improve the trans-splicing efficiency of a selected RTM, specific for intron 102 of COL7A1. Mutations in the COL7A1 gene underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa (DEB). We have shown that co-transfections of the RTM and a selected AS RNA, interfering with competitive splicing elements on a COL7A1-minigene (COL7A1-MG), lead to a significant increase of the RNA trans-splicing efficiency. Thereby, accurate trans-splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level. This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high trans-splicing efficiency is required.
PMCID: PMC4307297  PMID: 25569093
RNA trans-splicing; antisense oligonucleotides; RNA repair; genetic diseases
17.  Switched-capacitor realization of presynaptic short-term-plasticity and stop-learning synapses in 28 nm CMOS 
Synaptic dynamics, such as long- and short-term plasticity, play an important role in the complexity and biological realism achievable when running neural networks on a neuromorphic IC. For example, they endow the IC with an ability to adapt and learn from its environment. In order to achieve the millisecond to second time constants required for these synaptic dynamics, analog subthreshold circuits are usually employed. However, due to process variation and leakage problems, it is almost impossible to port these types of circuits to modern sub-100nm technologies. In contrast, we present a neuromorphic system in a 28 nm CMOS process that employs switched capacitor (SC) circuits to implement 128 short term plasticity presynapses as well as 8192 stop-learning synapses. The neuromorphic system consumes an area of 0.36 mm2 and runs at a power consumption of 1.9 mW. The circuit makes use of a technique for minimizing leakage effects allowing for real-time operation with time constants up to several seconds. Since we rely on SC techniques for all calculations, the system is composed of only generic mixed-signal building blocks. These generic building blocks make the system easy to port between technologies and the large digital circuit part inherent in an SC system benefits fully from technology scaling.
PMCID: PMC4313588  PMID: 25698914
switched-capacitor neuromorphic; stop-learning synapse; dynamic synapse; deep-submicron neuromorphic; low-leakage switched-capacitor circuits
18.  Plasticity in memristive devices for spiking neural networks 
Memristive devices present a new device technology allowing for the realization of compact non-volatile memories. Some of them are already in the process of industrialization. Additionally, they exhibit complex multilevel and plastic behaviors, which make them good candidates for the implementation of artificial synapses in neuromorphic engineering. However, memristive effects rely on diverse physical mechanisms, and their plastic behaviors differ strongly from one technology to another. Here, we present measurements performed on different memristive devices and the opportunities that they provide. We show that they can be used to implement different learning rules whose properties emerge directly from device physics: real time or accelerated operation, deterministic or stochastic behavior, long term or short term plasticity. We then discuss how such devices might be integrated into a complete architecture. These results highlight that there is no unique way to exploit memristive devices in neuromorphic systems. Understanding and embracing device physics is the key for their optimal use.
PMCID: PMC4345885  PMID: 25784849
memristive device; memristor; neuromorphic engineering; plasticity; hardware neural network
19.  Amino Alcohol- (NPS-2143) and Quinazolinone-Derived Calcilytics (ATF936 and AXT914) Differentially Mitigate Excessive Signalling of Calcium-Sensing Receptor Mutants Causing Bartter Syndrome Type 5 and Autosomal Dominant Hypocalcemia 
PLoS ONE  2014;9(12):e115178.
Activating calcium sensing receptor (CaSR) mutations cause autosomal dominant hypocalcemia (ADH) characterized by low serum calcium, inappropriately low PTH and relative hypercalciuria. Four activating CaSR mutations cause additional renal wasting of sodium, chloride and other salts, a condition called Bartter syndrome (BS) type 5. Until today there is no specific medical treatment for BS type 5 and ADH. We investigated the effects of different allosteric CaSR antagonists (calcilytics) on activating CaSR mutants.
All 4 known mutations causing BS type 5 and five ADH mutations were expressed in HEK 293T cells and receptor signalling was studied by measurement of intracellular free calcium in response to extracellular calcium ([Ca2+]o). To investigate the effect of calcilytics, cells were stimulated with 3 mM [Ca2+]o in the presence or absence of NPS-2143, ATF936 or AXT914.
All BS type 5 and ADH mutants showed enhanced signalling activity to [Ca2+]o with left shifted dose response curves. In contrast to the amino alcohol NPS-2143, which was only partially effective, the quinazolinone calcilytics ATF936 and AXT914 significantly mitigated excessive cytosolic calcium signalling of all BS type 5 and ADH mutants studied. When these mutants were co-expressed with wild-type CaSR to approximate heterozygosity in patients, ATF936 and AXT914 were also effective on all mutants.
The calcilytics ATF936 and AXT914 are capable of attenuating enhanced cytosolic calcium signalling activity of CaSR mutations causing BS type 5 and ADH. Quinazolinone calcilytics might therefore offer a novel treatment option for patients with activating CaSR mutations.
PMCID: PMC4266668  PMID: 25506941
20.  Human blood monocytes support persistence, but not replication of the intracellular pathogen C. pneumoniae 
BMC Immunology  2014;15:60.
Intracellular pathogens have devised various mechanisms to subvert the host immune response in order to survive and replicate in host cells. Here, we studied the infection of human blood monocytes with the intracellular pathogen C. pneumoniae and the effect on cytokine and chemokine profiles in comparison to stimulation with LPS.
Monocytes purified from peripheral blood mononuclear cells by negative depletion were infected with C. pneumoniae. While immunofluorescence confirmed the presence of chlamydial lipopolysaccharide (LPS) in the cytoplasm of infected monocytes, real-time PCR did not provide evidence for replication of the intracellular pathogen. Complementary to PCR, C. pneumoniae infection was confirmed by an oligonucleotide DNA microarray for the detection of intracellular pathogens. Raman microspectroscopy revealed different molecular fingerprints for infected and non-infected monocytes, which were mainly due to changes in lipid and fatty acid content. Stimulation of monocytes with C. pneumoniae or with LPS induced similar profiles of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6, but higher levels of IL-1β, IL-12p40 and IL-12p70 for C. pneumoniae which were statistically significant. C. pneumoniae also induced release of the chemokines MCP-1, MIP-1α and MIP-1β, and CXCL-8, which correlated with TNF-α secretion.
Infection of human blood monocytes with intracellular pathogens triggers altered cytokine and chemokine pattern as compared to stimulation with extracellular ligands such as LPS. Complementing conventional methods, an oligonucleotide DNA microarray for the detection of intracellular pathogens as well as Raman microspectroscopy provide useful tools to trace monocyte infection.
Electronic supplementary material
The online version of this article (doi:10.1186/s12865-014-0060-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4268907  PMID: 25488836
Intracellular pathogens; Endotoxin; Monocytes; Cytokines; DNA microarray
21.  Gestational Diabetes Mellitus Impairs Nrf2-Mediated Adaptive Antioxidant Defenses and Redox Signaling in Fetal Endothelial Cells In Utero 
Diabetes  2013;62(12):4088-4097.
In utero exposure to gestational diabetes mellitus (GDM) is associated with an increased risk of type 2 diabetes and cardiovascular disease in later life, yet the underlying mechanisms remain to be elucidated. We examined the effects of GDM on the proteome, redox status, and nuclear factor erythroid 2–related factor 2 (Nrf2)-mediated antioxidant gene expression in human fetal endothelial cells. Proteomic analysis revealed that proteins involved in redox homeostasis were significantly altered in GDM and associated with increased mitochondrial superoxide generation, protein oxidation, DNA damage, and diminished glutathione (GSH) synthesis. In GDM cells, the lipid peroxidation product 4-hydroxynonenal (HNE) failed to induce nuclear Nrf2 accumulation and mRNA and/or protein expression of Nrf2 and its target genes NAD(P)H:quinone oxidoreductase 1 (NQO1), Bach1, cystine/glutamate transporter, and glutamate cysteine ligase. Although methylation of CpG islands in Nrf2 or NQO1 promoters was unaltered by GDM, decreased DJ-1 and increased phosphorylated glycogen synthase kinase 3β levels may account for impaired Nrf2 signaling. HNE-induced increases in GSH and NQO1 levels were abrogated by Nrf2 small interfering RNA in normal cells, and overexpression of Nrf2 in GDM cells partially restored NQO1 induction. Dysregulation of Nrf2 in fetal endothelium may contribute to the increased risk of type 2 diabetes and cardiovascular disease in offspring.
PMCID: PMC3837032  PMID: 23974919
22.  Nanometer-resolved mechanical properties around GaN crystal surface steps 
The mechanical properties of surfaces and nanostructures deviate from their bulk counterparts due to surface stress and reduced dimensionality. Experimental indentation-based techniques present the challenge of measuring these effects, while avoiding artifacts caused by the measurement technique itself. We performed a molecular dynamics study to investigate the mechanical properties of a GaN step of only a few lattice constants step height and scrutinized its applicability to indentation experiments using a finite element approach (FEM). We show that the breakdown of half-space symmetry leads to an “artificial” reduction of the elastic properties of comparable lateral dimensions which overlays the effect of surface stress. Contact resonance atomic force microscopy (CR-AFM) was used to compare the simulation results with experiments.
PMCID: PMC4273285  PMID: 25551044
finite elements; gallium nitride; indentation; mechanical properties; molecular dynamics; nanostructures
23.  Leucocyte Telomere Length and Risk of Type 2 Diabetes Mellitus: New Prospective Cohort Study and Literature-Based Meta-Analysis 
PLoS ONE  2014;9(11):e112483.
Short telomeres have been linked to various age-related diseases. We aimed to assess the association of telomere length with incident type 2 diabetes mellitus (T2DM) in prospective cohort studies.
Leucocyte relative telomere length (RTL) was measured using quantitative polymerase chain reaction in 684 participants of the prospective population-based Bruneck Study (1995 baseline), with repeat RTL measurements performed in 2005 (n = 558) and 2010 (n = 479). Hazard ratios for T2DM were calculated across quartiles of baseline RTL using Cox regression models adjusted for age, sex, body-mass index, smoking, socio-economic status, physical activity, alcohol consumption, high-density lipoprotein cholesterol, log high-sensitivity C-reactive protein, and waist-hip ratio. Separate analyses corrected hazard ratios for within-person variability using multivariate regression calibration of repeated measurements. To contextualise findings, we systematically sought PubMed, Web of Science and EMBASE for relevant articles and pooled results using random-effects meta-analysis.
Over 15 years of follow-up, 44 out of 606 participants free of diabetes at baseline developed incident T2DM. The adjusted hazard ratio for T2DM comparing the bottom vs. the top quartile of baseline RTL (i.e. shortest vs. longest) was 2.00 (95% confidence interval: 0.90 to 4.49; P = 0.091), and 2.31 comparing the bottom quartile vs. the remainder (1.21 to 4.41; P = 0.011). The corresponding hazard ratios corrected for within-person RTL variability were 3.22 (1.27 to 8.14; P = 0.014) and 2.86 (1.45 to 5.65; P = 0.003). In a random-effects meta-analysis of three prospective cohort studies involving 6,991 participants and 2,011 incident T2DM events, the pooled relative risk was 1.31 (1.07 to 1.60; P = 0.010; I2 = 69%).
Low RTL is independently associated with the risk of incident T2DM. To avoid regression dilution biases in observed associations of RTL with disease risk, future studies should implement methods correcting for within-person variability in RTL. The causal role of short telomeres in T2DM development remains to be determined.
PMCID: PMC4229188  PMID: 25390655
24.  Analysis of FoxP3+ T-Regulatory Cells and CD8+T-Cells in Ovarian Carcinoma: Location and Tumor Infiltration Patterns Are Key Prognostic Markers 
PLoS ONE  2014;9(11):e111757.
Tumor infiltrating CD4+CD25+FoxP3+ regulatory immune cells (Treg) have been associated with impaired anti- tumor immune response and unfavorable prognosis for patients affected by ovarian carcinoma, whereas CD8+ T-cells have been found to positively influence survival rates in a large panel of solid tumors. Recently, density, location and tumor infiltration patterns of the respective immune cell subtypes have been identified as key prognostic factors for different types of tumors.
Patients and Methods
We stained 210 human ovarian carcinoma samples immunhistochemically for FoxP3 and CD8 to identify the impact different immune cell patterns have on generally accepted prognostic variables as well as on overall survival.
We found that FoxP3+ cells located within lymphoid aggregates surrounding the tumor were strongly associated with reduced survival time (P = 0.007). Central accumulation of CD8+ effector cells within the tumor bed shows a positive effect on survival (P = 0,001).
The distribution pattern of immune cells within the tumor environment strongly influences prognosis and overall survival time of patients with ovarian carcinoma.
PMCID: PMC4218808  PMID: 25365237
25.  Smoking prevalence, readiness to quit and smoking cessation in HIV+ patients in Germany and Austria 
Journal of the International AIDS Society  2014;17(4Suppl 3):19729.
Due to the interaction between smoking and the virus and the antiretroviral therapy, the excess health hazard due to smoking is higher in HIV+ patients than in the general population. International studies suggest a higher prevalence of smoking in HIV+ subjects compared to the general population. It was the aim of the study to assess prevalence of smoking, to analyze determinants of smoking, and to evaluate readiness to quit in HIV+ patients in Germany and Austria.
Material and Methods
Consecutive patients with positive tested HIV status, smokers and non-smokers, who are treated in seven different HIV care centres in Austria and Germany were included. Nicotine dependence was assessed with the Fagerström Test for Nicotine Dependency (FTND), and stages of change by a standardized readiness to quit questionnaire. Self-reported smoking status was objectified by measuring exhaled carbon monoxide levels. Smokers who wanted to quit were offered a structured smoking cessation programme, and those who did not want to quit received a 1-minute consultation. After six months, the smoking status of all included subjects was reassessed.
A total of 447 patients were included; the response rate was 92%. Prevalence of smoking was 49.4%. According to a multivariate logistic regression analysis, lower age, male sex, lower educational level, and smoking of the partner were significantly associated with the smoking status. According to the FTND, 25.3% showed a low (0–2 points), 27.6 a moderate (3–4 points) and 47.1% a high (5–10 points) dependency. Regarding stages of change, 15.4% of the smokers were in the stadium precontemplation, 48.4 in contemplation, 15.4 in preparation and 10.0 in the stadium action. 11.0% were not assignable in any stadium. Higher education level and lower grade of dependency were significantly associated with the wish to quit smoking. Six months after the baseline examination, smoking cessation visits (at least one session) was performed in 28.5% of the smokers. 13% of the smokers have quit smoking, 23% have reduced smoking and 63% did not change smoking habits positively 6 months after the first visit.
Prevalence rates for smoking in HIV+ subjects are higher than in the general population. Readiness to quit is, however, high, and 13% of smokers who have quit smoking after six months is a remarkable short-term success. This observation underlines the importance and feasibility of addressing smoking habits in HIV care.
PMCID: PMC4225294  PMID: 25397475

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