Search tips
Search criteria

Results 1-25 (52)

Clipboard (0)

Select a Filter Below

more »
Year of Publication
more »
1.  A biochemical marker panel in MRI-proven hyperacute ischemic stroke-a prospective study 
BMC Neurology  2012;12:14.
Computer tomography (CT) is still the fastest and most robust technique to rule out ICH in acute stroke. However CT-sensitivity for detection of ischemic stroke in the hyperacute phase is still relatively low. Moreover the validity of pure clinical judgment is diminished by several stroke imitating diseases (mimics). The "Triage® Stroke Panel", a biochemical multimarker assay, detects Brain Natriuretic Peptide (BNP), D-Dimers (DD), Matrix-Metalloproteinase-9 (MMP-9), and S100B protein and promptly generates a Multimarkerindex of these values (MMX). This index has been licensed for diagnostic purposes as it might increase the validity of the clinical diagnosis to differentiate between stroke imitating diseases and true ischemic strokes. Our aim was to prove whether the panel is a reliable indicating device for the diagnosis of ischemic stroke in a time window of 6 h to fasten the pre- and intrahospital pathway to fibrinolysis.
We investigated all consecutive patients admitted to our stroke unit during a time period of 5 months. Only patients with clinical investigation, blood sample collection and MRI within six hours from symptom onset were included. Values of biochemical markers were analyzed according to the results of diffusion weighted MR-imaging. In addition MMX-values in ischemic strokes were correlated with the TOAST-criteria. For statistical analysis the SAS Analyst software was used. Correlation coefficients were analyzed and comparison tests for two or more groups were performed. Statistical significance was assumed in case of p < 0.05. Finally a ROC-analysis was performed for the MMX-Index.
In total 174 patients were included into this study (n = 100 strokes, n = 49 mimics, n = 25 transitoric ischemic attacks). In patients with ischemic strokes the mean NIHSS was 7.6 ± 6.2, while the mean DWI-lesion volume was 20.6 ml (range 186.9 to 4.2 ml). According to the MMX or the individual markers there was no statistically significant difference between the group of ischemic strokes and the group of mimics. Moreover the correlation of the index and the DWI-lesion-volume was poor (p = 0.2).
In our setting of acute MRI-proven ischemic stroke the used multimarker-assay (Triage® Stroke Panel) was not of diagnostic validity. We do not recommend to perform this assay as this might lead to a unjustified time delay.
PMCID: PMC3380723  PMID: 22400994
Stroke; Biochemical marker; Brain natriuretic peptide; D-dimers; Matrix-metalloproteinase-9
2.  Characterization of the Murine Myeloid Precursor Cell Line MuMac-E8 
PLoS ONE  2014;9(12):e113743.
Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies.
PMCID: PMC4278753  PMID: 25546418
3.  Comparison of a standard CO2 pressure pneumoperitoneum insufflator versus AirSeal™: study protocol of a randomized controlled trial 
Trials  2014;15:239.
AirSeal™ is a novel class of valve-free insufflation system that enables a stable pneumoperitoneum with continuous smoke evacuation and carbon dioxide (CO2) recirculation during laparoscopic surgery. Comparison data to standard CO2 pressure pneumoperitoneum insufflators is scarce. The aim of this study is to evaluate the potential advantages of AirSeal™ compared to a standard CO2 insufflator.
This is a single center randomized controlled trial comparing elective laparoscopic cholecystectomy, colorectal surgery and hernia repair with AirSeal™ (group A) versus a standard CO2 pressure insufflator (group S). Patients are randomized using a web-based central randomization and registration system. Primary outcome measures will be operative time and level of postoperative shoulder pain by using the visual analog score (VAS). Secondary outcomes include the evaluation of immunological values through blood tests, anesthesiological parameters, surgical side effects and length of hospital stay. Taking into account an expected dropout rate of 5%, the total number of patients is 182 (n = 91 per group). All tests will be two-sided with a confidence level of 95% (P <0.05).
The duration of an operation is an important factor in reducing the patient’s exposure to CO2 pneumoperitoneum and its adverse consequences. This trial will help to evaluate if the announced advantages of AirSeal™, such as clear sight of the operative site and an exceptionally stable working environment, will facilitate the course of selected procedures and influence operation time and patients clinical outcome.
Trial registration NCT01740011, registered 23 November 2012.
PMCID: PMC4078359  PMID: 24950720
Pneumoperitoneum; AirSeal™; Laparoscopy; CO2 Insufflator; Shoulder pain
4.  Social Isolation Shortens Telomeres in African Grey Parrots (Psittacus erithacus erithacus) 
PLoS ONE  2014;9(4):e93839.
Telomeres, the caps of eukaryotic chromosomes, control chromosome stability and cellular senescence, but aging and exposure to chronic stress are suspected to cause attrition of telomere length. We investigated the effect of social isolation on telomere length in the highly social and intelligent African Grey parrot (Psittacus erithacus erithacus). Our study population consisted of single-housed (n = 26) and pair-housed (n = 19) captive individuals between 0.75 to 45 years of age. Relative telomere length of erythrocyte DNA was measured by quantitative real-time PCR. We found that telomere length declined with age (p<0.001), and socially isolated parrots had significantly shorter telomeres compared to pair-housed birds (p<0.001) – even among birds of similar ages. Our findings provide the first evidence that social isolation affects telomere length, which supports the hypothesis that telomeres provide a biomarker indicating exposure to chronic stress.
PMCID: PMC3976323  PMID: 24705445
5.  Comparison of ESBL – And AmpC Producing Enterobacteriaceae and Methicillin-Resistant Staphylococcus aureus (MRSA) Isolated from Migratory and Resident Population of Rooks (Corvus frugilegus) in Austria 
PLoS ONE  2013;8(12):e84048.
In order to test whether rooks (Corvus frugilegus) represent good indicators for the potential circulation of antibiotics in their native habitat, two populations with different migratory behavior were tested for the presence of beta-lactamase producing Enterobacteriaceae and methicillin-resistant Staphylococcus aureus (MRSA). In all, 54 and 102 samples of fresh feces of a migratory and a resident population were investigated. A total of 24 and 3 cefotaxime-resistant enterobacterial isolates were obtained from the migratory and resident population, respectively. In these isolates CTX-M-1 (n = 15), CTX-M-3 (n = 3), and CTX-M-15 (n = 3) genes were detected. TEM-1 and OXA-1 were associated with CTX-M in 3 and 2 isolates, respectively. In two E. coli isolates CMY-2 could be detected, where from one isolate displayed an overexpression of chromosomal AmpC as well. Among E. coli isolates the most common phylogenetic group was A (n = 11) and ST1683 (n = 5). In one E. coli of B2-ST131 the rfbO25b locus was detected. Three Enterobacter isolates were stably derepressed AmpC-producers. In five samples of the migratory population, PVL positive MRSA could be isolated. Two isolates were typed SCCmec IVa, spa type t127, and ST1. Three isolates carried a SCCmec type IVc, with spa type t852 and ST22. The highly significant difference of the occurrence of antibiotic resistance between the migratory population from eastern Europe compared to resident population in our study indicates that rooks may be good indicator species for the evaluation of environmental contamination with antibiotic resistant bacteria, especially due to their ecology, foraging behavior and differing migratory behavior.
PMCID: PMC3877145  PMID: 24391878
6.  Transformation 
RNA Biology  2012;9(12):1418-1423.
The textbook view that a primary sequence determines the unique fold of a given protein has been challenged by identification of proteins with variant structures, such as prions. Our recent studies revealed that the transcription factor RfaH simultaneously changes its topology and function. RfaH is a two-domain protein whose N-terminal domain binds to transcribing RNA polymerase, stimulating its processivity. The α-helical C-terminal domain masks the RNA polymerase-binding site of the N-terminal domain, preventing unwarranted recruitment to genes lacking a specific DNA sequence. Upon binding to its DNA target, RfaH domains dissociate, and the C-terminal domain refolds into a β-barrel. This dramatic transformation allows binding to the ribosomal protein S10 and subsequent recruitment of a ribosome, coupling transcription and translation. We define RfaH as first example of “transformer proteins”, in which two alternative structural states have distinct cellular functions and hypothesize that transformer proteins may be widespread in nature.
PMCID: PMC3679274  PMID: 23131843
RfaH; NusG; transcription factor; transformer protein; moonlighting protein; metamorphic protein; ops DNA; multifunctionality; protein folding; refolding
7.  Interdomain contacts control folding of transcription factor RfaH 
Nucleic Acids Research  2013;41(22):10077-10085.
Escherichia coli RfaH activates gene expression by tethering the elongating RNA polymerase to the ribosome. This bridging action requires a complete refolding of the RfaH C-terminal domain (CTD) from an α-helical hairpin, which binds to the N-terminal domain (NTD) in the free protein, to a β-barrel, which interacts with the ribosomal protein S10 following RfaH recruitment to its target operons. The CTD forms a β-barrel when expressed alone or proteolytically separated from the NTD, indicating that the α-helical state is trapped by the NTD, perhaps co-translationally. Alternatively, the interdomain contacts may be sufficient to drive the formation of the α-helical form. Here, we use functional and NMR analyses to show that the denatured RfaH refolds into the native state and that RfaH in which the order of the domains is reversed is fully functional in vitro and in vivo. Our results indicate that all information necessary to determine its fold is encoded within RfaH itself, whereas accessory factors or sequential folding of NTD and CTD during translation are dispensable. These findings suggest that universally conserved RfaH homologs may change folds to accommodate diverse interaction partners and that context-dependent protein refolding may be widespread in nature.
PMCID: PMC3905879  PMID: 23990324
8.  An α-helix to β-barrel domain switch transforms the transcription factor RfaH into a translationfactor 
Cell  2012;150(2):291-303.
NusG homologs regulate transcription and coupled processes in all living organisms. The Escherichia coli (E. coli) two-domain paralogs NusG and RfaH have conformationally identical N-terminal domains (NTDs) but dramatically different carboxy-terminal domains (CTDs), a β-barrel in NusG and an α-hairpin in RfaH. Both NTDs interact with elongating RNA polymerase (RNAP) to reduce pausing. In NusG, NTD and CTD are completely independent, and NusG-CTD interacts with termination factor Rho or ribosomal protein S10. In contrast, RfaH-CTD makes extensive contacts with RfaH-NTD to mask an RNAP-binding site therein. Upon RfaH interaction with its DNA target, the operon polarity suppressor (ops) DNA, RfaH-CTD is released, allowing RfaH-NTD to bind to RNAP. Here we show that the released RfaH-CTD completely refolds from an all-α to an all-β conformation identical to that of NusG-CTD. As a consequence, RfaH-CTD binding to S10 is enabled and translation of RfaH-controlled operons is strongly potentiated.
PMCID: PMC3430373  PMID: 22817892
9.  Label-Free Detection of Neuronal Differentiation in Cell Populations Using High-Throughput Live-Cell Imaging of PC12 Cells 
PLoS ONE  2013;8(2):e56690.
Detection of neuronal cell differentiation is essential to study cell fate decisions under various stimuli and/or environmental conditions. Many tools exist that quantify differentiation by neurite length measurements of single cells. However, quantification of differentiation in whole cell populations remains elusive so far. Because such populations can consist of both proliferating and differentiating cells, the task to assess the overall differentiation status is not trivial and requires a high-throughput, fully automated approach to analyze sufficient data for a statistically significant discrimination to determine cell differentiation. We address the problem of detecting differentiation in a mixed population of proliferating and differentiating cells over time by supervised classification. Using nerve growth factor induced differentiation of PC12 cells, we monitor the changes in cell morphology over days by phase-contrast live-cell imaging. For general applicability, the classification procedure starts out with many features to identify those that maximize discrimination of differentiated and undifferentiated cells and to eliminate features sensitive to systematic measurement artifacts. The resulting image analysis determines the optimal post treatment day for training and achieves a near perfect classification of differentiation, which we confirmed in technically and biologically independent as well as differently designed experiments. Our approach allows to monitor neuronal cell populations repeatedly over days without any interference. It requires only an initial calibration and training step and is thereafter capable to discriminate further experiments. In conclusion, this enables long-term, large-scale studies of cell populations with minimized costs and efforts for detecting effects of external manipulation of neuronal cell differentiation.
PMCID: PMC3579923  PMID: 23451069
10.  Results of the First Austrian Multidisciplinary Expert Panel on Controversies in Local Treatment of Breast Cancer 
Breast Care  2012;7(1):61-66.
At the first Austrian multidisciplinary expert panel on controversies in local treatment of breast cancer, 22 experts of all relevant disciplines discussed current areas of debate (surgery of the breast, surgery and pathology of the axilla, reconstructive surgery, radiotherapy, and imaging) in local therapy. The most controversial area of debate was the area of axillary surgery. The panel agreed that it was no longer necessary to perform completion axillary lymph node dissection (ALND) when micrometastases are diagnosed in the sentinel lymph node. The only prospective trial comparing patients with sentinel node macrometastases with or without completion ALND had to be terminated early due to failure in sufficient patient recruitment. As long as the frequently discussed issues have not been solved and in light of the lack of any clear level 1 evidence, the panel decided not to recommend omitting axillary dissection in patients with 1 or 2 macrometastases meeting the inclusion criteria of the ACOSOG Z0011 trial. The Austrian panel similarly decided not to recommend omitting axillary dissection in patients with macrometastases and low-risk breast cancer in general. These decisions reflect the increasing skepticism of the scientific community against rapidly shifting paradigms without sufficient and clear evidence.
PMCID: PMC3335360  PMID: 22553475
Breast cancer: local therapy, surgery, radiotherapy; Expert panel
11.  Brain metastases free survival differs between breast cancer subtypes 
British Journal of Cancer  2012;106(3):440-446.
Brain metastases (BM) are frequently diagnosed in patients with HER-2-positive metastatic breast cancer; in addition, an increasing incidence was reported for triple-negative tumours. We aimed to compare brain metastases free survival (BMFS) of breast cancer subtypes in patients treated between 1996 until 2010.
Brain metastases free survival was measured as the interval from diagnosis of extracranial breast cancer metastases until diagnosis of BM. HER-2 status was analysed by immunohistochemistry and reanalysed by fluorescent in situ hybridisation if a score of 2+ was gained. Oestrogen-receptor (ER) and progesterone-receptor (PgR) status was analysed by immunohistochemistry. Brain metastases free survival curves were estimated with the Kaplan–Meier method and compared with the log-rank test.
Data of 213 patients (46 luminal/124 HER-2/43 triple-negative subtype) with BM from breast cancer were available for the analysis. Brain metastases free survival differed significantly between breast cancer subtypes. Median BMFS in triple-negative tumours was 14 months (95% CI: 11.34–16.66) compared with 18 months (95% CI: 14.46–21.54) in HER-2-positive tumours (P=0.001) and 34 months (95% CI: 23.71–44.29) in luminal tumours (P=0.001), respectively. In HER-2-positive patients, co-positivity for ER and HER-2 prolonged BMFS (26 vs 15 m; P=0.033); in luminal tumours, co-expression of ER and PgR was not significantly associated with BMFS. Brain metastases free survival in patients with lung metastases was significantly shorter (17 vs 21 months; P=0.014).
Brain metastases free survival in triple-negative breast cancer, as well as in HER-2-positive/ER-negative, is significantly shorter compared with HER-2/ER co-positive or luminal tumours, mirroring the aggressiveness of these breast cancer subtypes.
PMCID: PMC3273356  PMID: 22233926
advanced breast cancer; brain metastases; carcinomatous meningitis; human epidermal growth factor receptor 2 (HER-2)-positive breast cancer; triple-negative disease
13.  Multidisciplinary Approach to Lifesaving Measures in the Elderly Individuals With Flail Chest Injury With ORIF of Rib Fractures 
In this case report, we present 2 cases of flail chest in geriatric patients after severe blunt chest trauma, which were treated at the University Hospital Innsbruck (Level I Trauma Center and Tyrolean Geriatric Fracture Center) by a multidisciplinary team of physicians from anesthesia, intensive care, trauma surgery, and acute geriatrics. We want to point out the benefit of a multidisciplinary approach in geriatric patients with flail chest.
PMCID: PMC3598516  PMID: 23569711
anesthesia; fragility fractures; geriatric medicine; geriatric trauma; trauma surgery
14.  Transformer proteins 
Cell Cycle  2012;11(23):4289-4290.
PMCID: PMC3552902  PMID: 23095672
RfaH; NusG; transcription factor; transformer protein; moonlighting; metamorphous; structural switch; multifunctionality; protein folding; refolding
16.  Overexpression of the Catalytically Impaired Taspase1T234V or Taspase1D233A Variants Does Not Have a Dominant Negative Effect in T(4;11) Leukemia Cells 
PLoS ONE  2012;7(5):e34142.
The chromosomal translocation t(4;11)(q21;q23) is associated with high-risk acute lymphoblastic leukemia of infants. The resulting AF4•MLL oncoprotein becomes activated by Taspase1 hydrolysis and is considered to promote oncogenic transcriptional activation. Hence, Taspase1’s proteolytic activity is a critical step in AF4•MLL pathophysiology. The Taspase1 proenzyme is autoproteolytically processed in its subunits and is assumed to assemble into an αββα-heterodimer, the active protease. Therefore, we investigated here whether overexpression of catalytically inactive Taspase1 variants are able to interfere with the proteolytic activity of the wild type enzyme in AF4•MLL model systems.
The consequences of overexpressing the catalytically dead Taspase1 mutant, Taspase1T234V, or the highly attenuated variant, Taspase1D233A, on Taspase1’s processing of AF4•MLL and of other Taspase1 targets was analyzed in living cancer cells employing an optimized cell-based assay. Notably, even a nine-fold overexpression of the respective Taspase1 mutants neither inhibited Taspase1’s cis- nor trans-cleavage activity in vivo. Likewise, enforced expression of the α- or β-subunits showed no trans-dominant effect against the ectopically or endogenously expressed enzyme. Notably, co-expression of the individual α- and β-subunits did not result in their assembly into an enzymatically active protease complex. Probing Taspase1 multimerization in living cells by a translocation-based protein interaction assay as well as by biochemical methods indicated that the inactive Taspase1 failed to assemble into stable heterocomplexes with the wild type enzyme.
Collectively, our results demonstrate that inefficient heterodimerization appears to be the mechanism by which inactive Taspase1 variants fail to inhibit wild type Taspase1’s activity in trans. Our work favours strategies targeting Taspase1’s catalytic activity rather than attempts to block the formation of active Taspase1 dimers to interfere with the pathobiological function of AF4•MLL.
PMCID: PMC3343046  PMID: 22570686
17.  Circulating Serologic and Molecular Biomarkers in Malignant Melanoma 
Mayo Clinic Proceedings  2011;86(10):981-990.
The worldwide incidence of malignant melanoma has been increasing during the past decade and is a public health concern because this disease accounts for up to 90% of deaths from cutaneous malignancies. It remains a devastating disease with few therapeutic options once in an advanced stage. Current methods of detection, prognostication, and monitoring of melanoma focus on clinical, morphologic, and histopathologic characteristics of measurable tumor. Although this information provides some insight into disease behavior and outcome, melanoma is still an unpredictable disease. Significant effort has been put into finding an informative serologic biomarker. However, the marker remains elusive, and investigations continue. Using the PubMed database, we reviewed the published literature on serologic melanoma biomarkers and present a synopsis of the extensive investigations that have been performed thus far, provide some insight into why most have failed to become incorporated into routine clinical use, and present an overview of innovative methods currently being explored.
PMCID: PMC3184027  PMID: 21964175
18.  Identification and evaluation of novel synovial tissue biomarkers in rheumatoid arthritis by laser scanning cytometry 
Suitable biomarkers are essential for therapeutic strategies in personalized medicine in terms of diagnosis as well as of prognosis. With highly specific biomarkers, it is possible, for example, to identify patients with poor prognosis, which enables early intervention and intensive treatment. The aim of this study was to identify and validate biomarkers and possible combinations for a prospective use in immunoscintigraphy, which may improve diagnosis of rheumatoid arthritis (RA) patients with consideration of inflammatory activity in the affected joints. Therefore, we tested several monoclonal antibodies (mAbs) directed against cellular-surface molecules on cells likely to be involved in the pathogenesis of RA.
Synovial tissue from patients with long-standing RA (accompanied by synovitis with varying states of current activity) and patients with acute non-RA arthritis were stained for surface molecules on different cell types by using fluorochrome-labeled antibodies. Tissue analysis was done by laser scanning cytometry (LSC), and statistical evaluation, by discriminant analysis and ROC analysis.
CD11b, HLA-DR, CD90, and CD64 revealed significant differences between tissues from patients with RA and acute non-RA arthritis. Especially with the expression of CD64, both patient cohorts could be discriminated with high sensitivity and specificity. RA classification was improved by simultaneously investigating the expression of two or three different surface proteins, such as HLA-DR, CD90, and CD29 in the tissue. The simultaneous analysis of CD64 together with CD304 or the combination of CD11b and CD38 was suitable for the identification of RA patients with high current activity in synovitis.
In this study, we showed that LSC is a novel reliable method in biomarker prevalidation in RA. Hence, identified mAbs in situ may allow their potential use in in vivo approaches. Moreover, we proved that biomarker-combination analysis resulted in better discrimination than did single-marker analysis. Combinations of these markers make a novel and reliable panel for the discrimination between RA and acute non-RA arthritis. In addition, further expedient combinations may be novel promising biomarker panels to identify current activity in synovitis in RA.
PMCID: PMC3392796  PMID: 22251373
19.  Human PAPS Synthase Isoforms Are Dynamically Regulated Enzymes with Access to Nucleus and Cytoplasm 
PLoS ONE  2012;7(1):e29559.
In higher eukaryotes, PAPS synthases are the only enzymes producing the essential sulphate-donor 3′-phospho-adenosine-5′-phosphosulphate (PAPS). Recently, PAPS synthases have been associated with several genetic diseases and retroviral infection. To improve our understanding of their pathobiological functions, we analysed the intracellular localisation of the two human PAPS synthases, PAPSS1 and PAPSS2. For both enzymes, we observed pronounced heterogeneity in their subcellular localisation. PAPSS1 was predominantly nuclear, whereas PAPSS2 localised mainly within the cytoplasm. Treatment with the nuclear export inhibitor leptomycin B had little effect on their localisation. However, a mutagenesis screen revealed an Arg-Arg motif at the kinase interface exhibiting export activity. Notably, both isoforms contain a conserved N-terminal basic Lys-Lys-Xaa-Lys motif indispensable for their nuclear localisation. This nuclear localisation signal was more efficient in PAPSS1 than in PAPSS2. The activities of the identified localisation signals were confirmed by microinjection studies. Collectively, we describe unusual localisation signals of both PAPS synthase isoforms, mobile enzymes capable of executing their function in the cytoplasm as well as in the nucleus.
PMCID: PMC3252339  PMID: 22242175
20.  A combination of a ribonucleotide reductase inhibitor and histone deacetylase inhibitors downregulates EGFR and triggers BIM-dependent apoptosis in head and neck cancer 
Oncotarget  2011;3(1):31-43.
Head and neck squamous cell carcinomas (HNSCCs) are the sixth most common malignant neoplasm and more than 50% of patients succumb to this disease. HNSCCs are characterized by therapy resistance, which relies on the overexpression of anti-apoptotic proteins and on the aberrant regulation of the epidermal growth factor receptor (EGFR). As inherent and acquired resistance to therapy counteracts improvement of long-term survival, novel multi-targeting strategies triggering cancer cell death are urgently required. We investigated how induction of replicational stress by the ribonucleotide reductase inhibitor hydroxyurea (HU) combined with histone deacetylase inhibitors (HDACi) exerts anti-tumor activity. We treated HNSCC cell lines and freshly isolated tumor cells with HDACi, such as the clinically approved anti-epileptic drug valproic acid (VPA), in combination with HU. Our data demonstrate that at clinically achievable levels VPA/HU combinations efficiently block proliferation as well as clonogenic survival, and trigger apoptosis of HNSCC cells. In the presence of VPA/HU, such tumor cells increase expression of the pro-apoptotic BCL-2 family protein BIM, independent of wild-type p53 signaling and in the absence of increased expression of the p53 targets PUMA and BAX. The pro-apoptotic activity of BIM in HNSCCs was found critical for tumor cell death; ectopic overexpression of BIM induced HNSCC apoptosis and RNAi-mediated depletion of BIM protected HNSCC cells from VPA/HU. Also, significantly elevated BIM levels (p<0.01) were detectable in the apoptotic tumor centers versus proliferating tumor margins in HNSCC patients (n=31), underlining BIM's clinical relevance. Importantly, VPA/HU treatment additionally reduces expression and cell surface localization of EGFR. Accordingly, in a xenograft mouse model, VPA/HU efficiently blocked tumor growth (P<0.001) correlating with BIM induction and EGFR downregulation. We provide a molecular rationale for the potent anti-cancer activities of this drug combination. Our data suggest its exploitation as a potential strategy for the treatment of HNSCC and other tumor entities characterized by therapy resistance linked to dysregulated EGFR activation.
PMCID: PMC3292890  PMID: 22289787
BH3-only protein; chemotherapy resistance; hydroxyurea; oral cancer; tumor xenograft; valproic acid
21.  Cholinergic modulation of the CAN current may adjust neural dynamics for active memory maintenance, spatial navigation and time-compressed replay 
Suppression of cholinergic receptors and inactivation of the septum impair short-term memory, and disrupt place cell and grid cell activity in the medial temporal lobe (MTL). Location-dependent hippocampal place cell firing during active waking, when the acetylcholine level is high, switches to time-compressed replay activity during quiet waking and slow-wave-sleep (SWS), when the acetylcholine level is low. However, it remains largely unknown how acetylcholine supports short-term memory, spatial navigation, and the functional switch to replay mode in the MTL. In this paper, we focus on the role of the calcium-activated non-specific cationic (CAN) current which is activated by acetylcholine. The CAN current is known to underlie persistent firing, which could serve as a memory trace in many neurons in the MTL. Here, we review the CAN current and discuss possible roles of the CAN current in short-term memory and spatial navigation. We further propose a novel theoretical model where the CAN current switches the hippocampal place cell activity between real-time and time-compressed sequential activity during encoding and consolidation, respectively.
PMCID: PMC3304506  PMID: 22435051
spatial navigation; short-term memory; calcium-activated non-specific cationic current; acetylcholine; encoding; consolidation; medial temporal lobe; hippocampus
22.  Identification of a low-risk subgroup of HER-2-positive breast cancer by the 70-gene prognosis signature 
British Journal of Cancer  2010;103(12):1788-1793.
Overexpression of HER-2 is observed in 15–25% of breast cancers, and is associated with increased risk of recurrence. Current guidelines recommend trastuzumab and chemotherapy for most HER-2-positive patients. However, the majority of patients does not recur and might thus be overtreated with adjuvant systemic therapy. We investigated whether the 70-gene MammaPrint signature identifies HER-2-positive patients with favourable outcome.
In all, 168 T1–3, N0–1, HER-2-positive patients were identified from a pooled database, classified by the 70-gene signature as good or poor prognosis, and correlated with long-term outcome. A total of 89 of these patients did not receive adjuvant chemotherapy.
In the group of 89 chemotherapy-naive patients, after a median follow-up of 7.4 years, 35 (39%) distant recurrences and 29 (33%) breast cancer-specific deaths occurred. The 70-gene signature classified 20 (22%) patients as good prognosis, with 10-year distant disease-free survival (DDFS) of 84%, compared with 69 (78%) poor prognosis patients with 10-year DDFS of 55%. The estimated hazard ratios (HRs) were 4.5 (95% confidence interval (CI) 1.1–18.7, P=0.04) and 3.8 (95% CI 0.9–15.8, P=0.07) for DDFS and breast cancer-specific survival (BCSS), respectively. In multivariate analysis adjusted for known prognostic factors and hormonal therapy, HRs were 5.8 (95% CI 1.3–26.7, P=0.03) and 4.7 (95% CI 1.0–21.7, P=0.05) for DDFS and BCSS, respectively.
The 70-gene prognosis signature is an independent prognostic indicator that identifies a subgroup of HER-2-positive early breast cancer with a favourable long-term outcome.
PMCID: PMC3008599  PMID: 21081926
breast cancer; gene expression profiling; MammaPrint; risk assessment; HER-2; adjuvant chemotherapy
23.  Evaluation of the preventive capacities of a topically applied azithromycin formulation against Lyme borreliosis in a murine model 
Journal of Antimicrobial Chemotherapy  2011;66(12):2814-2822.
Systemic antibiotic treatment of Lyme borreliosis is effective during the early stages of the infection, while chronic manifestations of the disease may remain refractory and difficult to treat. This study was carried out in order to evaluate the potential of topically applied azithromycin to eliminate the spirochaetal organisms in the skin of the freshly bitten host and thereby prevent Lyme borreliosis.
Laboratory mice were challenged with Borrelia burgdorferi sensu stricto by needle inoculation or via infected ticks as vectors. Then, an azithromycin-containing formulation was applied once daily to the sites of exposure for three consecutive days. In the case of needle inoculation, a 5% azithromycin formulation was applied starting 1 h, 3 days and 5 days after infection. In the case of tick exposure, 4%, 10% and 20% azithromycin formulations were applied, starting directly after the detachment of the engorged ticks. Subsequently, the infection status of the mice was determined.
Concentrations of azithromycin in murine skin were >3800-fold higher than the published minimal inhibitory concentration for B. burgdorferi as soon as 3 h after the first application. After needle inoculation, spirochaetes were not detectable in all infected mice after treatment, if the first application started 1 h or even after 3 days post-infection. Furthermore, no borrelial organisms were detected after topical treatment when ticks were used for spirochaete inoculation.
Our data indicate that topical treatment with a formulation containing azithromycin is a promising approach to prevent Lyme borreliosis shortly after a tick bite.
PMCID: PMC3215302  PMID: 21921078
Borrelia; preventive treatment; antibiotic treatment; spirochaetes; prophylaxis
24.  Asymptomatic Primary Merkel Cell Polyomavirus Infection among Adults 
Emerging Infectious Diseases  2011;17(8):1371-1380.
TOC Summary: This virus may be part of normal human flora and harmless in most adults.
Merkel cell polyomavirus (MCV) is a recently discovered virus that causes 80% of Merkel cell carcinomas. We examined data for 564 gay/bisexual male participants >18 years of age in the Multicenter AIDS Cohort Study in Pittsburgh, Pennsylvania, USA, and found that 447 (79.3%) were MCV-antibody positive at initial enrollment. Of the 117 MCV-seronegative men, 31 subsequently seroconverted over a 4-year follow-up period, corresponding to a 6.6% annual conversion rate. MCV immunoglobulin G levels remained detectable up to 25 years after exposure. No signs, symptoms, or routine diagnostic test results were associated with MCV infection, and no correlation between HIV infection or AIDS progression and MCV infection was noted. An initial correlation between chronic hepatitis B virus infection and MCV prevalence could not be confirmed among MCV seroconverters or in studies of a second hepatitis B virus–hyperendemic cohort from Qidong, China. In adults, MCV is typically an asymptomatic, common, and commensal viral infection that initiates rare cancers after virus (rather than host cell) mutations.
PMCID: PMC3381535  PMID: 21801612
viruses; MSM; men who have sex with men; Merkel cell polyomavirus; MCV; Merkel cell carcinoma; MCPyV; epidemiology; seroconversion; Pennsylvania; USA; China; HIV/AIDS and other retroviruses; research
25.  Bioassays to Monitor Taspase1 Function for the Identification of Pharmacogenetic Inhibitors 
PLoS ONE  2011;6(5):e18253.
Threonine Aspartase 1 (Taspase1) mediates cleavage of the mixed lineage leukemia (MLL) protein and leukemia provoking MLL-fusions. In contrast to other proteases, the understanding of Taspase1's (patho)biological relevance and function is limited, since neither small molecule inhibitors nor cell based functional assays for Taspase1 are currently available.
Efficient cell-based assays to probe Taspase1 function in vivo are presented here. These are composed of glutathione S-transferase, autofluorescent protein variants, Taspase1 cleavage sites and rational combinations of nuclear import and export signals. The biosensors localize predominantly to the cytoplasm, whereas expression of biologically active Taspase1 but not of inactive Taspase1 mutants or of the protease Caspase3 triggers their proteolytic cleavage and nuclear accumulation. Compared to in vitro assays using recombinant components the in vivo assay was highly efficient. Employing an optimized nuclear translocation algorithm, the triple-color assay could be adapted to a high-throughput microscopy platform (Z'factor = 0.63). Automated high-content data analysis was used to screen a focused compound library, selected by an in silico pharmacophor screening approach, as well as a collection of fungal extracts. Screening identified two compounds, N-[2-[(4-amino-6-oxo-3H-pyrimidin-2-yl)sulfanyl]ethyl]benzenesulfonamide and 2-benzyltriazole-4,5-dicarboxylic acid, which partially inhibited Taspase1 cleavage in living cells. Additionally, the assay was exploited to probe endogenous Taspase1 in solid tumor cell models and to identify an improved consensus sequence for efficient Taspase1 cleavage. This allowed the in silico identification of novel putative Taspase1 targets. Those include the FERM Domain-Containing Protein 4B, the Tyrosine-Protein Phosphatase Zeta, and DNA Polymerase Zeta. Cleavage site recognition and proteolytic processing of these substrates were verified in the context of the biosensor.
The assay not only allows to genetically probe Taspase1 structure function in vivo, but is also applicable for high-content screening to identify Taspase1 inhibitors. Such tools will provide novel insights into Taspase1's function and its potential therapeutic relevance.
PMCID: PMC3102056  PMID: 21647428

Results 1-25 (52)