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1.  The Antiapoptotic Function of miR-96 in Prostate Cancer by Inhibition of FOXO1 
PLoS ONE  2013;8(11):e80807.
microRNAs (miRNAs) are small molecules that regulate gene expression posttranscriptionally. In a previous study, we identified miR-96 to be upregulated in prostate cancer specimens in comparison to normal adjacent tissue and to be an independent marker of biochemical relapse in a multivariate prediction model. Therefore, we investigated the functional role of miR-96 in prostate carcinogenesis. LNCaP and DU145 prostate cancer cells were transiently transfected with miR-96 precursors and phenotypic changes were analyzed. The miR-96 increased proliferation and impaired apoptosis induced by camptothecine in these cells. In silico target prediction analysis identified FOXO1 as potential pro-apoptotic miR-96 target. miR-96 was able to bind to both bindings sites in the FOXO1 3’ UTR in a luciferase reporter gene assay. Overexpression of miR-96 in LNCaP cells resulted in a reduced FOXO1 expression. Overexpression of FOXO1 induced a strong apoptotic phenotype that was partially rescued by coexpression of miR-96. RT-qPCR and immunohistochemistry of 69 prostate cancer specimens revealed a downregulation of FOXO1 and an inverse correlation of miR-96 and FOXO1 protein expression. In conclusion, we show that miR-96 can regulate apoptosis in prostate cancer, by inhibiting the FOXO1 transcription factor.
doi:10.1371/journal.pone.0080807
PMCID: PMC3834337  PMID: 24260486
2.  Renal cell neoplasias: reversion-inducing cysteine-rich protein with Kazal motifs discriminates tumor subtypes, while extracellular matrix metalloproteinase inducer indicates prognosis 
Background
Matrix metalloproteinases can promote invasion and metastasis, which are very frequent in renal cell carcinoma even at the time of diagnosis. Knowing the reversion-inducing cysteine-rich protein with Kazal motifs (RECK) as an inhibitor of matrix metalloproteinases and the extracellular matrix metalloproteinase inducer (EMMPRIN) protein as inducer, we aimed to determine their expression, localization and possible antagonistic action in the pathogenesis and progression of renal cell tumors in a retrospective study.
Methods
Tumor and adjacent normal tissues of 395 nephrectomized patients were immunostained for RECK and EMMPRIN on a tissue microarray.
Results
RECK strongly decreased in renal cell carcinoma compared to normal counterparts (Wilcoxon signed rank test, P < 0.001), and it discriminated tumor entities showing the highest expression in oncocytomas. EMMPRIN, however, could be significantly correlated to pT stage and Fuhrman grading (Spearman’s correlation coefficient rs = 0.289 and rs = 0.382, respectively). Higher expression of EMMPRIN was associated with decreased overall survival in Kaplan-Meier analysis (P < 0.001), and the EMMPRIN level could independently predict survival for cases without metastasis and involvement of lymph nodes. Decreased RECK expression was confirmed by Western blotting in tissue of eight normal/tumor matches of patients after radical nephrectomy, whereas the EMMPRIN pattern appeared to be heterogeneous.
Conclusions
We propose RECK down regulation in renal cell carcinoma to be an early event that facilitates tumor formation and progression. EMMPRIN, however, as a prognostic tumor marker, increases only when aggressiveness is proceeding and could add an additional step to invasive properties of renal cell carcinoma.
doi:10.1186/1479-5876-11-258
PMCID: PMC3853196  PMID: 24131772
RECK; EMMPRIN; Renal cell carcinoma; TMA
3.  A New Algorithm for Integrated Analysis of miRNA-mRNA Interactions Based on Individual Classification Reveals Insights into Bladder Cancer 
PLoS ONE  2013;8(5):e64543.
Background
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. It has been proposed that miRNAs play an important role in cancer development and progression. Their ability to affect multiple gene pathways by targeting various mRNAs makes them an interesting class of regulators.
Methodology/Principal Findings
We have developed an algorithm, Classification based Analysis of Paired Expression data of RNA (CAPE RNA), which is capable of identifying altered miRNA-mRNA regulation between tissues samples that assigns interaction states to each sample without preexisting stratification of groups. The distribution of the assigned interaction states compared to given experimental groups is used to assess the quality of a predicted interaction. We demonstrate the applicability of our approach by analyzing urothelial carcinoma and normal bladder tissue samples derived from 24 patients. Using our approach, normal and tumor tissue samples as well as different stages of tumor progression were successfully stratified. Also, our results suggest interesting differentially regulated miRNA-mRNA interactions associated with bladder tumor progression.
Conclusions/Significance
The need for tools that allow an integrative analysis of microRNA and mRNA expression data has been addressed. With this study, we provide an algorithm that emphasizes on the distribution of samples to rank differentially regulated miRNA-mRNA interactions. This is a new point of view compared to current approaches. From bootstrapping analysis, our ranking yields features that build strong classifiers. Further analysis reveals genes identified as differentially regulated by miRNAs to be enriched in cancer pathways, thus suggesting biologically interesting interactions.
doi:10.1371/journal.pone.0064543
PMCID: PMC3663800  PMID: 23717626
4.  Mutated KRAS Results in Overexpression of DUSP4, a MAP-Kinase Phosphatase, and SMYD3, a Histone Methyltransferase, in Rectal Carcinomas 
Genes, chromosomes & cancer  2010;49(11):1024-1034.
Mutations of the KRAS oncogene are predictive for resistance to treatment with antibodies against the epithelial growth factor receptor in patients with colorectal cancer. Overcoming this therapeutic dilemma could potentially be achieved by the introduction of drugs that inhibit signaling pathways that are activated by KRAS mutations. To identify comprehensively such signaling pathways we profiled pretreatment biopsies and normal mucosa from 65 patients with locally advanced rectal cancer - 30 of which carried mutated KRAS - using global gene expression microarrays. By comparing all tumor tissues exclusively to matched normal mucosa, we could improve assay sensitivity, and identified a total of 22,297 features that were differentially expressed (adjusted P-value <0.05) between normal mucosa and cancer, including several novel potential rectal cancer genes. We then used this comprehensive description of the rectal cancer transcriptome as the baseline for identifying KRAS-dependent alterations. The presence of activating KRAS mutations is significantly correlated to an upregulation of 13 genes (adjusted P-value <0.05), among them DUSP4, a MAP-kinase phosphatase, and SMYD3, a histone methyltransferase. Inhibition of the expression of both genes has previously been shown using the MEK1-inhibitor PD98059 and the antibacterial compound Novobiocin, respectively. These findings suggest a potential approach to overcome resistance to treatment with antibodies against the epithelial growth factor receptor in patients with KRAS-mutant rectal carcinomas.
doi:10.1002/gcc.20811
PMCID: PMC3535184  PMID: 20725992
5.  Clinical judgment is the most important element in overhydration assessment of chronic hemodialysis patients 
Background
The assessment of hydration status remains a challenging task in hemodialysis (HD) management. There are only limited data available on the relevance of clinical decisions in the estimation of dialysis overhydration (OH). The objective of this study was to examine the significance of clinical judgment in the assessment of pre-dialysis OH.
Methods
We compared the performance of three methods of OH assessment: (1) clinical judgment guided by a single clinical examination with (2) multifrequency bioimpedance analysis (BIA) and (3) complex systematic clinical approach. We additionally studied the associations of these methods with selected laboratory and imaging parameters.
Results
Any of the single parameters alone reached a sufficient level of accuracy for reliable prediction of OH. Clinical judgment was the single most important factor in OH estimation, and also had the highest contribution when in combination with other parameters. BIA reliably measured extracellular fluid, but the automatically calculated OHBIA exhibited a substantial degree of inaccuracy that precludes the use of BIA as a standard at present. The combination of clinical judgment with additional clinical parameters had the highest prediction accuracy for OH. Among the parameters studied, vena cava collapsibility index and calf circumference showed the strongest association with OH. Echocardiography, cardiothoracic index, atrial natriuretic peptide levels and spirometry did not have acceptable sensitivity.
Conclusion
The systematic clinical approach combining physician and patient inputs, laboratory and imaging data enables an individualized decision and a superior accuracy in OH assessment.
Electronic supplementary material
The online version of this article (doi:10.1007/s10157-012-0745-9) contains supplementary material, which is available to authorized users.
doi:10.1007/s10157-012-0745-9
PMCID: PMC3751418  PMID: 23192771
Hemodialysis; Overhydration; Clinical judgment; Bioimpedance
6.  Influence of tibial slope asymmetry on femoral rotation in patients with lateral patellar instability 
Purpose
The geometry of the tibial plateau and its influence on the biomechanics of the tibiofemoral joint has gained increased significance. However, no quantitative data are available regarding the inclination of the medial and lateral tibial slope in patients with patellar instability. It was therefore the purpose of this study to evaluate tibial slope characteristics in patients with patellar dislocations and to assess the biomechanical effect of medial-to-lateral tibial slope asymmetry on lateral patellar instability.
Methods
Medial and lateral tibial slope was measured on knee magnetic resonance images in 107 patients and in 83 controls. The medial-to-lateral tibial slope asymmetry was assessed as the intra-individual difference between the medial and lateral tibial plateau inclination considering severity of trochlear dysplasia. The effect of tibial slope asymmetry on femoral rotation was calculated by means of radian measure.
Results
Severity of trochlear dysplasia was significantly associated with an asymmetric inclination of the tibial plateau. Whereas the medial tibial slope showed identical values between controls and study patients (n.s.), lateral tibial plateau inclination becomes flatter with increasing severity of trochlear dysplasia (p < 0.01). Consequently, the intra-individual tibial slope asymmetry increased steadily (p < 0.01) and increased internal femoral rotation in 20° and 90° of knee flexion angles in patients with severe trochlear dysplasia (p < 0.01). In addition, the extreme values of internal femoral rotation were more pronounced in patients with patellar instability, whereas the extreme values of external femoral rotation were more pronounced in control subjects (p = 0.024).
Conclusion
Data of this study indicate an association between tibial plateau configuration and internal femoral rotation in patients with lateral patellar instability and underlying trochlear dysplasia. Thereby, medial-to-lateral tibial slope asymmetry increased internal femoral rotation during knee flexion and therefore might aggravate the effect of femoral antetorsion in patients with patellar instability.
Level of evidence
III.
doi:10.1007/s00167-012-2247-4
PMCID: PMC3751338  PMID: 23096490
Patellar instability; Tibial slope; Femoral rotation; Biomechanics
7.  Selenoprotein P Status Correlates to Cancer-Specific Mortality in Renal Cancer Patients 
PLoS ONE  2012;7(10):e46644.
Selenium (Se) is an essential trace element for selenoprotein biosynthesis. Selenoproteins have been implicated in cancer risk and tumor development. Selenoprotein P (SePP) serves as the major Se transport protein in blood and as reliable biomarker of Se status in marginally supplied individuals. Among the different malignancies, renal cancer is characterized by a high mortality rate. In this study, we aimed to analyze the Se status in renal cell cancer (RCC) patients and whether it correlates to cancer-specific mortality. To this end, serum samples of RCC patients (n = 41) and controls (n = 21) were retrospectively analyzed. Serum Se and SePP concentrations were measured by X-ray fluorescence and an immunoassay, respectively. Clinical and survival data were compared to serum Se and SePP concentrations as markers of Se status by receiver operating characteristic (ROC) curve and Kaplan-Meier and Cox regression analyses. In our patients, higher tumor grade and tumor stage at diagnosis correlated to lower SePP and Se concentrations. Kaplan-Meier analyses indicated that low Se status at diagnosis (SePP<2.4 mg/l, bottom tertile of patient group) was associated with a poor 5-year survival rate of 20% only. We conclude that SePP and Se concentrations are of prognostic value in RCC and may serve as additional diagnostic biomarkers identifying a Se deficit in kidney cancer patients potentially affecting therapy regimen. As poor Se status was indicative of high mortality odds, we speculate that an adjuvant Se supplementation of Se-deficient RCC patients might be beneficial in order to stabilize their selenoprotein expression hopefully prolonging their survival. However, this assumption needs to be rigorously tested in prospective clinical trials.
doi:10.1371/journal.pone.0046644
PMCID: PMC3467258  PMID: 23056383
8.  Predictors of Renal Replacement Therapy in Acute Kidney Injury 
Nephron Extra  2012;2(1):247-255.
Backgrounds
Criteria that may guide early renal replacement therapy (RRT) initiation in patients with acute kidney injury (AKI) currently do not exist.
Methods
In 120 consecutive patients with AKI, clinical and laboratory data were analyzed on admittance. The prognostic power of those parameters which were significantly different between the two groups was analyzed by receiver operator characteristic curves and by leave-1-out cross validation.
Results
Six parameters (urine albumin, plasma creatinine, blood urea nitrogen, daily urine output, fluid balance and plasma sodium) were combined in a logistic regression model that estimates the probability that a particular patient will need RRT. Additionally, a second model without daily urine output was established. Both models yielded a higher accuracy (89 and 88% correct classification rate, respectively) than the best single parameter, cystatin C (correct classification rate 74%).
Conclusions
The combined models may help to better predict the necessity of RRT using clinical and routine laboratory data in patients with AKI.
doi:10.1159/000342257
PMCID: PMC3567877  PMID: 23599703
Acute kidney injury; Dialysis; Logistic regression model; Renal failure; Renal replacement therapy
9.  Reference miRNAs for miRNAome Analysis of Urothelial Carcinomas 
PLoS ONE  2012;7(6):e39309.
Background/Objective
Reverse transcription quantitative real-time PCR (RT-qPCR) is widely used in microRNA (miRNA) expression studies on cancer. To compensate for the analytical variability produced by the multiple steps of the method, relative quantification of the measured miRNAs is required, which is based on normalization to endogenous reference genes. No study has been performed so far on reference miRNAs for normalization of miRNA expression in urothelial carcinoma. The aim of this study was to identify suitable reference miRNAs for miRNA expression studies by RT-qPCR in urothelial carcinoma.
Methods
Candidate reference miRNAs were selected from 24 urothelial carcinoma and normal bladder tissue samples by miRNA microarrays. The usefulness of these candidate reference miRNAs together with the commonly for normalization purposes used small nuclear RNAs RNU6B, RNU48, and Z30 were thereafter validated by RT-qPCR in 58 tissue samples and analyzed by the algorithms geNorm, NormFinder, and BestKeeper.
Principal Findings
Based on the miRNA microarray data, a total of 16 miRNAs were identified as putative reference genes. After validation by RT-qPCR, miR-101, miR-125a-5p, miR-148b, miR-151-5p, miR-181a, miR-181b, miR-29c, miR-324-3p, miR-424, miR-874, RNU6B, RNU48, and Z30 were used for geNorm, NormFinder, and BestKeeper analyses that gave different combinations of recommended reference genes for normalization.
Conclusions
The present study provided the first systematic analysis for identifying suitable reference miRNAs for miRNA expression studies of urothelial carcinoma by RT-qPCR. Different combinations of reference genes resulted in reliable expression data for both strongly and less strongly altered miRNAs. Notably, RNU6B, which is the most frequently used reference gene for miRNA studies, gave inaccurate normalization. The combination of four (miR-101, miR-125a-5p, miR-148b, and miR-151-5p) or three (miR-148b, miR-181b, and miR-874,) reference miRNAs is recommended for normalization.
doi:10.1371/journal.pone.0039309
PMCID: PMC3380005  PMID: 22745731
10.  Detection of Simultaneous Group Effects in MicroRNA Expression and Related Target Gene Sets 
PLoS ONE  2012;7(6):e38365.
Expression levels of mRNAs are among other factors regulated by microRNAs. A particular microRNA can bind specifically to several target mRNAs and lead to their degradation. Expression levels of both, mRNAs and microRNAs, can be obtained by microarray experiments. In order to increase the power of detecting microRNAs that are differentially expressed between two different groups of samples, we incorporate expression levels of their related target gene sets. Group effects are determined individually for each microRNA, and by enrichment tests and global tests for target gene sets. The resulting lists of p-values from individual and set-wise testing are combined by means of meta analysis. We propose a new approach to connect microRNA-wise and gene set-wise information by means of p-value combination as often used in meta-analysis. In this context, we evaluate the usefulness of different approaches of gene set tests. In a simulation study we reveal that our combination approach is more powerful than microRNA-wise testing alone. Furthermore, we show that combining microRNA-wise results with ‘competitive’ gene set tests maintains a pre-specified false discovery rate. In contrast, a combination with ‘self-contained’ gene set tests can harm the false discovery rate, particularly when gene sets are not disjunct.
doi:10.1371/journal.pone.0038365
PMCID: PMC3378551  PMID: 22723856
11.  Immunoadsorption therapy in patients with multiple sclerosis with steroid-refractory optical neuritis 
Background
In multiple sclerosis relapses refractory to intravenous corticosteroid therapy, plasma exchange is recommended. Immunoadsorption (IA) is regarded as an alternative therapy, but its efficacy and putative mechanism of action still needs to be established.
Methods
We prospectively treated 11 patients with multiple sclerosis who had optical neuritis and fulfilled the indications for apheresis therapy (Trial registration DE/CA25/00007080-00). In total, five IA treatments were performed using tryptophan-IA. Clinical activity (visual acuity, Expanded Disability Status Scale, Incapacity Status Scale), laboratory values and visual evoked potentials were measured before, during and after IA, with a follow-up of six months. Moreover, proteomic analyses were performed to analyze column-bound proteins as well as corresponding changes in patients’ sera.
Results
After the third IA, we detected an improvement of vision in eight of eleven patients, whom we termed responders. Amongst these, the mean visual acuity improved from 0.15 ± 0.12 at baseline to 0.47 ± 0.32 after the third IA (P = 0.0252) up to 0.89 ± 0.15 (P < 0.0001) at day 180 ± 10 after IA. Soluble interleukin-2 receptor decreased in responders (P = 0.03), whereas in non-responders it did not. Proteomic analyses of proteins adsorbed to IA columns revealed that several significant immunological proteins as well as central nervous system protein fragments, including myelin basic protein, had been removed by IA.
Conclusions
IA was effective in the treatment of corticosteroid-refractory optic neuritis. IA influenced the humoral immune response. Strikingly, however, we found strong evidence that demyelination products and immunological mediators were also cleared from plasma by IA.
doi:10.1186/1742-2094-9-80
PMCID: PMC3418188  PMID: 22537481
Apheresis; Autoimmune diseases; Evoked potentials/visual; Immunoadsorption; Multiple sclerosis; Optic neuritis; Proteomics
12.  MiR-133b Targets Antiapoptotic Genes and Enhances Death Receptor-Induced Apoptosis 
PLoS ONE  2012;7(4):e35345.
Despite the importance of microRNAs (miRs) for regulation of the delicate balance between cell proliferation and death, evidence for their specific involvement during death receptor (DR)-mediated apoptosis is scarce. Transfection with miR-133b rendered resistant HeLa cells sensitive to tumor necrosis factor-alpha (TNFα)-induced cell death. Similarly, miR-133b caused exacerbated proapoptotic responses to TNF-related apoptosis-inducing ligand (TRAIL) or an activating antibody to Fas/CD95. Comprehensive analysis, encompassing global RNA or protein expression profiling performed by microarray experiments and pulsed stable isotope labeling with amino acids in cell culture (pSILAC), led to the discovery of the antiapoptotic protein Fas apoptosis inhibitory molecule (FAIM) as immediate miR-133b target. Moreover, miR-133b impaired the expression of the detoxifying protein glutathione-S-transferase pi (GSTP1). Expression of miR-133b in tumor specimens of prostate cancer patients was significantly downregulated in 75% of the cases, when compared with matched healthy tissue. Furthermore, introduction of synthetic miR-133b into an ex-vivo model of prostate cancer resulted in impaired proliferation and cellular metabolic activity. PC3 cells were also sensitized to apoptotic stimuli after transfection with miR-133b similar to HeLa cells. These data reveal the ability of a single miR to influence major apoptosis pathways, suggesting an essential role for this molecule during cellular transformation, tumorigenesis and tissue homeostasis.
doi:10.1371/journal.pone.0035345
PMCID: PMC3332114  PMID: 22532850
13.  Ultrasonic scalpel causes greater depth of soft tissue necrosis compared to monopolar electrocautery at standard power level settings in a pig model 
BMC Surgery  2012;12:3.
Background
Ultrasonic scalpel (UC) and monopolar electrocautery (ME) are common tools for soft tissue dissection. However, morphological data on the related tissue alteration are discordant. We developed an automatic device for standardized sample excision and compared quality and depth of morphological changes caused by UC and ME in a pig model.
Methods
100 tissue samples (5 × 3 cm) of the abdominal wall were excised in 16 pigs. Excisions were randomly performed manually or by using the self-constructed automatic device at standard power levels (60 W cutting in ME, level 5 in UC) for abdominal surgery. Quality of tissue alteration and depth of coagulation necrosis were examined histopathologically. Device (UC vs. ME) and mode (manually vs. automatic) effects were studied by two-way analysis of variance at a significance level of 5%.
Results
At the investigated power level settings UC and ME induced qualitatively similar coagulation necroses. Mean depth of necrosis was 450.4 ± 457.8 μm for manual UC and 553.5 ± 326.9 μm for automatic UC versus 149.0 ± 74.3 μm for manual ME and 257.6 ± 119.4 μm for automatic ME. Coagulation necrosis was significantly deeper (p < 0.01) when UC was used compared to ME. The mode of excision (manual versus automatic) did not influence the depth of necrosis (p = 0.85). There was no significant interaction between dissection tool and mode of excision (p = 0.93).
Conclusions
Thermal injury caused by UC and ME results in qualitatively similar coagulation necrosis. The depth of necrosis is significantly greater in UC compared to ME at investigated standard power levels.
doi:10.1186/1471-2482-12-3
PMCID: PMC3305372  PMID: 22361346
14.  Identification of Metastamirs as Metastasis-associated MicroRNAs in Clear Cell Renal Cell Carcinomas 
MicroRNAs (miRNAs) play a pivotal role in cancerogenesis and cancer progression, but their specific role in the metastasis of clear cell renal cell carcinomas (ccRCC) is still limited. Based on microRNA microarray analyses from normal and cancerous samples of ccRCC specimens and from bone metastases of ccRCC patients, we identified a set of 57 differentially expressed microRNAs between these three sample groups of ccRCC. A selected panel of 33 miRNAs was subsequently validated by RT-qPCR on total 57 samples. Then, 30 of the 33 examined miRNAs were confirmed to be deregulated. A stepwise down-regulation of miRNA expression from normal, over primary tumor to metastatic tissue samples, was found to be typical. A total of 23 miRNAs (miR-10b/-19a/-19b/-20a/-29a/-29b/-29c/-100/-101/-126/-127/-130/-141/-143/-145/-148a/-192/-194/-200c/-210/-215/-370/-514) were down-regulated in metastatic tissue samples compared with normal tissue. This down-regulated expression in metastatic tissue in comparison with primary tumor tissue was also present in 21 miRNAs. In cell culture experiments with 5-aza-2'-deoxycytidine and trichostatin A, epigenetic modifications were shown as one reason of this down-regulation. The altered miRNA profiles, comprising newly identified metastasis-associated miRNAs, termed metastamir and the predicted miRNA-target interactions together with the significant correlations of miRNAs that were either lost or newly appeared in the studied sample groups, afford a solid basis for further functional analyses of individual miRNAs in RCC metastatic progression.
doi:10.7150/ijbs.5106
PMCID: PMC3492794  PMID: 23139634
Renal cell carcinoma; Microarray; RT-qPCR; microRNAs; Metastasis.
15.  Central nervous system rather than immune cell-derived BDNF mediates axonal protective effects early in autoimmune demyelination 
Acta Neuropathologica  2011;123(2):247-258.
Brain-derived neurotrophic factor (BDNF) is involved in neuronal and glial development and survival. While neurons and astrocytes are its main cellular source in the central nervous system (CNS), bioactive BDNF is also expressed in immune cells and in lesions of multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). Previous data revealed that BDNF exerts neuroprotective effects in myelin oligodendrocyte glycoprotein-induced EAE. Using a conditional knock-out model with inducible deletion of BDNF, we here show that clinical symptoms and structural damage are increased when BDNF is absent during the initiation phase of clinical EAE. In contrast, deletion of BDNF later in the disease course of EAE did not result in significant changes, either in the disease course or in axonal integrity. Bone marrow chimeras revealed that the deletion of BDNF in the CNS alone, with no deletion of BDNF in the infiltrating immune cells, was sufficient for the observed effects. Finally, the therapeutic effect of glatiramer acetate, a well-characterized disease-modifying drug with the potential to modulate BDNF expression, was partially reversed in mice in which BDNF was deleted shortly before the onset of disease. In summary, our data argue for an early window of therapeutic opportunity where modulation of BDNF may exert neuroprotective effects in experimental autoimmune demyelination.
doi:10.1007/s00401-011-0890-3
PMCID: PMC3259380  PMID: 22009304
Neurotrophins; BDNF; EAE; Conditional knock-out mice; Neuroimmunology
16.  Fatty acid binding proteins (FABPs) in prostate, bladder and kidney cancer cell lines and the use of IL-FABP as survival predictor in patients with renal cell carcinoma 
BMC Cancer  2011;11:302.
Background
Fatty acid binding proteins (FABP) play an important role in carcinogenesis. Modified FABP expression patterns were described for prostate, bladder and for renal cell carcinoma. Studies on metabolic relationships and interactions in permanent cell lines allow a deeper insight into molecular processes. The aim of this study is therefore a systematic overview on mRNA and protein expressions of seven FABPs in frequently used urological cell lines.
Methods
Nine cell lines of renal carcinomas, seven of urinary bladder carcinomas, and five of prostate carcinomas were investigated. Quantitative RT-qPCR and western blotting were used to determine different FABPs. In addition, 46 paired cancerous and noncancerous tissue samples from nephrectomy specimen with renal cell carcinomas were investigated regarding the ileum FABP mRNA expression level and associated with survival outcome.
Results
General characteristics of all urological carcinoma cell lines were the expression of E-and IL-FABP on mRNA and protein level, while the expressions differed between the cell lines. The protein expression was not always congruent with the mRNA expression. Renal cell carcinoma cell lines showed expressions of L-, H- and B-FABP mRNA in addition to the general FABP expression in five out of the eight investigated cell lines. In bladder cancer cell lines, we additionally found the expression of A-FABP mRNA in six cell lines, while H-FABP was present only in three cell lines. In prostate cancer cell lines, a strong reduction of A- and E- FABP mRNA was observed. The expression of B-FABP mRNA and protein was observed only in the 22 RV-1 cells. IL-FABP mRNA was over-expressed in renal tumour tissue. The IL-FABP ratio was identified as an independent indicator of survival outcome.
Conclusions
Distinctly different FABP expression patterns were observed not only between the cell lines derived from the three cancer types, but also between the cell lines from the same cancer. The FABP patterns in the cell lines do not always reflect the real situation in the tumours. These facts have to be considered in functional studies concerning the different FABPs.
doi:10.1186/1471-2407-11-302
PMCID: PMC3199863  PMID: 21767383
17.  Reporting FDR analogous confidence intervals for the log fold change of differentially expressed genes 
BMC Bioinformatics  2011;12:288.
Background
Gene expression experiments are common in molecular biology, for example in order to identify genes which play a certain role in a specified biological framework. For that purpose expression levels of several thousand genes are measured simultaneously using DNA microarrays. Comparing two distinct groups of tissue samples to detect those genes which are differentially expressed one statistical test per gene is performed, and resulting p-values are adjusted to control the false discovery rate. In addition, the expression change of each gene is quantified by some effect measure, typically the log fold change. In certain cases, however, a gene with a significant p-value can have a rather small fold change while in other cases a non-significant gene can have a rather large fold change. The biological relevance of the change of gene expression can be more intuitively judged by a fold change then merely by a p-value. Therefore, confidence intervals for the log fold change which accompany the adjusted p-values are desirable.
Results
In a new approach, we employ an existing algorithm for adjusting confidence intervals in the case of high-dimensional data and apply it to a widely used linear model for microarray data. Furthermore, we adopt a concept of different relevance categories for effects in clinical trials to assess biological relevance of genes in microarray experiments. In a brief simulation study the properties of the adjusting algorithm are maintained when being combined with the linear model for microarray data. In two cancer data sets the adjusted confidence intervals can indicate significance of large fold changes and distinguish them from other large but non-significant fold changes. Adjusting of confidence intervals also corrects the assessment of biological relevance.
Conclusions
Our new combination approach and the categorization of fold changes facilitates the selection of genes in microarray experiments and helps to interpret their biological relevance.
doi:10.1186/1471-2105-12-288
PMCID: PMC3154206  PMID: 21756370
18.  Brain volume perfusion CT performed with 128-detector row CT system in patients with cerebral gliomas: A feasibility study 
European Radiology  2011;21(9):1811-1819.
Objectives
Validation of the feasibility and efficacy of volume perfusion computed tomography (VPCT) in the preoperative assessment of cerebral gliomas by applying a 128-slice CT covering the entire tumour.
Methods
Forty-six patients (25 men, 21 women; mean age 52.8 years) with cerebral gliomas were evaluated with VPCT. Two readers independently evaluated VPCT data, drawing volumes of interest (VOIs) around the tumour according to maximum intensity projection volumes, which were mapped automatically onto the cerebral blood volume (CBV), flow (CBF) and permeability (Ktrans) perfusion datasets. As control, a second VOI was placed in the contralateral healthy cortex. Correlation among perfusion parameters, tumour grade, hemisphere and VOIs was assessed. The diagnostic power of perfusion parameters was analysed by receiver operating characteristics curve analyses.
Results
VPCT was feasible in the assessment of the entire tumour extent. Mean values of Ktrans, CBV, CBF in high-grade gliomas were significantly higher compared with low-grade (p < 0.01). Ktrans demonstrated the highest diagnostic (97% sensitivity), positive (100%) and negative (94%) prognostic values.
Conclusions
VPCT was feasible in all subjects. All areas of different perfusion characteristics are depicted and quantified in colour-coded 3D maps. The derived parameters correlate well with tumour histopathology, differentiating low- from high-grade gliomas.
doi:10.1007/s00330-011-2150-2
PMCID: PMC3151396  PMID: 21573969
Cone-beam computed tomography; Brain neoplasms; Glioma; Feasibility studies
19.  Sequential interim analyses of survival data in DNA microarray experiments 
BMC Bioinformatics  2011;12:127.
Background
Discovery of biomarkers that are correlated with therapy response and thus with survival is an important goal of medical research on severe diseases, e.g. cancer. Frequently, microarray studies are performed to identify genes of which the expression levels in pretherapeutic tissue samples are correlated to survival times of patients. Typically, such a study can take several years until the full planned sample size is available.
Therefore, interim analyses are desirable, offering the possibility of stopping the study earlier, or of performing additional laboratory experiments to validate the role of the detected genes. While many methods correcting the multiple testing bias introduced by interim analyses have been proposed for studies of one single feature, there are still open questions about interim analyses of multiple features, particularly of high-dimensional microarray data, where the number of features clearly exceeds the number of samples. Therefore, we examine false discovery rates and power rates in microarray experiments performed during interim analyses of survival studies. In addition, the early stopping based on interim results of such studies is evaluated. As stop criterion we employ the achieved average power rate, i.e. the proportion of detected true positives, for which a new estimator is derived and compared to existing estimators.
Results
In a simulation study, pre-specified levels of the false discovery rate are maintained in each interim analysis, where reduced levels as used in classical group sequential designs of one single feature are not necessary. Average power rates increase with each interim analysis, and many studies can be stopped prior to their planned end when a certain pre-specified power rate is achieved. The new estimator for the power rate slightly deviates from the true power rate but is comparable to other estimators.
Conclusions
Interim analyses of microarray experiments can provide evidence for early stopping of long-term survival studies. The developed simulation framework, which we also offer as a new R package 'SurvGenesInterim' available at http://survgenesinter.R-Forge.R-Project.org, can be used for sample size planning of the evaluated study design.
doi:10.1186/1471-2105-12-127
PMCID: PMC3098786  PMID: 21527044
20.  Symptoms of the oral cavity and their association with local microbiological and clinical findings—a prospective survey in palliative care 
Supportive Care in Cancer  2011;20(3):531-537.
Purpose
Symptoms of the oral cavity clearly encompass more than radiation or chemotherapy-induced mucositis. Still, the burden of oral symptoms in palliative care has hardly been addressed directly, and considerations towards underlying disease processes have often been extrapolated from oncology patients.
Methods
We therefore conducted a prospective explorative survey with pilot character on patients on a specialized palliative care unit, describing symptom pattern (self assessment), clinical signs, bacteriological, mycological and virological findings, and correlating features of clinical history.
Results
Taste disturbances, dry mouth and the presence of Candida were found to be the most prevalent and correlating items. The broad spectrum of further symptoms, signs and findings did not show relevant correlations and did not permit any unilateral causal attributions.
Conclusions
Apart from the described focus on Candida colonisation/infections, a symptom-guided polypragmatic approach therefore seems to be justifiable for patients with oral symptoms in palliative care.
doi:10.1007/s00520-011-1114-z
PMCID: PMC3271219  PMID: 21336528
Palliative care; Oral symptoms; Taste disturbances; Dry mouth; Candida
21.  Suitable reference genes for relative quantification of miRNA expression in prostate cancer 
Experimental & Molecular Medicine  2010;42(11):749-758.
Real time quantitative PCR (qPCR) is the method of choice for miRNA expression studies. For relative quantification of miRNAs, normalization to proper reference genes is mandatory. Currently, no validated reference genes for miRNA qPCR in prostate cancer are available. In this study, the expression of four putative reference genes (hsa-miR-16, hsa-miR-130b, RNU6-2, SNORD7) was examined with regard to their use as normalizer. After SNORD7 was already shown an inappropriate reference gene in preliminary experiments using total RNA pools, we studied the expression of the putative reference genes in tissue and normal adjacent tissue sample pairs from 76 men with untreated prostate carcinoma collected after radical prostatectomy. hsa-miR-130b and RNU6-2 showed no significantly different expression between the matched malignant and non-malignant tissue samples, whereas hsa-miR-16 was significantly underexpressed in malignant tissue. Softwares geNorm and Normfinder predicted hsa-miR-130b and the geometric mean of hsa-miR-130b and RNU6-2 as the most stable reference genes. Normalization of the four miRNAs hsa-miR-96, hsa-miR-125b, hsa-miR-205, and hsa-miR-375, which were previously shown to be regulated, shows that normalization to hsa-mir-16 can lead to biased results. We recommend using hsa-miR-130b or the geometric mean of hsa-miR-130b and small RNA RNU6-2 for normalization in miRNA expression studies of prostate cancer.
doi:10.3858/emm.2010.42.11.076
PMCID: PMC2992854  PMID: 20890088
gene expression profiling; microRNA; polymerase chain reaction; prostate neoplasms
22.  Impact of RNA degradation on gene expression profiling 
BMC Medical Genomics  2010;3:36.
Background
Gene expression profiling is a highly sensitive technique which is used for profiling tumor samples for medical prognosis. RNA quality and degradation influence the analysis results of gene expression profiles. The impact of this influence on the profiles and its medical impact is not fully understood. As patient samples are very valuable for clinical studies, it is necessary to establish criteria for the RNA quality to be able to use these samples in later analysis.
Methods
To investigate the effects of RNA integrity on gene expression profiling, whole genome expression arrays were used. We used tumor biopsies from patients diagnosed with locally advanced rectal cancer. To simulate degradation, the isolated total RNA of all patients was subjected to heat-induced degradation in a time-dependent manner. Expression profiling was then performed and data were analyzed bioinformatically to assess the differences.
Results
The differences introduced by RNA degradation were largely outweighed by the biological differences between the patients. Only a relatively small number of probes (275 out of 41,000) show a significant effect due to degradation. The genes that show the strongest effect due to RNA degradation were, especially, those with short mRNAs and probe positions near the 5' end.
Conclusions
Degraded RNA from tumor samples (RIN > 5) can still be used to perform gene expression analysis. A much higher biological variance between patients is observed compared to the effect that is imposed by degradation of RNA. Nevertheless there are genes, very short ones and those with the probe binding side close to the 5' end that should be excluded from gene expression analysis when working with degraded RNA. These results are limited to the Agilent 44 k microarray platform and should be carefully interpreted when transferring to other settings.
doi:10.1186/1755-8794-3-36
PMCID: PMC2927474  PMID: 20696062
23.  Periostin is up-regulated in high grade and high stage prostate cancer 
BMC Cancer  2010;10:273.
Background
Expression of periostin is an indicator of epithelial-mesenchymal transition in cancer but a detailed analysis of periostin expression in prostate cancer has not been conducted so far.
Methods
Here, we evaluated periostin expression in prostate cancer cells and peritumoural stroma immunohistochemically in two independent prostate cancer cohorts, including a training cohort (n = 93) and a test cohort (n = 325). Metastatic prostate cancers (n = 20), hormone refractory prostate cancers (n = 19) and benign prostatic tissues (n = 38) were also analyzed.
Results
In total, strong epithelial periostin expression was detectable in 142 of 418 (34.0%) of prostate carcinomas and in 11 of 38 benign prostate glands (28.9%). Increased periostin expression in carcinoma cells was significantly associated with high Gleason score (p < 0.01) and advanced tumour stage (p < 0.05) in the test cohort. Whereas periostin expression was weak or absent in the stroma around normal prostate glands, strong periostin expression in tumour stroma was found in most primary and metastatic prostate cancers. High stromal periostin expression was associated with higher Gleason scores (p < 0.001). There was a relationship between stromal periostin expression and shortened PSA relapse free survival times in the training cohort (p < 0.05).
Conclusions
Our data indicate that periostin up-regulation is related to increased tumour aggressiveness in prostate cancer and might be a promising target for therapeutical interventions in primary and metastatic prostate cancer.
doi:10.1186/1471-2407-10-273
PMCID: PMC2903527  PMID: 20534149
24.  Stage II/III rectal cancer with intermediate response to preoperative radiochemotherapy: Do we have indications for individual risk stratification? 
Background
Response to preoperative radiochemotherapy (RCT) in patients with locally advanced rectal cancer is very heterogeneous. Pathologic complete response (pCR) is accompanied by a favorable outcome. However, most patients show incomplete response. The aim of this investigation was to find indications for risk stratification in the group of intermediate responders to RCT.
Methods
From a prospective database of 496 patients with rectal adenocarcinoma, 107 patients with stage II/III cancers and intermediate response to preoperative 5-FU based RCT (ypT2/3 and TRG 2/3), treated within the German Rectal Cancer Trials were studied. Surgical treatment comprised curative (R0) total mesorectal excision (TME) in all cases. In 95 patients available for statistical analyses, residual transmural infiltration of the mesorectal compartment, nodal involvement and histolologic tumor grading were investigated for their prognostic impact on disease-free (DFS) and overall survival (OS).
Results
Residual tumor transgression into the mesorectal compartment (ypT3) did not influence DFS and OS rates (p = 0.619, p = 0.602, respectively). Nodal involvement after preoperative RCT (ypN1/2) turned out to be a valid prognostic factor with decreased DFS and OS (p = 0.0463, p = 0.0236, respectively). Persistent tumor infiltration of the mesorectum (ypT3) and histologic tumor grading of residual tumor cell clusters were strongly correlated with lymph node metastases after neoadjuvant treatment (p < 0.001).
Conclusions
Advanced transmural tumor invasion after RCT does not affect prognosis when curative (R0) resection is achievable. Residual nodal status is the most important predictor of individual outcome in intermediate responders to preoperative RCT. Furthermore, ypT stage and tumor grading turn out to be additional auxiliary factors. Future clinical trials for risk-adapted adjuvant therapy should be based on a synopsis of clinicopathologic parameters.
doi:10.1186/1477-7819-8-27
PMCID: PMC2864265  PMID: 20388220
25.  Intra-oral compartment pressures: a biofunctional model and experimental measurements under different conditions of posture 
Clinical Oral Investigations  2010;15(2):165-176.
Oral posture is considered to have a major influence on the development and reoccurrence of malocclusion. A biofunctional model was tested with the null hypotheses that (1) there are no significant differences between pressures during different oral functions and (2) between pressure measurements in different oral compartments in order to substantiate various postural conditions at rest by intra-oral pressure dynamics. Atmospheric pressure monitoring was simultaneously carried out with a digital manometer in the vestibular inter-occlusal space (IOS) and at the palatal vault (sub-palatal space, SPS). Twenty subjects with normal occlusion were evaluated during the open-mouth condition (OC), gently closed lips (semi-open compartment condition, SC), with closed compartments after the generation of a negative pressure (CCN) and swallowing (SW). Pressure curve characteristics were compared between the different measurement phases (OC, SC, CCN, SW) as well as between the two compartments (IOS, SPS) using analysis of variance and Wilcoxon matched-pairs tests adopting a significance level of α = 0.05. Both null hypotheses were rejected. Average pressures (IOS, SPS) in the experimental phases were 0.0, −0.08 (OC); −0.16, −1.0 (SC); −48.79, −81.86 (CCN); and −29.25, −62.51 (SW) mbar. CCN plateau and peak characteristics significantly differed between the two compartments SPS and IOS. These results indicate the formation of two different intra-oral functional anatomical compartments which provide a deeper understanding of orofacial biofunctions and explain previous observations of negative intra-oral pressures at rest.
doi:10.1007/s00784-009-0367-0
PMCID: PMC3056003  PMID: 20127264
Intra-oral pressure; Biofunctional model; Functional intra-oral compartments; Swallowing; Oral posture

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