Postoperative motor deficits following elbow flexion reanimation by nerve transfer.

We aimed to assess the rate and type of postoperative motor deficits that might be encountered following elbow flexion reanimation using ulnar- and/or median-based side-to-end nerve transfers in patients with brachial plexus injuries. All patients who underwent elbow flexion reanimation between November 2015 and October 2017 at our facility by nerve transfer based on partial harvests of the median and/or ulnar nerves were included. Postoperative clinical assessment was conducted the day after surgery to identify motor deficits in the territory of the harvested nerves. If a clinically noticeable deficit was present, the type and extent of the deficit were noted, and postoperative clinical evaluations were conducted monthly to determine its progression. After reviewing the charts of 27 consecutive patients, 4 patients were found to have a postoperative motor deficit (15%). In all four cases, the deficit was limited to the anterior interosseous nerve (AIN) territory in patients who underwent a double transfer (i.e., ulnar-to-biceps and median-to-brachialis). With clinical impairments of the flexor pollicis longus and/or the flexor digitorum profundus of the index and third fingers initially ranging from grade-0 to grade-3 strength, full recovery to preoperative strength levels occurred in all cases after a mean of 7 months' follow-up. Transient motor deficits may be observed in the AIN territory following elbow flexion reanimation when a median-to-brachialis nerve transfer is associated with the original Oberlin procedure.

Correlative mass spectrometry imaging, applying time-of-flight secondary ion mass spectrometry and atmospheric pressure matrix-assisted laser desorption/ionization to a single tissue section.

RATIONALE: Mass spectrometry imaging (MSI) is a powerful tool for mapping the surface of a sample. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) and atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) offer complementary capabilities. Here, we present a workflow to apply both techniques to a single tissue section and combine the resulting data for the example of human colon cancer tissue. METHODS: Following cryo-sectioning, images were acquired using the high spatial resolution (1 µm pixel size) provided by TOF-SIMS. The same section was then coated with a para-nitroaniline matrix and images were acquired using AP-MALDI coupled to an Orbitrap mass spectrometer, offering high mass resolution, high mass accuracy and tandem mass spectrometry (MS/MS) capabilities. Datasets provided by both mass spectrometers were converted into the open and vendor-independent imzML file format and processed with the open-source software MSiReader. RESULTS: The TOF-SIMS and AP-MALDI mass spectra show strong signals of fatty acids, cholesterol, phosphatidylcholine and sphingomyelin. We showed a high correlation between the fatty acid ions detected with TOF-SIMS in negative ion mode and the phosphatidylcholine ions detected with AP-MALDI in positive ion mode using a similar setting for visualization. Histological staining on the same section allowed the identification of the anatomical structures and their correlation with the ion images. CONCLUSIONS: This multimodal approach using two MSI platforms shows an excellent complementarity for the localization and identification of lipids. The spatial resolution of both systems is at or close to cellular dimensions, and thus spatial correlation can only be obtained if the same tissue section is analyzed sequentially. Data processing based on imzML allows a real correlation of the imaging datasets provided by these two technologies and opens the way for a more complete molecular view of the anatomical structures of biological tissues.

In Situ Metabolomics in Cancer by Mass Spectrometry Imaging.

Metabolomics is a rapidly evolving and a promising research field with the expectation to improve diagnosis, therapeutic treatment prediction, and prognosis of particular diseases. Among all techniques used to assess the metabolome in biological systems, mass spectrometry imaging is the method of choice to qualitatively and quantitatively analyze metabolite distribution in tissues with a high spatial resolution, thus providing molecular data in relation to cancer histopathology. The technique is ideally suited to study tissues molecular content and is able to provide molecular biomarkers or specific mass signatures which can be used in classification or the prognostic evaluation of tumors. Recently, it was shown that FFPE tissue samples are also suitable for metabolic analyses. This progress in methodology allows access to a highly valuable resource of tissues believed to widen and strengthen metabolic discovery-driven studies.

Oral dosage form administration practice in children under 6 years of age: A survey study of paediatric nurses.

The purpose of this study was to interview paediatric nurses on administration issues using extemporaneous capsules and marketed capsules and tablets in children younger than 6 years old, based on most frequently administered drugs in six participating wards. The 59 responding nurses estimated respectively at 7.7±1.7 and 7.3±1.8years the age from which children would properly swallow extemporaneous capsules and marketed solids, with 33% and 37% of nurses considering that children under 6 would not get their prescribed treatment using these dosage forms. Refusal of the child to take the solid was the first reason to explain administration failure (85% of nurses for extemporaneous capsules, 89% for marketed solids). Although type of formulation and requirement of chewing were factors influencing the age at which children would take solid from nurses' experience, size of conventional tablets was not among these factors. All respondents use to crush tablets in children unable to swallow whole solids; 37% of nurses systematically split the tablets to ease the swallowing in children able to swallow. Only 11 nurses had an information tool at their disposal to guide manipulation of solids, with 7 of them using it in their daily practice. Providing specific-ward questionnaires, this study gives factual information on administration practices, perceptions and issues faced by paediatric nurses.

High fat diet-induced modifications in membrane lipid and mitochondrial-membrane protein signatures precede the development of hepatic insulin resistance in mice.

OBJECTIVE: Excess lipid intake has been implicated in the pathophysiology of hepatosteatosis and hepatic insulin resistance. Lipids constitute approximately 50% of the cell membrane mass, define membrane properties, and create microenvironments for membrane-proteins. In this study we aimed to resolve temporal alterations in membrane metabolite and protein signatures during high-fat diet (HF)-mediated development of hepatic insulin resistance. METHODS: We induced hepatosteatosis by feeding C3HeB/FeJ male mice an HF enriched with long-chain polyunsaturated C18:2n6 fatty acids for 7, 14, or 21 days. Longitudinal changes in hepatic insulin sensitivity were assessed via the euglycemic-hyperinsulinemic clamp, in membrane lipids via t-metabolomics- and membrane proteins via quantitative proteomics-analyses, and in hepatocyte morphology via electron microscopy. Data were compared to those of age- and litter-matched controls maintained on a low-fat diet. RESULTS: Excess long-chain polyunsaturated C18:2n6 intake for 7 days did not compromise hepatic insulin sensitivity, however, induced hepatosteatosis and modified major membrane lipid constituent signatures in liver, e.g. increased total unsaturated, long-chain fatty acid-containing acyl-carnitine or membrane-associated diacylglycerol moieties and decreased total short-chain acyl-carnitines, glycerophosphocholines, lysophosphatidylcholines, or sphingolipids. Hepatic insulin sensitivity tended to decrease within 14 days HF-exposure. Overt hepatic insulin resistance developed until day 21 of HF-intervention and was accompanied by morphological mitochondrial abnormalities and indications for oxidative stress in liver. HF-feeding progressively decreased the abundance of protein-components of all mitochondrial respiratory chain complexes, inner and outer mitochondrial membrane substrate transporters independent from the hepatocellular mitochondrial volume in liver. CONCLUSIONS: We assume HF-induced modifications in membrane lipid- and protein-signatures prior to and during changes in hepatic insulin action in liver alter membrane properties - in particular those of mitochondria which are highly abundant in hepatocytes. In turn, a progressive decrease in the abundance of mitochondrial membrane proteins throughout HF-exposure likely impacts on mitochondrial energy metabolism, substrate exchange across mitochondrial membranes, contributes to oxidative stress, mitochondrial damage, and the development of insulin resistance in liver.

CLIP2 as radiation biomarker in papillary thyroid carcinoma.

A substantial increase in papillary thyroid carcinoma (PTC) among children exposed to the radioiodine fallout has been one of the main consequences of the Chernobyl reactor accident. Recently, the investigation of PTCs from a cohort of young patients exposed to the post-Chernobyl radioiodine fallout at very young age and a matched nonexposed control group revealed a radiation-specific DNA copy number gain on chromosomal band 7q11.23 and the radiation-associated mRNA overexpression of CLIP2. In this study, we investigated the potential role of CLIP2 as a radiation marker to be used for the individual classification of PTCs into CLIP2-positive and -negative cases-a prerequisite for the integration of CLIP2 into epidemiological modelling of the risk of radiation-induced PTC. We were able to validate the radiation-associated CLIP2 overexpression at the protein level by immunohistochemistry (IHC) followed by relative quantification using digital image analysis software (P=0.0149). Furthermore, we developed a standardized workflow for the determination of CLIP2-positive and -negative cases that combines visual CLIP2 IHC scoring and CLIP2 genomic copy number status. In addition to the discovery cohort (n=33), two independent validation cohorts of PTCs (n=115) were investigated. High sensitivity and specificity rates for all three investigated cohorts were obtained, demonstrating robustness of the developed workflow. To analyse the function of CLIP2 in radiation-associated PTC, the CLIP2 gene regulatory network was reconstructed using global mRNA expression data from PTC patient samples. The genes comprising the first neighbourhood of CLIP2 (BAG2, CHST3, KIF3C, NEURL1, PPIL3 and RGS4) suggest the involvement of CLIP2 in the fundamental carcinogenic processes including apoptosis, mitogen-activated protein kinase signalling and genomic instability. In our study, we successfully developed and independently validated a workflow for the typing of PTC clinical samples into CLIP2-positive and CLIP2-negative and provided first insights into the CLIP2 interactome in the context of radiation-associated PTC.

Opposing role of Notch1 and Notch2 in a Kras(G12D)-driven murine non-small cell lung cancer model.

Lung cancer is the leading cause of cancer-related deaths worldwide. Recently, we have shown that Notch1 inhibition resulted in substantial cell death of non-small cell lung cancer (NSCLC) cells in vitro. New compounds targeting Notch signal transduction have been developed and are now being tested in clinical trials. However, the tumorigenic role of individual Notch receptors in vivo remains largely unclear. Using a Kras(G12D)-driven endogenous NSCLC mouse model, we analyzed the effect of conditional Notch1 and Notch2 receptor deletion on NSCLC tumorigenesis. Notch1 deficiency led to a reduced early tumor formation and lower activity of MAPK compared with the controls. Unexpectedly, Notch2 deletion resulted in a dramatically increased carcinogenesis and increased MAPK activity. These mice died significantly earlier due to rapidly growing tumor burden. We found that Notch1 regulates Ras/MAPK pathway via HES1-induced repression of the DUSP1 promoter encoding a phosphatase specifically suppressing pERK1/2. Interestingly, Notch1 but not Notch2 ablation leads to decreased HES1 and DUSP1 expression. However, Notch2-depleted tumors showed an appreciable increase in ß-catenin expression, a known activator of HES1 and important lung cancer oncogene. Characteristically for ß-catenin upregulation, we found that the majority of Notch2-deficient tumors revealed an undifferentiated phenotype as determined by their morphology, E-Cadherin and TTF1 expression levels. In addition, these carcinomas showed aggressive growth patterns with bronchus invasion and obstruction. Together, we show that Notch2 mediates differentiation and has tumor suppressor functions during lung carcinogenesis, whereas Notch1 promotes tumor initiation and progression. These data are further supported by immunohistochemical analysis of human NSCLC samples showing loss or downregulation of Notch2 compared with normal lung tissue. In conclusion, this is the first study characterizing the in vivo functions of Notch1 and Notch2 in Kras(G12D)-driven NSCLC tumorigenesis. These data highlight the clinical importance of a thorough understanding of Notch signaling especially with regard to Notch-targeted therapies.

A rapid ex vivo tissue model for optimising drug detection and ionisation in MALDI imaging studies.

The aim of this study was to establish an ex vivo model for a faster optimisation of sample preparation procedures, for example matrix choice, in matrix-assisted laser desorption/ionisation (MALDI) drug imaging studies. The ionisation properties of four drugs, afatinib, erlotinib, irinotecan and pirfenidone, were determined in an ex vivo tissue experiment by spotting decreasing dilution series onto liver sections. Hereby, the drug signals were distinctly detectable using different matrix compounds, which allowed the selection of the optimal matrix for each drug. The analysis of afatinib and erlotinib yielded high drug signals with α-cyano-4-hydroxycinnamic acid matrix, whereas 2,3-dihydroxybenzoic acid was identified as optimal matrix for irinotecan and pirfenidone detection. Our method was validated by a MALDI drug imaging approach of in vivo treated mouse tissue resulting in corresponding findings, indicating the spotting method as an appropriate approach to determine the matrix of choice. The present study shows the accordance between the detection of ex vivo spotted drugs and in vivo administered drugs by MALDI-TOF and MALDI-FT-ICR imaging, which has not been demonstrated so far. Our data suggest the ex vivo tissue spotting method as an easy and reliable model to optimise MALDI imaging measurements and to predict drug detection in tissue sections derived from treated mice prior to the recruitment of laboratory animals, which helps to save animals, time and costs.

Optoacoustic imaging and staging of inflammation in a murine model of arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is one of the most frequent inflammatory diseases, causing pain and disability in the affected joints. Early diagnosis is essential for the efficiency of symptom-targeting treatments, but its diagnosis requires careful clinical, serologic, and imaging examinations, such as magnetic resonance imaging (MRI), which is both expensive and time consuming. In an effort to provide the biomedical community with a more accessible way to assess the advancement of arthritis, this study sought to investigate the use of multispectral optoacoustic tomography (MSOT) in a murine arthritis model, to visualize the extent of inflammation in vivo through an L-selectin/P-selectin-targeting contrast agent. METHODS: Mice with collagen-induced arthritis were studied as a model of RA. MSOT was performed using an L-selectin/P-selectin-targeting contrast agent, polyanionic dendritic polyglycerol sulfate (dPGS) labeled with a near-infrared (NIR) fluorophore, to increase the contrast of the arthritic joint. The signal intensity ratios between healthy legs and arthritic legs were calculated. Findings on contrast-enhanced MRI, clinical observations, the lymphocyte:granulocyte ratio, and histologic findings served as referents for comparison. RESULTS: MSOT using an inflammation-targeting contrast agent, dPGS-NIR, allowed for accurate diagnosis of inflammation in the mouse joints. In addition, use of this technique resulted in significant differentiation of the inflamed joints from the healthy joints (P = 0.023). The observed advancement of arthritis on the MSOT images was confirmed by clinical observation, blood analysis, contrast-enhanced MRI, and ex vivo histologic examinations. CONCLUSION: This study demonstrates that the combination of an inflammation-targeting contrast agent and optoacoustic tomographic imaging presents a promising means for the diagnosis of RA and the staging of arthritis-related inflammation.

MiR-221/-222 differentiate prognostic groups in advanced breast cancers and influence cell invasion.

BACKGROUND: MiR-221/-222 are frequently overexpressed in breast cancer and are associated with increased malignancy. The specific modification of microRNAs (miRNAs) expression could be a promising strategy in breast cancer therapy, leading to the suppression of tumourigenic processes in tumour cells. METHODS: MiR-221/-222 expressions were analysed in 86 breast cancer tissues by quantitative RT-PCR and tested for correlation with immunohistochemistry data and clinical follow-up. In vitro assays were conducted using human breast cancer cell lines with lentiviral overexpression of miR-221/-222. RESULTS: In tumour tissues, miR-221/-222 were associated with the occurrence of distant metastases. In particular, high levels of miR-221 were revealed to have a high prognostic impact for the identification of significantly different groups with advanced tumours. MiR-221/-222 overexpression strongly increased cell proliferation and invasion in vitro. Following miR-221/-222 overexpression an increased uPAR expression and cell invasion were observed. CONCLUSION: This study demonstrates a significant role for highly expressed miR-221/-222 in advanced breast cancers allowing for the identification of significantly different prognostic groups, particularly for HER2-positive and lymph-node-positive breast cancers. Considering that miR-221/-222 are strongly involved in cell invasion, these miRNAs may be promising markers for breast cancer prognosis and therapy.

Super-resolution segmentation of imaging mass spectrometry data: Solving the issue of low lateral resolution.

In the last decade, imaging mass spectrometry has seen incredible technological advances in its applications to biological samples. One computational method of data mining in this field is the spatial segmentation of a sample, which produces a segmentation map highlighting chemically similar regions. An important issue for any imaging mass spectrometry technology is its relatively low spatial or lateral resolution (i.e. a large size of pixel) as compared with microscopy. Thus, the spatial resolution of a segmentation map is also relatively low, that complicates its visual examination and interpretation when compared with microscopy data, as well as reduces the accuracy of any automated comparison. We address this issue by proposing an approach to improve the spatial resolution of a segmentation map. Given a segmentation map, our method magnifies it up to some factor, producing a super-resolution segmentation map. The super-resolution map can be overlaid and compared with a high-res microscopy image. The proposed method is based on recent advances in image processing and smoothes the "pixilated" region boundaries while preserving fine details. Moreover, it neither eliminates nor splits any region. We evaluated the proposed super-resolution segmentation approach on three MALDI-imaging datasets of human tissue sections and demonstrated the superiority of the super-segmentation maps over standard segmentation maps.

[Update on protein analysis of fixed tissues]. / Neues zur Proteinanalytik archivierter Gewebeproben.

Tissue samples have been routinely used for decades to distinguish healthy from diseased tissue in histopathological characterization. While nucleic acid-based methodologies have been successfully in use for many years, protein-based techniques, in contrast, are at a very early stage (with the exception of immunohistochemistry). One reason for this delay may be that the scientific community has long thought that formalin-fixed and paraffin embedded (FFPE) tissues are unfit for protein analysis. However, recent reports demonstrate that many protein methods that are routinely used for frozen tissues can also be applied for FFPE tissues, including Western blot, protein microarray, matrix-assisted laser desorption/ionization (MALDI) imaging and 2D gel electrophoresis. The present article provides an overview of recent developments in this field, focussing particular attention on quantitative analysis and high throughput technologies that have the potential to be integrated into the routine workflow of clinical pathology laboratories.

[Analysis of signalling pathways in formalin-fixed breast cancer tissues]. / Analyse von Signalwegen in formalinfixierten Brustkrebsgeweben.

AIMS: The aim of our study was to develop and optimize methods for relative and absolute protein quantifications in formalin-fixed and paraffin-embedded (FFPE) tissues with special emphasis on HER mediated pathways in breast cancer. METHODS: Using a recently developed technology for extraction of full-length proteins from FFPE tissues, we evaluated >50 commercial antibodies for specificity using Western blots and protein microarrays. Purified HER receptor proteins were used to determine absolute protein concentrations. RESULTS: We confirmed specificity of 23 commercially available phosphospecific and non-phosphospecific antibodies using Western blots with protein extracts from cell lines and tissue extracts from breast cancer patients. Spiking known amounts of purified HER receptor proteins in HER receptor negative tissue extracts allowed us to precisely measure abundances of HER-receptors. CONCLUSIONS: Our results will provide a basis for the development of diagnostic techniques for the quantitative analysis of deregulated HER receptors and downstream signalling proteins in typical clinical tissues.

[MALDI imaging mass spectrometry for direct tissue analysis]. / In-situ-Proteomanalyse von Geweben: Mittels bildgebender Massenspektrometrie (MALDI Imaging).

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry is a new method for investigating the distribution of proteins and small molecules within biological systems through the in situ analysis of tissue sections using MALDI-TOF (time-of-flight) mass spectometry. The power of this technology lies in its capability to reliably combine protein data with specific cellular regions within the tissue. For the molecular image analysis, tissue samples are measured in a raster-like process in the mass spectrometer and for each spot a mass spectrum is obtained from molecules within the irradiated area. Software tools generate a mass spectrometry image or molecular weight-specific map of the sample at any desired molecular weight value and the presence and location of proteins and peptides are visualized in the tissue.There are three main fields of application, comprising molecular histology, screening for new biomarkers, and detection of drugs and their metabolites directly in the tissue.

Inflammation and mitochondrial fatty acid beta-oxidation link obesity to early tumor promotion.

Obesity is associated with increased risk for developing pancreatic cancer, and it is suggested that insulin resistance provides the missing link. Here we demonstrate that under the context of genetic susceptibility, a high fat diet (HFD) predisposes mice with oncogenic K-ras activation to accelerated pancreatic intraepithelial neoplasm (PanIN) development. Tumor promotion is closely associated with increased inflammation and abrogation of TNFR1 signaling significantly blocks this process underlining a central role for TNFalpha in obesity-mediated enhancement of PanIN lesions. Interestingly, however, despite increased TNFalpha levels, mice remain insulin sensitive. We show that, while aggravating tumor promotion, a HFD exerts dramatic changes in energy metabolism through enhancement of pancreatic exocrine insufficiency, metabolic rates, and expression of genes involved in mitochondrial fatty acid (FA) beta-oxidation that collectively contribute to improved glucose tolerance in these mice. While on one hand these findings provide significant evidence that obesity is linked to tumor promotion in the pancreas, on the other it suggests alterations in inflammatory responses and bioenergetic pathways as the potential underlying cause.

Prognostic value of protein tyrosine kinase 6 (PTK6) for long-term survival of breast cancer patients.

The cytoplasmic tyrosine kinase PTK6 (BRK) shows elevated expression in approximately two-thirds of primary breast tumours, and is implicated in EGF receptor-dependent signalling and epithelial tumorigenesis. Using immunohistochemistry, we performed a retrospective study on 426 archival breast cancer samples from patients with long-term follow-up and compared the protein expression levels of PTK6, the HER receptors, Sam68 (a substrate of PTK6), and signalling proteins including MAP kinase (MAPK), phosphorylated MAPK (P-MAPK), and PTEN. We show that PTK6 expression is of significant prognostic value in the outcome of breast carcinomas. In multivariate analysis, the disease-free survival of patients of >or=240 months was directly associated with the protein expression level of PTK6 (P

Array CGH demonstrates characteristic aberration signatures in human papillary thyroid carcinomas governed by RET/PTC.

The aim of this study is to investigate additional genetic alterations in papillary thyroid carcinomas (PTCs) with known RET/PTC rearrangements. We applied array-based comparative genomic hybridization (array CGH) to 33 PTC (20 PTC from adults, 13 post-Chernobyl PTC from children) with known RET/PTC status. Principal component analysis and hierarchical cluster analysis identified cases with similar aberration patterns. Significant deviations between tumour-groups were obtained by statistical testing (Fisher's exact test in combination with Benjamini-Hochberg FDR-controlling procedure). FISH analysis on FFPE sections was applied to validate the array CGH data. Deletions were found more frequently in RET/PTC-positive and RET/PTC-negative tumours than amplifications. Specific aberration signatures were identified that discriminated between RET/PTC-positive and RET/PTC-negative cases (aberrations on chromosomes 1p, 3q, 4p, 7p, 9p/q, 10q, 12q, 13q and 21q). In addition, childhood and adult RET/PTC-positive cases differ significantly for a deletion on the distal part of chromosome 1p. There are additional alterations in RET/PTC-positive tumours, which may act as modifiers of RET activation. In contrast, alterations in RET/PTC-negative tumours indicate alternative routes of tumour development. The data presented serve as a starting point for further studies on gene expression and function of genes identified in this study.

PTK (protein tyrosine kinase)-6 and HER2 and 4, but not HER1 and 3 predict long-term survival in breast carcinomas.

The HER receptors are of therapeutic and prognostic significance in breast cancer, and their function is modulated by cytoplasmic tyrosine kinases like PTK6 (brk). We performed a retrospective study on archival breast cancer samples from patients with long follow-up and compared the protein expression between individual HERs and between HERs and the PTK6. Univariate and multivariate analyses were used to study the prognostic value of parameters. Metastases-free survival of patients for longer than 240 months was inversely associated (P< or =0.05) with nodal status, tumour size, and oestrogen receptor status, but was also directly associated with high protein expression levels of HER4 and PTK6 in Kaplan-Meier analysis. In multivariate analysis for metastases-free survival of >240 months, the stepwise selected parameters were tumour size (relative risk 3.1), PTK6 expression (0.4), and number of positive lymph nodes (1.2). Furthermore, we demonstrated a timedependence of the prognostic value attributed to the parameters. The HER receptors (HER2,4), but not PTK6, were independent prognostic markers for metastases-free survival at 60 months, whereas at 240 months PTK6 is the strongest prognostic marker. We demonstrate that PTK6 is a prognostic marker of metastases-free survival in breast cancer, and is independent of the classical morphological and molecular markers of lymph node involvement, tumour size, and HER2 status.

[Aurora-a is a predictive marker for stage III epithelial ovarian cancers]. / Aurora-a ist ein prädiktiver Marker für Ovarialkarzinome des Stadiums III.

AIM: Overexpression of Aurora-A/STK15 kinase (hereafter AUKRA) is seen in a variety of epithelial cancers, such as gastrointestinal and gynaecological carcinomas. Its role as prognostic and/or predictive marker for adjuvant therapy of patients with advanced ovarian cancer is however still unclear. Therefore, the present study aimed at determining (1) the clinical value of AURKA expression (mRNA and protein) in 115 patients with ovarian carcinomas and (2) the basis of AURKA overexpression at the DNA level. METHODS: Formalin-fixed and Paraffin-embedded tissue samples (ovarian carcinoma: n=115; non-neoplastic ovaries: n=28) were processed for microdissection and quantitative RT-PCR as well as for semi-quantitative immunohistochemistry (IHC) of tissue microarrays according to standardised protocols. Fluorescence in Situ Hybridisation (FISH) was performed in a sub-set of cases (n=37) to analyse AURKA DNA copy numbers. RESULTS: The results demonstrate significantly elevated AURKA expression at the mRNA and protein level in ovarian carcinomas as compared to non-neoplastic ovaries (p < 0.0001). AURKA protein overexpression was observed in 68/107 (63.5%) of cases. For patients with stage III ovarian carcinoma having been optimally debulked and receiving adjuvant Taxane-based chemotherapy, AURKA overexpression was significantly linked to prolonged overall survival (p = 0.02). Finally AURKA overexpression was associated with increased AURKA DNA copy numbers (p = 0.01). CONCLUSION: In summary, AURKA overexpression, which is regulated at the DNA level, is a novel predictive marker for a subgroup of patients with stage III ovarian carcinomas.

Distribution pattern of inhaled ultrafine gold particles in the rat lung.

The role of alveolar macrophages in the fate of ultrafine particles in the lung was investigated. Male Wistar-Kyoto rats were exposed to ultrafine gold particles, generated by a spark generator, for 6 h at a concentration of 88 microg/m3 (4 x 10(6)/cm3, 16 nm modal mobility diameter). Up to 7 days, the animals were serially sacrificed, and lavaged cells and lung tissues were examined by transmission electron microscopy. The gold concentration/content in the lung, lavage fluid, and blood was estimated by inductively coupled plasma-mass spectrometry. Gold particles used were spherical and electron dense with diameters of 5-8 nm. The particles were individual or slightly agglomerated. By inductively coupled plasma-mass spectrometry analysis of the lung, 1945 +/- 57 ng (mean +/- SD) and 1512 +/- 184 ng of gold were detected on day 0 and on day 7, respectively, indicating that a large portion of the deposited gold particles was retained in the lung tissue. In the lavage fluid, 573 +/- 67 ng and 96 +/- 29 ng were found on day 0 and day 7, respectively, which means that 29% and 6% of the retained gold particles were lavageable on these days. A low but significant increase of gold (0.03 to 0.06% of lung concentration) was found in the blood. Small vesicles containing gold particles were found in the cytoplasm of alveolar macrophages. In the alveolar septum, the gold particles were enclosed in vesicles observed in the cytoplasm of alveolar type I epithelial cells. These results indicate that inhaled ultrafine gold particles in alveolar macrophages and type I epithelial cells are processed by endocytotic pathways, though the uptake of the gold particles by alveolar macrophages is limited. To a low degree, systemic particle translocation took place.