Effect of storage time on gene expression data acquired from unfrozen archived newborn blood spots.

Unfrozen archived newborn blood spots (NBS) have been shown to retain sufficient messenger RNA (mRNA) for gene expression profiling. However, the effect of storage time at ambient temperature for NBS samples in relation to the quality of gene expression data is relatively unknown. Here, we evaluated mRNA expression from quantitative real-time PCR (qRT-PCR) and microarray data obtained from NBS samples stored at ambient temperature to determine the effect of storage time on the quality of gene expression. These data were generated in a previous case-control study examining NBS in 53 children with cerebral palsy (CP) and 53 matched controls. NBS sample storage period ranged from 3 to 16years at ambient temperature. We found persistently low RNA integrity numbers (RIN=2.3±0.71) and 28S/18S rRNA ratios (~0) across NBS samples for all storage periods. In both qRT-PCR and microarray data, the expression of three common housekeeping genes-beta cytoskeletal actin (ACTB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and peptidylprolyl isomerase A (PPIA)-decreased with increased storage time. Median values of each microarray probe intensity at log2 scale also decreased over time. After eight years of storage, probe intensity values were largely reduced to background intensity levels. Of 21,500 genes tested, 89% significantly decreased in signal intensity, with 13,551, 10,730, and 9925 genes detected within 5years, > 5 to <10years, and >10years of storage, respectively. We also examined the expression of two gender-specific genes (X inactivation-specific transcript, XIST and lysine-specific demethylase 5D, KDM5D) and seven gene sets representing the inflammatory, hypoxic, coagulative, and thyroidal pathways hypothesized to be related to CP risk to determine the effect of storage time on the detection of these biologically relevant genes. We found the gender-specific genes and CP-related gene sets detectable in all storage periods, but exhibited differential expression (between male vs. female or CP vs. control) only within the first six years of storage. We concluded that gene expression data quality deteriorates in unfrozen archived NBS over time and that differential gene expression profiling and analysis is recommended for those NBS samples collected and stored within six years at ambient temperature.

Mimp/Mtch2, an Obesity Susceptibility Gene, Induces Alteration of Fatty Acid Metabolism in Transgenic Mice.

OBJECTIVE: Metabolic dysfunctions, such as fatty liver, obesity and insulin resistance, are among the most common contemporary diseases worldwide, and their prevalence is continuously rising. Mimp/Mtch2 is a mitochondrial carrier protein homologue, which localizes to the mitochondria and induces mitochondrial depolarization. Mimp/Mtch2 single-nucleotide polymorphism is associated with obesity in humans and its loss in mice muscle protects from obesity. Our aim was to study the effects of Mimp/Mtch2 overexpression in vivo. METHODS: Transgenic mice overexpressing Mimp/Mtch2-GFP were characterized and monitored for lipid accumulation, weight and blood glucose levels. Transgenic mice liver and kidneys were used for gene expression analysis. RESULTS: Mimp/Mtch2-GFP transgenic mice express high levels of fatty acid synthase and of ß-oxidation genes and develop fatty livers and kidneys. Moreover, high-fat diet-fed Mimp/Mtch2 mice exhibit high blood glucose levels. Our results also show that Mimp/Mtch2 is involved in lipid accumulation and uptake in cells and perhaps in human obesity. CONCLUSIONS: Mimp/Mtch2 alters lipid metabolism and may play a role in the onset of obesity and development of insulin resistance.

Pericyte coverage of differentiated vessels inside tumor vasculature is an independent unfavorable prognostic factor for patients with clear cell renal cell carcinoma.

BACKGROUND: The objective of this study was to evaluate the effect of pericyte coverage (PC) of differentiated tumor microvessels on the prognosis of patients with clear cell renal cell carcinoma (CCRCC). METHODS: Samples from 2 cohorts of patients with CCRCC (101 Asian patients and 524 US patients) were prepared using 2 different histologic approaches: routine sectioning versus tissue microarray. Then, the samples were immunohistochemically doubled-stained for a pericyte marker (alpha smooth muscle actin [α-SMA]) and a differentiated vessel marker (cluster of differentiation 34 [CD34]), followed by multispectral image capturing and computerized image analyses to quantify the microvessel density (MVD) and the PC of differentiated vessels. The correlations of PC and the MVD:PC ratio with clinicopathologic characteristics were analyzed. RESULTS: There was an inverse correlation between differentiated MVD and PC. Higher PC correlated with more aggressive clinicopathologic characteristics of CCRCC in both cohorts, including more advanced T-classification, higher pathologic grades, and the occurrence of tumor necrosis. The MVD:PC ratio was an independent favorable prognostic factor for overall and recurrence-free survival in the Asian cohort and for recurrence-free survival in the US cohort. PC also was an independent prognostic factor, with higher PC predicting a poorer outcome. The combination of PC and MVD was better at distinguishing the outcome of patients with CCRCC. PC combined with differentiated MVD or with the MVD:PC ratio provided additional, independent prognostic information to the Leibovich risk model, and that information was used to generate improved risk models. CONCLUSIONS: The authors consistently observed that higher PC was correlated with more aggressive clinicopathologic characteristics. PC was an independent unfavorable prognostic factor. The authors concluded that pericytes should be considered for therapeutic targeting.

Mycoplasmosis in ferrets.

We report an outbreak of severe respiratory disease associated with a novel Mycoplasma species in ferrets. During 2009-2012, a respiratory disease characterized by nonproductive coughing affected ≈8,000 ferrets, 6-8 weeks of age, which had been imported from a breeding facility in Canada. Almost 95% became ill, but almost none died. Treatments temporarily decreased all clinical signs except cough. Postmortem examinations of euthanized ferrets revealed bronchointerstitial pneumonia with prominent hyperplasia of bronchiole-associated lymphoid tissue. Immunohistochemical analysis with polyclonal antibody against Mycoplasma bovis demonstrated intense staining along the bronchiolar brush border. Bronchoalveolar lavage samples from 12 affected ferrets yielded fast-growing, glucose-fermenting mycoplasmas. Nucleic acid sequence analysis of PCR-derived amplicons from portions of the 16S rDNA and RNA polymerase B genes failed to identify the mycoplasmas but showed that they were most similar to M. molare and M. lagogenitalium. These findings indicate a causal association between the novel Mycoplasma species and the newly recognized pulmonary disease.

Evaluation of sex-specific gene expression in archived dried blood spots (DBS).

Screening newborns for treatable serious conditions is mandated in all US states and many other countries. After screening, Guthrie cards with residual blood (whole spots or portions of spots) are typically stored at ambient temperature in many facilities. The potential of archived dried blood spots (DBS) for at-birth molecular studies in epidemiological and clinical research is substantial. However, it is also challenging as analytes from DBS may be degraded due to preparation and storage conditions. We previously reported an improved assay for obtaining global RNA gene expression from blood spots. Here, we evaluated sex-specific gene expression and its preservation in DBS using oligonucleotide microarray technology. We found X inactivation-specific transcript (XIST), lysine-specific demethylase 5D (KDM5D) (also known as selected cDNA on Y, homolog of mouse (SMCY)), uncharacterized LOC729444 (LOC729444), and testis-specific transcript, Y-linked 21 (TTTY21) to be differentially-expressed by sex of the newborn. Our finding that trait-specific RNA gene expression is preserved in unfrozen DBS, demonstrates the technical feasibility of performing molecular genetic profiling using such samples. With millions of DBS potentially available for research, we see new opportunities in using newborn molecular gene expression to better understand molecular pathogenesis of perinatal diseases.

Genomic characterization of explant tumorgraft models derived from fresh patient tumor tissue.

BACKGROUND: There is resurgence within drug and biomarker development communities for the use of primary tumorgraft models as improved predictors of patient tumor response to novel therapeutic strategies. Despite perceived advantages over cell line derived xenograft models, there is limited data comparing the genotype and phenotype of tumorgrafts to the donor patient tumor, limiting the determination of molecular relevance of the tumorgraft model. This report directly compares the genomic characteristics of patient tumors and the derived tumorgraft models, including gene expression, and oncogenic mutation status. METHODS: Fresh tumor tissues from 182 cancer patients were implanted subcutaneously into immune-compromised mice for the development of primary patient tumorgraft models. Histological assessment was performed on both patient tumors and the resulting tumorgraft models. Somatic mutations in key oncogenes and gene expression levels of resulting tumorgrafts were compared to the matched patient tumors using the OncoCarta (Sequenom, San Diego, CA) and human gene microarray (Affymetrix, Santa Clara, CA) platforms respectively. The genomic stability of the established tumorgrafts was assessed across serial in vivo generations in a representative subset of models. The genomes of patient tumors that formed tumorgrafts were compared to those that did not to identify the possible molecular basis to successful engraftment or rejection. RESULTS: Fresh tumor tissues from 182 cancer patients were implanted into immune-compromised mice with forty-nine tumorgraft models that have been successfully established, exhibiting strong histological and genomic fidelity to the originating patient tumors. Comparison of the transcriptomes and oncogenic mutations between the tumorgrafts and the matched patient tumors were found to be stable across four tumorgraft generations. Not only did the various tumors retain the differentiation pattern, but supporting stromal elements were preserved. Those genes down-regulated specifically in tumorgrafts were enriched in biological pathways involved in host immune response, consistent with the immune deficiency status of the host. Patient tumors that successfully formed tumorgrafts were enriched for cell signaling, cell cycle, and cytoskeleton pathways and exhibited evidence of reduced immunogenicity. CONCLUSIONS: The preservation of the patient's tumor genomic profile and tumor microenvironment supports the view that primary patient tumorgrafts provide a relevant model to support the translation of new therapeutic strategies and personalized medicine approaches in oncology.

Predictive value of intratumoral microvascular density in patients with advanced non-small cell lung cancer receiving chemotherapy plus bevacizumab.

INTRODUCTION: The use of bevacizumab combined with chemotherapy represents a recent advance in clinical oncology for significantly improving the survival of patients who have non-small cell lung cancer (NSCLC). There is an unmet need for biomarkers that can predict response to such treatment and identify patients sensitive to it. Our study was designed to investigate the predictive value of intratumoral microvascular density (MVD) in patients with NSCLC treated with bevacizumab. METHODS: Sixteen patients with NSCLC who underwent chemotherapy combined with bevacizumab were included into this study. Paraffin-embedded tumor samples were sectioned and stained immunohistochemically for the blood vessel markers CD34 and CD31 to characterize the intratumoral vasculature. A computerized image analysis program was used to quantitatively calculate the intratumoral MVD. Treatment response was evaluated by computed tomography scanning. RESULTS: Two types of blood vessels, undifferentiated (CD31*/CD34*) and differentiated (CD34*), were identified. A positive correlation was found between the largest percentage of tumor shrinkage and the MVD of undifferentiated (CD31*/CD34*) vessels, with Spearman correlation coefficient being 0.576 (p = 0.019). No correlation between tumor shrinkage and differentiated vessel MVD (CD34*) was found. Moreover, seven of the eight patients with more undifferentiated vessels showed a partial response, versus only one of the seven patients with fewer undifferentiated vessels (p = 0.009). CONCLUSIONS: There are two major types of microvessel in lung cancer vasculature. The MVD of undifferentiated vessels is a favorable predictor for patients with NSCLC treated with a chemotherapy regimen plus bevacizumab, with a higher MVD value correlating with better treatment response. Further studies are needed to verify the predictive role of MVD in treatment of NSCLC with bevacizumab.

Identification and mechanism of 10-carbon fatty acid as modulating ligand of peroxisome proliferator-activated receptors.

Peroxisome proliferator-activated receptors (PPARα, -ß/δ, and -γ) are a subfamily of nuclear receptors that plays key roles in glucose and lipid metabolism. PPARγ is the molecular target of the thiazolidinedione class of antidiabetic drugs that has many side effects. PPARγ is also activated by long chain unsaturated or oxidized/nitrated fatty acids, but its relationship with the medium chain fatty acids remains unclear even though the medium chain triglyceride oils have been used to control weight gain and glycemic index. Here, we show that decanoic acid (DA), a 10-carbon fatty acid and a major component of medium chain triglyceride oils, is a direct ligand of PPARγ. DA binds and partially activates PPARγ without leading to adipogenesis. Crystal structure reveals that DA occupies a novel binding site and only partially stabilizes the AF-2 helix. DA also binds weakly to PPARα and PPARß/δ. Treatments with DA and its triglyceride form improve glucose sensitivity and lipid profiles without weight gain in diabetic mice. Together, these results suggest that DA is a modulating ligand for PPARs, and the structure can aid in designing better and safer PPARγ-based drugs.

Plasma-based circulating MicroRNA biomarkers for Parkinson's disease.

BACKGROUND: The current "gold-standard" for Parkinson's disease (PD) diagnosis is based primarily on subjective clinical rating scales related with motor features. Molecular biomarkers that are objective and quantifiable remain attractive as clinical tools to detect PD prior to its motor onsets. OBJECTIVE: Here, we aimed to identify, develop, and validate plasma-based circulating microRNA (miRNAs) as biomarkers for PD. METHODS: Global miRNA expressions were acquired from a discovery set of 32 PD/32 controls using microarrays. k-Top Scoring Pairs (k-TSP) algorithm and significance analysis of microarrays (SAM) were applied to obtain comprehensive panels of PD-predictive biomarkers. TaqMan miRNA-specific real-time PCR assays were performed to validate the microarray data and to evaluate the biomarker performance using a new replication set of 42 PD/30 controls. Data was analyzed in a paired PD-control fashion. The validation set was composed of 30 PD, 5 progressive supranuclear palsy, and 4 multiple system atrophy samples from a new clinical site. RESULTS: We identified 9 pairs of PD-predictive classifiers using k-TSP analysis and 13 most differentially-expressed miRNAs by SAM. A combination of both data sets produced a panel of PD-predictive biomarkers: k-TSP1 (miR-1826/miR-450b-3p), miR-626, and miR-505, and achieved the highest predictive power of 91% sensitivity, 100% specificity, 100% positive predicted value, and 88% negative predicted value in the replication set. However, low predictive values were shown in the validation set. CONCLUSIONS: This proof-of-concept study demonstrates the feasibility of using plasma-based circulating miRNAs as biomarkers for neurodegenerative disorders such as PD and shows the challenges of molecular biomarker research using samples from multiple clinical sites.

Serum adiponectin, resistin, and circulating soluble receptor for advanced glycation end products in colectomy patients.

AIM: Surgical trauma and associated complications are frequently related to physiological stress during colectomy. This study evaluated the response of adiponectin, resistin, and circulating soluble receptor for advanced glycation end products (sRAGE) in colectomy patients with or without an enhanced recovery protocol. METHOD: Serum samples were collected from 44 colectomy patients at 3 timframes. The surgical procedures were laparoscopic (LAP), hand-assisted laparoscopic (HALS), or open colectomy (OPEN). Adiponectin, resistin, and sRAGE levels were determined by ELISA. Repeated measures ANOVA was applied and P values < 0.05 were considered significant. RESULTS: A total of 132 (44 × 3) sera were used for analysis. Levels of adiponectin was significantly decreased between PREOP and POD3 (P < 0.001). Conversely, concentrations of resistin significantly increased from PREOP to POD1 and returned to baseline value by POD3 (P < 0.001). Serum sRAGE levels were significantly higher in LAP in comparison with HALS (P = 0.004) and OPEN (P < 0.001). sRAGE levels were significantly higher in sera of patients that underwent ERP (P < 0.001). CONCLUSIONS: Serum adiponectin, resistin, and sRAGE have the potential to develop into a panel of stress markers. Higher sRAGE levels in sera of LAP and ERP patients may be indicative of a protective and syngeristic role for colectomy recovery.

Host response transcriptional profiling reveals extracellular components and ABC (ATP-binding cassette) transporters gene enrichment in typhoid fever-infected Nigerian children.

BACKGROUND: Salmonella enterica serovar Typhi (S. Typhi) is a human-specific pathogen that causes typhoid fever, and remains a global health problem especially in developing countries. Its pathogenesis is complex and host response is poorly understood. In Africa, typhoid fever can be a major cause of morbidity in young infected children. The onset of the illness is insidious and clinical diagnosis is often unreliable. Gold standard blood culture diagnostic services are limited, thus rapid, sensitive, and affordable diagnostic test is essential in poor-resourced clinical settings. Routine typhoid fever vaccination is highly recommended but currently licensed vaccines provide only 55-75% protection. Recent epidemiological studies also show the rapid emergence of multi-drug resistant S. Typhi strains. High-throughput molecular technologies, such as microarrays, can dissect the molecular mechanisms of host responses which are S. Typhi-specific to provide a comprehensive genomic component of immunological responses and suggest new insights for diagnosis and treatment. METHODS: Global transcriptional profiles of S. Typhi-infected young Nigerian children were obtained from their peripheral blood and compared with that of other bacteremic infections using Agilent gene expression microarrays. The host-response profiles of the same patients in acute vs. convalescent phases were also determined. The top 96-100 differentially-expressed genes were identified and four genes were validated by quantitative real-time PCR. Gene clusters were obtained and functional pathways were predicted by DAVID (Database for Annotation, Visualization and Integrated Discovery). RESULTS: Transcriptional profiles from S. Typhi-infected children could be distinguished from those of other bacteremic infections. Enriched gene clusters included genes associated with extracellular peptides/components such as lipocalin (LCN2) and systemic immune response which is atypical in bacterial invasion. Distinct gene expression profiles can also be obtained from acute vs. convalescent phase during typhoid fever infection. We found novel down-regulation of ABC (ATP-binding cassette) transporters genes such as ABCA7, ABCC5, and ABCD4 and ATPase activity as the highest enriched pathway. CONCLUSIONS: We identified unique extracellular components and ABC transporters gene enrichments in typhoid fever-infected Nigerian children, which have never been reported. These enriched gene clusters may represent novel targeted pathways to improve diagnostic, prognostic, therapeutic and next-generation vaccine strategies for typhoid fever in Africa.

Identification of a lysosomal pathway that modulates glucocorticoid signaling and the inflammatory response.

The antimalaria drug chloroquine has been used as an anti-inflammatory agent for treating systemic lupus erythematosus and rheumatoid arthritis. We report that chloroquine promoted the transrepression of proinflammatory cytokines by the glucocorticoid receptor (GR). In a mouse collagen-induced arthritis model, chloroquine enhanced the therapeutic effects of glucocorticoid treatment. By inhibiting lysosome function, chloroquine synergistically activated glucocorticoid signaling. Lysosomal inhibition by either bafilomycin A1 (an inhibitor of the vacuolar adenosine triphosphatase) or knockdown of transcription factor EB (TFEB, a master activator of lysosomal biogenesis) mimicked the effects of chloroquine. The abundance of the GR, as well as that of the androgen receptor and estrogen receptor, correlated with changes in lysosomal biogenesis. Thus, we showed that glucocorticoid signaling is regulated by lysosomes, which provides a mechanistic basis for treating inflammation and autoimmune diseases with a combination of glucocorticoids and lysosomal inhibitors.

Reduction of protein adsorption and macrophage and astrocyte adhesion on ventricular catheters by polyethylene glycol and N-acetyl-L-cysteine.

Cellular obstruction of poly(dimethyl)siloxane (PDMS) catheters is one of the most prevalent causes of shunt failure in the treatment of hydrocephalus. By modifying PDMS using short- and long-chain mono-functional polyethylene glycol (PEG604 and PEG5K, respectively) and N-acetyl-L-cysteine via adsorption and covalent binding (NAC and NAC/EDC/NHS, respectively), we increased surface wettability. We hypothesized that these surface modifications would inhibit protein adsorption and decrease host macrophage and astrocyte adhesion. Tested in a bioreactor set to mimic physiological flow, all modified surfaces significantly decreased albumin adsorption compared with PDMS (p < 0.05) except for PEG604-modified PDMS (p = 0.14). All four modification strategies significantly reduced (p < 0.01) fibronectin adsorption. PEG604, PEG5K, NAC, and NAC/EDC/NHS reduced the average level of macrophage adhesion by 53%, 63%, 40%, and 58% (p <.0.05 except when comparing PDMS with NAC) and astrocyte adhesion by 47%, 83%, 91%, and 72% (p < 0.05 except when comparing PDMS with PEG604), respectively. Combined with saline soak results which suggest that the surface wettability is stable over 30 days for each modification, our results are consistent with the hypothesis that these modifications decrease cell adhesion on catheters in vitro for the treatment of hydrocephalus.

Effects of surface wettability, flow, and protein concentration on macrophage and astrocyte adhesion in an in vitro model of central nervous system catheter obstruction.

While silicone devices have vastly improved an array of medical treatments, reactions at the tissue-substrate interface often impede their functionality. Insertion of a poly(dimethyl)siloxane (PDMS) catheter into the cerebral ventricles to drain excess cerebrospinal fluid (CSF) is the most common treatment of hydrocephalus, but shunting often fails because inflammatory tissue, choroid plexus cells, and debris grow into these central nervous system catheters and obstruct flow. We hypothesized that plasma oxidation of PDMS would inhibit macrophage and astrocyte adhesion under flow (0 to 0.3 mL/min) and protein (20.8 to 240 mg/dL) conditions similar to those observed in the physiological state. Oxidation (to increase wettability) had an inhibitory effect on macrophage cell binding (yielding a significant 88% change) that was generally more pronounced than the effect of flow (22% change) or protein concentration (3% change). In contrast, greater flow increased binding of astrocytes in most cases (yielding a significant 97% change); plasma oxidation (19% change), and protein concentration (60% change) had less pronounced effects. This study is the initial indicator that plasma oxidation of PDMS catheters may inhibit macrophage adhesion during CSF outflow but may not be as effective at inhibiting astrocyte binding.

Chromosomal amplification of leucine-rich repeat kinase-2 (LRRK2) is required for oncogenic MET signaling in papillary renal and thyroid carcinomas.

The receptor tyrosine kinase MET is frequently amplified in human tumors, resulting in high cell surface densities and constitutive activation even in the absence of growth factor stimulation by its endogenous ligand, hepatocyte growth factor (HGF). We sought to identify mechanisms of signaling crosstalk that promote MET activation by searching for kinases that are coordinately dysregulated with wild-type MET in human tumors. Our bioinformatic analysis identified leucine-rich repeat kinase-2 (LRRK2), which is amplified and overexpressed in papillary renal and thyroid carcinomas. Down-regulation of LRRK2 in cultured tumor cells compromises MET activation and selectively reduces downstream MET signaling to mTOR and STAT3. Loss of these critical mitogenic pathways induces cell cycle arrest and cell death due to loss of ATP production, indicating that MET and LRRK2 cooperate to promote efficient tumor cell growth and survival in these cancers.

VX680/MK-0457, a potent and selective Aurora kinase inhibitor, targets both tumor and endothelial cells in clear cell renal cell carcinoma.

Aurora kinases are key regulators of cell mitosis and have been implicated in the process of tumorigenesis. In recent years, the Aurora kinases have attracted much interest as promising targets for cancer treatment. Here we report on the roles of Aurora A and Aurora B kinases in clear cell renal cell carcinoma (ccRCC). Using genomewide expression array analysis of 174 patient samples of ccRCC, we found that expression levels of Aurora A and B were significantly elevated in ccRCC compared to normal kidney samples. High expression levels of Aurora A and Aurora B were significantly associated with advanced tumor stage and poor patient survival. Inhibition of Aurora kinase activity with the drug VX680 (also referred to as MK-0457) inhibited ccRCC cell growth in vitro and led to ccRCC cell accumulation in the G2/M phase and apoptosis. Growth of ccRCC xenograft tumors was also inhibited by VX680 treatment, accompanied by a reduction of tumor microvessel density. Analysis of endothelial cell lines demonstrated that VX680 inhibits endothelial cell growth with effects similar to that seen in ccRCC cells. Our findings suggest that VX680 inhibits the growth of ccRCC tumors by targeting the proliferation of both ccRCC tumor cells and tumor-associated endothelial cells. Aurora kinases and their downstream cell cycle proteins have an important role in ccRCC and may be potent prognostic markers and therapy targets for this disease.

Mechanical contributions to astrocyte adhesion using a novel in vitro model of catheter obstruction.

Drainage and diversion of cerebrospinal fluid (CSF) through shunt systems is the most common treatment for hydrocephalus, but complications due to tissue obstruction of the catheter occur in up to 61% of patients. Although shunt systems have undergone limited technological advancements to resist mammalian cell adhesion, there is a need to further reduce adhesion that can exacerbate obstruction. The high intrinsic variability in clinical studies and an inability to predict chronic adhesion of host cells in vitro while maintaining the environmental conditions observed in hydrocephalus have impeded progress. We designed the hydrocephalus shunt catheter bioreactor (HSCB) to measure inflammatory cell adhesion under experimentally manipulated conditions of CSF pressure, pulsation rate, and flow rates. For a 20-h period, astrocytes were perfused through the pulsatile flow system, and adhesion on silicone catheters was recorded. These results were compared with those obtained under static cell culture conditions. Astrocyte adhesion was significantly increased under conditions of increased flow rate (0.25 and 0.30 mL/min), and a trend toward increased adhesion was observed under conditions of elevated pressure and pulsation rate. Because the HSCB represents physiologic conditions more accurately than static cell culture, our results suggest that standard static cell culturing techniques are insufficient to model inflammatory cell adhesion on catheters used in the treatment of hydrocephalus and that changes to the ventricular microenvironment can alter the mechanisms of cellular adhesion. The HSCB represents a relevant test system and is an effective model system for the analysis of cellular adhesion and occlusion of shunt catheters.

Activation of the PI3K/AKT pathway induces urothelial carcinoma of the renal pelvis: identification in human tumors and confirmation in animal models.

Urothelial carcinoma of the renal pelvis is a deadly disease with an unclear tumorigenic mechanism. We conducted gene expression profiling on a set of human tumors of this type and identified a phosphatidylinositol 3-kinase (PI3K)/AKT activation expression signature in 76.9% (n = 13) of our samples. Sequence analysis found both activating mutations of PIK3CA (13.6%, n = 22) and loss of heterozygosity at the PTEN locus (25%, n = 8). In contrast, none of the other subtypes of kidney neoplasms (e.g., clear-cell renal cell carcinoma) harbored PIK3CA mutations (n = 87; P < 0.001). Immunohistochemical analysis of urothelial carcinoma samples found loss of PTEN protein expression (36.4%, n = 11) and elevation of phosphorylated mammalian target of rapamycin (mTOR; 63.6%, n = 11). To confirm the role of the PI3K/AKT pathway in urothelial carcinoma, we generated mice containing biallelic inactivation of Pten in the urogenital epithelia. These mice developed typical renal pelvic urothelial carcinomas, with an incidence of 57.1% in mice older than 1 year. Laser capture microdissection followed by PCR confirmed the deletion of Pten exons 4 and 5 in the animal tumor cells. Immunohistochemical analyses showed increased phospho-mTOR and phospho-S6K levels in the animal tumors. Renal lymph node metastases were found in 15.8% of the animals with urothelial carcinoma. In conclusion, we identified and confirmed an important role for the PI3K/AKT pathway in the development of urothelial carcinoma and suggested that inhibitors of this pathway (e.g., mTOR inhibitor) may serve as effective therapeutic agents.

Archived unfrozen neonatal blood spots are amenable to quantitative gene expression analysis.

BACKGROUND: State laws in the USA mandate that blood be drawn from all newborn infants to screen for health-threatening conditions. These screening assays consume only a small portion of the blood samples, which are collected on filter paper ('Guthrie') cards. Many states archive unused blood spots, often in unrefrigerated storage. OBJECTIVES: While individual RNA transcripts have been identified from archived neonatal blood spots, no study to date has performed quantitative analysis of archived blood spot RNA. METHODS: We demonstrate that RNA can be isolated and amplified from newborn blood spots stored unfrozen for as long as 9 years, and can be analyzed by microarray and qPCR. RESULTS: Microarray assays of archived neonatal blood spots consistently detected 3,000-4,000 expressed genes with correlations of 0.90 between replicates. Blood spot mRNA is amenable to qPCR and we detected biologically relevant expression levels of housekeeping and immune-mediating genes. CONCLUSIONS: These experiments demonstrate the feasibility of using blood spots as a source of RNA which can be analyzed using quantitative microarray and qPCR assays. The application of these methods to the analysis of widely collected biological specimens may be a valuable resource for the study of perinatal determinants of disease development.

A special key for unlocking the door to targeted therapies of breast cancer.

Personalized medicine and targeted therapy is the best hope for patients with cancer. C-Met-HGF/SF inhibition may be an effective cancer treatment for ductal carcinoma of the breast as it is expected to be an important signalling target in a large number of malignancies.