An on-line SPE-LC-MS/MS method for quantification of nucleobases and nucleosides present in biological fluids.

Solid phase extraction (SPE) and liquid chromatographic (LC) separation of nucleobases and nucleosides are challenging due to the high hydrophilicity of these compounds. Herein we report a novel on-line SPE-LC-MS/MS method for their quantification after pre-column derivatization with chloroacetaldehyde (CAA). The method proposed is selective and sensitive with limits of detection at the nano-molar level. Analysis of urine and saliva samples by using this method is demonstrated. Adenine, guanine, cytosine, adenosine, guanosine, and cytidine were found in the range from 0.19 (guanosine) to 1.83 µM (cytidine) in urine and from 0.015 (guanosine) to 0.79 µM (adenine) in saliva. Interestingly, methylation of cytidine was found to be significantly different in urine from that in saliva. While 5-hydroxymethylcytidine was detected at a very low level (<0.05 µM) in saliva, it was found to be the most prominent methylated cytidine in urine at a high level of 3.33 µM. Since on-line SPE is deployed, the proposed LC-MS/MS quantitative assay is convenient to carry out and offers good assay accuracy and repeatability.

A novel one-pot fluorescence tagging and depyrimidination strategy for quantification of global DNA methylation.

DNA methylation is intensively studied in medical science. Current HPLC methods for quantification of global DNA methylation involve digestion of a DNA sample and HPLC determination of both cytosine (C) and 5-methylcytosine (5mC) so that percentage of 5mC in total cytosine can be calculated as DNA methylation level. Herein we report a novel HPLC method based on a one-pot fluorescence tagging and depyrimidination reaction between DNA and chloroacetaldehyde (CAA) for highly sensitive quantification of global DNA methylation. In the one-pot reaction, C and 5mC residues in a DNA sequence react with CAA, forming fluorescent etheno-adducts that are then released from the sequence through depyrimidination. Interestingly, etheno-5mC (ε-5mC) is ∼20 times more fluorescent than ε-C and other ε-nucleobases resulting from the reaction, which greatly facilitates the quantification. Further, due to the tagging-induced increase in structural aromaticity, ε-nucleobases are far more separable by HPLC than intact nucleobases. The proposed HPLC method with fluorescence detection (HPLC-FD) is quick (i.e., < 1h per assay) and highly sensitive with a detection limit of 0.80 nM (or 250 fg on column) for 5mC. Using the method, DNA samples isolated from yeast, HCT-116 cells, and tissues were analyzed. Global DNA methylation was measured to be in the range from 0.35% to 2.23% in the samples analyzed. This sensitive method allowed accurate analyses of minute DNA samples (∼100 ng) isolated from milligrams of tissues.

The association of prostatic lipids with progression, racial disparity and discovery of biomarkers in prostate cancer.

BACKGROUND: It remains under-investigated whether prostatic lipid profiles are associated with pathogenesis, progression, racial disparity, and discovery of biomarkers in prostate cancer (PCa). METHODS: The electrospray ionization-tandem mass spectrometry was applied to quantitate prostatic lipids in human and mouse PCa and non-cancer prostatic tissues. Biostatistics and bioinformatics were used to compare the concentrations of prostatic lipids at levels of total lipid, group, class and individual species between PCa and benign prostatic tissues, between races, and among pathological conditions of PCa. RESULTS: Prostatic concentrations of total lipids as well as neutral lipids were significantly higher in PCa than in benign prostatic tissues in all population and Caucasian American population, but not in African American population. The prostatic phospholipid were not statistically different between PCa and benign prostatic tissues in all study populations. Cholesteryl ester is the only lipid class significantly higher in PCa than in benign prostatic tissues in all study populations. A panel of prostatic lipid parameters in each study population was identified as diagnostic and prognostic biomarkers with >60% of sensitivity, specificity and accuracy simultaneously. Lipid profiling on mouse prostatic tissues further confirmed correlation of prostatic lipid profiles to the pathogenesis and progression of PCa. In addition, a few prostatic lipids in mouse can serve as prognostic biomarkers in differentiation of indolent from aggressive PCa. CONCLUSION: The prostatic lipids are widely associated with the pathogenesis, progression and racial disparity of PCa. A panel of prostatic lipids can serve as diagnostic, prognostic and race-specific biomarkers for PCa.

Distribution and clinical relevance of phospholipids in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common liver cancer and featured with prominent disparity in incidence and mortality rate between male and female. It remains unclear whether alterations of phospholipids (PL) in hepatic tissues contribute to the pathogenesis, progression, and disparity of HCC. METHODS: Using electrospray ionization mass spectrometry (ESI-MS), PL profiles including 320 individual phospholipid species in 13 PL classes were determined in paired samples from HCC and adjacent benign hepatic tissues (BHT). RESULTS: (1) Concentrations of PLs in most of individual species, in subgroups and in total were decreased in HCC than in BHT in all studied population; (2) the number of individual PL species significantly different between HCC and BHT, and the number of PLs in six subgroups and in total decreased in HCC were more in male population than in female population; (3) panels of PL parameters (more in male population than in female population) were identified as biomarkers in differentiation of HCC from BHT, and in the prediction of pathological grade and clinical stage of HCC with high sensitivity, specificity, and accuracy. CONCLUSION: It is concluded that alterations of PLs in hepatic tissues play important roles in pathogenesis, progression, and gender disparity of HCC.

Racial differences in distribution of fatty acids in prostate cancer and benign prostatic tissues.

BACKGROUND: It remains controversial whether and which fatty acids are different between prostate cancer (PCa) and benign prostatic tissues (BPT) in association with occurrence, progression and racial disparity between African American (AA) and Caucasian American (CA) populations. METHODS: Total fatty acids (TFA) and free fatty acid (FFA) were determined on fresh frozen prostatic tissues including 26 PCa and 21 BPT from AA and CA patients by Gas chromatography with flame ionization detection (GC-FID) and Electrospray Ionization Mass Spectrometry (ESI-MS), respectively. RESULTS: In all studied population, TFA in 8 out of 16 individual species, in total and in groups of saturated total fatty acid (STFA), mono-unsaturated total fatty acid (MUTFA), poly-unsaturated total fatty acid (PUTFA) and n-6 TFA were significantly higher in PCa than in BPT; FFA in 4 out of 10 individual species, in total and in groups of MUFFA, PUFFA, n-6 FFA and n-3 FFA were significantly higher in PCa than in BPT. The concentrations of most fatty acid parameters correlated with Gleason's grade and clinical stage of PCa. As compared with CA men, AA men had higher concentrations of TFA, especially TFA with chains of 14-18 carbons than in BPT, and lower concentrations of TFA in PCa. CONCLUSIONS: Increasing in prostatic fatty acids in the form of TFA and FFA correlated to occurrence, progression and racial disparity of PCa.

Multi-Institutional, Multidisciplinary Study of the Impact of Course-Based Research Experiences.

Numerous national reports have called for reforming laboratory courses so that all students experience the research process. In response, many course-based research experiences (CREs) have been developed and implemented. Research on the impact of these CREs suggests that student benefits can be similar to those of traditional apprentice-model research experiences. However, most assessments of CREs have been in individual courses at individual institutions or across institutions using the same CRE model. Furthermore, which structures and components of CREs result in the greatest student gains is unknown. We explored the impact of different CRE models in different contexts on student self-reported gains in understanding, skills, and professional development using the Classroom Undergraduate Research Experience (CURE) survey. Our analysis included 49 courses developed and taught at seven diverse institutions. Overall, students reported greater gains for all benefits when compared with the reported national means for the Survey of Undergraduate Research Experiences (SURE). Two aspects of these CREs were associated with greater student gains: 1) CREs that were the focus of the entire course or that more fully integrated modules within a traditional laboratory and 2) CREs that had a higher degree of student input and results that were unknown to both students and faculty.

Quantification of Small Extracellular Vesicles by Size Exclusion Chromatography with Fluorescence Detection.

Chemical analysis of small extracellular vesicles (sEVs) circulating in body fluids holds potentials in noninvasive diagnosis of diseases and evaluation of therapeutic treatments. However, quantification of sEVs remains a challenge due to lacking of cost-effective analytical protocols. Herein we report a facile method based on size exclusion chromatography with fluorescence detection (SEC-FD) for sEVs quantification. After removal of cells and cell debris, a 0.50 mL sample (e.g., cell culture medium) is incubated with CM-Dil dye to fluorescently label sEVs. The incubation solution is then separated on a SEC column packed with Sepharose CL-4B. The eluent is monitored fluorescently at Ex553 nm/Em570 nm by using a fluorometer equipped with a 50-µL flow through cuvette. Separation efficiency of the proposed SEC-FD method was evaluated by analyzing 100 nm liposomes and albumin-FITC conjugate. Liposomes were eluted out in less than 6 min, about 10 min before albumin-FITC. A separation repeatability (RSD in retention time) of 1.4% (n = 5) was obtained for liposomes. In analysis of cell culture media, linear calibration curves based on SEC-FD peak height versus sEVs concentration were obtained with r2 value of 0.996. Intraday quantification repeatability (RSD in peak height) was 3.2% (n = 5). The detection limit was estimated to be 2.9 × 107 exosome particles/mL. The proposed assay was applied to the first study of sEVs secretion from TK6 cells cultured in serum-free medium for a culturing period from 1 to 48 h.

Systemic distribution, subcellular localization and differential expression of sphingosine-1-phosphate receptors in benign and malignant human tissues.

AIMS: Five sphingosine-1-phosphate receptors (S1PR): S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5 (S1PR1-5) have been shown to be involved in the proliferation and progression of various cancers. However, none of the S1PRs have been systemically investigated. In this study, we performed immunohistochemistry (IHC) for S1PR1-S1PR5 on different tissues, in order to simultaneously determine the systemic distribution, subcellular localization and expression level of all five S1PRs. METHODS: We constructed tissue microarrays (TMAs) from 384 formalin-fixed paraffin-embedded (FFPE) blocks containing 183 benign and 201 malignant tissues from 34 human organs/systems. Then we performed IHC for all five S1PRs simultaneously on these TMA slides. The distribution, subcellular localization and expression of each S1PR were determined for each tissue. The data in benign and malignant tissues from the same organ/tissue were then compared using the Student's t-test. In order to reconfirm the subcellular localization of each S1PR as determined by IHC, immunocytochemistry (ICC) was performed on several malignant cell lines. RESULTS: We found that all five S1PRs are widely distributed in multiple human organs/systems. All S1PRs are expressed in both the cytoplasm and nucleus, except S1PR3, whose IHC signals are only seen in the nucleus. Interestingly, the S1PRs are rarely expressed on cellular membranes. Each S1PR is unique in its organ distribution, subcellular localization and expression level in benign and malignant tissues. Among the five S1PRs, S1PR5 has the highest expression level (in either the nucleus or cytoplasm), with S1PR1, 3, 2 and 4 following in descending order. Strong nuclear expression was seen for S1PR1, S1PR3 and S1PR5, whereas S1PR2 and S1PR4 show only weak staining. Four organs/tissues (adrenal gland, liver, brain and colon) show significant differences in IHC scores for the multiple S1PRs (nuclear and/or cytoplasmic), nine (stomach, lymphoid tissues, lung, ovary, cervix, pancreas, skin, soft tissues and uterus) show differences for only one S1PR (cytoplasmic or nuclear), and twenty three organs/tissues show no significant difference in IHC scores for any S1PR (cytoplasmic or nuclear) between benign and malignant changes. CONCLUSION: This is the first study to evaluate the expression level of all S1PRs in benign and malignant tissues from multiple human organs. This study provides data regarding the systemic distribution, subcellular localization and differences in expression of all five S1PRs in benign and malignant changes for each organ/tissue.

The C-terminal common to group 3 POTES (CtG3P): a newly discovered nucleolar marker associated with malignant progression and metastasis.

A gene family expressed in prostate, ovary, testis and placenta (POTEs) is newly defined and primate-specific. POTE genes have 13 paralogs, which are dispersed in 8 chromosomes and divided into three groups. The proteins encoded by these genes contain three domains: An N-terminal, ankyrin repeats and a C-terminus. Previous studies suggest that POTE proteins are localized in the inner aspect of cellular membrane and are considered as cancer-testis antigens, because they expressed widely in cancers, but in limited benign tissues. In this study, we will study the subcellular distribution of all POTE proteins and their associations with the progress and metastasis of malignancies. By performing Immunohistochemistry, Immunocytochemistry and immunofluorescence assay on tissue microarray slides containing tissues with different pathology and origins or on cell lines, we found that the epitopes of N- and C-terminals of all detected POTEs were widely expressed in benign and malignant tissues. Among these epitopes, C-terminal common to group 3 POTEs (CtG3P) was the only portion localized in nucleoli. The nucleolar IHC scores for CtG3P was lowest in benign tissues (4.47 ± 3.43), significantly higher in localized malignancies (5.32 ± 3.36, p = 3.63E-02), and highest in metastatic malignancies (7.90 ± 2.29, p = 8.13E-12). The CtG3P was better in differentiation of benign from malignant changes, and/or in differentiation of localized from metastatic cancers as compared with Ki-67 and AgNORs. In addition, transient transfection of siRNA against mRNA of group 3 POTEs influences the growth and survival of MCF-7 cells in vitro in a dose dependent manner.

Integrated lipidomics and transcriptomic analysis of peripheral blood reveals significantly enriched pathways in type 2 diabetes mellitus.

BACKGROUND: Insulin resistance is a key element in the pathogenesis of type 2 diabetes mellitus. Plasma free fatty acids were assumed to mediate the insulin resistance, while the relationship between lipid and glucose disposal remains to be demonstrated across liver, skeletal muscle and blood. METHODS: We profiled both lipidomics and gene expression of 144 total peripheral blood samples, 84 from patients with T2D and 60 from healthy controls. Then, factor and partial least squares models were used to perform a combined analysis of lipidomics and gene expression profiles to uncover the bioprocesses that are associated with lipidomic profiles in type 2 diabetes. RESULTS: According to factor analysis of the lipidomic profile, several species of lipids were found to be correlated with different phenotypes, including diabetes-related C23:2CE, C23:3CE, C23:4CE, ePE36:4, ePE36:5, ePE36:6; race-related (African-American) PI36:1; and sex-related PE34:1 and LPC18:2. The major variance of gene expression profile was not caused by known factors and no significant difference can be directly derived from differential gene expression profile. However, the combination of lipidomic and gene expression analyses allows us to reveal the correlation between the altered lipid profile with significantly enriched pathways, such as one carbon pool by folate, arachidonic acid metabolism, insulin signaling pathway, amino sugar and nucleotide sugar metabolism, propanoate metabolism, and starch and sucrose metabolism. The genes in these pathways showed a good capability to classify diabetes samples. CONCLUSION: Combined analysis of gene expression and lipidomic profiling reveals type 2 diabetes-associated lipid species and enriched biological pathways in peripheral blood, while gene expression profile does not show direct correlation. Our findings provide a new clue to better understand the mechanism of disordered lipid metabolism in association with type 2 diabetes.

Identification of plasma lipid biomarkers for prostate cancer by lipidomics and bioinformatics.

BACKGROUND: Lipids have critical functions in cellular energy storage, structure and signaling. Many individual lipid molecules have been associated with the evolution of prostate cancer; however, none of them has been approved to be used as a biomarker. The aim of this study is to identify lipid molecules from hundreds plasma apparent lipid species as biomarkers for diagnosis of prostate cancer. METHODOLOGY/PRINCIPAL FINDINGS: Using lipidomics, lipid profiling of 390 individual apparent lipid species was performed on 141 plasma samples from 105 patients with prostate cancer and 36 male controls. High throughput data generated from lipidomics were analyzed using bioinformatic and statistical methods. From 390 apparent lipid species, 35 species were demonstrated to have potential in differentiation of prostate cancer. Within the 35 species, 12 were identified as individual plasma lipid biomarkers for diagnosis of prostate cancer with a sensitivity above 80%, specificity above 50% and accuracy above 80%. Using top 15 of 35 potential biomarkers together increased predictive power dramatically in diagnosis of prostate cancer with a sensitivity of 93.6%, specificity of 90.1% and accuracy of 97.3%. Principal component analysis (PCA) and hierarchical clustering analysis (HCA) demonstrated that patient and control populations were visually separated by identified lipid biomarkers. RandomForest and 10-fold cross validation analyses demonstrated that the identified lipid biomarkers were able to predict unknown populations accurately, and this was not influenced by patient's age and race. Three out of 13 lipid classes, phosphatidylethanolamine (PE), ether-linked phosphatidylethanolamine (ePE) and ether-linked phosphatidylcholine (ePC) could be considered as biomarkers in diagnosis of prostate cancer. CONCLUSIONS/SIGNIFICANCE: Using lipidomics and bioinformatic and statistical methods, we have identified a few out of hundreds plasma apparent lipid molecular species as biomarkers for diagnosis of prostate cancer with a high sensitivity, specificity and accuracy.

The expression level of lysophosphatidylcholine acyltransferase 1 (LPCAT1) correlates to the progression of prostate cancer.

BACKGROUND: Lysophosphatidylcholine acyltransferase 1 (LPCAT1), the enzyme catalyzing the reaction in remodeling of phosphatidylcholine (PC) has been reported to express in prostate. However, its diagnostic and prognostic values remain unclear. METHODS: Immunohistochemistry (IHC) for LPCAT1 was performed on the tissue microarray (TMA) slides containing 251 samples from 148 patients with various prostatic disorders. The association of expression level of LPCAT1 with the progression of prostate cancer was analyzed. RESULTS: LPCAT1 IHC mean score was the highest in metastatic prostate cancer (8.00±1.28), which was significantly higher than that in primary prostate cancer (4.63±3.00, p=9.73E-07), in high grade prostatic intraepithelial neoplasia (HGPIN, 2.72±2.47, p=1.02E-12), and in benign prostate (2.68, p=6.17E-12). The mean score in primary prostate cancer was significantly higher than that in HGPIN (p=4.09E-04) and in benign prostate (p=2.74E-04). There was no significant difference in the mean score between HGPIN and benign prostate (p=0.951). LPCAT1 IHC score also correlated to the tumor grade and stage of prostate cancer. Patients who underwent prostatectomy for prostate cancer and developed biochemical recurrence or clinical metastasis had higher LPCAT1 IHC score than those who underwent prostatectomy for prostate cancer and did not develop biochemical recurrence and clinical metastasis. The association of LPCAT1 with the progression of prostate cancer was independent of patient race and age, PSA level and positivity of surgical resection margins. CONCLUSIONS: LPCAT1 correlates with the progression of prostate cancer and could be a new biomarker in diagnosis, prognosis and studying the pathogenesis of prostate cancer.

Transcriptomic profiles of peripheral white blood cells in type II diabetes and racial differences in expression profiles.

BACKGROUND: Along with obesity, physical inactivity, and family history of metabolic disorders, African American ethnicity is a risk factor for type 2 diabetes (T2D) in the United States. However, little is known about the differences in gene expression and transcriptomic profiles of blood in T2D between African Americans (AA) and Caucasians (CAU), and microarray analysis of peripheral white blood cells (WBCs) from these two ethnic groups will facilitate our understanding of the underlying molecular mechanism in T2D and identify genetic biomarkers responsible for the disparities. RESULTS: A whole human genome oligomicroarray of peripheral WBCs was performed on 144 samples obtained from 84 patients with T2D (44 AA and 40 CAU) and 60 healthy controls (28 AA and 32 CAU). The results showed that 30 genes had significant difference in expression between patients and controls (a fold change of <-1.4 or >1.4 with a P value <0.05). These known genes were mainly clustered in three functional categories: immune responses, lipid metabolism, and organismal injury/abnormaly. Transcriptomic analysis also showed that 574 genes were differentially expressed in AA diseased versus AA control, compared to 200 genes in CAU subjects. Pathway study revealed that "Communication between innate and adaptive immune cells"/"Primary immunodeficiency signaling" are significantly down-regulated in AA patients and "Interferon signaling"/"Complement System" are significantly down-regulated in CAU patients. CONCLUSIONS: These newly identified genetic markers in WBCs provide valuable information about the pathophysiology of T2D and can be used for diagnosis and pharmaceutical drug design. Our results also found that AA and CAU patients with T2D express genes and pathways differently.

High-throughput next-generation sequencing technologies foster new cutting-edge computing techniques in bioinformatics.

The advent of high-throughput next generation sequencing technologies have fostered enormous potential applications of supercomputing techniques in genome sequencing, epi-genetics, metagenomics, personalized medicine, discovery of non-coding RNAs and protein-binding sites. To this end, the 2008 International Conference on Bioinformatics and Computational Biology (Biocomp) - 2008 World Congress on Computer Science, Computer Engineering and Applied Computing (Worldcomp) was designed to promote synergistic inter/multidisciplinary research and education in response to the current research trends and advances. The conference attracted more than two thousand scientists, medical doctors, engineers, professors and students gathered at Las Vegas, Nevada, USA during July 14-17 and received great success. Supported by International Society of Intelligent Biological Medicine (ISIBM), International Journal of Computational Biology and Drug Design (IJCBDD), International Journal of Functional Informatics and Personalized Medicine (IJFIPM) and the leading research laboratories from Harvard, M.I.T., Purdue, UIUC, UCLA, Georgia Tech, UT Austin, U. of Minnesota, U. of Iowa etc, the conference received thousands of research papers. Each submitted paper was reviewed by at least three reviewers and accepted papers were required to satisfy reviewers' comments. Finally, the review board and the committee decided to select only 19 high-quality research papers for inclusion in this supplement to BMC Genomics based on the peer reviews only. The conference committee was very grateful for the Plenary Keynote Lectures given by: Dr. Brian D. Athey (University of Michigan Medical School), Dr. Vladimir N. Uversky (Indiana University School of Medicine), Dr. David A. Patterson (Member of United States National Academy of Sciences and National Academy of Engineering, University of California at Berkeley) and Anousheh Ansari (Prodea Systems, Space Ambassador). The theme of the conference to promote synergistic research and education has been achieved successfully.

Transcriptome profiling of Saccharomyces cerevisiae mutants lacking C2H2 zinc finger proteins.

BACKGROUND: The budding yeast Saccharomyces cerevisiae is a eukaryotic organism with extensive genetic redundancy. Large-scale gene deletion analysis has shown that over 80% of the ~6200 predicted genes are nonessential and that the functions of 30% of all ORFs remain unclassified, implying that yeast cells can tolerate deletion of a substantial number of individual genes. For example, a class of zinc finger proteins containing C2H2 zinc fingers in tandem arrays of two or three is predicted to be transcription factors; however, seven of the thirty-one predicted genes of this class are nonessential, and their functions are poorly understood. In this study we completed a transcriptomic profiling of three mutants lacking C2H2 zinc finger proteins, ypr013cDelta,ypr015cDelta and ypr013cDeltaypr015cDelta. RESULTS: Gene expression patterns were remarkably different between wild type and the mutants. The results indicate altered expression of 79 genes in ypr013cDelta, 185 genes in ypr015cDelta and 426 genes in the double mutant when compared with that of the wild type strain. More than 80% of the alterations in the double mutants were not observed in either one of the single deletion mutants. Functional categorization based on Munich Information Center for Protein Sequences (MIPS) revealed up-regulation of genes related to transcription and down-regulation of genes involving cell rescue and defense, suggesting a decreased response to stress conditions. Genes related to cell cycle and DNA processing whose expression was affected by single or double deletions were also identified. CONCLUSION: Our results suggest that microarray analysis can define the biological roles of zinc finger proteins with unknown functions and identify target genes that are regulated by these putative transcriptional factors. These findings also suggest that both YPR013C and YPR015C have biological processes in common, in addition to their own regulatory pathways.

Expression of fibronectin and HIF-1alpha in renal cell carcinomas: relationship to von Hippel-Lindau gene inactivation.

The von Hippel-Lindau (VHL) tumor suppressor gene (TSG) at 3p25 is mutated in approximately 50% of conventional (clear cell) renal cell carcinomas (cRCC). VHL normally regulates the ubiquitin-mediated proteolysis of hypoxia-inducible factor 1alpha (HIF-1alpha), and VHL inactivation results in increased cellular HIF-1alpha expression. VHL protein (pVHL) also interacts with fibronectin (Fn) and VHL inactivation results in defective Fn extracellular matrix assembly. The present study investigated the immunohistochemical (IHC) staining for Fn and HIF-1alpha in 11 cRCC and the relationship of the staining to VHL inactivation by gene deletion, mutation, or hypermethylation. Evidence for VHL inactivation by 3p deletions and VHL mutations were found in six tumors. Fn-positive IHC staining of tumor cells and negative to weak staining of extracellular stroma was found in five cases having exon 1 or exon 2 mutations. In contrast, Fn staining was absent in tumor cells and positive in the stroma of five tumors without VHL inactivation and in one tumor with a C-terminal exon 3 mutation. HIF-1alpha tumor cell staining was present in the cRCC with VHL inactivation but was also present in two tumors having 3p deletions but neither mutation nor hypermethylation of VHL. These two cRCC showed a tumor cell-negative and stroma-positive pattern of Fn staining. The findings indicate that VHL inactivation plays a role in the development of some cRCC by altering Fn cell--stroma relationships. They also suggest that some C-terminal mutations may not interfere with Fn assembly and that a 3p TSG in addition to VHL influences HIF-1alpha degradation.

Genomic sequence of a ranavirus (family Iridoviridae) associated with salamander mortalities in North America.

Disease is among the suspected causes of amphibian population declines, and an iridovirus and a chytrid fungus are the primary pathogens associated with amphibian mortalities. Ambystoma tigrinum virus (ATV) and a closely related strain, Regina ranavirus (RRV), are implicated in salamander die-offs in Arizona and Canada, respectively. We report the complete sequence of the ATV genome and partial sequence of the RRV genome. Sequence analysis of the ATV/RRV genomes showed marked similarity to other ranaviruses, including tiger frog virus (TFV) and frog virus 3 (FV3), the type virus of the genus Ranavirus (family Iridoviridae), as well as more distant relationships to lymphocystis disease virus, Chilo iridescent virus, and infectious spleen and kidney necrosis virus. Putative open reading frames (ORFs) in the ATV sequence identified 24 genes that appear to control virus replication and block antiviral responses. In addition, >50 other putative genes, homologous to ORFs in other iridoviral genomes but of unknown function, were also identified. Sequence comparison performed by dot plot analysis between ATV and itself revealed a conserved 14-bp palindromic repeat within most intragenic regions. Dot plot analysis of ATV vs RRV sequences identified several polymorphisms between the two isolates. Finally, a comparison of ATV and TFV genomic sequences identified genomic rearrangements consistent with the high recombination frequency of iridoviruses. Given the adverse effects that ranavirus infections have on amphibian and fish populations, ATV/RRV sequence information will allow the design of better diagnostic probes for identifying ranavirus infections and extend our understanding of molecular events in ranavirus-infected cells.

Diagnostic and molecular evaluation of three iridovirus-associated salamander mortality events.

In 1998 viruses were isolated from tiger salamander larvae (Ambystoma tigrinum diaboli and A. tigrinum melanostictum) involved in North Dakota and Utah (USA) mortality events and spotted salamander (A. maculatum) larvae in a third event in Maine (USA). Although sympatric caudates and anurans were present at all three sites only ambystomid larvae appeared to be affected. Mortality at the North Dakota site was in the thousands while at the Utah and Maine sites mortality was in the hundreds. Sick larvae were lethargic and slow moving. They swam in circles with obvious buoyancy problems and were unable to remain upright. On the ventral surface, near the gills and hind limbs, red spots or swollen areas were noted. Necropsy findings included: hemorrhages and ulceration of the skin, subcutaneous and intramuscular edema, swollen and pale livers with multifocal hemorrhage, and distended fluid-filled intestines with areas of hemorrhage. Light microscopy revealed intracytoplasmic inclusions, suggestive of a viral infection, in a variety of organs. Electron microscopy of ultra thin sections of the same tissues revealed iridovirus-like particles within the inclusions. These viruses were isolated from a variety of organs, indicating a systemic infection. Representative viral isolates from the three mortality events were characterized using molecular assays. Characterization confirmed that the viral isolates were iridoviruses and that the two tiger salamander isolates were similar and could be distinguished from the spotted salamander isolate. The spotted salamander isolate was similar to frog virus 3, the type species of the genus Ranavirus, while the tiger salamander isolates were not. These data indicate that different species of salamanders can become infected and die in association with different iridoviruses. Challenge assays are required to determine the fish and amphibian host range of these isolates and to assess the susceptibility of tiger and spotted salamanders to heterologous virus isolates.