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For decades, cell biologists and cancer researchers have taken advantage of non-murine species to increase our understanding of the molecular processes that drive normal cell and tissue development, and when perturbed, cause cancer. The advent of whole-genome sequencing has revealed the high genetic homology of these organisms to humans. Seminal studies in non-murine organisms such as Drosophila melanogaster, Caenorhabditis elegans, and Danio rerio identified many of the signaling pathways involved in cancer. Studies in these organisms offer distinct advantages over mammalian cell or murine systems. Compared to murine models, these three species have shorter lifespans, are less resource intense, and are amenable to high-throughput drug and RNA interference screening to test a myriad of promising drugs against novel targets. In this review, we introduce species-specific breeding strategies, highlight the advantages of modeling brain tumors in each non-mammalian species, and underscore the successes attributed to scientific investigation using these models. We conclude with an optimistic proposal that discoveries in the fields of cancer research, and in particular neuro-oncology, may be expedited using these powerful screening tools and strategies.

The five most common species of Pacific salmon, Rainbow Trout (steelhead) Oncorhynchus spp., and Atlantic Salmon Salmo salar intermingle in the North Pacific Ocean and its freshwater tributaries. Efficient morphological methods for distinguishing among these species are sometimes limited by condition of the specimen (degraded or missing morphology), life history stage, or training of the observer. Researchers have successfully applied various genetic methods to distinguish among these species when morphological analyses are not possible, but they cannot easily incorporate these methods into standard fish and wildlife population monitoring analysis workflows. Here we test five 5′–3′ exonuclease (TaqMan) assays developed from mitochondrial genes and provide novel methods that take advantage of TaqMan output to distinguish among these species. We found that combinations of as few as two of the five assays were adequate to distinguish all species. TaqMan chemistry is designed to interrogate a single nucleotide locus. We also explore the basis for the variation in the observed scatter plot distributions (variation in florescent signals) and show that this variation is due to nucleotide diversity in and near the probe site. Because the SNPs underlying the assays developed here are all physically close to one another along the mitochondrial genome, the potential exists to develop a single DNA sequence-based assay to discriminate among salmon species. This single assay can be added to a genotyping-by-sequencing panel to identify and exclude nontarget species from analyses.

Knowledge of the distribution of genetic resources within and among taxa is prerequisite for development of management strategies which facilitate conservation of those resources. We used restriction-site associated DNA (RAD) sequencing to survey genetic variation in Alvord Chub and Borax Lake Chub from the Alvord Basin in southeastern Oregon and northern Nevada, USA. Our specific goals were to gain an understanding of the population genetic structure of Alvord Chub and the relationship between this species and Borax Lake Chub. Despite the fact that our collections were taken from ponds and streams, isolated from one another by kilometers of desert, our results revealed that diversity in Alvord Chub was primarily distributed among two regional groupings, which we hypothesize to be related to habitat changes following the end of the most recent glaciation event, approximately 10 thousand years ago. Our results further revealed that Alvord Chub and Borax Lake Chub shared common ancestors during that glaciation event, and that divergence between the two may have been sympatric. Finally, we observed evidence that the clade containing Alvord Chub and Borax Lake Chub was isolated from its closest extant relative, the Tui chub, during the Miocene Epoch, when volcanic flows were forming the mountains surrounding what became the Alvord Basin. These results will be useful for informing conservation strategies for Alvord Chub, and provide new insights regarding the role of these species in the evolutionary legacy of the region.

Patient-derived xenografts retain the genotype of the parent tumors more readily than tumor cells maintained in culture. The two previously reported clival chordoma xenografts were derived from recurrent tumors after radiation. To study the genetics of clival chordoma in the absence of prior radiation exposure we established a patient-derived xenograft at primary resection of a clival chordoma. Epicranial grafting of clival chordoma collected during surgery was performed. Tumor growth was established in a nonobese diabetic/severe combined immunodeficiency mouse and tumors have been passaged serially for seven generations. Physaliferous cell architecture was shown in the regenerated tumors, which stained positive for Brachyury, cytokeratin, and S100 protein. The tumors showed bone invasion. Single-nucleotide polymorphism analysis of the tumor xenograft was compared with the parental tumor. Copy number gain of the T gene (brachyury) and heterozygous loss of cyclin dependent kinase inhibitor 2A (CDKN2A) was observed. Heterozygous loss of the tumor-suppressor fragile histidine triad (FHIT) gene also was observed, although protein expression was preserved. Accumulation of copy number losses and gains as well as increased growth rate was observed over three generations. The patient-derived xenograft reproduces the phenotype of clival chordoma. This model can be used in the future to study chordoma biology and to assess novel treatments.

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults, with a median survival of just over one year. The failure of available treatments to achieve remission in patients with GBM has been attributed to the presence of cancer stem cells (CSCs), which are thought to play a central role in tumor development and progression and serve as a treatment-resistant cell repository capable of driving tumor recurrence; in fact, the property of “stemness” itself may be responsible for treatment resistance. In this study, we identify a novel lncRNA, Cancer stem cell associated distal enhancer of SOX2 (CASCADES) that functions as an epigenetic regulator in glioma CSCs (GSCs). CASCADES is expressed in IDH-wild type GBM and significantly enriched in GSCs. Knockdown of CASCADES in GSCs results in differentiation towards a neuronal lineage in a cell- and cancer-specific manner. Bioinformatics analysis reveals that CASCADES functions as a super-enhancer associated lncRNA epigenetic regulator of SOX2. Our findings identify CASCADES as a critical regulator of stemness in GSCs and represent a novel epigenetic and therapeutic target for disrupting the cancer stem cell compartment in GBM.

Atypical Teratoid/Rhabdoid Tumours (ATRT) are highly malignant neoplasms arising primarily in the CNS of children. They are defined by loss of function mutations in smarcb1, a gene serving a vital role in neurogenesis and differentiation. In order to recapitulate ATRT in the mouse, we used a Cre-Lox recombination system to conditionally knockout smarcb1 in specific cell compartments. Loss of smarcb1 in BLBP-expressing cells of the developing brain led to severe neurologic defects. Mice exhibited seizures, ataxia, and median 12-day survival. Histological analysis revealed severe thinning of the cerebral cortex and cerebellum. Temporally-targeted smarcb1 loss in BLBP/Nestin-expressing embryonic compartments did not result in tumour formation. Similarly, BLBP-expressing, smarcb1-deficient neural stem/progenitor cells (NSC/NPCs) were isolated and allografted but did not form tumours. These cells demonstrated decreased proliferation, higher apoptosis, and upregulation of p53, CDKN1A, and CDKN2A. In contrast, ubiquitous smarcb1 loss at the earlier embryonic day 6.5 produced widespread tumorigenicity in the forebrain, hindbrain, skullbase, and spine; each with unique phenotypes, survival, and morphology. We employed a clinically-relevant Nanostring gene-panel screen to stratify tumours into genetically distinct subgroups. Our findings indicate that smarcb1 plays an important role in CNS development. Loss of smarcb1 in NSC/NPCs is lethal, and its developmental context influences cell fate. Targeted smarcb1 loss likely plays a tumorigenic role at an earlier developmental stage than previously determined, in a diverse array of primitive stem cells. These data support the generation of a murine ATRT model capable of producing distinct tumour entities that recapitulate the human disease.

Proliferating cancer cells are characterized by high rates of glycolysis, lactate production, and altered mitochondrial metabolism. This metabolic reprogramming provides important metabolites for proliferation of tumor cells, including glioblastoma. These biological processes, however, generate oxidative stress that must be balanced through detoxification of reactive oxygen species (ROS). Using an unbiased retroviral loss-of-function screen in nontransformed human astrocytes, we demonstrate that mitochondrial PTEN-induced kinase 1 (PINK1) is a regulator of the Warburg effect and negative regulator of glioblastoma growth. We report that loss of PINK1 contributes to the Warburg effect through ROS-dependent stabilization of hypoxia-inducible factor-1A and reduced pyruvate kinase muscle isozyme 2 activity, both key regulators of aerobic glycolysis. Mechanistically, PINK1 suppresses ROS and tumor growth through FOXO3a, a master regulator of oxidative stress and superoxide dismutase 2. These findings highlight the importance of PINK1 and ROS balance in normal and tumor cells. PINK1 loss was observed in a significant number of human brain tumors including glioblastoma (n > 900) and correlated with poor patient survival. PINK1 overexpression attenuates in vivo glioblastoma growth in orthotopic mouse xenograft models and a transgenic glioblastoma model in Drosophila. Cancer Res; 76(16); 4708–19. ©2016 AACR.

Protecting genetic diversity throughout the range of a species is important for conservation, as doing so provides for long-term evolutionary potential and persistence under a changing environment. Conservation of diversity at the intraspecific level requires identification of all genetically distinct population segments within species; i.e., conservation units (CUs). Silene spaldingii occurs in grasslands of the Columbia Plateau region of western North America and is listed as threatened under the Federal Endangered Species Act. The recovery plan identified five physiographic regions across the range of the species to use as surrogates for genetic CUs. We collected leaf samples from an average of 26 plants from each of 19 of the largest populations across all five physiographic regions and used variable microsatellite and chloroplast DNA markers to determine how genetic variation is distributed across the range of the species and how well physiographic regions reflect population structure within this species. Results of several multivariate analyses clustered our samples into four genetic groups which did not correspond well with the physiographic regions. We observed little genetic differentiation among populations in the main range of the species which encompasses nearly all of four contiguous physiographic regions. However, three other distinct genetic groups were identified: two in the disjunct northeast corner and one at the southeast edge of the main range. Modification of the CUs to reflect the genetic groups rather than the physiographic regions would result in CUs which better reflect historical patterns of population structure. Moreover, use of the genetic units to inform translocation and genetic rescue efforts could improve our ability to mimic natural patterns of gene flow. Our results suggest that physiographic regions may not always be an accurate reflection of population structure for threatened or endangered species.

Genotypic sex identification assays can provide valuable information about fish populations when phenotypic sex determination is difficult. Here we describe the development of a TaqMan® assay (Ots_SexID) designed to identify the genotypic sex by targeting a region previously examined in the growth hormone pseudogene for winter-run Chinook salmon (Oncorhynchus tshawytscha) collected from the Sacramento River and spawned at the Livingston Stone National Fish Hatchery. Accuracy of the marker was assessed by comparing genotypic sex assignments for Chinook salmon spawned at Livingston Stone National Fish hatchery in 2012 (n = 84) with phenotypic sex recorded during spawning. Genotypic sex was observed to be concordant with phenotypic sex identified using Ots_SexID in 83/84 individuals, suggesting that the assay could be used to predict phenotypic sex with ∼︀99% accuracy. To evaluate the utility of the TaqMan assay in other parts of the species’ range, we examined collections from 29 other populations ranging from Alaska to California. Genotypic sex assignments based on the assay were generally concordant with observed phenotypes, but there were some strong exceptions. These results suggest that the new assay will be very useful for Sacramento River winter-run Chinook salmon, but also highlight the importance of thoroughly testing any genotypic sex identification assay before application in a population of interest.

Patient-derived xenografts (PDXs) retain the genotype of the parental tumors more readily than tumor cells maintained in culture. In order to study the genetics of clival chordoma in the absence of prior radiation we sought to establish a PDX following the primary resection of a clival chordoma. Epicranial grafting of a primary clival chordoma was performed in NOD/SCID mice. Tumors have been passaged serially in mice for 7 generations. Physaliferous cell architecture was demonstrated in the regenerated tumors, which stained positive for Brachyury, Cytokeratin, and S100. The tumors demonstrated bone invasion. Single-nucleotide polymorphism analysis of the tumor xenograft was compared to the parental tumor. We identified homozygous amplification of the T-gene (brachury) and heterozygous loss of CDKN2A. We also identified heterozygous loss of the tumor suppressor FHIT gene, although protein expression was preserved. Accumulation of copy number losses and gains over three generations showed for the first time that there is inherent genomic instability in chordoma. In conclusion, this PDX reproduces the phenotype of clival chordoma. This study is the first to show chromosomal genomic instability in a chordoma that is serially propagated through multiple generations. The genomic instability observed in chordoma may serve as the biological basis for resistance to chemotherapy as is observed for other forms of cancer.

Urbanization presents numerous challenges to aquatic species. For fish, typical threats include altered flow dynamics, poor water quality, and degraded food webs. These threats can be detrimental to fish and whether urban environments can support healthy populations is unclear. Tryon Creek is one of the largest urban watersheds in Portland, Oregon where developed land accounts for 55.6% of total land use. We used demographic, life history, disease, and genetic indices to assess the overall health of the Coastal Cutthroat Trout (Oncorhynchus clarkii) population in Tryon Creek. Results of these population indicators were within ranges for streams with Coastal Cutthroat Trout populations not influenced by urbanization and considered healthy. Coastal Cutthroat Trout in Tryon Creek exhibited a mean condition factor of 1.07 (95% CI 0.95–1.19, range 0.94–1.24), four size-classes, an estimated density of 0.01–0.03 individuals/m2, and migratory behavior. The population did not exhibit excessive deviation from Hardy–Weinberg equilibrium. Genetic diversity in Tryon Creek (He = 0.76; allelic richness = 6.6) was comparable to that observed in non-urbanized streams in the Mt. Hood National Forest (He = 0.72; allelic richness = 5.6). Moreover, allele frequencies appeared stable, and no genetic divergence was detected among generations (F ST ~0.00; non-significant allele frequency heterogeneity tests). The population tested negative for 12 different pathogens and results for Renibacterium salmoninarum were inconclusive. The characteristics of Coastal Cutthroat Trout in Tryon Creek were similar to populations not influenced by urbanization which suggests an urban stream can support a healthy fish population.

Most coho salmon Oncorhynchus kisutch in Washington state spawn at 3 y of age, creating the potential for three temporal populations or “broodlines” at each spawning site. This is generally prevented by a portion of males in each site that mature and reproduce at 2 y of age, resulting in population structure in which the geographic component is stronger than the temporal component. The Quilcene National Fish Hatchery, located on Big Quilcene River in the Hood Canal region of Washington state, selected against late returning coho salmon by excluding all but the earliest returning fish from its broodstock for an unknown number of generations, and restricted gene flow among broodlines by excluding 2-y-old males for 27 generations. The resulting hatchery population exhibited three distinct broodlines that returned in alternating years: an “early” broodline that arrived 1 mo before the wild fish, a “late” broodline that arrived at the same time as the wild fish, and a “middle” broodline that arrived in between these two broodlines. We evaluated temporal and geographic components of population genetic structure in coho salmon from the Quilcene National Fish Hatchery and nine other sites from Puget Sound and the Strait of Juan de Fuca using 10 microsatellite loci. Genetic diversity at the Quilcene National Fish Hatchery was lowest in the early broodline and highest in the late broodline. Divergence among broodlines at the Quilcene National Fish Hatchery was greater than that observed at any other site, and was also greater than that observed between any of the sites. This apparent reversal of the relative magnitudes of temporal and geographic components for this species emphasizes the importance of variable age-at-maturity in shaping population genetic structure.

Balancing the disparate objectives of fishery augmentation and conservation of an endemic fish population presents a substantial challenge. In the case of Warm Springs National Fish Hatchery (Warm Springs Hatchery), strategies for achieving both objectives included incorporation of natural fish into the hatchery broodstock and restricting proportions of hatchery fish on the spawning grounds. The hatchery has been more successful in implementing the latter than the former. We analyzed 76 single nucleotide polymorphism markers in Spring Chinook Salmon Oncorhynchus tshawytscha collected from the Warm Springs River in 1976–1977 (prior to hatchery production) and 2001–2011 (posthatchery) to examine whether the genetic characteristics of the endemic population had changed during that time. Pre- and posthatchery collections clustered together when compared with those from Round Butte Hatchery, which has a nearby segregated program, and other Columbia River populations. The difference between pre- and pos...

We examined whether a supplementation program for steelhead Oncorhynchus mykiss in southwestern Washington could produce hatchery fish that contained genetic characteristics of the endemic population from which it was derived and simultaneously meet a production goal. Hatchery fish were produced for three consecutive years by using broodstock comprised of endemic juveniles that were caught in the wild and raised to maturity, and then the program transitioned to an integrated broodstock comprised of wild and hatchery adults that returned to spawn. Importantly, some auxiliary conservation-based husbandry protocols were attempted (i.e., pairwise mating between males and females) but not always completed due to insufficient broodstock and conflict between production and conservation goals. The hatchery met production goals in 6 of 9 years, but wild-type genetic integrity of hatchery fish was degraded every year. Specifically, we analyzed 10 microsatellites and observed a 60% reduction in the effective...

Summary The application of DNA-based markers toward the task of discriminating among alternate salmon runs has evolved in accordance with ongoing genomic developments and increasingly has enabled resolution of which genetic markers associate with important life-history differences. Accurate and efficient identification of the most likely origin for salmon encountered during ocean fisheries, or at salvage from fresh water diversion and monitoring facilities, has far-reaching consequences for improving measures for management, restoration and conservation. Near-real-time provision of high-resolution identity information enables prompt response to changes in encounter rates. We thus continue to develop new tools to provide the greatest statistical power for run identification. As a proof of concept for genetic identification improvements, we conducted simulation and blind tests for 623 known-origin Chinook salmon (Oncorhynchus tshawytscha) to compare and contrast the accuracy of different population sampling baselines and microsatellite loci panels. This test included 35 microsatellite loci (1266 alleles), some known to be associated with specific coding regions of functional significance, such as the circadian rhythm cryptochrome genes, and others not known to be associated with any functional importance. The identification of fall run with unprecedented accuracy was demonstrated. Overall, the top performing panel and baseline (HMSC21) were predicted to have a success rate of 98%, but the blind-test success rate was 84%. Findings for bias or non-bias are discussed to target primary areas for further research and resolution.

Many working on large carnivore conservation over the last four decades have seen how frequently the controversies associated with wolves, grizzly bears and mountain lions appear more like trench w...

The dynamic Quaternary geology of the Pacific Ring of Fire created substantial challenges for biogeography. Fish life history and population genetic variation were shaped by climate change, repeated formation and subsidence of ice sheets, sea-level change, volcanism and tectonics, isostatic rebound, and now human activities. It is widely recognized in Chinook salmon (Oncorhynchus tshawytscha) that parallel evolution and phenotypic plasticity have obscured range-wide patterns of life-history segregation with evolutionary lineage, yet the idea of the lineages themselves persists. We employed a large, internationally standardized, microsatellite data set to explore population structure at coast-wide scale and test for two divergent lineages, whether or not related to life history. We found at least 27 distinct lineages. However, relationships among groups were poorly resolved — essentially a star phylogeny. We found pervasive isolation by distance among groups, complicating cluster analysis. Only in the interior Columbia River (east of the Cascade Mountains) is there a deep genetic bifurcation that supports both the two-lineage hypothesis and the life-history segregation hypothesis. This broad-scale perspective helps reconcile different views of Chinook salmon phylogeography and life-history distribution.

Glioblastoma (GBM) is the most common primary brain tumor in adults. Despite current advances in therapy consisting of surgery followed by chemotherapy and radiation, the overall survival rate still remains poor. Therapeutic failures are partly attributable to the highly infiltrative nature of tumor adjacent to normal brain parenchyma. Recently, evidence is mounting to suggest that actin cytoskeleton dynamics are critical components of the cell invasion process. Drebrin is an actin-binding protein involved in the regulation of actin filament organization, and plays a significant role in cell motility; however, the role of drebrin in glioma cell invasiveness has not yet been fully elucidated. Therefore, this study was aimed to clarify the role of drebrin in glioma cell morphology and cell motility. Here we show that drebrin is expressed in glioma cell lines and in operative specimens of GBM. We demonstrate that stable overexpression of drebrin in U87 cells leads to alterations in cell morphology, and induces increased invasiveness in vitro while knockdown of drebrin in U87 cells by small interfering RNA (siRNA) decreases invasion and migration. In addition, we show that depletion of drebrin by siRNA alters glioma cell morphology in A172 GBM cell line. Our results suggest that drebrin contributes to the maintenance of cell shape, and may play an important role in glioma cell motility.

Medulloblastoma is a malignant embryonic brain tumor arising in the posterior fossa and typically occurring in pediatric patients. Current multimodal treatment regimes have significantly improved the survival rates; however, a marked heterogeneity in therapy response is observed, and one third of all patients die within 5 years after diagnosis. Large-scale genetic and transcriptome analysis revealed four medulloblastoma subgroups (WNT, SHH, Group 3, and Group 4) associated with different demographic parameters, tumor manifestation, and clinical behavior. Future treatment protocols will integrate molecular classification schemes to evaluate subgroup-specific intensification or de-escalation of adjuvant therapies aimed to increase tumor control and reduce iatrogenic induced morbidity. Furthermore, the identification of genetic drivers allows assessing target therapies in order to increase the chemotherapeutic armamentarium. This review highlights the biology behind the current classification system and elucidates relevant aspects of the disease influencing forthcoming clinical trials.

The malignant glioma is the most common primary human brain tumor, and its migration and invasiveness away from the primary tumor mass are considered a leading cause of tumor recurrence and treatment failure. Recently, gene expression profiling revealed that the transmembrane glycoprotein CD99 is more highly expressed in malignant glioma than in normal brain. Although its function is not completely understood, CD99 is implicated in cell adhesion and migration in a variety of different cell types. CD99 has wild-type and splice variant isoforms. Previous studies have shown that wild-type CD99 may be an oncosuppressor in some tumors, distinct from the role of the splice variant isoform. In this study, our data reveal that only wild-type CD99 is expressed in human glioma cells and tissues. Using a tissue microarray, we validated that gliomas demonstrate higher expression of CD99 compared with nonneoplastic brain. To assess the role of CD99 in glioma migration and invasion, we inhibited CD99 expression by siRNA and demonstrated decreased glioma migration and invasion. In contrast, when CD99 was overexpressed in glioma cells, we observed enhancement of cell migration and invasiveness. An orthotopic brain tumor model demonstrates that CD99 overexpression significantly increases invasiveness and decreases survival rate. Interestingly, Rac activity was decreased and Rho activity was increased in CD99 overexpressing glioma cells, and the proportion of amoeboid cells to mesenchymal cells was significantly increased. Taken together, our findings suggest that CD99 may play an important role in the migration and invasion of human gliomas independent of Akt, ERK, or JNK signaling pathways. Moreover, CD99 might be involved in amoeboid-mesenchymal transition in glioma migration. CD99 may be an important future target to inhibit migration and invasion, especially in CD99-expressing gliomas.

Malignant astrocytomas are highly invasive brain tumors. The Rho family of cytoskeletal GTPases are key regulators of astrocytoma migration and invasion; expression of the guanine nucleotide exchange factor ECT2 is elevated in primary astrocytomas and predicts both survival and malignancy. Mice bearing orthotopically implanted astrocytoma cells with diminished ECT2 levels following ECT2 knockdown exhibit longer survival. Although ECT2 is normally expressed in the nucleus, we show that ECT2 is aberrantly localized to the cytoplasm in both astrocytoma cell lines and primary human astrocytomas, and colocalizes with RAC1 and CDC42 at the leading edge of migrating astrocytoma cells. Inhibition of ECT2 expression by RNA interference resulted in decreased RAC1 and CDC42 activity, but no change in RHO activity, suggesting that ECT2 is capable of activating these pro-migratory Rho family members. ECT2 overexpression in astrocytoma cells resulted in a transition to an amoeboid phenotype that was abolished with the ROCK inhibitor, Y-27632. Cytoplasmic fractionation of astrocytoma cells followed by ECT2 immunoprecipitation and mass spectrometry were used to identify protein-binding partners that modulate the activity of ECT2 toward RAC1 and RHO/ROCK. We identified RASAL2 as an ECT2-interacting protein that regulates RHO activity in astrocytoma cells. RASAL2 knockdown leads to a conversion to an amoeboid phenotype. Our studies reveal that ECT2 has a novel role in mesenchymal-amoeboid transition in human astrocytoma cells.

The hepatocyte growth factor (HGF)/MET pathway plays a critical role in the development of the nervous system and has been implicated in medulloblastoma pathogenesis. Recent studies have shown a role for gene amplification of activators of this pathway, as well as silencing of its inhibitors in medulloblastoma pathogenesis. We analyzed exon array data from a cohort of 103 primary medulloblastomas to show that HGF/MET pathway elements are dysregulated in tumors compared to normal cerebellum. To determine if mutation of HGF/MET pathway genes is a mechanism for pathway dysregulation, we conducted a mutational analysis by exon resequencing of three key components of this pathway, including serine protease inhibitor Kunitz-type 1 (SPINT1), serine protease inhibitor Kunitz-type 2 (SPINT2), and MET, in 32 primary human medulloblastoma specimens. From this analysis, we identified multiple coding synonymous and nonsynonymous single nucleotide polymorphisms in these genes among the 32 tumor samples. Interestingly, we also discovered two unreported sequence variants in SPINT1 and SPINT2 in two tumors that resulted in Arginine to Histidine amino acid substitutions at codons 418 and 233, respectively. However, conservation assessment and functional assays of these two variants indicate that they involve nonconserved residues and that they do not affect the function of SPINT1 and SPINT2 as tumor suppressor genes. In conclusion, our data suggest that mutation alone plays a minor role in causing aberrancies of the HGF/MET pathway in medulloblastoma in comparison with other malignancies such as breast, hepatocellular, renal, and lung carcinomas.

Ecological theory predicts that individual survival should vary between sex and age categories due to differences in allocation of nutritional resources for growth and reproductive activities. During periods of environmental stress, such relationships may be exacerbated, and affect sex and age classes differently. We evaluated support for hypotheses about the relative roles of sex, age, and winter and summer climate on the probability of mountain goat (Oreamnos americanus) survival in coastal Alaska. Specifically, we used known-fates analyses (Program MARK) to model the effects of age, sex, and climatic variation on survival using data collected from 279 radio-marked mountain goats (118 M, 161 F) in 9 separate study areas during 1977–2008. Models including age, sex, winter snowfall, and average daily summer temperature (during Jul–Aug) best explained variation in survival probability of mountain goats. Specifically, our findings revealed that old animals (9+ yr) have lower survival than younger a...

The cofilin pathway plays a central role in the regulation of actin polymerization and the formation of cell membrane protrusions that are essential for cell migration. Overexpression of cofilin has been linked to the aggressiveness of a variety of different cancers. In these cancers, the phosphorylation of cofilin at Ser3 is a key regulatory mechanism modulating cofilin activity. The activation status of cofilin has been directly linked to tumor invasion. Accordingly, in this study, we examined the expression of cofilin and its activation status in astrocytoma cell lines and astrocytic tumors. We show that cofilin expression was increased and correlated with increasing grade malignant astrocytoma. In addition, both cofilin and LIMK had elevated expression in astrocytoma cell lines. Knockdown of cofilin by siRNA altered astrocytoma cell morphology and inhibited astrocytoma migration and invasion. Conversely, overexpression of a cofilin phosphorylation mutant in an in vivo intracranial xenograft model resulted in a more highly invasive phenotype than those xenographs expressing wild-type cofilin. Animals harboring astrocytomas stably expressing the cofilin phosphorylation mutant (cofilin-S3A) demonstrated marked local invasiveness and spread across the corpus callosum to the contralateral hemisphere in all animals. Taken together, these data indicate that the cofilin activity pathway may represent a novel therapeutic target to diminish the invasion of these highly malignant tumors.

Pediatric brain tumors are the leading cause of cancer-related death in children, and among them, embryonal tumors represent the largest group with an associated poor prognosis and long-term morbidity for survivors. The field of cancer epigenetics has emerged recently as an important area of investigation and causation of a variety of neoplasms, and is defined as alterations in gene expression without changes in DNA sequence. The best studied epigenetic modifications are DNA methylation, histone modifications, and RNA-based mechanisms. These modifications play an important role in normal development and differentiation but their dysregulation can lead to altered gene function and cancer. In this review the authors describe the mechanisms of normal epigenetic regulation, how they interplay in neuroembryogenesis, and how these can cause brain tumors in children when dysregulated. The potential use of epigenetic markers to design more effective treatment strategies for children with malignant brain tumors is also discussed.

The Modoc sucker Catostomus microps received legal protection in the United States based partially on concerns that anthropogenic environmental changes had restricted migration among populations and catalyzed hybridization with a more abundant congener, the Sacramento sucker Catostomus occidentalis. We applied eight microsatellite markers to samples of both species collected from two tributaries to the Pit River, California (Ash Creek and Turner Creek), and one tributary of Goose Lake, Oregon (Thomas Creek). Modoc sucker populations in these three tributaries seemed to be largely isolated from one another: gene flow between Ash Creek and Turner Creek was no greater than that among these two creeks and Thomas Creek. In contrast, divergence estimates among collections of Sacramento suckers indicated greater gene flow between Ash Creek and Turner Creek than between either of these creeks and Thomas Creek. Samples collected at a single site (Ash Valley) were identified based on morphology as Modoc suckers, but genetic data suggested they were much more similar to Sacramento suckers. Interspecific hybrids were detected in all three tributaries. Collections of Modoc suckers yielded 0.0–3.9% hybrids, and collections of Sacramento suckers yielded 0.0–80.0% hybrids. The two collections with the greatest proportions of hybrids (54.5 and 80.0%) were both from tributaries to lower Thomas Creek, and neither of these tributaries is thought to have upstream populations of Modoc suckers. Based on 1) low levels of hybrid detection in all three tributaries, 2) the absence of hybrids from typical parental habitats (upstream habitats for Modoc suckers and Pit River mainstem for Sacramento suckers), and 3) highly significant RST (variance in allele size) values between the species, we conclude that hybridization is common but that significant introgression (i.e., loss of parental genotypes) has not occurred. We also note that hybridization, and subsequent introgression, may become a conservation concern in such cases when the habitat of one or both of these species is eliminated or modified.

Populations of fall Chinook salmon Oncorhynchus tshawytscha in the Columbia River are divided among two evolutionarily significant units: lower Columbia River fall Chinook salmon (or “tules”) in the lower portion of the river and upriver “brights” (URBs) in the upper portion. The two lineages migrate together through portions of the lower Columbia River but spawn allopatrically. Little White Salmon National Fish Hatchery has been releasing URBs adjacent to what was historically exclusively tule spawning habitat in the White Salmon River for approximately 22 years. Tules and URBs now spawn in sympatry in the adjacent White Salmon River, and potential introgression between these two lineages has been identified as a genetic risk of the hatchery program. To assess the extent of introgression, we analyzed 13 microsatellite loci in juveniles taken from the White Salmon River in three consecutive years. Assignment tests revealed that juveniles leaving the White Salmon River from March to early May rese...

The strict homing of sockeye salmon Oncorhynchus nerka results in reproductively isolated populations that often spawn in close proximity and share rearing habitat. High spawning fidelity enables these populations to adapt to local conditions, resulting in a wide range of life history characteristics and genetic variation within individual watersheds. The Wood River system in southwestern Alaska provides a pristine, well-studied system in which to examine fine-scale population structure and its influences on juvenile life histories. Adult sockeye salmon spawn in lake beaches, rivers, and small tributaries throughout this watershed, and juveniles rear in five nursery lakes. We genotyped 30 spawning populations and 6,066 migrating smolts at 45 single nucleotide polymorphism loci, two of which are candidates for positive selection in the study system. We show that there is significant genetic structure (F ST = 0.032) in the Wood River lakes and that divergence is generally related to spawning rather...

The bull trout Salvelinus confluentus is a broadly distributed char in northwestern North America that has undergone significant population declines. This species is currently protected under the Endangered Species Act across its range in the coterminous United States. To clarify patterns of phylogenetic structure and to assist with identification of conservation units, we examined genetic variation within and among 75 representative bull trout populations sampled throughout the USA. Genealogies from a 520-base-pair portion of the mitochondrially encoded NADH dehydrogenase 1 gene (ND-1) revealed reciprocal monophyly between coastal and interior lineages that differed by 1.34% in DNA sequence. The geographic distribution of the two lineages was divided by the Cascade Mountains, a pattern that likely reflects postglacial dispersal from separate glacial refugia. Analysis of microsatellite variation revealed that 76% of populations had an estimated effective population size less than 50 and indicated...

Adult Chinook salmon Oncorhynchus tshawytscha navigate in river systems using olfactory cues that may be influenced by hydrologic factors such as flow and the number, size and spatial distribution of tributaries. Thus, river hydrology may influence both homing success and the level of straying (gene flow), which in turn influences population structure. In this study, two methods of multivariate analysis were used to examine the extent to which four indicators of hydrology and waterway distance explained population structure of O. tshawytscha in the Yukon River. A partial Mantel test showed that the indicators of hydrology were positively associated with broad-scale (Yukon basin) population structure, when controlling for the influence of waterway distance. Multivariate multiple regression showed that waterway distance, supplemented with the number and flow of major drainage basins, explained more variation in broad-scale population structure than any single indicator. At an intermediate spatial scale, indicators of hydrology did not appear to influence population structure after accounting for waterway distance. These results suggest that habitat changes in the Yukon River, which alter hydrology, may influence the basin-wide pattern of population structure in O. tshawytscha. Further research is warranted on the role of hydrology in concert with waterway distance in influencing population structure in Pacific salmon.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Dysregulation of WNT signaling occurs in up to 20% of cases. Using a genome-wide approach, we identified the secreted frizzled-related protein 1, 2 and 3 (SFRP1, SFRP2 and SFRP3) family of WNT inhibitors as putative tumor suppressor genes silenced by promoter region methylation in MB. SFRP1, SFRP2 and SFRP3 expression increased after 5-aza-2′-deoxycytidine treatment. SFRP1, SFRP2 and SFRP3 methylation was identified in 23.5, 3.9 and 15.7% of primary MB specimens, respectively, by methylation-specific PCR. Stable SFRP1, SFRP2 and SFRP3 expression reduced phospho-DVL2 levels and hindered MB cell proliferation and colony formation in soft agar in vitro. In 60% of primary tumors, SFRP1 was expressed at levels twofold lower than that in normal cerebellum. SFRP1 expression impaired tumor formation in vivo in flank and orthotopic intracerebellar xenograft models and conferred a significant survival advantage (P

Although medulloblastoma is the most common pediatric malignant brain tumor, its molecular underpinnings are largely unknown. We have identified rare, recurrent homozygous deletions of Kruppel-like Factor 4 (KLF4) in medulloblastoma using high-resolution single nucleotide polymorphism arrays, digital karyotyping, and genomic real-time polymerase chain reaction (PCR). Furthermore, we show that there is loss of physiological KLF4 expression in more than 40% of primary medulloblastomas both at the RNA and protein levels. Medulloblastoma cell lines drastically increase the expression of KLF4 in response to the demethylating agent 5-azacytidine and demonstrate dense methylation of the promoter CpG island by bisulfite sequencing. Methylation-specific PCR targeting the KLF4 promoter demonstrates CpG methylation in approximately 16% of primary medulloblastomas. Reexpression of KLF4 in the D283 medulloblastoma cell line results in significant growth suppression both in vitro and in vivo. We conclude that KLF4 is inactivated by either genetic or epigenetic mechanisms in a large subset of medulloblastomas and that it likely functions as a tumor suppressor gene in the pathogenesis of medulloblastoma.

Short tandem repeat (STR) markers, which exhibit many alleles per locus, are commonly used to assign fish to their populations of origin. Single-nucleotide polymorphisms (SNPs), which have many technical advantages over STRs, typically exhibit only two alleles per locus. Simulation studies have indicated that the number of independent alleles is a good predictor of the accuracy of genetic markers for fishery applications. Extant STR baselines for salmon Oncorhynchus spp. contain hundreds of alleles, and it has been estimated that hundreds of SNP markers will need to be developed before the SNP baselines will be comparable to the STR baselines. We compared 15 STRs exhibiting 349 independent alleles with 61 SNPs exhibiting 66 independent alleles for accuracy in assigning individuals to closely related populations of chum salmon O. keta. The SNP baseline yielded slightly higher mean accuracies for proportional assignment and comparable accuracies for individual assignment. Based on the number of ind...

Single-nucleotide polymorphisms (SNPs) exhibit several attributes that make them appealing as a class of genetic markers for applications in ecology and evolution. Two commonly cited limitations of SNPs in this capacity are that ascertainment bias and natural selection may shape allele frequencies of these markers, thus biasing estimates of population structure. The impacts of ascertainment bias and selection on estimates of population parameters have been demonstrated in a few model species, but their impacts relative to locus-specific variability and other potential complications on structure inferences in wild populations are unclear. We examined 22 allozymes, 9 short tandem repeats (STRs), and 41 SNPs in approximately 1,300 Chinook salmon Oncorhynchus tshawytscha representing 16 collections. We used plots of the genetic differentiation index F ST versus heterozygosity and sequence criteria to identify SNPs that might be under natural selection. We then calculated several measures of populatio...

An international multi-laboratory project was conducted to develop a standardized DNA database for Chinook salmon (Oncorhynchus tshawytscha ). This project was in response to the needs of the Chinook Technical Committee of the Pacific Salmon Commission to identify stock composition of Chinook salmon caught in fisheries during their oceanic migrations. Nine genetics laboratories identified 13 microsatellite loci that could be reproducibly assayed in each of the laboratories. To test that the loci were reproducible among laboratories, blind tests were conducted to verify scoring consistency for the nearly 500 total alleles. Once standardized, a dataset of over 16,000 Chinook salmon representing 110 putative populations was constructed ranging throughout the area of interest of the Pacific Salmon Commission from Southeast Alaska to the Sacramento River in California. The dataset differentiates the major known genetic lineages of Chinook salmon and provides a tool for genetic stock identification of ...

We characterize 38 single nucleotide polymorphism genotyping assays for chum salmon ( Oncorhynchus keta ), an important species for both commercial and subsistence fisheries in western Alaska. These assays are based on the 5′′ -nuclease reaction and thus facilitate high-throughput genotyping with minimal optimization time. Minor allele frequency differences (∆ ∆ ∆ q ) among collections were between 0.01 and 0.50 resulting in per locus F ST estimates of 0.00–0.08 with an average of 0.03.

The intentional introduction of red king crab, Paralithodes camtschatica (Tilesius, 1815) in the Barents Sea represent one of a few successful cases and one that now supports a commercial fishery. Introductions of alien species into new environments are often associated with genetic bottlenecks, which cause a reduction in the genetic variation, and this could be important for the spreading potential of the species in the Atlantic Ocean. Red king crab samples collected in the Varangerfjord located on the Barents Sea (northern Norway) were compared with reference crab samples collected from the Bering Sea and Kamchatka regions in the Pacific Ocean. All samples were screened for eleven microsatellite loci, based on the development of species-specific primers. The observed number of alleles per locus was similar, and no reduction in genetic variation, including gene diversity and allelic richness, was detected between the Varangerfjord sample and the reference sample from Okhotsk Sea near Kamchatka, indicating no genetic bottlenecking at least for the microsatellite loci investigated. The same results were found in comparison with the sample from Bering Sea. The level of genetic differentiation among the samples, measured as overall F ST across all loci, was relatively low (0.0238) with a range of 0.0035–0.1000 for the various loci investigated. The largest pairwise F ST values were found between the Bering Sea and Varangerfjord/Barents Sea samples, with a value of 0.0194 across all loci tested. The lowest value (0.0101) was found between the Varangerfjord and Kamchatka samples. Genetic differentiation based on exact tests on allele frequencies revealed highly significant differences between all pairwise comparisons. The high level of genetic variation found in the Varangerfjord/Barents Sea sample could be of significance with respect to further spreading of the species to other regions in the North Atlantic Ocean.

Today, microbial drinking water quality is monitored through either time-consuming laboratory methods or indirect on-line measurements. Results are thus either delayed or insufficient to support proactive action. A novel, optical, on-line bacteria sensor with a 10-minute time resolution has been developed. The sensor is based on 3D image recognition and the obtained pictures are analyzed with algorithms considering 59 quantified image parameters. The sensor counts individual suspended particles and classifies them as either bacteria or abiotic particles. The technology is capable of distinguishing and quantifying bacteria and particles in pure and mixed suspensions and the quantification correlates with total bacterial counts. Several field applications have demonstrated that the technology can monitor changes in the concentration of bacteria and is thus well suited for rapid detection of critical conditions such as pollution events in drinking water.

Stress granules are small RNA-protein granules that modify the translational landscape during cellular stress to promote survival. The RhoGTPase RhoA is implicated in the formation of RNA stress granules. Our data demonstrate that the cytokinetic proteins epithelial cell transforming 2 and Aurora kinase B (AurkB) are localized to stress granules in human astrocytoma cells. AurkB and its downstream target histone-3 are phosphorylated during arsenite-induced stress. Chemical (AZD1152-HQPA) and siRNA inhibition of AurkB results in fewer and smaller stress granules when analyzed using high-throughput fluorescent-based cellomics assays. RNA immunoprecipitation with the known stress granule aggregates TIAR and G3BP1 was performed on astrocytoma cells, and subsequent analysis revealed that astrocytoma stress granules harbor unique mRNAs for various cellular pathways, including cellular migration, metabolism, translation, and transcriptional regulation. Human astrocytoma cell stress granules contain mRNAs that are known to be involved in glioma signaling and the mammalian target of rapamycin pathway. These data provide evidence that RNA stress granules are a novel form of epigenetic regulation in astrocytoma cells, which may be targetable by chemical inhibitors and enhance astrocytoma susceptibility to conventional therapy, such as radiation and chemotherapy.

BACKGROUND: (blind field). METHODS: Expression profiling, molecular subgrouping and analysis of somatic copy number alterations were conducted on multiple independent cohorts of patient tumour samples to examine intermediates of the MET signaling pathway in medulloblastoma. To examine the in vitro and in vivo effects of foretinib treatment; MET signalling biochemical analysis; migration and invasion assays; and foretinib pharmacokinetic studies were performed. Medulloblastoma xenografts and transgenic mouse models were used to evaluate foretinib treatment in vivo. RESULTS: We analyzed three large non-overlapping cohorts of medulloblastoma patients (discovery cohort, n = 199; validation cohort 1, n = 439; validation cohort 2, n = 285) and demonstrated that cMET, known to be involved in tumor progression and dissemination, is a marker of sonic hedgehog (SHH) medulloblastoma. Importantly, immunohistochemical analysis of activated cMET (phosphorylated cMET) in another independent patient cohort (n = 385) revealed that cMET activation correlates with increased tumor relapse and a poor survival in pediatric patients with SHH medulloblastomas, thus defining a subset of patients that may benefit from cMET targeted therapy. We show that foretinib, an FDA approved inhibitor of cMET, suppresses cMET activation, decreases proliferation and induces apoptosis, both in medulloblastoma cell lines and in SHH medulloblastoma xenografts. Furthermore foretinib penetrates the blood-brain barrier and is effective both in the primary and in the metastatic compartments. Treatment of mouse xenografts and of an aggressive transgenic model of metastatic SHH medulloblastoma with foretinib reduced primary medulloblastoma growth, decreased the incidence of metastases by 36% and increased survival by 45%. CONCLUSIONS: Our results provide strong rationale for advancing foretinib into clinical trials for SHH-driven medulloblastomas. SECONDARY CATEGORY: Tumor Biology.

Brain tumors are the most common pediatric solid tumors, comprising 20–25 % of all childhood cancers. Although recent advances in the multidisciplinary care of the child with a brain tumor have taken place, brain tumors remain the leading cause of cancer-related death in the pediatric population. About 60–70 % of all the pediatric brain tumors occur in the posterior fossa. These tumors are clinically, pathologically, and genetically diverse and heterogeneous. While our understanding of the molecular mechanisms of these tumors is far from complete, the application of next generation lab-based strategies to pediatric brain tumors is closing our knowledge gap in a tangible way.

We characterize 32 single nucleotide polymorphism genotyping assays for resolving genotypic variation in sockeye salmon Oncorhynchus nerka in the Pacific Rim. These assays are based on the 5′′ -nuclease reaction and thus facilitate high-throughput genotyping with minimal optimization time. Minor allele frequency differences (∆ q) among collections were between 4.7% and 97.9%, resulting in per locus F ST estimates of 0.02–0.71 with an average of 0.22.

We report 39 single nucleotide polymorphisms (SNPs) observed in 23 nuclear DNA sequencesin coho salmon Oncorhynchus kisutch . High-throughput genotyping assays based on the5 ′′′ -nuclease reaction were developed for 17 of these nuclear SNPs and for two previouslypublished mitochondrial DNA SNPs. Minor allele frequency differences ( ∆ q ) amongcollections were between 5.2% and 51.2%, resulting in per locus F ST estimates of 0.00–0.24with an average of 0.09. Keywords :5 ′ -nuclease reaction, coho salmon, Oncorhynchus kisutch , SNP, Taq Man Received 22 November 2005; revision received 8 December 2005; accepted 8 January 2006 Coho salmon Oncorhynchus kisutch are anadromous, spawn-ing in streams and tributaries of rivers that drain into thenorth Pacific Ocean from Asia and North America. Cohosalmon are harvested in freshwater and near-shore areasthat often do not coincide with spawning sites and thuscontain mixtures of individuals from many populations.The sustainability of these harvests depends on the abilityof resource managers to estimate the origins of the fishharvested, a task increasingly done using genetic data.Genetic markers requiring electrophoresis of proteins(Utter

Single nucleotide polymorphisms (SNPs) are a class of genetic markers that are well suited to a broad range of research and management applications. Although advances in genotyping chemistries and analysis methods continue to increase the potential advantages of using SNPs to address molecular ecological questions, the scarcity of available DNA sequence data for most species has limited marker development. As the number and diversity of species being targeted for large-scale sequencing has increased, so has the potential for using sequence from sister taxa for marker development in species of interest. We evaluated the use of Oncorhynchus mykiss and Salmo salar sequence data to identify SNPs in three other species (Oncorhynchus tshawytscha, Oncorhynchus nerka and Oncorhynchus keta). Primers designed based on O. mykiss and S. salar alignments were more successful than primers designed based on Oncorhynchus-only alignments for sequencing target species, presumably due to the much larger number of potential targets available from the former alignments and possibly greater sequence conservation in those targets. In sequencing approximately 89 kb we observed a frequency of 4.30 x 10(-3) SNPs per base pair. Approximately half (53/101) of the subsequently designed validation assays resulted in high-throughput SNP genotyping markers. We speculate that this relatively low conversion rate may reflect the duplicated nature of the salmon genome. Our results suggest that a large number of SNPs could be developed for Pacific salmon using sequence data from other species. While the costs of DNA sequencing are still significant, these must be compared to the costs of using other marker classes for a given application.

As anadromous Chinook salmon Oncorhynchus tshawytscha bound for U.S. and Canadian spawning grounds migrate through the U.S. portion of the Yukon River, they are targeted by several fisheries. To fulfill treaty obligations between the two countries, fishery managers need to know what portion of fish caught in the United States are of Canadian origin. Allozyme markers have been used to assign individuals in mixed fishery samples to U.S. and Canadian portions of the Yukon River; however, these markers are limited by sampling difficulties and by the number of available loci. Microsatellite DNA markers have been considered as an alternative; however, microsatellite data are not readily transportable among laboratories or countries. Here we present the use of single nucleotide polymorphism (SNP) markers that combine the ease of sampling and large potential number of loci of other DNA markers with universally transportable data. Simulations and analyses of known fish suggest that the SNP baseline can be...

Tanner crabs Chionoecetes bairdi and snow crabs C. opilio are both important commercial species for Alaskan fisheries. The geographical ranges of Tanner and snow crabs overlap in the southeastern Bering Sea where a commercial fishery targets the more abundant snow crabs. Morphological identification of these species and of hybrids has proven difficult and impedes monitoring of the harvests. Techniques for the genetic identification of these species have been developed, but it may not be possible to process the desired number of samples because of throughput limitations and sample requirement constraints. Here we present a rapid, high-throughput assay for identifying these species based on single nucleotide polymorphisms (SNPs) in the nuclear rRNA internal transcribed spacer 1 region and the mitochondrial DNA 16S rRNA gene. These SNPs can also be used to infer the direction of hybridization (i.e., the species of each gender).

We describe 27 single nucleotide polymorphisms (SNPs) in a commercially important bivalve, the weathervane scallop ( Patinopecten caurinus ), identified using a targeted-gene approach. We further characterize 12 of these using 5′′ -nuclease and allele-specific PCR assays. Polymorphisms were identified in both mitochondrial and nuclear genes. These are the first SNPs developed for delineating population structure in the weathervane scallop and will provide a useful complement to currently available genetic markers.