Landscape resistance mediates native fish species distribution shifts and vulnerability to climate change in riverscapes.

A broader understanding of how landscape resistance influences climate change vulnerability for many species is needed, as is an understanding of how barriers to dispersal may impact vulnerability. Freshwater biodiversity is at particular risk, but previous studies have focused on popular cold-water fishes (e.g., salmon, trout, and char) with relatively large body sizes and mobility. Those fishes may be able to track habitat change more adeptly than less mobile species. Smaller, less mobile fishes are rarely represented in studies demonstrating effects of climate change, but depending on their thermal tolerance, they may be particularly vulnerable to environmental change. By revisiting 280 sites over a 20 year interval throughout a warming riverscape, we described changes in occupancy (i.e., site extirpation and colonization probabilities) and assessed the environmental conditions associated with those changes for four fishes spanning a range of body sizes, thermal and habitat preferences. Two larger-bodied trout species exhibited small changes in site occupancy, with bull trout experiencing a 9.2% (95% CI = 8.3%-10.1%) reduction, mostly in warmer stream reaches, and westslope cutthroat trout experiencing a nonsignificant 1% increase. The small-bodied cool water slimy sculpin was originally distributed broadly throughout the network and experienced a 48.0% (95% CI = 42.0%-54.0%) reduction in site occupancy with declines common in warmer stream reaches and areas subject to wildfire disturbances. The small-bodied comparatively warmer water longnose dace primarily occupied larger streams and increased its occurrence in the lower portions of connected tributaries during the study period. Distribution shifts for sculpin and dace were significantly constrained by barriers, which included anthropogenic water diversions, natural step-pools and cascades in steeper upstream reaches. Our results suggest that aquatic communities exhibit a range of responses to climate change, and that improving passage and fluvial connectivity will be important climate adaptation tactics for conserving aquatic biodiversity.

Cottus schitsuumsh, a new species of sculpin (Scorpaeniformes: Cottidae) in the Columbia River basin, Idaho-Montana, USA.

Fishes of the genus Cottus have long been taxonomically challenging because of morphological similarities among species and their tendency to hybridize, and a number of undescribed species may remain in this genus. We used a combination of genetic and morphological methods to delineate and describe Cottus schitsuumsh, Cedar Sculpin, a new species, from the upper Columbia River basin, Idaho-Montana, USA. Although historically confused with the Shorthead Sculpin (C. confusus), the genetic distance between C. schitsuumsh and C. confusus (4.84-6.29%) suggests these species are distant relatives. Moreover, the two species can be differentiated on the basis of lateral-line pores on the caudal peduncle, head width, and interpelvic width. Cottus schitsuumsh is also distinct from all other Cottus in this region in having a single small, skin-covered, preopercular spine. Haplotypes of mtDNA cytochrome oxidase c subunit 1 of C. schitsuumsh differed from all other members of the genus at three positions, had interspecific genetic distances typical for congeneric fishes (1.61-2.74% to nearest neighbors), and were monophyletic in maximum-likelihood trees. Microsatellite analyses confirmed these taxonomic groupings for species potentially sympatric with C. schitsuumsh and that fish used in morphological comparisons were unlikely to be introgressed. Its irregular distribution, in the Spokane River basin in Idaho and portions of the Clark Fork River basin in Montana, may have resulted from human-assisted translocation.

Sustained angiopoietin-2 expression disrupts vessel formation and inhibits glioma growth.

Systematic analyses of the expression of angiogenic regulators in cancer models should yield useful information for the development of novel therapies for malignant gliomas. In this study, we analyzed tumor growth, vascularization, and angiopoietin-2 (Ang2) expression during the development of U-87 MG xenografts. We found that tumoral angiogenesis in this model follows a multistage process characterized by avascular, prolific peripheral angiogenesis, and late vascular phases. On day 4, we observed an area of central necrosis, a peripheral ring of Ang2-positive glioma cells, and reactive Ang2-positive vascular structures in the tumor/brain interface. When the tumor had developed a vascular network, Ang2 was expressed only in peripheral vascular structures. Because Ang2 expression was downmodulated in the late stages of development, probably to maintain a stable tumoral vasculature, we next studied whether sustained Ang2 expression might impair vascular development and, ultimately, tumor growth. Ang2 prevented the formation of capillary-like structures by and impaired angiogenesis in a chorioallantoic membrane chicken model. Finally, we tested the effect of sustained Ang2 expression on U-87 MG xenograft development. Ang2 significantly prolonged the survival of intracranial U-87 MG tumor-bearing animals. Examination of Ang2-treated xenografts revealed areas of tumor necrosis and vascular damage. We therefore conclude that deregulated Ang2 expression during gliomagenesis hindered successful angiogenesis and that therapies that sustain Ang2 expression might be effective against malignant gliomas.

Downmodulation of E1A protein expression as a novel strategy to design cancer-selective adenoviruses.

Oncolytic adenoviruses are being tested as potential therapies for human malignant tumors, including gliomas. Here we report for the first time that a mutation in the E1A gene results in low levels of E1A protein, conditioning the replication of mutant adenoviruses specifically to cancer cells. In this study, we compared the oncolytic potencies of three mutant adenoviruses encompassing deletions within the CR1 (Delta-39), CR2 (Delta-24) regions, or both regions (Delta-24/39) of the E1A protein. Delta-39 and Delta-24 induced a cytopathic effect with similar efficiency in glioma cells and a comparable capacity for replication. Importantly, the activity of Delta-39 was significantly attenuated compared to Delta-24 in proliferating normal human astrocytes. Direct analyses of the activation of E2F-1 promoter demonstrated the inability of Delta-39 to induce S-phase-related transcriptional activity in normal cells. Interestingly, E1A protein levels in cells infected with Delta-39 were remarkably downmodulated. Furthermore, protein stability studies revealed enhanced degradation of CR1 mutant E1A proteins, and inhibition of the proteasome activity resulted in the striking rescue of E1A levels. We conclude that the level of E1A protein is a critical determinant of oncolytic phenotype and we propose a completely novel strategy for the design and construction of conditionally replicative adenoviruses.

Robust infectivity and replication of Delta-24 adenovirus induce cell death in human medulloblastoma.

The diverse advanced treatment modalities currently available to children with medulloblastoma, including surgery and radiotherapy, are associated with deleterious side effects and often with an unfavorable prognosis. A mutant adenovirus, Delta-24, which has a 24-base pair deletion in the Rb-binding region of the E1A gene, demonstrates selective replication and oncolysis in various malignant phenotypes. Here we report the ability of Delta-24 to kill medulloblastoma cells. Flow cytometric analyses of cell receptors demonstrated expression of the coxsackie adenovirus receptor and RGD-related integrins in the assessed medulloblastoma cell lines. Infectivity assays using a replication-deficient adenovirus to transduce the green fluorescence protein gene showed that the Delta-24 adenovirus infects 99% of Daoy and 46% of D283 Med medulloblastoma cells at a multiplicity of infection (MOI) of 50. Within 4 days after infecting medulloblastoma cells with Delta-24, a noticeable cytopathic effect was produced. Delta-24 induced a total cytopathic effect in Daoy and D283 Med medulloblastoma cells after 6 and 8 days of infection, respectively. In the infected population of cells, cell death correlated with the accumulation of cells in the S phase. At 5 days post-infection with 2.5 MOIs of Delta-24 adenovirus, the percentage of Daoy medulloblastoma cells in the S phase increased to 71.9+/-5.5%, compared with control values of 20.5+/-1.4%. The release of viral progeny was quantified as being increased by two orders of magnitude, indicating efficient replication of Delta-24 in medulloblastoma cells. This is the first report of the ability of oncolytic adenoviruses to infect and kill medulloblastoma cells, the findings of which suggest the potential efficacy of Delta-24 as a therapy for human medulloblastoma tumors.

A novel E1A-E1B mutant adenovirus induces glioma regression in vivo.

Malignant gliomas are the most frequently occurring primary brain tumors and are resistant to conventional therapy. Conditionally replicating adenoviruses are a novel strategy in glioma treatment. Clinical trials using E1B mutant adenoviruses have been reported recently and E1A mutant replication-competent adenoviruses are in advanced preclinical testing. Here we constructed a novel replication-selective adenovirus (CB1) incorporating a double deletion of a 24 bp Rb-binding region in the E1a gene, and a 903 bp deleted region in the E1b gene that abrogates the expression of a p53-binding E1B-55 kDa protein. CB1 exerted a potent anticancer effect in vitro in U-251 MG, U-373 MG, and D-54 MG human glioma cell lines, as assessed by qualitative and quantitative viability assays. Replication analyses demonstrated that CB1 replicates in vitro in human glioma cells. Importantly, CB1 acquired a highly attenuated replicative phenotype in both serum-starved and proliferating normal human astrocytes. In vivo experiments using intracranially implanted D-54 MG glioma xenografts in nude mice showed that a single dose of CB1 (1.5 x 10(8) PFU/tumor) significantly improved survival. Immunohistochemical analyses of expressed adenoviral proteins confirmed adenoviral replication within the tumors. The CB1 oncolytic adenovirus induces a potent antiglioma effect and could ultimately demonstrate clinical relevance and therapeutic utility.

Preclinical characterization of the antiglioma activity of a tropism-enhanced adenovirus targeted to the retinoblastoma pathway.

BACKGROUND: Oncolytic adenoviruses are promising therapies for the treatment of gliomas. However, untargeted viral replication and the paucity of coxsackie-adenovirus receptors (CARs) on tumor cells are major stumbling blocks for adenovirus-based treatment. We studied the antiglioma activity of the tumor-selective Delta-24 adenovirus, which encompasses an early 1 A adenoviral (E1A) deletion in the retinoblastoma (Rb) protein-binding region, and of the Delta-24-RGD adenovirus. Delta-24-RGD has an RGD-4C peptide motif inserted into the adenoviral fiber, which allows the adenovirus to anchor directly to integrins. METHODS: CAR and integrin expression were examined by flow cytometry in six glioma cell lines and in normal human astrocytes (NHAs). Adenoviral vectors containing green fluorescent protein (GFP) (AdGFP and AdGFP-RGD) were used to infect glioma cell lines with high or low CAR expression. Viability of glioma cells infected with different adenoviruses was assessed by trypan blue staining. Adenovirus replication was quantified with the infection-dose replication assay. Athymic mice carrying glioma xenografts received intratumoral injections of Delta-24-RGD or Delta-24 and were followed for survival, which was analyzed by the Kaplan-Meier method and the log-rank test. All statistical tests were two-sided. RESULTS: Half the glioma cell lines expressed low levels of CAR (defined as <50% of cells expressing detectable CAR); all lines expressed integrins in more than 50% of cells. Infection of U-87 MG cells (a low-CAR-expressing line) with AdGFP-RGD resulted in approximately six times more GFP-positive cells than infection with AdGFP. Delta-24-RGD was more cytopathic to both low- and high-CAR-expressing glioma lines than Delta-24, and it replicated more efficiently in both cell lines. In the xenografted mice, intratumoral injection of Delta-24-RGD was associated with longer survival than intratumoral injection of Delta-24 (P<.001, log-rank test). Furthermore, 60% of Delta-24-RGD-treated mice but only 15% of Delta-24-treated mice survived more than 4 months (difference = 45%, 95% CI = 21% to 68%). CONCLUSIONS: The antitumor activity of Delta-24-RGD suggests that it has the potential to be an effective agent in the treatment of gliomas.