Molecular Manipulation of MicroRNA397 Abundance Influences the Development and Salt Stress Response of Arabidopsis thaliana.

Arabidopsis thaliana (Arabidopsis) has been used extensively as a heterologous system for molecular manipulation to genetically characterize both dicotyledonous and monocotyledonous plant species. Here, we report on Arabidopsis transformant lines molecularly manipulated to over-accumulate the small regulatory RNA microRNA397 (miR397) from the emerging C4 monocotyledonous grass model species Setaria viridis (S. viridis). The generated transformant lines, termed SvMIR397 plants, displayed a range of developmental phenotypes that ranged from a mild, wild-type-like phenotype, to a severe, full dwarfism phenotype. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR)-based profiling of the SvMIR397 transformant population revealed a strong correlation between the degree of miR397 over-accumulation, repressed LACCASE (LAC) target gene expression, reduced lignin content, and the severity of the developmental phenotype displayed by SvMIR397 transformants. Further, exposure of SvMIR397 transformants to a 7-day regime of salt stress revealed the SvMIR397 transformant lines to be more sensitive to the imposed stress than were wild-type Arabidopsis plants. Taken together, the findings reported here via the use of Arabidopsis as a heterologous system show that the S. viridis miR397 small regulatory RNA is able to repress the expression of three Arabidopsis LAC genes which led to reduced lignin content and increased salt stress sensitivity.

Dry and wet periods drive rapid shifts in community assembly in an estuarine ecosystem.

The impacts of changing climate regimes on emergent processes controlling the assembly of ecological communities remain poorly understood. Human alterations to the water cycle in the western United States have resulted in greater interannual variability and more frequent and severe extremes in freshwater flow. The specific mechanisms through which such extremes and climate regime shifts may alter ecological communities have rarely been demonstrated, and baseline information on current impacts of environmental variation is widely lacking for many habitats and communities. Here, we used observations and experiments to show that interannual variation in winter salinity levels in San Francisco Bay controls the mechanisms determining sessile invertebrate community composition during the following summer. We found consistent community changes in response to decadal-scale dry and wet extremes during a 13-year period, producing strikingly different communities. Our results match theoretical predictions of major shifts in species composition in response to environmental forcing up to a threshold, beyond which we observed mass mortality and wholesale replacement of the former community. These results provide a window into potential future community changes, with environmental forcing altering communities by shifting the relative influences of the mechanisms controlling species distributions and abundances. We place these results in the context of historical and projected future environmental variation in the San Francisco Bay Estuary.

Using targeted virotherapy to treat a resistant Ewing sarcoma model: from the bedside to the bench and back.

UNLABELLED: Metastatic Ewing sarcoma (EWS) is often resistant to current multimodal chemotherapeutic regimens. Oncolytic virus therapy (OV) is a novel therapeutic platform whereby viruses can selectively infect as well as replicate in and kill tumor cells, while sparing normal tissues. The purpose of this study is to investigate the efficacy of the biotherapeutic oncolytic agent, vesicular stomatitis virus (VSVΔM51), to kill EWS cells that are resistant to conventional therapy. Our hypothesis is that systemic delivery of VSVΔM51 can demonstrate tumor-specific killing of resistant EWS cells, as well as a significant decrease of tumor burden in EWS bearing mice. METHODS: A biopsy sample was obtained from a patient with metastatic EWS and was used to establish a novel EWS cell line. In vitro assays evaluated the oncolytic effect of vesicular stomatitis virus (VSVΔM51) on this cell line. EWS xenograft mice model bearing either lung or subcutaneous tumors was established to evaluate the antitumor specific oncolytic effect of VSVΔM51 after local and systemic delivery. RESULTS: The established EWS cell line shared similar molecular and genetic traits to the patient's original tumor specimen. VSVΔM51 effectively infected and killed EWS cells in vitro. In vivo, VSVΔM51 selectively infected and killed EWS and led to significant delay in tumor growth. CONCLUSION: This study has been designed to implement a translational link between the bedside and the bench, where a specific challenging clinical scenario guided this basic science research. This research demonstrated that a sarcoma, which is resistant to current conventional standard therapies, is still susceptible to an alternative therapeutic platform, such as OV. Adding OV to the armamentarium of sarcoma treatment can enhance the future therapeutic approach towards these cancer patients.

Range-wide persistence of the endangered arroyo toad (Anaxyrus californicus) for 20+ years following a prolonged drought.

Prolonged drought due to climate change has negatively impacted amphibians in southern California, U.S.A. Due to the severity and length of the current drought, agencies and researchers had growing concern for the persistence of the arroyo toad (Anaxyrus californicus), an endangered endemic amphibian in this region. Range-wide surveys for this species had not been conducted for at least 20 years. In 2017-2020, we conducted collaborative surveys for arroyo toads at historical locations. We surveyed 88 of the 115 total sites having historical records and confirmed that the arroyo toad is currently extant in at least 61 of 88 sites and 20 of 25 historically occupied watersheds. We did not detect toads at almost a third of the surveyed sites but did detect toads at 18 of 19 specific sites delineated in the 1999 Recovery Plan to meet one of four downlisting criteria. Arroyo toads are estimated to live 7-8 years, making populations susceptible to prolonged drought. Drought is estimated to increase in frequency and duration with climate change. Mitigation strategies for drought impacts, invasive aquatic species, altered flow regimes, and other anthropogenic effects could be the most beneficial strategies for toad conservation and may also provide simultaneous benefits to several other native species that share the same habitat.

Synergistic interaction between oncolytic viruses augments tumor killing.

A major barrier to all oncolytic viruses (OVs) in clinical development is cellular innate immunity, which is variably active in a spectrum of human malignancies. To overcome the heterogeneity of tumor response, we combined complementary OVs that attack cancers in distinct ways to improve therapeutic outcome. Two genetically distinct viruses, vesicular stomatitis virus (VSV) and vaccinia virus (VV), were used to eliminate the risk of recombination. The combination was tested in a variety of tumor types in vitro, in immunodeficient and immunocompetent mouse tumor models, and ex vivo, in a panel of primary human cancer samples. We found that VV synergistically enhanced VSV antitumor activity, dependent in large part on the activity of the VV B18R gene product. A recombinant version of VSV expressing the fusion-associated small-transmembrane (p14FAST) protein also further enhanced the ability of VV to spread through an infected monolayer, resulting in a "ping pong" oncolytic effect wherein each virus enhanced the ability of the other to replicate and/or spread in tumor cells. Our strategy is the first example where OVs are rationally combined to utilize attributes of different OVs to overcome the heterogeneity of malignancies and demonstrates the feasibility of combining complementary OVs to improve therapeutic outcome.

Environmental signatures associated with cholera epidemics.

The causative agent of cholera, Vibrio cholerae, has been shown to be autochthonous to riverine, estuarine, and coastal waters along with its host, the copepod, a significant member of the zooplankton community. Temperature, salinity, rainfall and plankton have proven to be important factors in the ecology of V. cholerae, influencing the transmission of the disease in those regions of the world where the human population relies on untreated water as a source of drinking water. In this study, the pattern of cholera outbreaks during 1998-2006 in Kolkata, India, and Matlab, Bangladesh, and the earth observation data were analyzed with the objective of developing a prediction model for cholera. Satellite sensors were used to measure chlorophyll a concentration (CHL) and sea surface temperature (SST). In addition, rainfall data were obtained from both satellite and in situ gauge measurements. From the analyses, a statistically significant relationship between the time series for cholera in Kolkata, India, and CHL and rainfall anomalies was determined. A statistically significant one month lag was observed between CHL anomaly and number of cholera cases in Matlab, Bangladesh. From the results of the study, it is concluded that ocean and climate patterns are useful predictors of cholera epidemics, with the dynamics of endemic cholera being related to climate and/or changes in the aquatic ecosystem. When the ecology of V. cholerae is considered in predictive models, a robust early warning system for cholera in endemic regions of the world can be developed for public health planning and decision making.

The p14 FAST protein of reptilian reovirus increases vesicular stomatitis virus neuropathogenesis.

The fusogenic orthoreoviruses express nonstructural fusion-associated small transmembrane (FAST) proteins that induce cell-cell fusion and syncytium formation. It has been speculated that the FAST proteins may serve as virulence factors by promoting virus dissemination and increased or altered cytopathology. To directly test this hypothesis, the gene encoding the p14 FAST protein of reptilian reovirus was inserted into the genome of a heterologous virus that does not naturally form syncytia, vesicular stomatitis virus (VSV). Expression of the p14 FAST protein by the VSV/FAST recombinant gave the virus a highly fusogenic phenotype in cell culture. The growth of this recombinant fusogenic VSV strain was unaltered in vitro but was significantly enhanced in vivo. The VSV/FAST recombinant consistently generated higher titers of virus in the brains of BALB/c mice after intranasal or intravenous infection compared to the parental VSV/green fluorescent protein (GFP) strain that expresses GFP in place of p14. The VSV/FAST recombinant also resulted in an increased incidence of hind-limb paralysis, it infected a larger volume of brain tissue, and it induced more extensive neuropathology, thus leading to a lower maximum tolerable dose than that for the VSV/GFP parental virus. In contrast, an interferon-inducing mutant of VSV expressing p14 was still attenuated, indicating that this interferon-inducing phenotype is dominant to the fusogenic properties conveyed by the FAST protein. Based on this evidence, we conclude that the reovirus p14 FAST protein can function as a bona fide virulence factor.

A let-7 MicroRNA-sensitive vesicular stomatitis virus demonstrates tumor-specific replication.

Creation of potent oncolytic viruses (OVs) suitable for the clinic may require new strategies in virus design. Replication-competent viruses facilitate a variety of approaches to achieving tumor specificity. Altered expression of microRNAs is a common hallmark of cancer that we demonstrate can be used to alter expression of a potent wild-type viral gene to achieve tumor-specific replication of an engineered vesicular stomatitis virus (VSV). Incorporation of let-7 microRNA complementary sequences within VSV eliminates undesirable replication and associated toxicity in normal cells but permits growth in cancer cells in vitro and in vivo. This is proof of concept that viruses designed to exploit the differential microRNA expression in cancer cells is a viable approach, potentially useful in optimizing oncolytic viral gene expression for maximal antitumor activity and safety.

The role of containerships as transfer mechanisms of marine biofouling species.

Fouling of ships is an important historical and enduring transfer mechanism of marine nonindigenous species (NIS). Although containerships have risen to the forefront of global maritime shipping since the 1950s, few studies have directly sampled fouling communities on their submerged surfaces, and little is known about differences in the fouling characteristics among commercial ship types. Twenty-two in-service containerships at the Port of Oakland (San Francisco Bay, California) were sampled to test the hypothesis that the extent and taxonomic richness of fouling would be low on this type of ship, resulting from relatively fast speeds and short port durations. The data showed that the extent of macroorganisms (invertebrates and algae) was indeed low, especially across the large surface areas of the hull. Less than 1% of the exposed hull was colonized for all apart from one vessel. These ships had submerged surface areas of >7000 m(2), and fouling coverage on this area was estimated to be <17 m(2) per vessel, with zero biota detected on the hulls of many vessels. The outlying smaller vessel (4465 m(2)) had an estimated coverage of 90% on the hull and also differed substantially from the other ships in terms of its recent voyage history, shorter voyage range and slower speeds. Despite the low extent of fouling, taxonomic richness was high among vessels. Consistent with recent studies, a wide range of organisms were concentrated at more protected and heterogeneous (non-hull) niche areas, including rudders, stern tubes and intake gratings. Green algae and barnacles were most frequently sampled among vessels, but hydroids, bryozoans, bivalves and ascidians were also recorded. One vessel had 20 different species in its fouling assemblage, including non-native species (already established in San Francisco Bay) and mobile species that were not detected in visual surveys. In contrast to other studies, dry dock block areas did not support many organisms, despite little antifouling deterrence in some cases. Comparisons with previous studies suggest that the accumulation of fouling on containerships may be lower than on other ship types (eg bulkers and general cargo vessels), but more data are needed to determine the hierarchy of factors contributing to differences in the extent of macrofouling and non-native species vector risks within the commercial fleet.

Concentrated sodium chloride brine solutions as an additional treatment for preventing the introduction of nonindigenous species in the ballast tanks of ships declaring no ballast on board.

Currently, seawater flushing is the only management strategy for reducing the number of viable organisms in residual sediments and water of ballast tanks of vessels declaring no ballast on board (NOBOB) that traffic ports of the eastern United States. Previously, we identified several species of freshwater and brackish-water peracarid crustaceans able to survive the osmotic shock that occurs during open-ocean ballast water exchange and, potentially, to disperse over long distances via ballasted ships and NOBOB vessels. We tested the efficacy of concentrated sodium chloride brine solutions as an additional treatment for eradicating the halotolerant taxa often present in the ballast tanks of NOBOB ships. The lowest brine treatments (30 ppt for 1 h) caused 100% mortality in several species of cladocerans and copepods collected from oligohaline habitats. Several brackish-water peracarid crustaceans, however, including some that can survive in freshwater as well, required higher brine concentrations and longer exposure durations (45-60 ppt for 3-24 h). The most resilient animals were widely introduced peracarid crustaceans that generally prefer mesohaline habitats but do not tolerate freshwater (required brine treatments of 60-110 ppt for 3-24 h). Brine treatments (30 ppt) also required less time to cause 100% mortality for eight taxa compared with treatments using 34 ppt seawater. Based on these experiments and published data, we present treatment strategies for the ballast tank biota often associated with NOBOB vessels entering the Great Lakes region. We estimate the lethal dosage of brine for 95% of the species in our experiments to be 110 ppt (95% confidence interval, 85-192 ppt) when the exposure time is 1 h and 60 ppt (95% confidence interval, 48-98 ppt) when the exposure duration is 6 h or longer.

Complex rearrangement of chromosomes 19, 21, and 22 in Ewing sarcoma involving a novel reciprocal inversion-insertion mechanism of EWS-ERG fusion gene formation: a case analysis and literature review.

EWS-ERG Ewing sarcoma (ES) gene fusions often result from complex chromosomal rearrangements. We report an unusually aggressive case of ES with an EWS-ERG fusion gene that appeared to be a result of a simple balanced and reciprocal translocation, t(19;22)(q13.2;q12.2). Subsequent molecular investigation of the primary tumor, the metastasis, and a cell line generated from this ES permitted reconstruction of each genomic step in the evolution of this complex EWS-ERG fusion. We elucidated a new mechanism of reciprocal insertion inversion between chromosome 21 and 22, involving cryptic alterations to both the ERG and EWS genes. Molecular cytogenetic investigation, using systematic analysis with locus-specific probes, identified the cognate genomic breakpoints within chromosome 21 and 22, mandatory for the excision and exchange of both 3'ERG and 3'EWS, resulting in the formation of the EWS-ERG fusion gene present on the der(22). Array comparative genomic hybridization and fluorescence in situ hybridization studies of the ES cell line derived from this tumor identified additional acquired chromosomal and genomic abnormalities, likely associated with establishment and adaptation to in vitro growth. Notably, the cell line had lost one copy of the RB1 gene within the 13q13.1 approximately q14.2 region, and also had a near-tetraploid karyotype. The significance of these findings and their relationship to other reports of variant and complex ES translocations involving the ERG gene are reviewed.

Behavioral approach system sensitivity and risk taking interact to predict left-frontal EEG asymmetry.

The Behavioral Approach System (BAS) hypersensitivity theory of bipolar disorder (BD; Alloy & Abramson, 2010; Depue & Iacono, 1989) suggests that hyperreactivity in the BAS results in the extreme fluctuations of mood characteristic of BD. In addition to risk conferred by BAS hypersensitivity, cognitive and personality variables may play a role in determining risk. We evaluated relationships among BAS sensitivity, risk taking, and an electrophysiological correlate of approach motivation, relative left-frontal electroencephalography (EEG) asymmetry. BAS sensitivity moderated the relationship between risk taking and EEG asymmetry. More specifically, individuals who were high in BAS sensitivity showed left-frontal EEG asymmetry regardless of their level of risk-taking behavior. However, among individuals who were moderate in BAS sensitivity, risk taking was positively associated with asymmetry. These findings suggest that cognitive and personality correlates of bipolar risk may evidence unique contributions to a neural measure of trait-approach motivation. Clinical implications of these findings are discussed.