Somatic mutagenesis with a Sleeping Beauty transposon system leads to solid tumor formation in zebrafish

Large-scale sequencing of human cancer genomes and mouse transposon-induced tumors has identified a vast number of genes mutated in different cancers. One of the outstanding challenges in this field is to determine which genes, when mutated, contribute to cellular transformation and tumor progression. To identify new and conserved genes that drive tumorigenesis we have developed a novel cancer model in a distantly related vertebrate species, the zebrafish, Danio rerio. The Sleeping Beauty (SB) T2/Onc transposon system was adapted for somatic mutagenesis in zebrafish. The carp ß-actin promoter was cloned into T2/Onc to create T2/OncZ. Two transgenic zebrafish lines that contain large concatemers of T2/OncZ were isolated by injection of linear DNA into the zebrafish embryo. The T2/OncZ transposons were mobilized throughout the zebrafish genome from the transgene array by injecting SB11 transposase RNA at the 1-cell stage. Alternatively, the T2/OncZ zebrafish were crossed to a transgenic line that constitutively expresses SB11 transposase. T2/OncZ transposon integration sites were cloned by ligation-mediated PCR and sequenced on a Genome Analyzer II. Between 700–6800 unique integration events in individual fish were mapped to the zebrafish genome. The data show that introduction of transposase by transgene expression or RNA injection results in an even distribution of transposon re-integration events across the zebrafish genome. SB11 mRNA injection resulted in neoplasms in 10% of adult fish at ∼10 months of age. T2/OncZ-induced zebrafish tumors contain many mutated genes in common with human and mouse cancer genes. These analyses validate our mutagenesis approach and provide additional support for the involvement of these genes in human cancers. The zebrafish T2/OncZ cancer model will be useful for identifying novel and conserved genetic drivers of human cancers.     

Alzheimer's disease and non-demented high pathology control nonagenarians: comparing and contrasting the biochemistry of cognitively successful aging

The amyloid cascade hypothesis provides an economical mechanistic explanation for Alzheimer's disease (AD) dementia and correlated neuropathology. However, some nonagenarian individuals (high pathology controls, HPC) remain cognitively intact while enduring high amyloid plaque loads for decades. If amyloid accumulation is the prime instigator of neurotoxicity and dementia, specific protective mechanisms must enable these HPC to evade cognitive decline. We evaluated the neuropathological and biochemical differences existing between non-demented (ND)-HPC and an age-matched cohort with AD dementia. The ND-HPC selected for our study were clinically assessed as ND and possessed high amyloid plaque burdens. ELISA and Western blot analyses were used to quantify a group of proteins related to APP/Aβ/tau metabolism and other neurotrophic and inflammation-related molecules that have been found to be altered in neurodegenerative disorders and are pivotal to brain homeostasis and mental health. The molecules assumed to be critical in AD dementia, such as soluble or insoluble Aβ40, Aβ42 and tau were quantified by ELISA. Interestingly, only Aβ42 demonstrated a significant increase in ND-HPC when compared to the AD group. The vascular amyloid load which was not used in the selection of cases, was on the average almost 2-fold greater in AD than the ND-HPC, suggesting that a higher degree of microvascular dysfunction and perfusion compromise was present in the demented cohort. Neurofibrillary tangles were less frequent in the frontal cortices of ND-HPC. Biochemical findings included elevated vascular endothelial growth factor, apolipoprotein E and the neuroprotective factor S100B in ND-HPC, while anti-angiogenic pigment epithelium derived factor levels were lower. The lack of clear Aβ-related pathological/biochemical demarcation between AD and ND-HPC suggests that in addition to amyloid plaques other factors, such as neurofibrillary tangle density and vascular integrity, must play important roles in cognitive failure.     

Effects of engineered nanoparticles on the assembly of exopolymeric substances from phytoplankton

The unique properties of engineered nanoparticles (ENs) that make their industrial applications so attractive simultaneously raise questions regarding their environmental safety. ENs exhibit behaviors different from bulk materials with identical chemical compositions. Though the nanotoxicity of ENs has been studied intensively, their unintended environmental impacts remain largely unknown. Herein we report experimental results of EN interactions with exopolymeric substances (EPS) from three marine phytoplankton species: Amphora sp., Ankistrodesmus angustus and Phaeodactylum tricornutum. EPS are polysaccharide-rich anionic colloid polymers released by various microorganisms that can assemble into microgels, possibly by means of hydrophobic and ionic mechanisms. Polystyrene nanoparticles (23 nm) were used in our study as model ENs. The effects of ENs on EPS assembly were monitored with dynamic laser scattering (DLS). We found that ENs can induce significant acceleration in Amphora sp. EPS assembly; after 72 hours EN-EPS aggregation reached equilibrium, forming microscopic gels of ～4-6 μm in size. In contrast, ENs only cause moderate assembly kinetic acceleration for A. angustus and P. tricornutum EPS samples. Our results indicate that the effects of ENs on EPS assembly kinetics mainly depend on the hydrophobic interactions of ENs with EPS polymers. The cycling mechanism of EPS is complex. Nonetheless, the change of EPS assembly kinetics induced by ENs can be considered as one potential disturbance to the marine carbon cycle.     

A hypomethylating variant of MTHFR, 677C>T, blunts the neural response to errors in patients with schizophrenia and healthy individuals

Background

Responding to errors is a critical first step in learning from mistakes, a process that is abnormal in schizophrenia. To gain insight into the neural and molecular mechanisms of error processing, we used functional MRI to examine effects of a genetic variant in methylenetetrahydrofolate reductase (MTHFR 677C>T, rs1801133) that increases risk for schizophrenia and that has been specifically associated with increased perseverative errors among patients. MTHFR is a key regulator of the intracellular one-carbon milieu, including DNA methylation, and each copy of the 677T allele reduces MTHFR activity by 35%.

Methodology/Principal Findings

Using an antisaccade paradigm, we found that the 677T allele induces a dose-dependent blunting of dorsal anterior cingulate cortex (dACC) activation in response to errors, a pattern that was identical in healthy individuals and patients with schizophrenia. Further, the normal relationship between dACC activation and error rate was disrupted among carriers of the 677T allele.

Conclusions/Significance

These findings implicate an epigenetic mechanism in the neural response to errors, and provide insight into normal cognitive variation through a schizophrenia risk gene.     

Age-related differences in amplification of covalently closed circular DNA at early times after duck hepatitis B virus infection of ducks

Inoculation of 3-day-old (3D) or 3-week-old (3W) ducklings with duck hepatitis B virus results in chronic or transient infection, respectively. We previously showed that rapid production of neutralizing antibody following inoculation of 3W ducklings prevents virus from spreading in the liver and leads to a transient infection (Y.-Y. Zhang and J. Summers, J. Virol. 78:1195-1201, 2004). In this study we further investigated early events of viral infection in both 3D and 3W ducks. We present evidence that a lower level of virus replication in the hepatocytes of 3W birds is an additional factor that probably favors transient infection. We suggest that lower virus replication is due to a less rapid covalently closed circular DNA amplification, leading to lower viremias and a slower spread of infection in the liver, and that the slower spread of infection in 3W ducks makes the infection more sensitive to interruption by the host immune responses.     

Receptor-induced conformational changes in the SU subunit of the avian sarcoma/leukosis virus A envelope protein: implications for fusion activation

The avian sarcoma/leukosis virus (ASLV) is activated for fusion by a two-step mechanism. For ASLV subgroup A (ASLV-A), association with its receptor (Tva) at neutral pH converts virions to a form that can bind target membranes and, in some assays, induce the lipid-mixing stage of fusion. Low pH is necessary to complete the fusion reaction. ASLV-A env (EnvA) exists on the viral surface as a trimer of heterodimers consisting of receptor binding (SU-A) and fusion-mediating (TM-A) subunits. As the receptor binding and fusion-mediating functions reside in separate subunits, we hypothesize that SU-A and TM-A are conformationally coupled. To begin to understand the effect of the binding of a soluble 47-residue domain of the receptor (sTva) on this coupling and the subsequent function of low pH, we prepared recombinant proteins representing full-length SU-A and a nested set of deletion mutant proteins. Full-length SU-A binds sTva with high affinity, but even small deletions at either the N or the C terminus severely impair sTva binding. We have purified the full-length SU-A subunit and characterized its interactions with sTva and the subsequent effect of low pH on the complex. sTva binds SU-A with an apparent KD of 3 pM. Complex formation occludes hydrophobic surfaces and tryptophan residues and leads to a partial loss of alpha-helical structure in SU-A. Low pH does not alter the off rate for the complex, further alter the secondary structure of SU-A, or induce measurable changes in tryptophan environment. The implications of these findings for fusion are discussed.