Developing rapid in vivo assays to investigate structure response relationships

Incorporation of nanoparticles (NPs) into consumer products is on the rise and human exposure to NPs is unavoidable. Currently, there is insufficient data to assess the safety of nanoparticles. I conducted a series of five studies using the zebrafish model to determine which NP components (i.e., core material or surface functionalization) contribute to biological responses and how ionic strength influences these results. The first study employed a systematic, rapid embryonic zebrafish assay to identify specific responses to precisely engineered lead sulfide (PbS-NPs) and gold nanoparticles (AuNPs) functionalized with different surface ligands. Lead sulfide nanoparticles functionalized with either 3-mercaptopropanesulfane (MT) or sodium 2,3-dimercaptopropanesulfonate (DT) ligands with nearly identical core sizes caused differential responses at the same concentration. I determined that the different responses were because MT-functionalized NPs released more soluble lead ions than DT-functionalized NPs due to different decomposition and oxidation rates. The second study investigated the different biological responses of three NPs identified during toxicity screening of a gold nanoparticle library. AuNPs functionalized with 2-mercaptoethanesulfonic acid (MES), N,N,N-trimethylammoniumethanethiol (TMAT), or 2-(2-(2-mercaptoethoxy)ethoxy)ethanol (MEEE), induced differential biological responses in embryonic zebrafish at the same concentration. Exposure to MES-AuNPs induced sublethal effects, while TMAT-AuNPs were embryo-lethal and MEEE-AuNPs were benign. Gold tissue concentration was confirmed to be similar in exposed embryos using inductively coupled-mass spectrometry. Microarrays were used to gain insight to the causes of the different responses. This approach identified that MES- and TMAT-AuNPs perturbed inflammatory and immune responses. These differential biological responses may be due to misregulated transport mechanisms causing numerous downstream defects unique to each surface functional group‟s property. In the next study, I tested the long-term consequences of developmental exposure to TMAT-, MES, and MEEE-AuNPs, and showed that MES- and TMAT-AuNPs affected larval behavior that persisted into adulthood. During the course of these investigations, I found that high ion concentration in exposure solutions results in NP agglomeration, presenting a problem for NP testing in the zebrafish model. For the fourth study, I focused on solving this by determining that zebrafish can be raised in nearly ion-free media without adverse consequences. When 3-MPA-AuNPs were dispersed in this new low ionic media, I observed adverse responses in the embryonic zebrafish toxicity assay, but not when the NPs were suspended in high ionic media. Thus, I demonstrated that the media greatly influences both agglomeration rates and biological responses, but most importantly, that the zebrafish is insensitive to external ions. The fifth study focused on the adverse response observed when embryonic zebrafish were exposed to 3-MPA-AuNPs. Exposed larvae failed to respond to a touch in the caudal fin at 120 hours post fertilization (hpf). Addition of a neuromuscular stimulus, nicotine, revealed the exposed embryos were not paralyzed, but experienced a reduction in axonal projections. A global genomic analysis (RNA-seq) using embryos exposed to 3-MPA-AuNP and MEEE-AuNPs (non-toxic control) from 6 to 120 hpf suggested that neurophysiological and signal transduction processes were perturbed. Functional analysis of the data led to the hypothesis that the most elevated gene, early growth response 1 (EGR-1), impacts axonogenesis in the caudal fin, interfering with glutaminergic synapses and preventing the connection of sensory neurons and touch perception. Although MEEE-AuNPs did not cause morphological defects, the RNA-seq analysis identified that these NPs perturbed immune and inflammatory system processes. Collectively, these results suggest that surface functional groups drive the differential responses to nanomaterials. The five studies summarized here confirm that a systems toxicological approach using the zebrafish model enables the rapid identification of structure-activity relationships, which will facilitate the design of safer nano-containing products

Assessments at multiple levels of biological organization allow for an integrative determination of physiological tolerances to turbidity in an endangered fish species.

Turbidity can influence trophic levels by altering species composition and can potentially affect fish feeding strategies and predator-prey interactions. The estuarine turbidity maximum, described as an area of increased suspended particles, phytoplankton and zooplankton, generally represents a zone with higher turbidity and enhanced food sources important for successful feeding and growth in many fish species. The delta smelt (Hypomesus transpacificus) is an endangered, pelagic fish species endemic to the San Francisco Estuary and Sacramento-San Joaquin River Delta, USA, where it is associated with turbid waters. Turbidity is known to play an important role for the completion of the species' life cycle; however, turbidity ranges in the Delta are broad, and specific requirements for this fish species are still unknown. To evaluate turbidity requirements for early life stages, late-larval delta smelt were maintained at environmentally relevant turbidity levels ranging from 5 to 250 nephelometric turbidity units (NTU) for 24 h, after which a combination of physiological endpoints (molecular biomarkers and cortisol), behavioural indices (feeding) and whole-organism measures (survival) were determined. All endpoints delivered consistent results and identified turbidities between 25 and 80 NTU as preferential. Delta smelt survival rates were highest between 12 and 80 NTU and feeding rates were highest between 25 and 80 NTU. Cortisol levels indicated minimal stress between 35 and 80 NTU and were elevated at low turbidities (5, 12 and 25 NTU). Expression of stress-related genes indicated significant responses for gst, hsp70 and glut2 in high turbidities (250 NTU), and principal component analysis on all measured genes revealed a clustering of 25, 35, 50 and 80 NTU separating the medium-turbidity treatments from low- and high-turbidity treatments. Taken together, these data demonstrate that turbidity levels that are either too low or too high affect delta smelt physiological performance, causing significant effects on overall stress, food intake and mortality. They also highlight the need for turbidity to be considered in habitat and water management decisions

An exploratory trial implementing a community-based child oral health promotion intervention for Australian families from refugee and migrant backgrounds: a protocol paper for Teeth Tales

Introduction: Inequalities are evident in early childhood caries rates with the socially disadvantaged experiencing greater burden of disease. This study builds on formative qualitative research, conducted in the Moreland/Hume local government areas of Melbourne, Victoria 2006–2009, in response to community concerns for oral health of children from refugee and migrant backgrounds. Development of the community-based intervention described here extends the partnership approach to cogeneration of contemporary evidence with continued and meaningful involvement of investigators, community, cultural and government partners. This trial aims to establish a model for child oral health promotion for culturally diverse communities in Australia.<p></p> Methods and analysis: This is an exploratory trial implementing a community-based child oral health promotion intervention for Australian families from refugee and migrant backgrounds. Families from an Iraqi, Lebanese or Pakistani background with children aged 1–4 years, residing in metropolitan Melbourne, were invited to participate in the trial by peer educators from their respective communities using snowball and purposive sampling techniques. Target sample size was 600. Moreland, a culturally diverse, inner-urban metropolitan area of Melbourne, was chosen as the intervention site. The intervention comprised peer educator led community oral health education sessions and reorienting of dental health and family services through cultural Competency Organisational Review (CORe).<p></p> Ethics and dissemination: Ethics approval for this trial was granted by the University of Melbourne Human Research Ethics Committee and the Department of Education and Early Childhood Development Research Committee. Study progress and output will be disseminated via periodic newsletters, peer-reviewed research papers, reports, community seminars and at National and International conferences.<p></p&gt

The role of drug resistance in poor viral suppression in rural South Africa: findings from a population-based study.

BACKGROUND:Understanding factors driving virological failure, including the contribution of HIV drug resistance mutations (DRM), is critical to ensuring HIV treatment remains effective. We examine the contribution of drug resistance mutations for low viral suppression in HIV-positive participants in a population-based sero-prevalence survey in rural South Africa. METHODS:We conducted HIV drug resistance genotyping and ART analyte testing on dried blood spots (DBS) from HIV-positive adults participating in a 2014 survey in North West Province. Among those with virologic failure (> 5000 copies/mL), we describe frequency of DRM to protease inhibitors (PI), nucleoside reverse transcriptase inhibitors (NRTI), and non-nucleoside reverse transcriptase inhibitors (NNRTI), report association of resistance with antiretroviral therapy (ART) status, and assess resistance to first and second line therapy. Analyses are weighted to account for sampling design. RESULTS:Overall 170 DBS samples were assayed for viral load and ART analytes; 78.4% of men and 50.0% of women had evidence of virologic failure and were assessed for drug resistance, with successful sequencing of 76/107 samples. We found ≥1 DRM in 22% of participants; 47% were from samples with detectable analyte (efavirenz, nevirapine or lopinavir). Of those with DRM and detectable analyte, 60% showed high-level resistance and reduced predicted virologic response to ≥1 NRTI/NNRTI typically used in first and second-line regimens. CONCLUSIONS:DRM and predicted reduced susceptibility to first and second-line regimens were common among adults with ART exposure in a rural South African population-based sample. Results underscore the importance of ongoing virologic monitoring, regimen optimization and adherence counseling to optimize durable virologic suppression

Predation by zooplankton on Batrachochytrium dendrobatidis: Biological control of the deadly amphibian chytrid fungus?

Batrachochytrium dendrobatidis (hereafter Batrachochytrium), a fungal pathogen of amphibians, causes the disease chytridiomycosis which is responsible for unprecedented population declines and extinctions globally. Host defenses against chytridiomycosis include cutaneous symbiotic bacteria and anti-microbial peptides, and proposed treatment measures include use of fungicides and bioaugmentation. Efforts to eradicate the fungus from localized areas of disease outbreak have not been successful. Instead, control measures to mitigate the impacts of the disease on host populations, many of which are already persisting with Batrachochytrium in an endemic state, may be more realistic. The infective stage of the fungus is an aquatic zoospore, 3-5µm in diameter. Here we show that zoospores of Batrachochytrium are consumed by the zooplankter Daphnia magna. This species inhabits amphibian breeding sites where Batrachochytrium transmission occurs, and consumption of Batrachochytrium zoospores may lead to effective biological control of Batrachochytrium.KEYWORDS: Batrachochytrium dendrobatidis, Biological control, Zooplankton, Amphibian chytrid fungus, Daphnia magnaThis is the authors’ post-peer review version of the final article. The final published version is copyrighted by Springer and can be found at: http://www.springer.com/life+sciences/evolutionary+%26+developmental+biology/journal/10531Keywords: Batrachochytrium dendrobatidis, Biological control, Zooplankton, Amphibian chytrid fungus, Daphnia magn

Persistent resistance to HIV-1 infection in CD4 T cells from exposed uninfected Vietnamese individuals is mediated by entry and post-entry blocks

BACKGROUND: We have previously reported that CD4 T cells from some exposed uninfected (EU) Vietnamese intravenous drug users are relatively resistant to HIV infection in vitro. Here, we further characterized the restriction of viral replication in CD4 T cells from five EUs and assessed its persistence in serial samples. RESULTS: CD4 T cells and/or PBMC sampled during a period of between 2 and 6 years were challenged with replication-competent HIV-1 and other retroviral particles pseudotyped with envelope proteins of various tropisms. CCR5 expression and function in resistant CD4 T cells was evaluated. The step at which HIV-1 replication is restricted was investigated by real-time PCR quantification of HIV-1 reverse transcripts. We identified three patterns of durable HIV-1 restriction in EU CD4 T cells. CD4 T cells from four of the five EU subjects were resistant to HIV-1 R5 infection. In two cases this resistance was associated with low CCR5 surface expression, which was itself associated with heterozygous CCR5 mutations. In the other two cases, CD4 T cells were resistant to HIV-1 R5 infection despite normal CCR5 expression and signaling function, and normal β-chemokine secretion upon CD4 T cell activation. Instead, restriction appeared to be due to enhanced CD4 T cell sensitivity to β-chemokines in these two subjects. In the fifth EU subject the restriction involved post-entry steps of viral replication and affected not only HIV-1 but also other lentiviruses. The restriction was not overcome by a high viral inoculum, suggesting that it was not mediated by a saturable inhibitory factor. CONCLUSION: Various constitutive mechanisms of CD4 T cell resistance to HIV-1 infection, affecting entry or post-entry steps of viral replication, are associated with resistance to HIV-1 in subjects who remain uninfected despite long-term high-risk behavior

Case studies : Using the zebrafish to evaluate neurobehavioral phenotypes

The use of zebrafish in behavioral neuroscience is rapidly growing. Zebrafish can be assessed for alterations in multiple behavioral endpoints, creating opportunities to use this powerful model to identify chemicals that alter behavioral phenotypes. To evaluate the utility of zebrafish for neurotoxicity research, we designed custom instrumentation to evaluate numerous embryonic and adult zebrafish behaviors. PRAT or Photomotor Response Analysis Tool was used to analyze the embryonic photomotor response (EPR) behavior in embryonic zebrafish (24 hours post fertilization). Shuttleboxes were used to evaluate learning and active avoidance conditioning and a zebrafish Visual Imaging System (zVIS) was used to measure fear responses. Social behavior was observed using Viewpoint tracking software. Startle responses were also analyzed using taps and Noldus Ethiovision XT tracking software. EPR results showed differential movement activities throughout development of larval zebrafish. Highest movement peaks were seen in 35-37 hours post fertilization fish. Using these custom analysis tools, we also evaluated the impact of Vitamin E deficiency and developmental Benzo[a]pyrene exposure on complex adult behaviors. Generational effects of BaP exposure were also tested. Zebrafish were fed defined-diets that either had sufficient or deficient levels of Vitamin E. The vitamin E deficient zebrafish had a ~30% decrease in learning rate relative to the fish with sufficient levels of Vitamin E. Startle response data showed that vitamin E deficient fish do not get desensitized to tap stimulus. Three exposure groups and generations were reared and spawned for the BaP study (0.1% DMSO controls, 1.25 ppm BaP, 2.5 ppm BaP). The zVIS system consists of an array of 8 tanks with only single side views of video projections on LCD monitors. This allows individual fish to visualize either a group of swimming zebrafish or single predator fish. For the socialization assay zebrafish were tracked using Viewpoint tracking software. Distances apart from each other were measured and analyzed in BaP exposed fish. For the predator test, zebrafish were expected to move away from the screen. The proximity of the zebrafish is tracked relative to the LCD screen projections. The preliminary results from BaP exposed zebrafish and 0.1% DMSO controls showed the percent of time spent away from the screen during the predator test or fear response assay was in the high 70% range for all fish. The 2.5 ppm BaP fish had on average the highest percentage (65% vs 50%) time spent away from the screen. Although it is uncertain as of now if there are any generational effects because further analysis is needed. Preliminary shoaling data shows that shoaling speed may be affected by DMSO exposure. The use of DMSO controls may not be optimal for this study. Disassociation is seen in both 1.25 ppm BaP and 2.5 ppm BaP exposure groups in the F2 generation. Collectively, these data demonstrate that custom behavioral systems are able to measure complex behavioral phenotypes and suggests that there are enormous opportunities for translation neurotoxicity research using zebrafish

Pressure of the hot gas in simulations of galaxy clusters

14siWe analyse the radial pressure profiles, the intracluster medium (ICM) clumping factor and the Sunyaev-Zel'dovich (SZ) scaling relations of a sample of simulated galaxy clusters and groups identified in a set of hydrodynamical simulations based on an updated version of the treepm-SPH GADGET-3 code. Three different sets of simulations are performed: the first assumes non-radiative physics, the others include, among other processes, active galactic nucleus (AGN) and/or stellar feedback. Our results are analysed as a function of redshift, ICM physics, cluster mass and cluster cool-coreness or dynamical state. In general, the mean pressure profiles obtained for our sample of groups and clusters show a good agreement with X-ray and SZ observations. Simulated cool-core (CC) and non-cool-core (NCC) clusters also show a good match with real data. We obtain in all cases a small (if any) redshift evolution of the pressure profiles of massive clusters, at least back to z = 1. We find that the clumpiness of gas density and pressure increases with the distance from the cluster centre and with the dynamical activity. The inclusion of AGN feedback in our simulations generates values for the gas clumping (√{C}_{ρ }˜ 1.2 at R200) in good agreement with recent observational estimates. The simulated YSZ-M scaling relations are in good accordance with several observed samples, especially for massive clusters. As for the scatter of these relations, we obtain a clear dependence on the cluster dynamical state, whereas this distinction is not so evident when looking at the subsamples of CC and NCC clusters.openopenPlanelles, S.; Fabjan, D.; Borgani, S.; Murante, G.; Rasia, E.; Biffi, V.; Truong, N.; Ragone-Figueroa, C.; Granato, G. L.; Dolag, K.; Pierpaoli, E.; Beck, A. M.; Steinborn, Lisa K.; Gaspari, M.Planelles, S.; Fabjan, D.; Borgani, Stefano; Murante, G.; Rasia, E.; Biffi, Veronica; Truong, N.; Ragone Figueroa, C.; Granato, G. L.; Dolag, K.; Pierpaoli, E.; Beck, A. M.; Steinborn, Lisa K.; Gaspari, M