Effects of genetic mutations and chemical exposures on Caenorhabditis elegans feeding: evaluation of a novel, high-throughput screening assay

Background

Government agencies have defined a need to reduce, refine or replace current mammalian-based bioassays with testing methods that use alternative species. Invertebrate species, such as Caenorhabditis elegans, provide an attractive option because of their short life cycles, inexpensive maintenance, and high degree of evolutionary conservation with higher eukaryotes. The C. elegans pharynx is a favorable model for studying neuromuscular function, and the effects of chemicals on neuromuscular activity, i.e., feeding. Current feeding methodologies, however, are labor intensive and only semi-quantitative.

Methodology/Principal Findings

Here a high-throughput assay is described that uses flow cytometry to measure C. elegans feeding by determining the size and intestinal fluorescence of hundreds of nematodes after exposure to fluorescent-labeled microspheres. This assay was validated by quantifying fluorescence in feeding-defective C. elegans (eat mutants), and by exposing wild-type nematodes to the neuroactive compounds, serotonin and arecoline. The eat mutations previously determined to cause slow pumping rates exhibited the lowest feeding levels with our assay. Concentration-dependent increases in feeding levels after serotonin exposures were dependent on food availability, while feeding levels decreased in arecoline-exposed nematodes regardless of the presence of food. The effects of the environmental contaminants, cadmium chloride and chlorpyrifos, on wild-type C. elegans feeding were then used to demonstrate an application of the feeding assay. Cadmium exposures above 200 µM led to a sharp drop in feeding levels. Feeding of chlorpyrifos-exposed nematodes decreased in a concentration-dependent fashion with an EC₅₀ of 2 µM.

Conclusions/Significance

The C. elegans fluorescence microsphere feeding assay is a rapid, reliable method for the assessment of neurotoxic effects of pharmaceutical drugs, industrial chemicals or environmental agents. This assay may also be applicable to large scale genetic or RNAi screens used to identify genes that are necessary for the development or function of the pharynx or other neuromuscular systems.     

Myosin light chain 3f attenuates age-induced decline in contractile velocity in MHC type II single muscle fibers

Aging is characterized by a progressive loss of muscle mass and impaired contractility (e.g., decline in force, velocity, and power). Although the slowing of contraction speed in aging muscle is well described, the underlying molecular mechanisms responsible for the decrement in speed are unknown. Myosin heavy chain (MHC) isoforms are the primary molecules determining contractile velocity; however, the contraction speed of single fibers within a given MHC isoform type is variable. Recent evidence proposes that the decline in shortening velocity (Vo) with aging is associated with a decrease in the relative content of essential myosin light chain 3f (MLC(3f) ) isoform. In the current study, we first evaluated the relative content of MLC(3f) isoform and Vo in adult and old rats. We then used recombinant adenovirus (rAd) gene transfer technology to increase MLC(3f) protein content in the MHC type II semimembranosus muscle (SM). We hypothesized that (i) aging would decrease the relative MLC(3f) content and Vo in type II fibers, and (ii) increasing the MLC(3f) content would restore the age-induced decline in Vo. We found that there was an age-related decrement in relative MLC(3f) content and Vo in MHC type II fibers. Increasing MLC(3f) content, as indicated by greater % MLC(3f) and MLC(3f) /MLC(2f) ratio, provided significant protection against age-induced decline in Vo without influencing fiber diameter, force generation, MHC isoform distribution, or causing cellular damage. To the best of our knowledge, these are the first data to demonstrate positive effects of MLC(3f) against slowing of contractile function in aged skeletal muscle.     

Perturbations in nuclear factor-kappaB or c-Jun N-terminal kinase pathways in pancreatic beta cells confer susceptibility to cytokine-induced cell death

Pro-inflammatory cytokines have been implicated in the death of pancreatic beta cells leading to type 1 diabetes. NIT-1 cells are an insulinoma cell line derived from mice expressing the SV40 large T antigen. These cells are a useful tool in analysis of beta cell death. NIT-1 cells are highly susceptible to caspase-dependent apoptosis induced by TNF-alpha alone. Primary islets are not susceptible to cell death induced by TNF-alpha alone; however, they are killed by TNF-alpha and IFN-gamma in a nitric oxide-dependent manner. We examined signal transduction in NIT-1 cells in response to cytokines to determine the mechanism for TNF-alpha-induced apoptosis. We found that NIT-1 cells are defective in the activation of nuclear factor-kappaB (NFkappaB) as a result of functionally deficient RelA activity, because overexpression of RelA protected NIT-1 cells from apoptosis. TNF-alpha also did not induce phosphorylation of c-Jun N-terminal kinase in NIT-1 cells. Together, these defects prevent expression of anti-apoptotic genes in NIT-1 cells and make them susceptible to TNF-alpha. To determine whether similar defects in primary beta cells would induce the same effect, we examined TNF-alpha-induced apoptosis in islets isolated from mice deficient in NFkappaB p50. These islets were as susceptible as wild-type islets to TNF-alpha and IFN-gamma-induced cell death. In contrast to wild-type islets, cell death was not prevented by inhibition of nitric oxide in p50-deficient islets. Blocking NFkappaB has been proposed as a mechanism for protection of beta cells from cytokine-induced cell death in vivo. Our results suggest that this would make beta cells equally or more sensitive to cytokines.     

Application of a Mathematical Model to Describe the Effects of Chlorpyrifos on Caenorhabditis elegans Development

Background

The nematode Caenorhabditis elegans is being assessed as an alternative model organism as part of an interagency effort to develop better means to test potentially toxic substances. As part of this effort, assays that use the COPAS Biosort flow sorting technology to record optical measurements (time of flight (TOF) and extinction (EXT)) of individual nematodes under various chemical exposure conditions are being developed. A mathematical model has been created that uses Biosort data to quantitatively and qualitatively describe C. elegans growth, and link changes in growth rates to biological events. Chlorpyrifos, an organophosphate pesticide known to cause developmental delays and malformations in mammals, was used as a model toxicant to test the applicability of the growth model for in vivo toxicological testing.

Methodology/Principal Findings

L1 larval nematodes were exposed to a range of sub-lethal chlorpyrifos concentrations (0–75 µM) and measured every 12 h. In the absence of toxicant, C. elegans matured from L1s to gravid adults by 60 h. A mathematical model was used to estimate nematode size distributions at various times. Mathematical modeling of the distributions allowed the number of measured nematodes and log(EXT) and log(TOF) growth rates to be estimated. The model revealed three distinct growth phases. The points at which estimated growth rates changed (change points) were constant across the ten chlorpyrifos concentrations. Concentration response curves with respect to several model-estimated quantities (numbers of measured nematodes, mean log(TOF) and log(EXT), growth rates, and time to reach change points) showed a significant decrease in C. elegans growth with increasing chlorpyrifos concentration.

Conclusions

Effects of chlorpyrifos on C. elegans growth and development were mathematically modeled. Statistical tests confirmed a significant concentration effect on several model endpoints. This confirmed that chlorpyrifos affects C. elegans development in a concentration dependent manner. The most noticeable effect on growth occurred during early larval stages: L2 and L3. This study supports the utility of the C. elegans growth assay and mathematical modeling in determining the effects of potentially toxic substances in an alternative model organism using high-throughput technologies.     

Role of echocardiography in the diagnosis and management of infective endocarditis

Infective endocarditis is one of the most common causes of serious infection and carries a high risk of morbidity and mortality. It represents the fourth leading cause of life-threatening infections after urosepsis, pneumonia, and intra-abdominal sepsis. There is still a continuous rise in the incidence of infective endocarditis, with a rate of about 20,000 new cases in the United States alone. This rise in incidence of infective endocarditis is mainly caused by increasing numbers of intravenous drug abusers, patients with artificial valves and elderly patients. In this paper, we will briefly review the crucial role of echocardiography in the diagnosis and management of infective endocarditis.     

Non-Additive Effects of Genotypic Diversity Increase Floral Abundance and Abundance of Floral Visitors

Background

In the emerging field of community and ecosystem genetics, genetic variation and diversity in dominant plant species have been shown to play fundamental roles in maintaining biodiversity and ecosystem function. However, the importance of intraspecific genetic variation and diversity to floral abundance and pollinator visitation has received little attention.

Methodology/Principal Findings

Using an experimental common garden that manipulated genotypic diversity (the number of distinct genotypes per plot) of Solidago altissima, we document that genotypic diversity of a dominant plant can indirectly influence flower visitor abundance. Across two years, we found that 1) plant genotype explained 45% and 92% of the variation in flower visitor abundance in 2007 and 2008, respectively; and 2) plant genotypic diversity had a positive and non-additive effect on floral abundance and the abundance of flower visitors, as plots established with multiple genotypes produced 25% more flowers and received 45% more flower visits than would be expected under an additive model.

Conclusions/Significance

These results provide evidence that declines in genotypic diversity may be an important but little considered factor for understanding plant-pollinator dynamics, with implications for the global decline in pollinators due to reduced plant diversity in both agricultural and natural ecosystems.     

Change within and among forest communities: the influence of historic disturbance,environmental gradients,and community attributes

Understanding how ecological communities change over time is critical for biodiversity conservation, but few long‐term studies directly address decadal‐scale changes in both the within‐ and among‐community components of diversity. In this study, we use a network of permanent forest vegetation plots, established in Great Smoky Mountains National Park (USA) in 1978, to examine the factors that influence change in community composition within and among communities. In 2007, we resampled 15 plots that were logged in the late 1920s and 15 plots that had no documented history of intensive human disturbance. We found that understory species richness decreased by an average of 4.3 species over the 30‐yr study period in the logged plots, but remained relatively unchanged in the unlogged plots. In addition, tree density decreased by an average of 145 stems ha^?1 in the logged plots, but was relatively stable in the unlogged plots. However, we found that historic logging had no effect on within‐community understory or tree compositional turnover during this time period. Instead, sites at lower elevations and sites with lower understory biomass in 1978 had higher understory compositional turnover than did sites at higher elevations and sites with higher understory biomass. In addition, sites with lower soil cation exchange capacity (CEC) and with lower tree basal area in 1978 had higher tree compositional turnover than did sites with higher soil CEC and higher tree basal area. Among‐community similarity was unchanged from 1978 to 2007 for both the logged and unlogged plots. Overall, our results indicate that human disturbance can affect plant communities for decades, but the extent of temporal change in community composition may nevertheless depend more on environmental gradients and community attributes.