Axon degeneration assays in Superior Cervical Ganglion explant cultures

The ability of peripheral nervous system neurons to extend long, axon-like neurites in vitro makes them ideally suited for studies on mechanisms of axon survival and degeneration. In this chapter we describe how to prepare explant cultures of sympathetic neurons of the superior cervical ganglion (SCG). We also describe how to induce and assess axon degeneration with an injury or a chemical insult

The MEK-ERK pathway negatively regulates bim expression through the 3' UTR in sympathetic neurons

Background: Apoptosis plays a critical role during neuronal development and disease. Developing sympathetic neurons depend on nerve growth factor (NGF) for survival during the late embryonic and early postnatal period and die by apoptosis in its absence. The proapoptotic BH3-only protein Bim increases in level after NGF withdrawal and is required for NGF withdrawal-induced death. The regulation of Bim expression in neurons is complex and this study describes a new mechanism by which an NGF-activated signalling pathway regulates bim gene expression in sympathetic neurons.Results: We report that U0126, an inhibitor of the prosurvival MEK-ERK pathway, increases bim mRNA levels in sympathetic neurons in the presence of NGF. We find that this effect is independent of PI3-K-Akt and JNK-c-Jun signalling and is not mediated by the promoter, first exon or first intron of the bim gene. By performing 3' RACE and microinjection experiments with a new bim-LUC+3'UTR reporter construct, we show that U0126 increases bim expression via the bim 3' UTR. We demonstrate that this effect does not involve a change in bim mRNA stability and by using PD184352, a specific MEK1/2-ERK1/2 inhibitor, we show that this mechanism involves the MEK1/2-ERK1/2 pathway. Finally, we demonstrate that inhibition of MEK/ERK signalling independently reduces cell survival in NGF-treated sympathetic neurons.Conclusions: These results suggest that in sympathetic neurons, MEK-ERK signalling negatively regulates bim expression via the 3' UTR and that this regulation is likely to be at the level of transcription. This data provides further insight into the different mechanisms by which survival signalling pathways regulate bim expression in neurons

Microinjection of superior cervical ganglion neurons for studying axon degeneration

Primary cultures of neurons of the peripheral nervous system have been successfully used for studying many aspects of neuronal development and survival, including investigations into the mechanisms of axon degeneration. In this chapter we describe how to prepare and microinject dissociated cultures of sympathetic neurons of the superior cervical ganglion (SCG) specifically for use in highly controlled and targeted assays of axon survival and degeneration

Differential Survival of Non-O157 Shiga Toxigenic Escherichia coli in Simulated Cattle Feedlot Runoff

Environmental survival time is important when evaluating adverse health outcomes from foodborne pathogens. Although outbreaks associated with manure-impacted irrigation or runoff water are relatively infrequent, their broad scope, regulatory importance, and severe health outcomes highlight the need to better understand the environmental survival of manure-borne pathogens. Shiga toxigenic Escherichia coli (STEC) are excreted in feces and persist in the environment until they die or recolonize a new host. Surface waters contaminated with manure-borne STEC can infect humans through drinking and recreational water use or irrigated crops that are minimally cooked. In this study, manure-impacted water microcosms mimicking beef cattle feedlot runoff were used to assess survival of STEC strains representing seven STEC serotypes (O26, O45, O103, O111, O121, O145, and O157) and persistence of target O antigen genes. Microcosms were sampled over the course of 1 year, and the entire experiment was repeated in a second year. Culture and polymerase chain reaction (PCR)- based techniques were used for detection and enumeration. Serotype-specific survival results were observed. Both STEC O26 and O45 declined slowly and remained culturable at 24 months. In contrast, STEC O121 and O145 decreased rapidly (-0.84 and -1.99 log10 abundance per month, respectively) and were unculturable by months 4 and 5, but detectable by PCR for a mean of 4.5 and 8.3 months, respectively. STEC O103, O111, and O157 remained culturable for a mean of 11.6, 5.5, and 15 months and detectable by PCR for a mean of 12, 13.8, and 18.6 months after inoculation, respectively. Results document that some STEC serotypes have the biological potential to survive in manure-impacted waters for extended periods of time when competing microflora are eliminated. Serotype-specific differences in survival of target bacteria and persistence of target genes were observed in this sample set, with STEC O26 and O45 strains appearing the most robust in these microcosm studies

Sediment Movement Within a Strip Intercropping System

This study was conducted to identify sediment movement within a strip intercropping system in southwestern Iowa during the third year of a three-year crop rotation. Soil loss, resulting from the application of simulated rainfall to a Monona silt loam soil, was measured from individual corn (Zea mays L.), soybean (Glycine max (L.) Merr), and winter wheat (Triticum aestivum L.) strips, and from multiple strips which included all three crops. Because of the crop rotation and residue management procedures, used at the study site, a substantial amount of surface cover and vegetative mass was present on each of the strips. As a result, soil loss resulting from simulated rainfall applied for a one-hour duration at an intensity of approximately 64 mm/hr (2.5 in/hr) was less than or equal to 1.5 Mg/ha (0.67 tons/acre) from each of the individual and multiple strips. Thus, the strip intercropping system established on this highly erodible site provided effective erosion control

Manure Impacts on Interrill Erosion

This study was conducted to measure runoff and erosion from interrill areas as affected by the long-term application of manure and fertilizer to a Tripp sandy loam soil located near Mitchell, Nebraska. Soil that had been removed from the top 0.1 m of the soil profile was placed in a 1 m2 soil pan. Rainfall was then applied to the soil pan during initial and wet simulation events. Total runoff was similar on the manure and no-manure treatments. The long-term application of manure (55 years) at a rate of 27 Mg ha-1 (wet basis) per year did not significantly influence interrill erosion on this sandy loam soil. Interrill erosion was also unaffected by the addition of manure immediately before rainfall simulation tests to soils on which manure had been applied in previous years. No significant differences in runoff and erosion were found among plots receiving varying amounts of fertilizer. Selected soil properties were generally unaffected by the varying manure and fertilizer treatments

Narrow grass hedge effects on microbial transport following variable applications of beef cattle manure

The effectiveness of a 1.4 m wide grass hedge in reducing microbial transport following manure application was examined in this study. Beef cattle manure was applied to 0.75 m wide by 4.0 m long plots established on an Aksarben silty clay loam located in southeast Nebraska. Manure was added at rates required to meet none or the 1-, 2-, or 4-year nitrogen requirements for corn. The transport of phages, total coliforms, E. coli, and enterococci was measured for three 30 min simulated rainfall events, which were separated by approximately 24 h intervals. The narrow grass hedge reduced total counts of phages, E. coli, and enterococci from 10.8 to 9.01 log PFU ha-1, from 12.4 to 11.9 log CFU ha-1, and from 11.8 to 11.2 log CFU ha-1, respectively. For the plots that received manure, no significant differences in transport of phages or enterococci were found among the three manure application rates. Rainfall simulation run significantly affected measurements of phages, total coliforms, and enterococci, with measurements during the three runs varying from 8.91 to 10.5 log PFU ha-1, from 12.7 to 13.3 log CFU ha-1, and from 11.2 to 11.7 log CFU ha-1, respectively. Counts for phages, total coliforms, and enterococci were significantly less for the first than the second and third rainfall simulation runs. All four of the microbial constituents were significantly correlated to dissolved P, particulate P, total P, and total N. A narrow grass hedge placed on the contour significantly reduced microbial transport following variable applications of beef cattle manure

A Simplified Equation for Modeling Sediment Transport Capacity

Sediment transport capacity for shallow overland flow was represented as a quadratic function of downslope distance using the assumption of a linear increase in overland flow discharge with downslope distance and an approximation to the Yalin equation for sediment transport capacity. The simplified equation for sediment transport applies to complex topography having uniform soil and management characteristics. The simplified equation accurately approximated the Yalin equation when calibrated using the average of the hydraulic shear stresses at the end of a constant slope reference profile and the end of the actual profile. The simplified equation is useful in deriving closed-form solutions to the governing erosion equations for steady state conditions and reduces the computational time when numerical solutions are required

A Simplified Equation for Modeling Sediment Transport Capacity

Sediment transport capacity for shallow overland flow was represented as a quadratic function of downslope distance using the assumption of a linear increase in overland flow discharge with downslope distance and an approximation to the Yalin equation for sediment transport capacity. The simplified equation for sediment transport applies to complex topography having uniform soil and management characteristics. The simplified equation accurately approximated the Yalin equation when calibrated using the average of the hydraulic shear stresses at the end of a constant slope reference profile and the end of the actual profile. The simplified equation is useful in deriving closed-form solutions to the governing erosion equations for steady state conditions and reduces the computational time when numerical solutions are required

Microbial transport as affected by residue cover and manure application rate

Manure is applied to cropland areas with varying surface cover to meet single- or multiple-year crop nutrient requirements. The objectives of this field study were to (1) examine microbial transport following land application of manure to sites with and without wheat residue, (2) compare microbial loads following land application to meet the 0, 1, 2, 4, and 8-year P-based requirements for corn, and (3) evaluate the effects of rainfall simulation run on microbial transport. Manure was added and incorporated by disking plots that were 0.75 m wide by 2.0 m long. Three 30 min simulated rainfall events, separated by 24 h intervals, were then applied at an intensity of 70 mm h-1. Plots containing wheat residue had a total coliform load of 12.6 log CFU ha-1, which was significantly greater than the 12.4 log CFU ha-1 measured on the plots without wheat residue. The plots with and without wheat residue had transport rates of E. coli and enterococci that were not significantly different. The plots on which manure was added at rates varying from 5.4 to 42.8 Mg ha-1 had counts of total coliforms and enterococci that were not significantly different. Rainfall simulation run did not significantly affect measurements of phages, total coliforms, or enterococci. Transport of selected microbes was found to be significantly affected by residue cover, manure application rate, and rainfall simulation run