The purpose of this study is to find out that if antibiotic streptomycin decreases neuronal network activity or affects the pharmacological responses. The experiments in this study were conducted via MEA (multi-electrode array) technology which records neuronal activity from devices that have multiple small electrodes, serve as neural interfaces connecting neurons to electronic circuitry. The result of this study shows that streptomycin lowered the spike production of neuronal network, and also, sensitization was seen when neuronal network pre-exposed to streptomycin.
Subsidiary mother cell (SMC) divisions during stomatal complex formation in Zea mays are asymmetric generating a small subsidiary cell (SC) and a larger epidermal cell. Mutants with a high number of abnormally shaped subsidiary cells include the brick1 (brk1) and discordia1 (dcd1) mutants. BRK1 is homologous to HSPC300, an ARP2/3 complex activator, and is involved in actin nucleation while DCD1 is a regulatory subunit of the PP2A phosphatase needed for microtubule generation (Frank and Smith, 2002; Wright et al. 2009). Possible causes of the abnormal SCs in brk1 mutants include a failure of the SMC nucleus to polarize in advance of mitosis, no actin patch, and transverse and/or no PPBs (Gallagher and Smith, 2000; Panteris et al 2006). The abnormal subsidiary mother cell division in dcd1 is due to correctly localized, but disorganized preprophase bands (PPBs; Wright et al. 2009). The observation that brk1 has defects in PPB formation and that the dcd1 phenotype is enhanced by the application of actin inhibitors led us to examine the dcd1; brk1 double mutant (Gallagher and Smith, 1999). We found that dcd1; brk1 double mutants demonstrate a higher percentage of aberrant SCs than the single mutants combined suggesting that these two mutations have a synergistic and additive effect on SC formation. Our observations and results are intriguing and the future step will be to quantitate the abnormal PPBs and phragmoplasts in the double and single mutants using immunolocalization of tubulin and actin as well as observations of live cells expressing tubulin-YFP.
The Attwater’s prairie-chicken (Tympanuchus cupido attwateri; APC) is a species of grouse native to Texas coastal prairies and is on the critically endangered species list as a result of habitat destruction and overhunting. All of the current populations were captively bred and released into the wild. Survivorship for released APCs is very low, and individuals seldom survive to reproduce in the wild. One factor contributing to this may be an alteration in the gut microbiota as a result of captivity. Factors potentially influencing the gut microbial composition in captivity include antibiotic therapy, stress, and a predominantly commercially formulated diet. Recent studies have begun to shed light on the importance of the host microbial endosymbionts. Antibiotic administration, stress, diet, age, genotype and other factors have been shown to influence microbial populations in the gastrointestinal tracts of many different vertebrates. Sequencing of 16S rRNA gene amplicons on the Ion Torrent™ platform was used in this study to identify groups of bacteria in the cloacas as a surrogate for the gut microbiota in the APC. Antibiotic-treated and untreated birds, wild-hatched and captive-bred birds, and individuals sampled before and after release to the wild were examined. Significant differences were found between wild-hatched and captive raised birds both pre- and post release. In addition, there was extensive variation among the populations at the lower taxonomic ranks between individuals for each group of APCs. Principal coordinate analysis based on the weighted UniFrac distance metric further exhibited some clustering of individuals by treatment. These data suggest that captive breeding may have long-term effects on the cloacal microbiota of APCs with unknown consequences to their long-term health and survivorship.
A method was developed for extending a fine-scaled forest gap model to a watershed-scaled landscape, using the Eastern Cross Timbers ecoregion as a case study for the method. A topographic wetness index calculated from digital elevation data was used as a measure of hydrologic across the modeled landscape, and the gap model modified to have with a topographically-based hydrologic input parameter. The model was parameterized by terrain type units that were defined using combinations of USDA soil series and classes of the topographic wetness index. A number of issues regarding the sources, grid resolutions, and processing methods of the digital elevation data are addressed in this application of the topographic wetness index. Three different grid sizes, 5, 10, and 29 meter, from both LiDAR-derived and contour-derived elevation grids were used, and the grids were processed using both single-directional flow algorithm and bi-directional flow algorithm. The result of these different grids were compared and analyzed in context of their application in defining terrain types for the forest gap model. Refinements were made in the timescale of gap model’s weather model, converting it into a daily weather generator, in order to incorporate the effects of the new topographic/hydrologic input parameter. The precipitation model was converted to use a Markov model to initiate a sequence of wet and dry days for each month, and then daily precipitation amounts were determined using a gamma distribution. The output of the new precipitation model was analyzed and compared with a 100-year history of daily weather records at daily, monthly, and annual timescales. Model assumptions and requirements for biological parameters were thoroughly investigated and questioned. Often these biological parameters are based on little more than assumptions and intuition. An effort to base as many of the model’s biological parameters on measured data was made, including a new …
Municipalities protect human health and environmental resources from impacts of urban natural gas drilling through setback distances; the regulation of distances between well sites and residences, freshwater wells, and other protected uses. Setback distances have increased over time, having the potential to alter the amount and geographical distribution of drillable land within a municipality, thereby having implications for future land use planning and increasing the potential for future incompatible land uses. This study geographically applies a range of setback distances to protected uses and freshwater wells in the city limits of Denton, Texas to investigate the effect on the amount of land remaining for future gas well development and production. Denton lies on the edge of a productive region of the Barnett Shale geological formation, coinciding with a large concentration of drillable land in the southwestern region of the study area. This region will have the greatest potential for impacts to future municipal development and land use planning as a result of future gas well development and higher setback standards. Given the relatively high acreage of drillable land in industrially zoned subcategory IC-G and the concern regarding gas well drilling in more populated areas, future drilling in IC-G, specifically in IC-G land cover classes mowed/grazed/agriculture and herbaceous, would have the least impact on residential uses and tree cover, as well as decreasing the potential for future incompatible land uses.
The City of Dallas Environmental Education Initiative (EEI) is a hands-on, inquiry-based, K-12 water conservation education program that teaches students concepts about water and specific water conservation behaviors. Few descriptions and evaluations, especially quantitative in nature, of water conservation education programs have previously been conducted in the literature. This research measured the quantitative effects and impacts of the education program on water use in single-family homes in Dallas, Texas. A total of 2,122 students in 104 classrooms at three schools in the Dallas Independent School District received hands-on, inquiry-based water conservation education lessons and the average monthly water use (in gallons) in single-family homes was analyzed to measure whether or not there was a change in water use. The results showed that over a period of one calendar year the water use in the single-family homes within each school zone and throughout the entire research area in this study experienced a statistically significant decrease in water use of approximately 501 gallons per home per month (independent, t-test, p>0.001). Data from this research suggests that EEI is playing a role in decreasing the amount of water used for residential purposes. Additionally, this research demonstrates the use of a quantitative tool by which a water conservation education program’s effect on behavior change can be measured. This research shows great promise for reducing use and increasing the conservation of our world’s most precious resource.
The goals of captive breeding programs for endangered species include preserving genetic diversity and avoiding inbreeding. Typically this is accomplished by minimizing population mean kinship; however, this approach becomes less effective when errors in the pedigree exist and may result in inbreeding depression, or reduced survival. Here, both pedigree- and DNA-based methods were used to assess inbreeding depression in the critically endangered Attwater’s prairie-chicken (Tympanuchus cupido attwateri). Less variation in the pedigree-based inbreeding coefficients and parental relatedness values were observed compared to DNA-based measures suggesting that errors exist in the pedigree. Further, chicks identified with high parental DNA-based relatedness exhibited decreased survival at both 14- and 50-days post-hatch. A similar pattern was observed in later life stages (> 50 days post-hatch) with birds released to the wild; however, the pattern varied depending on the time post-release. While DNA-based inbreeding coefficient was positively correlated with mortality to one month post-release, an opposite pattern was observed at nine months suggesting purging of deleterious alleles. I also investigated whether immunocompetence, or the ability to produce a normal immune response, was correlated with survival; however, no significant correlation was observed suggesting that inbreeding was a more important factor influencing survival. Pairing individuals for breeding by minimizing DNA-based parental relatedness values resulted in a significant increase in chick survival. This study highlights the importance of using DNA-based methods to avoid inbreeding depression when errors exist in the pedigree.
Current energy and environmental challenges are driving the use of cellulosic materials for biofuel production. A major obstacle in this pursuit is poor ethanol tolerance among cellulolytic Clostridium species. The first objective of this work was to establish a potential upper boundary of ethanol tolerance for the cellulosome itself. The hydrolytic function of crude cellulosome extracts from C. cellulolyticum on carboxymethyl cellulose (CMC) with 0, 5, 10, 15, 20 and 25% (v/v) ethanol was determined. Results indicated that the endoglucanase activity of the cellulosome incubated in 5% and 10% ethanol was significantly different from a control without ethanol addition. Furthermore a significant difference was observed in endoglucanase activity for cellulosome incubated in 5%, 10%, 15%, 20% and 25% ethanol in a standalone experiment. Endoglucanase activity continued to be observed for up to 25% ethanol, indicating that cellulosome function in ethanol will not be an impediment to future efforts towards engineering increasing production titers to levels at least as high as the current physiological limits of the most tolerant ethanologenic microbes. The second objective of this work was to study bioethanol production by a microbial co-culture involving Clostridium cellulolyticum and a recombinant Zymomonas mobilis engineered for the utilization of oligodextrans. The recombinant Z. mobilis ZM4 pAA1 and wild type ZM4 were first tested on RM medium (ATCC 1341) containing 2% cellobiose as the carbon source. Ethanol production from the recombinant Z. mobilis was three times that observed from the wild type Z. mobilis. Concomitant with ethanol production was the reduction in OD from 2.00 to 1.580, indicating the consumption of cellobiose. No such change in OD was observed from the wild type. The recombinant ZM4 was then co-cultured with C. cellulolyticum using cellobiose and microcrystalline cellulose respectively as carbon sources. Results indicate that the recombinant ZM4 acted synergistically with C. cellulolyticum …
Legumes play an important role in agriculture as major food sources for humans and as feed for animals. Bioavailable nitrogen is a limiting nutrient for crop growth. Legumes are important because they can form a symbiotic relationship with soil bacteria called rhizobia that results in nitrogen-fixing root nodules. In this symbiosis, rhizobia provide nitrogen to the legumes and the legumes provide carbon sources to the rhizobia. The Medicago truncatula NPF1.7/NIP/LATD gene is essential for root nodule development and also for proper development of root architecture. Work in our lab on the MtNPF1.7/MtNIP/LATD gene has established that it encodes a nitrate transporter and strongly suggests it has another function. Mtnip-1/latd mutants have pleiotropic defects, which are only partially explained by defects in nitrate transport. MtNPF1.7/NIP/LATD is a member of the large and diverse NPF/NRT1(PTR) transporter family. NPF/NRT1(PTR) members have been shown to transport other compounds in addition to nitrate: nitrite, amino acids, di- and tri-peptides, dicarboxylates, auxin, abscisic acid and glucosinolates. In Arabidopsis thaliana, the AtNPF6.3/NRT1.1( CHL1) transporter was shown to transport auxin as well as nitrate. Atchl1 mutants have defects in root architecture, which may be explained by defects in auxin transport and/or nitrate sensing. Considering the pleiotropic phenotypes observed in Mtnip-1/latd mutant plants, it is possible that MtNPF1.7/NIP/LATD could have similar activity as AtNPF6.3/NRT1.1(CHL1). Experimental evidence shows that the MtNPF1.7/NIP/LATD gene is able to restore nitrate-absent responsiveness defects of the Atchl1-5 mutant. The constitutive expression of MtNPF1.7/NIP/LATD gene was able to partially, but not fully restore the wild-type phenotype in the Atchl1-5 mutant line in response to auxin and cytokinin. The constitutive expression of MtNPF1.7/NIP/LATD gene affects the lateral root density of wild-type Col-0 plants differently in response to IAA in the presence of high (1mM) or low (0.1 mM) nitrate. MtNPF1.7/NIP/LATD gene expression is not regulated by nitrate …
Neuronal networks, derived from mouse embryonic frontal cortex (FC) tissue grown on microelectrode arrays, were used to investigate effects of gentamicin pretreatment on pharmacological response to the L-type calcium channel blocker, verapamil. Gentamicin is a broad spectrum antibiotic used to control bacterial contamination in cell culture. The addition of gentamicin directly to medium affects the pharmacological and morphological properties of the cells in culture. A reproducible dose response curve to verapamil from untreated cultures was established and the mean EC50 was calculated to be 1.5 ± 0.5 μM (n=10). 40 μM bicuculline was added to some cell cultures to stabilize activity and verapamil dose response curves were performed in presence of bicuculline, EC50 1.4 ± 0.1 μM (n=9). Statistical analysis showed no significant difference in verapamil EC50s values obtained in presence of bicuculline and hence the data was combined and a standard verapamil EC50 was calculated as 1.4 ± 0.13 μM (n=19). This EC50 was then used to compare verapamil EC50s obtained from neuronal cell cultures with chronic and acute exposures to gentamicin. FC cultures (21- 38 days old) were found to be stable in presence of 2300 μM gentamicin. The recommended concentration of gentamicin for contamination control is 5uL /1 ml medium (108 μM). At this concentration, the verapamil EC50 shifted from 1.4 ± 0.13 μM to 0.9 ± 0.2 μM. Given the limited data points and only two complete CRCs, statistical comparison was not feasible. However, there is a definite trend that shows sensitization of cells to verapamil in presence of gentamicin. The cultures exposed to 108 μM gentamicin for 5 days after seeding showed loss of adhesion and no data could be collected for pharmacological analysis. To conclude, acute gentamicin exposure of neuronal cell cultures causes increased sensitivity to verapamil and chronic or long term exposure to …
Forensic STR analysis is limited by the quality and quantity of DNA. Significant damage or alteration to the molecular structure of DNA by depurination, crosslinking, base modification, and strand breakage can impact typing success. Two methods that could potentially improve STR typing of challenged samples were explored: an in vitro DNA repair assay (PreCR™ Repair Mix) and whole genome amplification. Results with the repair assay showed trends of improved performance of STR profiling of bleach-damaged DNA. However, the repair assay did not improve DNA profiles from environmentally-damaged bloodstains or bone, and in some cases resulted in lower RFU values for STR alleles. The extensive spectrum of DNA damage and myriad combinations of lesions that can be present in forensic samples appears to pose a challenge for the in vitro PreCR™ assay. The data suggest that the use of PreCR™ in casework should be considered with caution due to the assay’s varied results. As an alternative to repair, whole genome amplification (WGA) was pursued. The DOP-PCR method was selected for WGA because of initial primer design and greater efficacy for amplifying degraded samples. Several modifications of the original DOP-PCR primer were evaluated. These modifications allowed for an overall more robust amplification of damaged DNA from both contemporary and historical skeletal remains compared with that obtained by standard DNA typing and a previously described DOP-PCR method. These new DOP-PCR primers show promise for WGA of degraded DNA.
Dried blood spots is an alternative method to collect blood samples from research subjects. However, little is known about how hemoglobin and hematocrit affect bead-based multiplex assay performance. The purpose of this study was to determine how bead-based multiplex assays perform when analyzing dried blood spot samples. A series of four experiments outline the study each with a specific purpose. A total of 167 subject samples were collected and 92 different biomarkers were measured. Median fluorescence intensity results show a positive correlation between filtered and non-filtered samples. Utilizing a smaller quantity of sample results in a positive correlation to a larger sample. Removal of hemoglobin from the dried blood spot sample does not increase detection or concentration of biomarkers. Of the 92 different biomarkers measured 56 were detectable in 100-75% of the attempted samples. We conclude that blood biomarkers can be detected using bead-based multiplex assays. In addition, it is possible to utilize a smaller quantity of sample while avoiding the use of the entire sample, and maintaining a correlation to the total sample. While our method of hemoglobin was efficient it also removed the biomarkers we wished to analyze. Thus, an alternative method is necessary to determine if removing hemoglobin increases concentration of biomarkers. More research is necessary to determine if the biomarkers measured in this study can be measured over time or within an experimental model.
Macrophage-derived foam cells play a predominant role in the deposition of arterial plaques during the early stages of atherosclerosis. The deposition of arterial plaques is known to be effected by several factors, including a person’s dietary habits. The consumption of a high-fat (>60% of calories from fat) meal is known to elevate serum LDL and triglycerides, which have been previously implicated in the formation pf foam cells. One limitation of current research models is that it is not possible to directly measure foam cells in vivo. Thus, the purpose of the present study was to validate the use of blood derived monocytes as a proxy measure of foam cells. In order to complete this objective, we evaluated monocyte oxLDL phagocytosis capacity following consumption of a high-fat meal. Eight men and women participated in the present study and venous blood samples were collected prior to the meal, 1-h, 3-h, and 5-h post-meal. Monocytes (CD14+/16- and CD14+/16+) were evaluated for adhesion molecule expression (CD11a, CD11b, and CD18), scavenger R (CD36) expression, and oxLDL phagocytosis using an image-based flow cytometry method developed in our laboratory for this purpose. Data was statistically analyzed for significance using a single-factor ANOVA with repeated measures and a p < 0.05. Consumption of a high-fat meal caused an increase significant increase in the proportion of pro-inflammatory monocytes (CD14+/16+) and a decrease in classic monocytes (CD14+/16-), with the greatest difference occurring at 5 h post prandial (p = 0.038). We also found that pro-inflammatory monocyte expression of adhesion molecules and CD36 increased in a manner that would promote in vivo movement of monocytes into the subendothelial space. Finally, over the course of the 5 h postprandial period, the majority of oxLDL uptake occurred in pro-inflammatory compared to classic monocytes. These results suggest that consuming a high-fat meal increases the …
Copepods comprise an ecologically important role in freshwater and marine ecosystems, which is why they are often considered an important ecotoxicological model organism. The International Organization for Standardization’s (ISO) 14669 protocol is the only guideline for the determination of acute toxicity in three European marine copepod species: Acartia tonsa. The goal of this project was to assess the feasibility of establishing and maintaining cultures of Acartia tonsa, as well as to refine current culturing and egg separation methods. Initial culture methodology proved difficult for consistent production of eggs and collection of nauplii. The development of an airlift system for the separation of eggs from nauplii and adults, based on size, successfully increased the availability of eggs, nauplii and adults. The sensitivity and relative conditions of the copepod species was assessed by running a series of 48h acute toxicity tests with the reference toxicants 3,5-dichlorophenol, 4,4’-methylenebis(2,6-di-tert-butylphenol. The acute 48 hour median lethal dose concentration (LC50), the no observed effect concentration (NOEC), and the lowest observed effect concentration (LOEC) was analyzed for the three reference compounds for of A. tonsa.
Rhodopseudomonas palustris is a metabolically versatile phototrophic α-proteobacterium. The organism experiences a wide range of stresses in its environment and during metabolism. The oxidative an pH stresses of four ECF (extracytoplasmic function) σ-factors are investigated. Three of these, σ0550, σ1813, and σ1819 show responses to light-generated singlet oxygen and respiration-generated superoxide reactive oxygen species (ROS). The EcfG homolog, σ4225, shows a high response to superoxide and acid stress. Two proteins, one containing the EcfG regulatory sequence, and an alternative exported catalase, KatE, are presented to be regulated by σ4225. Transcripts of both genes show similar responses to oxidative stress compared to σ4225, indicating it is the EcfG-like σ-factor homolog and controls the global stress response in R. palustris.
Stomata are specialized plant structures required for gaseous exchange with the outer environment. During stomata formation, the cytoskeleton plays an important role in controlling the division of the individual cells leading to the generation of the stomata complex. Two mutants that affect microfilament and microtubule organization in subsidiary mother cells include brk1 and dcd1. While only 20% of the subsidiary cells in the brk1 and dcd1 single mutants are abnormally shaped, it was reported that there is a synergistic effect between the brk1 and dcd1 mutations in the brk1; dcd1 double mutant since 100% of the subsidiary cells are abnormal. The focus of this research is to try to understand this synergistic effect by investigating the actin cytoskeleton and nuclear position in the single and double mutants. The reported results include the observation that the size of actin patch was largest in the wild-type subsidiary mother cells (SMCs) and smallest in dcd1 and brk1; dcd1 SMCs and that brk1 and brk1; dcd1 double mutants had fewer actin patches than wild-type and dcd1 SMCs. Additionally, we observed that some SMCs that did not have actin patches still underwent nuclear migration suggesting that nuclear migration may not be solely dependent on actin patch formation. Finally, during SMC cytokinesis, a large percentage of double mutant (brk1; dcd1) cells showed an off-track development of the phragmoplast as compared to the single mutants and the wild-type plant explaining the large number of abnormally shaped subsidiary cells in the double mutants.
A common measurement of body temperature during exercise in a hot, humid environment is mean skin temperature collected from 3-12 sites on the body. However, such an approach fails to demonstrate localized differences in skin temperature that are likely to exist as a function of gender. The purpose of this study was to examine potential differences in skin temperature between men and women at 17 different locations on the body. Young women (21 ± 1 y; n = 11) and men (23 ± 3; n = 10) were recruited to complete a 60-min walk/jog interval protocol in a hot (34 ± 1 °C), humid (64 ± 8%) environment while skin temperature was measured. Data was analyzed using a repeated-measures ANOVA (p < 0.05) and location of interaction effects determined using a Fisher’s least squares difference test. We observed a higher change (p < 0.05) from baseline skin temperatures (ΔTsk) for women in three locations: left upper back (women: avg. ΔTsk = 4.12 ± 0.20 °C; men: avg. ΔTsk = 2.70 ± 0.10 °C), right upper back (women: avg. ΔTsk = 4.19 ± 0.07 °C; men: avg. ΔTsk = 2.92 ± 0.05 °C), and right mid-back (women: avg. ΔTsk = 4.62 ± 0.14 °C; men: avg. ΔTsk =3.55 ± 0.09 °C). Individual time differences between genders occurred after 7- (left upper back) and 15-min (right upper back, right mid-back) of exercise and were maintained until the end of exercise. Women have a greater increase in skin temperature at three locations on the back following the onset of exercise in a hot, humid environment. This report provides important information regarding the implications of women exercising in a hot, humid environment.
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