Browse the Biology/Ecology Collections
The Investigation of Shrinkage in Apoptotic Bodies03/11/2015The Investigation of Shrinkage in Apoptotic Bodies By Ashley Ilinkoski and Michael Model Objectives Apoptosis is programmed cell death where the cell shrinks then eventually fragments and dies, also considered the point of no return. This method can possibly be controlled by putting cells in different media to ensure ions pouring into the cell rather than out. Shrinkage is one of the most universal and specific signs of apoptotic cell death. (Some data suggest that shrinkage due to water loss is the requirement for apoptosis). However, in some apoptotic systems shrinkage occurs late, after irreversible cell damage has occurred. Such systems provide a good model to investigate the exact role of shrinkage in apoptotic development. One specific hypothesis we wish to address is that shrinkage is necessary for cell fragmentation into “apoptotic bodies”. Methods Apoptosis is induced in HeLa cells with camptothecin or RNA polymerase actinomycin D. After the onset of irreversible changes (i.e., morphological blebbing, mitochondrial depolarization or cytochrome c release) treatments designed to inhibit cell water loss are applied. Aqua porin inhibitors are also being introduced to the cells to see if the porins close to sustain cell life by cutting off water transport. The results include formation of apoptotic bodies and DNA fragmentation. The loss of cell material is monitored by the TIE-TTD microscopy and DNA fragmentation by the TUNEL assay. Results Preliminary data indicates that high-potassium medium that opposes water loss prevents formation of apoptotic bodies. Conversely, high-sodium medium that activates water loss results in active cell fragmentation. Future work will clarify these results. |
The effects of oxytocin variants on grooming behavior in mice03/11/2015Oxytocin (Oxt) is a nine amino acid neuropeptide that was thought to be invariant in its sequence across species. However, recent work in primates has found that in some New World Monkeys there can be one or two amino acid substitutions in the protein. So, to assess whether or not these alternative sequences are functional in mice, we set out to perform a grooming bioassay. We hypothesized that these alternative forms of Oxt would have differential effects on grooming compared to the native protein. To test this hypothesis we performed stereotaxic surgery on mice and implanted guide cannulae aimed at the third ventricle. Following recovery from surgery, mice were injected with two µl of each of four treatments over the course of four days: Saline, Oxytocin, Peptide 1, and Peptide 2. Following each microinjection subjects were videotaped for 30 minutes and the amount of grooming scored by an observer blind to each treatment. At the completion of the study site checks were performed to verify the location of the microinjections. While we are still in the process of analyzing our data- all of our treatments were successfully delivered to the lateral ventricle; thus, no animals will need to be excluded from the study. We predict that treatment with Peptide 1 and 2 will result in less grooming compared to treatment with Oxt. |
The Effect of Leaf Litter Species and Habitat Conditions on Vernal Pool Invertebrates and Communities03/11/2015Vernal pools fill during the spring when snowmelt and rainwater gather in depressions in the ground. Undecomposed leaf litter from previous years will fall into these depressions and become both a shelter and a food source for many invertebrates. Leaf litter from different tree species were tested to compare the invertebrate communities that colonized in each and, ultimately, to determine whether one leaf species was colonized quicker or more abundantly than another. Invertebrates were identified to the family level. The family Asellidae was the most abundant invertebrate type in all leaf species litters, accounting for 56% of the total invertebrate count, followed by the family Chironomidae with 26% of the total. A multivariate analysis showed that there was no significant difference between invertebrate communities among leaf litter types. Samples were heavily dominated by a few families of invertebrates and were colonized by very few other families. The similarity between communities showed that the invertebrates likely perform many of the same ecological functions on different leaf types. As a whole community, this ecological function is primarily to breakdown larger leaf litter so that smaller organisms can continue the decomposition process. |
T3 Concentration Due to Calorie Restriction03/11/2015My abstract talks about obesity as a problem and how triiodothyronine (T3) is changed due to calorie restriction in rat plasma. |
Root decomposition in forest ecosystems: chemical or morphological control?03/11/2015Problem: Roots are the major type of plant tissue that contributes to soil organic carbon. Our study was designed to test whether variation in root chemical and morphological traits change decomposition and soil carbon sequestration rates. Compared to tulip roots (Liriodendron tulipifera), elm roots (Ulmus americana) have higher lignin:Nitrogen ratio, but finer diameter, as well as greater root tip abundance. Based on morphological traits, we expect elm roots to decompose faster because of their higher surface area and fine morphology causing them to easily break into the soil. Based on chemical properties, we expect that tulip roots will decompose faster because they have lower lignin:nitrogen ratio. Since microbial communities can adapt to the quality of locally available nutrients, it is expected that decay rates will be accelerated for tissues that have a ‘home field advantage,’ being more similar to neighboring tree species. Methods: Litterbags filled with soil and elm or tulip roots, including treatment groups of either 1st and 2nd order roots, 3rd and 4th order roots, or entire root systems, were left to decompose for 42 weeks in under trees of both species in riparian forest at Jennings Woods. Results: Tulip roots decomposed faster than elm roots, implying that root tissue chemistry has a greater effect on decomposition than morphological characteristics. The strength of our predicted relationships varied between treatment groups. Decomposition occurred faster under tulip trees regardless of the identity of the roots, giving evidence against ‘home field advantage’ theory. |
Rhododendron Roots03/11/2015The Rhododendron genus can be found in many different habitats around the world but scarce in desserts, and dry forests as well as tundra regions. . Rhododendrons are studied from the xylem to the leaves, but we believe the secret is in the roots. Kong et al., found two different dimensions of root trait diameter across 96 subtropical woody species: a diameter related dimension that may integrate root construction, and possibly maintenance and persistence, with a branching density dimension that may express difference in root plastic responses to environment. We would like to address the question of what really goes on in the roots and if the anatomy and morphology of the roots are connected to the temperature tolerance of different rhododendron species. We also believe that Rhododendron with similar cold tolerances have similar fungi on their roots. The study site was the Helen S. Layer Rhododendron garden at Holden Arboretum. We studied the morphology, anatomy of six species of Rhododendron from three sections: Ponticum section (Maximum, Degronianum), Pentanthera section (Austrinum, Molle), and Tsutsuti (Yedoense, Indicum). |
Recombination rate and nucleotide diversity in recombination hot-spots in HIV-1 Pol gene from low-income and high-income countries03/11/2015Identification of recombination hotspots in HIV-1 genome is animportant tool that can be used to predict evolution of the virus and to inform the design of an antiviral drug that avoids targeting the recombination hotspots (e.g. Gao, 2009). Depending on the different selective pressures acting on different genomic regions, recombination can facilitate the accumulation of mutations and accelerate the emergence of resistance to therapies, dissociate favorable combinations of mutations or contribute to emergence of multi-drug resistance bycombining multiple drug resistance mutations in a single recombinant product. The latter is mostly a problem in low- to middle-income countries as multiple subtypes tend to co-circulate in these countries (e.g. Rajaram et al, 2007). For example, under conditions of fast spreading of the virus, such as in standing social intravenous drug user networks, the chances of super-infection, and thus recombination, are greater than that of other transmission modes, suggesting that such epidemics may have a higher rate of virus recombination (Berry et al,2009). We hypothesize that differences in socioeconomic status between countries likely to influence difference in recombination rate of HIV-1 genes that are under strong purifying selection pressure (e.g. Pol gene), which in turn can influence the levels of nucleotide diversity of the virus. In this project we have examined and contrasted the correlationcoefficients between recombination rate and nucleotide diversity of recombination hotspots in Pol gene for low-income and high-income countries. Our study shows that there is a significant difference in recombination events and thus nucleotide diversity in HIV-1 pol gene accounting to socioeconomic status. |
Overview of Ebola History and Spread03/11/2015Survey of evolutionary rate estimates in Ebola virus Ebola virus first appeared in 1976 in Sudan and Zaire as an illness characterized by fever, severe diarrhea, vomiting and a high fatality rate. Since then there were multiple Ebola outbreaks, some deadlier than others. The ongoing epidemic begun in 2013 in Guinea, and then spread to neighboring countries, leading to over 4900 estimated deaths in the region by late 2014, and continuing to ravage the region. The disease is highly contagious when there is a skin-to-skin contact, with the burial practices exasperating the problem of transmission. Recent spread of epidemic outside of remote villages underscores the critical need to develop vaccination and/or treatment options to be broadly available in potentially affected areas. However, up to date vaccine development has been challenging , in part because previous outbreaks were relatively small, thus, little was understood about the evolution of the Ebola virus. Currently, more genomes of Ebola are sequenced and available, thus, enabling phylogenetic and molecular evolutionary studies. In order to know whether vaccine or antibody treatments will maintain efficacy, the nature of genetic change in the virus (especially in the targeted regions) must be ascertained. Thus, in this project we survey published estimates of evolutionary rates of various Ebola genes and/or strains to determine (a) the most conserved genes as the most promising vaccine candidates, and (b) whether the rate of evolutionary changes in the virus has increased in recent epidemics compared to previous ones. |
Investigating Chemical Stress and Antibiotic Sensitivity of Escherichia coli and Staphylococcus epidermidis03/11/2015Previous lab work has investigated the effects of thermal stress and antibiotic sensitivity on bacteria used in microbiology teaching labs concentrating on Pseudomonas fluorescens. This investigation, however, focuses on how chemical stress affects the antibiotic sensitivity of two common laboratory strains: Escherichia coli and Staphylococcus epidermidis. Literature searches revealed that potassium acetate is a common additive in sporulation media. Using the Kirby Bauer procedure – a technique used in clinical labs to characterize antibiotic sensitivity of clinical isolates – we studied how the addition of sodium chloride, potassium chloride, sodium acetate and potassium acetate to growth media affects the sensitivity of E. coli and S. epidermidis. After measuring zones of inhibition created by the antibiotic disks, we found that sodium and potassium acetate increased the sensitivity of the two bacteria significantly, while sodium and potassium chloride had negligible effects. Further studies will characterize the relationship between acetate and potential antibiotic sensitivity. |
Hypoxic tumor microenvironment alters Vascular Endothelial Growth Factor isoform expression in cancer cells03/11/2015Vascular endothelial growth factor (VEGF) is necessary for angiogenesis and tumor growth. VEGF splice isoforms have distinct characteristics, with VEGF121 being more diffusible, thus allowing for more distant angiogenic signaling, and VEGF165 and VEGF189 being found within the cell and the extracellular matrix. We hypothesized that VEGF expression in cancer cells would increase under hypoxia and that the VEGF 121 isoform would be enhanced. We examined VEGF mRNA levels and relative isoform ratios in cells grown in hypoxia or treated with CoCl2 to mimic hypoxia. Prostate cancer (PC3, LNCaP) and leukemia (MOLT-4, K562) cells were initially cultured under normoxic conditions with 20% O2 and then placed in a hypoxia chamber with 1% O2 or treated with CoCl2. RNA was extracted from treated and control cells, and quantitative real-time PCR analysis was performed to analyze VEGF expression under the different conditions. Our findings suggest that hypoxia increased both VEGF121 and VEGF165 expression in prostate cancer and leukemia cells. These results of elevated VEGF121 indicate the potential for lethal metastatic tumor growth distant from the primary tumor site. Overall, this work highlights the role of the hypoxic tumor microenvironment in regulating the functionally distinct VEGF isoforms in cancer cells. |
How marmoset monkeys (Callithrix jacchus) cope with stability challenges during arboreal locomotion03/11/2015For arboreal mammals, problems of stability increase as branches become smaller and more compliant (i.e., move more under the animal’s weight). Changing the size and compliancy of a substrate should cause arboreal mammals to change their gait to facilitate balance. We tested this by having two marmosets (Callithrix jacchus) cross differently sized stable and compliant arboreal supports (5cm, 2.5cm, and 1.25cm in diameter). We filmed the marmosets with four high-speed cameras, testing how they would change their gait in order to maximize stability on thin and unsteady branches. Overall, the marmosets put more limbs on the substrate and for longer periods of time when substrate diameter decreased and when the substrate was compliant. Specifically, duty factors (the percent of time that a limb contacts the support during a stride) increased when the substrate size decreased. There was also a difference in duty factor between the stable and compliant substrates, such that the duty factor increased when the marmosets were on the compliant substrate. The average number of supporting limbs contacting the substrate at any one time also increased when the size of the substrate decreased and when the substrate became compliant. These findings show that marmosets respond to variation in substrate size and substrate compliancy in similar ways, in both cases increasing the average number of limb contacts and the duration of limb contact on a substrate in order to maintain stability. |
Fibroblast Growth Factor 8 Hypomorphy Does Not Affect Astrocyte Arborization Complexity in the Corpus Callosum of Adult Mice03/11/2015Fibroblast growth factor 8 (FGF8) has an array of functions in relation to a developing organism. Previous studies examined the role of FGF8 and its involvement with the formation of the corpus callosum in Fgf8 hypomorphic mice. Astrocytic glial cells are critical for the interhemispheric crossing of callosal fibers, and thus the formation of the corpus callosum. Using GFAP immunohistochemistry (IHC), we showed that midline astrocyte cell number was significantly reduced in heterozygous (+/neo) and homozygous (neo/neo) Fgf8 hypomorphic as compared to wildtype (WT) mice. These results suggest FGF8 signaling is critical for late gestational astrocyte cell development, possibly by acting on the progenitor cells that are destined to become astrocytes. Recently, it was shown that FGF8 increased astrocyte arborization complexity in cell culture (Kang et al., 2014). These findings led us to ask whether FGF8 also affected GFAP immunoreactive astrocyte arborization complexities in the corpus callosum of adult WT and Fgf8+/neo mouse brains using a computerized Sholl analysis. Our results showed, that unlike astrocytes in cell culture, corpus callosal astrocytes do not differ in complexity between WT and Fgf8+/neo adult mice. Currently, we are designing experiments that are designed to elucidate the incongruence between the in vitro and in vivo data. Indeed, cell culture astrocytes are derived from late embryonic animals. Therefore, instead of studying astrocyte arborization in adult, we will use newborn Fgf8 hypomorphic mice. Furthermore, we will study whether FGF8 signaling deficits affect the development of other glial cell types, such as oligodendrocytes and microglia using the IHC markers Olig2 and IBA-1. Future studies will address if FGF8-dependent disruption of midline glial cells development have functional consequences on the ability of midline glial cells to guide extending callosal fibers. |
Evaluating Stereoscopic Effects with EEG of Memory Formation and Regional Brain Activity03/11/2015A current line of research is focusing on the processes of spatial learning. Previous studies have indicated that students have trouble learning concepts which have a spatial component to them. Topics such as molecular geometry within the field of chemistry are notably difficult. Recent research has suggested that stereoscopic presentation of these objects may promote better learning. To understand the processes which contribute to spatial learning a neurophysiological approach is necessary. Therefore, in order to investigate the effectiveness of stereoscopic presentation and the neural correlates of it, an Electroencephalograph (EEG) was used. Participants were fitted with an EEG cap and had electrical scalp activity recorded using Brain Vision software. Participants were presented with images in both 3-D and 2-D of the organic molecule Hemoglobin. During the tasks the molecule rotated across three different planes, changed colors, and changed focal distance. The data collected was then analyzed using EEGLAB for MATLAB. Individual components associated with visual processing (visual cortex), executive processing (pre-frontal cortex), and memory (hippocampus) were identified using an Independent Components Analysis (ICA) and dipole modeling. The brainwave activity in these components were recorded and the components were correlated with one another. Preliminary data collected indicated an increased amount of activation in the visual cortex for 3-D processing as compared with its 2-D counterpart. The goal of these findings is to identify areas of the brain associated with spatial learning through stereoscopic presentation and to identify the optimal conditions for learning various spatial objects and molecules. |
Does sex, age, or species drive gut microbial community similarities between captive lorikeets?03/11/2015Gut microbial communities are known to be associated with immune defense. Changes to this microbial ecosystem can have negative effects on animal health. The Cleveland Metroparks Zoo has recently experienced an abnormally high mortality rate in their captive lorikeet colony, particularly in their younger animals. We hypothesized that lorikeet health may be associated with differences in gut microbial communities. We predicted that differences in gut microbiota between animals may be correlated with particular individual traits including bird age, sex, and species. Amplifying the 16S rRNA gene region of bacterial DNA and using a fragment analysis technique, we assessed gut microbial community structure across 34 birds. Analysis revealed two distinct groupings of similarly structured gut microbial communities across our samples; however, bird sex, age or species did not correlate with these groupings. In future laboratory studies, we will examine whether these two distinct groupings are driven by known deleterious microbes. |
Developmental allometry of the effective mechanical advantage in eastern cottontail rabbits (Sylvilagus floridanus)03/11/2015Juvenile mammals must often compete in the same ecological niche as adults, despite smaller absolute body size and muscle force capacity. However, because limb joints operate as levers, greater muscle mechanical advantage (i.e., ‘leverage’) in juveniles may permit greater force production despite these disadvantages. Previous research on mammals has shown that bony proxies for extensor muscle leverage are greater early in development and decrease throughout growth. However, no previous study of mammalian musculoskeletal development has collected the locomotor data needed to examine changes in actual (i.e., “effective”) mechanical advantage. We used high-speed videography and force plate measurements, combined with anatomical data on muscle lever arm lengths, to calculate effective mechanical advantage (EMA), a measure of limb muscle leverage, exploring the relationship between body size (proxy for age) and EMA. We predicted that if rabbits maintain proportionality during growth, then the length variables that determine muscle leverage should scale to the cube root of overall volume, and therefore the cube root of overall body mass (i.e., Mb0.33). However, we found that muscle lever arms scaled to Mb0.27, whereas the lever arms of the loading forces that the limb muscles had to resist scaled to Mb0.45. Therefore EMA, as the quotient of these two measures, scaled to Mb-0.18, such that muscle leverage decreased by 36% over the course of rabbit growth. Greater leverage early in life may permit young rabbits to attain adult-like levels of locomotor performance, despite smaller size and reduced muscle mass. |
Circadian Analysis on Human Population Entrainment: Inferences from the Power Grid03/11/2015Few, if any studies have focused on the daily rhythmic nature of modern industrialized populations. The present study involved actigraphic analyses of continuously streaming electrical load data from a human subject pool of approximately 43 million primarily residential users in the U.S. Pacific Northwest as a reflection of daily household activity. Rhythm analyses reveal striking seasonal and intra-week differences in human activity patterns, largely devoid of manufacturing and automated load interference. Length of the diurnal activity period (alpha) is longer during the spring than the summer (16.64 h versus 15.98 h, respectively; p < 0.01). As expected, significantly more activity occurs in the solar dark phase during the winter than during the summer (6.29 h versus 2.03 h, respectively; p < 0.01). Interestingly, throughout the year a “weekend effect” is evident, where morning activity onset occurs approximately 1 h later during the weekend than during the work week (5:54 am versus 6:52 am, respectively; p < 0.01). This indicates a general phase-delaying response to the absence of job-related or other weekday morning arousal cues, substantiating a preference or need to sleep longer on weekends. Finally, a shift in onset time can be seen during the transition to Daylight Saving Time, but not the transition back to Standard Time. The use of grid power load as a means for human actimetry assessment thus offers new insights into the collective diurnal activity patterns of large human populations. |
Beta Synemin is a Dual AKAP03/11/2015b-Synemin is a cytoskeletal that also functions to targets PKA to specific subcellular locations, thus it also functions as an A-kinase anchoring protein (AKAP). We have recently confirmed that b-synemin is capable of binding both PKA type I and PKA type II using yeast two-hybrid, FACS-FRET, and co-immunoprecipitation analysis, which therefore classifies it as a dual AKAP. Most interestingly, our co-immunoprecipitation data also suggests that the binding of PKA type I to b-synemin only occurs only upon stimulation of the b-adrenergic pathway. Currently we are carrying out co-immunoprecipitation studies to determine the binding status of PKA type II and the catalytic subunit of PKA under both basal and stimulated conditions. These data are exciting because they suggest temporal compartmentalization of PKA type I and II at a single subcellular location, b-synemin. |