The 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.

Oxytocin (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.

Vernal 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.

My abstract talks about obesity as a problem and how triiodothyronine (T3) is changed due to calorie restriction in rat plasma.

Problem: 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.

The 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).

Identification 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.

Survey 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.

Previous 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.

Vascular 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.