AJAS 2020 Delegates

Investigating the Effect of Up-regulating the GABA and NO/cGMP/PKG Pathways as a Potential Therapy for Epilepsy Using Caenorhabditis elegans as a Model Organism

Around the world, 65 million people have been diagnosed with some type of epilepsy. Of them, 1/3 have uncontrollable seizures from ineffective treatments. Epilepsy is a common neurological disorder in which nerve cell activity is disturbed, causing recurrent seizures. A seizure is a brief disruption in normal brain activity that interferes with brain function. Having a seizure does not mean you have epilepsy, but having epilepsy means you will have seizures. This particular study focuses on treating tonic-clonic, or convulsive, seizures by augmenting the gamma-aminobutyric acid (GABA) and protein kinase G (PKG) pathways. This study hopes to provide an improved treatment for epilepsy by using various drugs that interact with the GABA and PKG pathway. A seizure was induced on the worms by electroshock. Then, recovery time of the worms and the percentage of worms convulsing over time were recorded. To ensure accuracy, ten trials were conducted for each treatment with six worms in each trial. The data collected supported a 75 mM dosage of vigabatrin as the most effective treatment for epileptic seizures (data significant with p < 0.0005). These findings can be applied in the real world as a treatment for epilepsy patients who currently have ineffective antiepileptic drugs. In the future, different combinations of drugs and drugs that interact with different pathways, should be tested using a similar study. This will allow for a greater amount of antiepileptic drugs to be developed to help the 12.7 million people who have uncontrollable seizures live better lives.

Using 25-Hydroxyvitamin D3 to Protect the Epigenetic Modifications Leading to Type 2 Diabetes in Drosophila melanogaster 

Currently, over 415 million people worldwide have diabetes and within that population, 90% - 95% have type 2 diabetes; increasingly more children and young adults are also developing it. The research conducted in this study aimed to assess the role of 25-Hydroxyvitamin D3 solution, at varying concentrations, in protecting diabetic-like phenotypes from being present in and passed on from the parents to offspring as well as to limit DNA methyltransferase activity in fruit flies. It was hypothesized that the highest concentration of vitamin D solution (0.055 ul/ml) would be most effective in having a protective role, with the exception of that in the DHR96 mutant flies, as they lack the vitamin D receptor (negative control). Fruit flies were suitable model organisms, as they share 75% of the disease-causing genes with humans and can develop diet-induced insulin resistance. All fly groups (including parents and offspring) were utilized for assays including measuring adult body mass, wing size, glucose and sugars content, and a DNMT kit used to quantify DNMT activity. The results indicated that the hypothesis was supported; the highest concentration of vitamin D solution was most effective in the offspring of both the wildtype and InR mutant fly groups. Therefore, the research conducted can be applied to further studies to show the role of 25-Hydroxyvitamin D3 as a potential DNMT inhibitor (specific to genes associated with type 2 diabetes with further studies) and as a potential treatment for type 2 diabetes.

The Effects of Strains of Pseudomonas Bacteria on the Production of Polyhydroxyalkanoates (PHA) from Polyethylene Terephthalate (PET)

Polyethylene terephthalate (PET) is widely used industrially due to its optimal properties, including extreme durability. The goal of this study was to test the optimal conditions in which a certain strain of  Pseudomonas bacteria can produce polyhydroxyalkaonates (PHA) via catalysis, particularly through the fermentation of terephthalic acid (a component of PET). If successfully completed, PET, which is often found in nondegradable substances like water bottles and tires, can be recycled into PHA, which is known to have optimal characteristics that allow for use in various fields. It was hypothesized that if Pseudomonas Putida is allowed to ferment terephthalic acid, then it will produce the greatest ratio of collected PHA to PET. This hypothesis was made based of off the lack of research on P. Aeruginosa and Fluorescens. It was further hypothesized that the resulting PHA from Pseudomonas Fluorescens will in turn be the slowest degrading polymer. First, Pseudomonas aeruginosa produced the highest amount of PHA, with an average of 56.363% of the expected level. The distribution of Pseudomonas A. lied above that to Pseudomonas P. and F., thus not supporting the initial hypothesis. Furthermore, Pseudomonas F. resulted in a high rate of degradation of 0.8108 while the lowest was Pseudomonas A. with a rate of 0.0266. Thus, both hypotheses were rejected with Pseudomonas A. proving to be optimal in production of PHA while Pseudomonas F. seemed to show higher durability. Overall, the stark differences in durability allow for a novel mechanism of recycling PET into a substance of great use.

Adult Stem Cells for Musculoskeletal Tissue Regeneration

Adult mesenchymal stem cells (MSC) have been cited to have immunoregulatory effects in addition to their regenerative and multipotency capabilities, meaning they contribute to a regenerative environment that can help repair injuries in vivo and secrete bioactive macromolecules that regulate the immune response after injury. MSCs’ abilities include regulating inflammation, apoptosis, and angiogenesis by affecting immune cell lineages. A subset of MSCs of perivascular niche origin are characterized by the presence of the CD146 surface antigen. These CD146+ MSCs possess greater differential capabilities and immunoregulatory potential. This research compares the effectiveness of bone marrow (BM) MSCs and fat pad (FP) MSCs in responding to an inflammatory environment as well as pericytic MSCs compared to other MSCs. For each subset of stem cells tested, the inflammatory and growth factor profile was quantified in basal, non-inflammatory, conditions and after exposure to an inflammatory environment induced by human inflammatory cytokines TNFα and IFNγ. RT-PCR for the GAPDH, CD146, IL-6, IL-8, and IDO genes and ELISA human growth factor and inflammation antibody arrays were used to quantify changes in response to inflammation. Qualitative comparison of quantitative results suggests that CD146+ FP MSCs are the most effective subset of MSCs at responding to inflammation in vitro. As a result, CD146+ FP MSCs are the best stem cell subset to use for the millions of musculoskeletal injury treatments each year in order to regenerate injured tissue to shorten the healing process as well as prevent prolonged inflammation, scarring, and chronic tissue damage at injury sites. 

Developing a red/far-red light responsive PLEKHA7 expression plasmid to repress the proliferation of anchorage-independent colorectal cells via adeno-associated virus and electroporation transmission routes.

Colorectal cancer is a leading cause of death among men and women in the United States whose survival rates exponentially decrease as it progresses and metastasizes without diagnosis or treatment. By genetically re-engineering cancerous tissue, gene therapy seeks to revert its malignant status and reestablish proper functionality.

This study aimed to reestablish the integrity of the zonula adherens in colorectal cancer cells through the re-introduction of the deficient PLEKHA7 protein, a key regulator of anchorage-independent growth. To do this, this study implemented genetic engineering to transfect targeted colorectal cancer cells with the PLEKHA7 gene and compensate for its down-regulated state during cancer. To minimize potential medical complications and maximize the manipulability, cost-efficiency, and site-specificity of gene expression, a red-light inducible and far-red-light repressible plasmid with the PLEKHA7 gene was constructed to restore the PLEKHA7 concentrations in the colorectal cancer cells, and ultimately thwart their proliferation and metastasis.

The Development of a Two Pronged Treatment Targeting the NF-kB and MGMT Pathways by Creating a Novel Drug Cocktail Using Temozolomide, Disulfiram, and Zinc for Newly Diagnosed Glioblastoma

Grade IV glioblastoma is the most lethal and aggressive brain tumor in adults. Despite treatment advances combining maximal surgical resection with radiotherapy and concurrent adjuvant chemotherapy (temozolomide), a patient prognosis remains marginal and survival is limited to 14.6 months. Furthermore, higher end therapticual treatments such as gene therapy are financially crippling, with costs starting at around $500,000 and rising to 1.5 million dollars. As repositioned drugs inflict minimal harm on the human body and, compared to newly developed drugs, are 170 million times less expensive to bring into market and keep it in market, researchers are becoming increasingly more interested in this area of treatment. 

With this problem in mind, various combinations of zinc, disulfiram (Antabuse), and temozolomide were tested to treat glioblastoma. A combinatorial matrix of the three drugs was created and alamar blue, proteasome-glo, and cell proliferation assays were conducted on newly diagnosed glioblastoma cell line (ATCC 73M). The alamar blue test demonstrated a time dependent statistically significant cytotoxic effect of various drug combinations. When analyzing both the proteasome-glo and cell proliferation data, temozolomide and disulfiram alone were the least effective in inducing apoptosis. However, when these drugs were combined with zinc, their activity showed a synergistic cytotoxic effect, thus resulting in a sharp decline in proliferation and proteasome activity in the glioblastoma cells. 
If the most effective drug combinations are able to succeed in clinical trials, a viable treatment would be introduced to glioblastoma patients, thus potentially increasing patient prognosis.

The advent of artificially engineering tissues promises large-scale replacement of damaged vital organs which can also help to transcend the alarming ratios of recipients in need of transplants to donors available. The current clinical success of tissue engineering are limited to low-metabolism, acellular, prevascularized, and thin tissues. The primary factor limiting the progression of tissue engineering is hypoxia. Given that a toxic oxidative milieu can be generated via hypoxia, cytokines, and inflammation, cellular transplants are particularly susceptible to oxidative damage, resulting in increased cell death and decreased efficacy of implants. Hence, this investigation reports the design and validation of a scaffold which can generate its own supply of oxygen for prospective applications in tissue engineering constructs. In response to the need for economically translatable scaffolds, cellulose producing pristine bacterial cultures and kombucha tea derived cultures were repurposed and cultivated for harvesting cellulose membranes. The verification of a one step modulation to alter the entire structure of the scaffold offers an efficient methodology which enhances porosity. Additionally, the two component system of the bacterial cellulose biomaterial and INS-1 cells demonstrates the first step towards bacterial cellulose based platforms to support cell and regulate cell morphology on implanted biomaterials using only the scaffold and cells, without added adhesion promoters. The ability to cultivate this novel scaffolding platform presents the possibility to further fine tune structural characteristics of the scaffold to cater to an array of cell types and be utilized to treat a variety of conditions previously thought to be incurable and chronic.

DODDER GROWTH CHANGES INDUCED BY AUXIN THROUGH A TOMATO HOST

Abstract: Our research is essential to agriculture and understanding the basic biology of parasitic weeds. We studied growth changes in Cuscuta pentagona by venous connection to host tomato. Two methods that we used included measuring dodder stem length and time-lapse photography of stem movement using a GoPro Hero 4 action camera. Time-lapse video recordings, representing more than 1400 hours of experimental interaction between dodder and its host were watched to gather data. We made different concentrations of IAA (auxin) in an agar media and applied two of each concentration to sandpapered stems of tomato. We used a bridge like system to apply dodder to the juvenile tomato. The system we used involved removing the free-growing stem of Ludwigia peruviana with the haustorium-bearing stem region of Cuscuta pentagona. We took cuttings of Ludwigia peruviana near the treated tomatoes under grow lights and watered as needed. During the beginning of the “searching” behavior, 3 stems created a successful bridge to hosts 6, 7, and 8 in our 2nd trial. This bridging flourished with 9 stems of dodder starting a whipping motion, soon looping far enough to be recognized as circumnutation. A curious observation made during the experiment was that the only three dodder that bridged to tomato occurred in tomatoes with low or no auxin agar treatment. A behavior observed in tomato hosts was the hypersensitive response to haustorial connections. Although it is not yet determined, it is hypothesized to be due to either disease transmitted by dodder or by the haustorium intrusion itself.

A Novel Approach to Combating Obesity: Targeting the Antigen Presentation of Adipocytes to Reduce Inflammation in the Obese Adipose Tissue

Obesity is associated with chronic inflammation of the adipose tissue. This inflammation aggravates the current state of obesity, making it harder to treat, and leads to life-threatening side effects. To combat obesity, it is crucial to combat its inflammation first. Unfortunately, inflammation of the adipose tissue is not well understood. This study aims to establish fat cells can present antigens to induce an inflammatory response and stimulate immune cells to secrete pathogenic IgG autoimmune antibodies as novel mechanisms behind obesity-induced inflammation, and to target these mechanisms with potential anti-inflammatories--anti-TNF, metformin, or Vitamin D--to ultimately combat obesity from an innovative immunological approach. To conduct this study, RT-qPCR, immunofluorescence, and ELISAs were performed to quantify antigen presentation, visually display protein expression, and quantify autoimmune antibody secretion respectively. The results suggest that fat cells can present antigens, a process normally performed by immune cells, and induce the secretion of pathogenic antibodies that are directed to attack the fatty tissue. These results also established that anti-TNF, metformin, and Vitamin D are potential anti-inflammatory agents that can target the newfound immunological properties of fat cells to ultimately combat obesity. In conclusion, this study sheds light on mechanisms behind obesity-induced inflammation and therapeutic agents to combat it.  All data was statistically significant when a t-test and an ANOVA was run (ɑ=0.05).

The Effect of Glyphosate on Gene Expression in Saccharomyces cerevisiae

Glyphosate is a widely used chemical herbicide and the active ingredient in the weedkiller “Roundup.” Glyphosate recently came under public scrutiny with the World Health Organization’s reclassification of glyphosate from noncarcinogenic to “probably carcinogenic to humans.” This assessment references a series of surveys identifying correlations between glyphosate exposure and Non-Hodgkin’s Lymphoma. This study used Saccharomyces cerevisiae, baker’s yeast, as a model to assess the effect of glyphosate on gene expression. If the increased occurrence of Non-Hodgkin’s Lymphoma is the result of glyphosate-induced mutations, then upregulation of genes associated with DNA maintenance and repair would be expected in yeast cells exposed to higher concentrations of glyphosate. For this experiment, yeast cells were cultured in solutions of varying concentration of glyphosate. Protocols were followed to extract total RNA from the yeast cells, standardize total RNA, and convert total RNA to cDNA. PCR was performed using select primers for specific yeast genes with human homologs: TEL1, RAD54, TDH1, POR1, and PFY1. Gel electrophoresis qualitatively assessed the relative production of each gene via the intensity of each band. Images of the gels were loaded into a python program, which calculated the intensity of each band. TDH1 is a gene involved in glycolysis, a critical cellular function; its production was conserved in all samples. TEL1 and RAD54 are genes involved in DNA maintenance and repair; they experienced a 2.5x upregulation in the highest glyphosate concentration, 1000mg/L. POR1 and PFY1 failed to resolve. This supports the hypothesis and necessitates further research on glyphosate’s carcinogenicity. 

A Pilot Study on the Clinical Utility of SPECT-CT

Nuclear imaging is a popular procedure used by hospitals to investigate the function of a patient’s internal organs non-invasively. One common procedure is Single Photon Emission Computed Tomography (SPECT). There are many advantages and disadvantages to each procedure. PET imaging is able to take high resolution images but is expensive to operate. SPECT imaging is less expensive but is not able to produce as high-resolution images as PET’s. Because SPECT imaging is less costly, it is more widely available in hospitals. Additionally, SPECT imaging can be less accurately interpreted due to motion artifact

This study uses a low-resolution Computer Tomography (CT) scan in conjunction with SPECT scan to correct for motion artifact, also known as attenuation correction. The purpose of doing so is to see whether or not the hybridization of these two procedures will procure a more accurate image for a doctor to identify cardiac ischemia, decreased blood pressure to the heart. In our case, we will be using SPECT-CT scans targeted at heart disease patients to see whether or not the CT scan changed the diagnosis of the patient.