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Research in the lab
We have several different research projects. Some of these projects are intertwined, while others stand alone. Most of my research students work in more than one area.
Project #1: The Phytochemicals of Pseudognathalium obtusifolium
Studying the regional plant, Pseudognaphalium obtusifolum, an annual herb that is a member of the Asteraceae family, may lead to the discovery of possible medical uses. Historically, this Eastern North American plant has uses in traditional medicine among many Native American groups and early settlers of our region, yet little scientific research has delved into its actual medicinal value.
The ability to extract phytochemicals having specific chemical properties from plant material is important for scientists wishing to study the secondary metabolites found in the extract material. Some secondary metabolites have the potential of playing several roles within the cell including acting in conjunction with defense mechanisms and as chemical messengers. In recent years, it has been determined that the phytochemicals found in some plant species have anti-carcinogenic properties and many researchers have focused their work on uncovering those compounds. Their ability to possess antioxidant, anti-inflammatory, antimicrobial, and anti-tumorigenic properties has placed significance on their pharmacological effects.
Flavonoids are some of the most abundant secondary metabolites and are known for their biochemical activity involving the introduction of reactive oxygen species (ROS) into the cell. Their anti-tumorigenic effects have been linked to their ability to introduce ROS which can induce cell cycle arrest and alter protein kinase activity. This can potentially cause a gradual decrease in cellular proliferation or self-induced apoptosis. Currently, there are approximately 37 flavonol, 28 flavone, and 1 flavanone derivatives that can be found within the Asteraceae family and Gnaphalium genus. At least three flavenoids are specific to P. obtusifolium.
Crude Pseudognaphalium obtusifolum extracts were initially obtained from collected plants by treating with liquid nitrogen and homogenization, followed by treatment with different solvents – ethanol, hexane, and dichloromethane. Obtained extracts were suspended in DMSO. Our recent research has improved upon the previous extraction method. We hope to analyze each of the derived extracts in order to identify specific phytochemicals, especially flavenoids, which may have effects on eukaryotic cells. Of the available derived extracts, dichloromethane (DCM)-derived extract is the most likely to contain flavenoid molecules based on the known structure of flavenoids.
Project #2: The Effects on Pseudognathalium obtusifolium Phytochemicals on Saccharomyces cerevisiae
Saccharomyces cerevisiae (budding yeast) is considered a model eukaryotic organism and is being utilized to investigate the general effects of each of the derived extracts on eukaryotic cells. Changes in cell proliferation are determined by spectrophotometric methods, plate colony counting, and hemocytometry. In both quantitative and qualitative assessments, yeast cultured with a particular dilution of hexane-derived extract were shown to have an increase in proliferation. Yeast treated with ethanol- and dichloromethane-derived extracts have decreased proliferation.
Additionally, treated cells are being observed by immunofluorescence microscopy to help determine changes in mitochondrial morphology an distribution. Apoptotic mitochondrial morphology was observed in cells treated with dichloromethane-derived extract.
Currently, we are repeating experiments to determine proliferation changes by flow cytometry and to assess possible ROS-protection for cells treated with hexane-derived extracts.
Project #3: The Effects on Pseudognathalium obtusifolium Dichloromethane-derived Phytochemicals on MCF-7 Human Breast Ductal Adenocarcinoma Cells
This research aims to determine if dichloromethane-derived extracts from P. obtusifolium, likely to contain flavenoids, affect the proliferation, viability, and induction of apoptosis in MCF-7 human breast ductal adenocarcinoma cells. MCF-7 is an estrogen-dependent cell line. Estrogen is synthesized from testosterone irreversibly via the enzyme aromatase, which is vital for human tissues such as the uterus, breast, and the placenta.
We used dichloromethane-derived extract to treat MCF-7 cells. Results from flow cytometry analysis revealed that proliferation was significantly decreased in both a time and dilution-dependent manner. LDH toxicity assays revealed the extract was cytotoxic, and cells exhibited signs of apoptosis by light microscopy. Changes in expression of 43 apoptotic signaling proteins were determined using antibody arrays. We have utilized this data to construct a proposed signaling pathway, indicating increased or decreased expression of the factors involved in apoptosis of MCF-7 cells treated with dichloromethane-derived extract.
We are currently studying changes in expression of specific MCF-7 apoptotic factors by qPCR to help further deduce the apoptosis signaling pathway that is induced by treatment with dichloromethane-derived extract. Also, since some phytochemicals can be used as aromatase inhibiters, we are conducting studies to determine if anti-aromatase activity of phytochemicals in dichloromethane-derived extract could be the cause of decreasing proliferation and apoptosis of MCF-7 cells.
Project #4: Detecting and Exploring Alu Frequencies using PCR and Bioinformatics
We are using polymerase chain reaction (PCR) to examine a nuclear DNA molecular genotype and learn about population genetics. In our experiments, we are detecting the presence (or absence) of a length polymorphism, the chromosome 16 Alu insertion allele. The Alu "jumping gene" is a transposon that is dependent upon the L1 transposon for movement within the human genome. The two-allele Alu and L1 genetic system has three genotypes.
Using Allele Server (www.bioservers.org),we are able to integrate our results with bioinformatics. We are testing Hardy-Weinberg equilibrium, relating our data to worldwide allele and genotype frequencies, and exploring sequence data.
Project #5: Detecting and Exploring mtDNA Haplotypes I and HV
Separate from the DNA found in the nucleus of our cells (nDNA), mitochondria have their own DNA (mtDNA). This genetic material is passed to each of us along a matrilineal lineage (our mothers). Over time, single nucleotide changes occur in mtDNA and are inherited by future generations in a given population. These changes, called single nucleotide polymorphisms (SNPs), mostly occur in a region of the mtDNA known as the hypervariable region. Human mtDNA haplogroups are defined by differences in the SNPs of mtDNA. Each haplogroup represents a major branch point in the mtDNA phylogenetic tree.
Population geneticists use information about the history of female lineage throughout human history to trace the inheritance of mtDNA. This mtDNA evidence indicates that modern humans, now spread around Earth, originated in Africa. All mtDNA haplogroups, when traced back to our origins in Africa, lead back to the matrilineal most recent common ancestor (MRCA) for all humans currently living on our planet. This woman is commonly referred to as Mitochondrial Eve.
Using polymerase chain reaction (PCR) to amplify a specific regions of mitochondrial DNA (mtDNA) found in the hypervariable region, we are determining the presence (or absence) of single nucleotide polymorphisms (SNPs) indicating the I and HV haplotypes. Additionally, Analysis for the HV haplotype requires restriction analysis. Using bioinformatics, our data and DNA sequences of the amplified region are compared with that of other populations.
According to population geneticists, the most successful maternal lineage in Europe and the Near East is Haplogroup HV. Most Europeans (over half) and many Near Easterners (about 25% - 40%) are descendents of a common female ancestor. This single common ancestor, and founder of the HV lineage, lived from 25,000 - 40,000 years ago. The majority of HV haplogroup Europeans belong to a branch of HV, the H haplogroup. Some other Europeans belong to a smaller branch of HV, the V haplogroup. The I Haplogroup is rare, being found in about 2% of Europeans, and for Near Easterners, less than 1% of the population.
Project #6: Effects of Bromelain on the ATCC HTB-55 Cell Line and Mucin Secretion
These studies focus on use of the Calu-3 cell line (American Type Culture Collection, ATCC HTB-55) as a respiratory model for studying the effects of natural plant enzymes, collectively referred to as bromelain, on human airway epithelial cells. Calu-3 cells are derived from human lung bronchial submucosal glands that have the most impact on airway surface liquid relative to other pulmonary cell types.
Calu-3 monolayers polarize at an ALI (air-liquid interface), produce mucins, and secrete substances that are immunologically active in a manner characteristic of bronchiolar epithelium. When exposed to therapeutics, including pharmacological agents, integrity and permeability of the monolayer, cell viability, and histology can be used to measure response. These studies may be beneficial in understanding the physiology of mucin secreting glands and their roles in a variety of respiratory disorders.
Our focus is how bromelain may be utilized to provide symptomatic relief to those suffering from abnormal secretion of mucins, including that found in cystic fibrosis (CF). A CF genetic mutation leads to damage in many areas of the body, including the lungs, due to abnormal transport of chloride and sodium ions across the epithelium. The up-regulation of mucin-expressing genes found in lung cells in CF results in mucin secretions of increased viscosity. Over time, the lungs become permanently damaged. We have determined the effects of bromelain on Calu-3 cells through the study of bromelain toxicity (TSH assay), studies of cell viability and metabolism (MTS assay). We further aim to study the ability of bromelain to breakdown secreted mucins (spectrophotometry) and learn more about the mechanisms using immunofluorescence microsopy to track cellular uptake of bromelain, measuring changes in mucin secretion or production, and monitoring changes in Calu-3 production of reactive oxygen species.
Since epithelium of the airway is involved in the pathogenesis of many disorders, understanding the role of the airway epithelium, how mucin secretion is regulated and may be reduced, and the Calu-3 response to bromelain, this work may facilitate the development of a novel therapy for certain respiratory disorders.
PEER REVIEWED RESEARCH JOURNAL ARTICLES AND REVIEWS
Whittom, A., Villarreal, A., Soni, M., Owusu-Duku, B., Meshram, A., Rajkowska, G., Stockmeier, C., Miguel-Hidalgo, J. (2014) Markers of apoptosis induction and proliferation in the orbitofrontal cortex in alcohol dependence. Alcohol Clin Exp Res. Nov; 38(11):2790-9. #
Rana, S., Verdin, L., Verdin, L., Giuffria, B., Beasley, J., Bridges, L., Cooper, A., Dhawan, G., Dolia, R., Downing, J. M., Ferger, Z., Hasan, S., Lawson, W., Nguyen, Y., Sipin, A., Shnaydruk, A., Waquad, A., Wilbert, G., Williams, Z., Windham, J., Rosado, D. J., and Whittom Reiken, A. (2014). Use of Pseudognaphalium obtusifolium in Native American Traditional Medicine and its Possible Health Benefits. NAAAS & Affiliates 2014 Monograph Series. #
Miguel-Hidalgo, J.J., Whittom, A., Villarreal, A., Soni, M., Meshram, A., Pickett, J.C., Rajkowska, G., and Stockmeier, C.A. (2014) Apoptosis-related proteins and proliferation markers in the orbitofrontal cortex in major depressive disorder. J. Affect. Disord. 2014 Apr; 158:62-70. #
Miguel-Hidalgo, J. J., Whittom, A., Soni, M., Stockmeier, C. A. (2010) Caspase 8 in the orbitofrontal cortex in major depression, alcoholism and comorbid depression and alcoholism. Alcohol Clin Exp Res. 34(S2): 151A.
Miguel-Hidalgo, J.J., Waltzer, R., Whittom, A. A., Austin, M. C., Rajkowska, G., Stockmeier, C. A. (2010). Glial and glutamatergic markers in depression, alcoholism, and their comorbidity. J. Affective Disord. 127(1-3): 230-40.
Bidwell, III, L., Whittom, A. A., Thomas, E., Lyons, D., Hebert, M. D., and Raucher, D. (2010). A thermally targeted peptide inhibitor of symmetrical dimethylation inhibits cancer-cell proliferation. Peptides. 31(5): 834-841.
Hearst, S. M., Gilder, A. S., Negi, S. S., Davis, M. D., George, E. M., Whittom, A. A., Toyota, C. G., Husedzinovic, A., Gruss, O. J., and Hebert, M. D. (2009). Cajal-body formation correlates with differential coilin phosphorylation in primary and transformed cell lines. J. Cell Sci. 122 (11): 1872-1881.
Whittom, A. A., Xu, H., and Hebert, M. D. (2008). Coilin levels and modifications influence artificial reporter splicing. Cell. Mol. Life Sci. 65 (7-8): 1256-1271.
Hebert, M. D. and Whittom, A. A. (2007). Gene-based approaches toward Friedreich ataxia therapeutics. Cell. Mol. Life Sci. 64 (23): 3034-3043.
ABSTRACT PUBLICATIONS WITH ASSOCIATED PRESENTATIONS
Whittom Reiken, A., McDonnell, M., Thompson, A. , Witherspoon, J., Suller, A., Pickering, A., Alqahtani, O., Baik, C., Bedi, A., Bodige, K., Mao, Y., Peavy, L., Rijal, A., Thapa, P., Uttiramerur, A., and Walker, J. (2016). Dichloromethane-derived Pseudognathalium obtusifolum extract induces MCF-7 human breast cancer cell apoptosis. Abstract (and poster presentation by A. Reiken, A. Uttiramerur, A. Suller, and C. Bidgood), XCelligence Cancer & Immunotherapy Symposium, Friday, April 15, 2016 - Saturday, April 16, 2016, New Orleans, LA.
Thompson, A., Johnston, J., Witherspoon, J., Boduggam, T., Burri, S., Dhawan, G., Dixon, J., Paden, S., Price, T., Salinas, F., Singh, N., and Whittom Reiken, A. (2016). Analysis of changes in MCF7 human breast cancer apoptosis protein expression following treatment with dichloromethane-derived Pseudognaphalium obtusifolium extract. Abstract for Presentation by Ambreanna Thompson, Mississippi Academy of Sciences 80th Annual Research Symposium. J. Miss. Acad. Sci. 61 (1):42. #
Suller, A., Bodige, K., Lavallais, C., Peavy, L., Penumaka, G., Price, T., Sindhu, R., Weber, N., Bernius, K., Felix, S., Hancock, R., Hart, H., Hoye, J., Irby, T., Lumpkin, D., McPhail, A., Mosley, A., Rittenhouse, J., Tibbs, E., Witherspoon, J., and Whittom Reiken, A. (2016). The effects of Pseudognathalium obtusifolum extracts on Saccharomyces cerevisiae HBO strain proliferation and mitochondrial morphology. Abstract for Presentation by Amanda Suller, Mississippi Academy of Sciences 80th Annual Research Symposium. J. Miss. Acad. Sci. 61 (1):37. #
Carter, K., Alqahtani, O., Boduggam, T., Campbell, D., Long, L., Patel, N., Thompson, A., McDonnell, M., Witherspoon, J., and Whittom Reiken, A. (2015). Diminished MCF7 Human Breast Cancer Adenocarcinoma Cell Proliferation Consequent to Treatment with Whole Leaf Pseudognaphalium obtusifolium Extract. Long and Short Abstracts and Oral Presentation, Presented by Kaylan Carter. 12th Annual Mississippi College -Tougaloo College Undergraduate Research Symposium, April 9-10, Mississippi College, Department of Biological Sciences, Clinton, MS. #
McDonnell, M., Carter, K., Algahtani, O., and Whittom Reiken, A. (2015). Dichloromethane-derived Pseudognathalium obtusifoilum extract decreases proliferation of breast cancer ductal epithelial adenocarcinoma cell line. Abstract, Oral Presentation, and Poster Presentation, Presented by Mary McDonnell, Mississippi Academy of Sciences 79th Annual Research Symposium and the Mississippi INBRE Graduate Scholars Symposium, February 26-27, 2015, Hattiesburg, MS, Health Sciences Division. J. Miss. Acad. Sci. 60 (1):114. #
Hirt, S., Young, R., Johnson, J., Fu, E., Tolbert, K., Thomas, D., Girimaji, A., Takkallapelly, S., Townes, J., Wright, J., McDonnell, M., Rosado, D., and Whittom Reiken, A. (2015). Effects of Pseudognathalium obtusifolium extracts on proliferation of Saccharomyces cerevesia. Abstract and Oral Presentation, Presented by Mary McDonnell, Mississippi Academy of Sciences 79th Annual Research Symposium, February 26-27, 2015, Hattiesburg, MS, Cellular, Molecular and Developmental Biology Division. J. Miss. Acad. Sci. 60 (1):51. #
Long, L., Bridges, L., Zenon, A., Rana, S., Rosado, D., and Whittom, A. (2015). Pseudognathalium obstusifoilum: revealing the mysterious secrets of a commonplace native weed. Abstract and Oral Presentation, Presented by Leanna Long, Mississippi Academy of Sciences 79th Annual Research Symposium, February 26-27, 2015, Hattiesburg, MS, Agriculture and Plant Science Division. J. Miss. Acad. Sci. 60 (1):42. #
Carter, K., McDonnell, M., and Whittom Reiken, A. (2015). Effects of whole leaf Pseudognathalium obstusifoilum chemical extracts on proliferation of MCF7 adenocarcinoma cells. Abstract and Oral Presentation, Presented by Kaylan Carter, Mississippi Academy of Sciences 79th Annual Research Symposium, February 26-27, 2015, Hattiesburg, MS, Health Sciences Division. J. Miss. Acad. Sci. 60 (1):114. #
McDonnell, M., Carter, K., Algahtani, O., and Whittom Reiken, A. (2015). Proliferation, cell viability, toxicity, and protein expression analysis of MCF7 breast ductal epithelial adenocarcinoma cells cultured with Pseudognaphalium obtusifolium dichloromethane-derived extract indicates a possible anti-carcinogenic treatment alternative. Poster presentation, Presented by Mary McDonnell. Mississippi INBRE Division, 31st Annual Southern Biomedical Engineering Conference, April 30- May 2, 2015, Kenner, LA. 31st Southern Biomedical Engineering Conference Program, 36. #
Patel, N., Thomas, D., Carter, K., Samaradivakara, N., Zenon, A., St. Fleur, V., and Whittom Reiken, A. (2015). Community services shadow education projects and activities to reinforce basic science principles. Abstract and Oral Presentation, Presented by Neil Patel, Mississippi Academy of Sciences 79th Annual Research Symposium, February 26-27, 2015, Hattiesburg, MS, Science Education Division. J. Miss. Acad. Sci. 60 (1):147. #
Rana, S., and Reiken, A. W. (2014). Usage of Pseudognaphalium obtusifolium in Native American Culture And its Possible Health Benefits. Abstract and oral presentation for NAAAS and Affiliates International Research Forum, Mississippi College and Universidad Autónoma de Coahuila, April 27-May 1, 2014. #
Downing, J. M., Lawson, W. J., Williams, Z. C., Baig, S. B., Haong, W., Waquad, A. S., Dolia, R. N., Shnaydruk, A., Goski, V., and Whittom Reiken, A. A. (2014) Effects of bromelain on mucins of the ATCC HTB-55 cell line: possibilities for a novel therapeautic. Abstract and poster presentation for Mississippi College and Tougaloo College 11th Annual research Symposium, Tougaloo College, Jackson, MS, spring 2014. #
Verdin, L., Cooper, A., Dolia, R., Downing, J. M., Rosado, D., Sipin, A., Verdin, L., Waquad, A., and Whittom Reiken, A. (2013). Possible ROS inhibition or antioxidant production of Pseudognaphalium obtusifolium hexane extract. Abstract No. 1564. Abstract for Poster Presentation, Presented by Lindsey Verdin, American Society for Cell Biology Conference, New Orleans, LA, fall 2013; Mol. Biol. Cell 24:24 655 (suppl) Abstract No. 1564. #
Verdin, L., Cooper, A., Dolia, R., Downing, J. M., Rosado, D., Sipin, A., Verdin, L., Waquad, A., Williams, Z., and Whittom Reiken, A. (2013). P. obtusifolium hexane extract promotes cell proliferation of HBO yeast strain and demonstrates possible antioxidant effects. Abstract No. 1554. Abstract for Poster Presentation, Presented by Leah Verdin. American Society for Cell Biology Conference, New Orleans, LA, fall 2013; Mol. Biol. Cell 24:24 649-650 (suppl) Abstract No. 1554. #
Dramis, N., Cannon, P., Sullivan, S., and Whittom Reiken, A. (2012). Identifying the mtDNA HV Haplotype. Extended Abstract, Short Abstract, and Oral Presentation, Presented by Nicole Dramis. Mississippi College and Tougaloo College 9th Annual Research Symposium, Tougaloo College, Jackson, MS. #
Giuffria, B., Bridges, L., Ferger, Z., Windham, J., Rosado, D. J. and Whittom Reiken, A. (2012). The Effects of Pseudognathalium obtusifolum Extracts on Eukaryotic and Bacterial Cell Proliferation. Extended Abstract, Short Abstract, and Oral Presentation, Presented by Benjamin Giuffria. Mississippi College and Tougaloo College 9th Annual Research Symposium, Tougaloo College, Jackson, MS. #
Whittom, A. A., Miguel-Hidalgo, J. J., and Soni, M. Caspase 8 in the orbitofrontal cortex in major depression, alcoholism and comorbid depression and alcoholism. Abstract and poster presentation, presented by J.J. Hidalgo. 33rd Research Society on Alcoholism Annual Scientific Conference, June 26-30, 2010 in San Antonio, Texas.
Reiken, A. A., Wolfe, C. L., and Norcum, M. T. (2004). Incorporation and intracellular mapping of his-tagged p43/EMAP II within the multienzyme aminoacyl-tRNA synthetase complex. Abstract and oral presentation for Mississippi Academy of Sciences 68th Annual Meeting, J. Miss. Acad. Sci. 49 (1): 24.
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