Biological Sciences 2017

Ergot alkaloids in bioactive Metarhizium species fungi | Caroline E. Leadmon and Daniel G. Panaccione 

Division of Plant and Soil Sciences, Davis College of Agriculture, Natural Resources, and Design, West Virginia University, Morgantown, WV 26505 

Fungi in the genus Metarhizium have recently been discovered to have the genetic capacity to produce lysergic acid-derived ergot alkaloids. Metarhizium species are known to be natural insecticides; however, production of ergot alkaloids in insects has yet to be connected with insect fatality. Production of ergot alkaloids appears to be tightly regulated. We grew Metarhizium species on different media and in inoculated insects, and found that alkaloid accumulation depended on fungus species, growth medium, and method of inoculation into insects. To investigate the role of these chemicals in insect pathogenesis we are taking a gene knockout approach. The gene dmaW is known in other fungi to be the first gene in the ergot alkaloid pathway; thus it was selected as our target gene because eliminating it should prevent all production of ergot alkaloids. We are creating the dmaW gene knock-out by replacing the target gene with the gene conferring resistance to hygromycin, an antibiotic. Understanding ergot alkaloids in Metarhizium species fungi may lead to better strategies for biocontrol of insects. 

Elevated nitrogen shifts microbial community structure and function in forest soils | Abby Rosiello, Ashley Henderson, Joseph Carrara, Rajanikanth Govindarajulu, Edward Brzostek and Jennifer Hawkins 

Department of Biology, West Virginia University, Morgantown, WV 26506 

Atmospheric nitrogen deposition owing to the burning of fossil fuels has enhanced the storage of carbon in soils. The role that soil microbial communities play in this process is important but not well understood. In this project, two distinct forest sites (Bear Brook, Maine, and Fernow, West Virginia) that have undergone similar long-term nitrogen fertilization treatments were used to study how soil microbes respond to elevated nitrogen. DNA and RNA were extracted and sequenced from three soil layers (bulk, organic horizon, and rhizosphere) to determine the structure and function of the associated microbial communities. Preliminary metatranscriptomic results show both decreases and increases in transcriptional activity for most major fungi and bacteria under elevated nitrogen, suggesting that elevated N alters microbial activity. It is expected that shifts in microbial community structure will reflect these changes in transcriptional activity. Collectively, these results indicate that shifts in microbial community activity due to N deposition are a dominant driver of greater soil carbon sequestration, and may therefore provide findings that can be used in diverse settings, such as agriculture. 

Functional analysis of a gene involved in synthesis of fungal lysergic acid amides | Jessi K. Tyo and Daniel G. Panaccione 

Biochemistry program and Division of Plant and Soil Science, Davis College of Agriculture, Natural Resources and Design, West Virginia University, Morgantown, WV 26506 

Ergot alkaloids are chemicals that are produced by certain fungi and are important in agriculture and medicine. Many important ergot alkaloids are lysergic acid amides, such as ergonovine and lysergic acid alpha-hydroxyethylamide (LAH). The pathway to create ergonovine is established, but the pathway from ergonovine to LAH is unknown. Based on genome sequence comparisons we hypothesized that a FAD monooxygenase gene ( easO) was involved in the production of LAH from ergonovine. Using PCR methods, we prepared a gene knock out construct and introduced it into the LAH-producing fungus Metarhizium anisopliae by protoplast transformation . Four independent knock outs were identified by PCR strategies that showed the knock-out construct had integrated into the easO locus. High performance liquid chromatography (HPLC) analysis demonstrated that the knock-out fungal strains lacked LAH and retained ergonovine. The data supported our hypothesis that easO was involved in the production of LAH from ergonovine. An understanding of the production of lysergic acid amides is beneficial because of the medical purposes of lysergic acid derivatives. 

Genetic diversity and morphological variation in a vulnerable WV native orchid, Corallorhiza bentleyi | Nicole Fama, Brandon Sinn and Craig Barrett 

Department of Biology, West Virginia University, Morgantown, WV 26506 

Corallorhiza bentleyi is a rare orchid species endemic to five counties along the West Virginia/Virginia border. This recently described species has approximately 10 populations, and is both geographically restricted and locally rare. More research is needed to characterize the genetic diversity the orchid requires for long-term survival . We took a three-fold approach to quantify diversity among populations of Corallorhiza bentleyi. First, microsatellite markers were developed to identify length polymorphisms within C. bentleyi. Fungal host DNA was amplified and sequenced to quantify the degree of specificity towards the orchid’s ectomycorrhizal fungal hosts. Finally, morphological diversity was analyzed with principal components analysis by measuring sizes of flower parts from different individuals. It is expected that, due to small populations sizes, reliance on self-pollination, and reliance on one species as a fungal host, that genetic diversity will be low among populations. The goal of this research is to obtain more information concerning genetic diversity, relationships with its fungal host, and environmental requirements in order to influence management C. bentleyi populations and their habitats in southern Appalachia. 

Mitochondrial DNA evolution in mycoheterotrophic orchid Hexalectris | Ali Ranjbaran, Brandon Sinn and Craig Barrett 

Department of Biology, West Virginia University, Morgantown, WV 26506 

Mycoheterotrophs are a group of plants that parasitize fungi. Partial mycoheterotrophs can still carry out photosynthesis, while others—holomycotrophs—have lost this ability. About half of all mycoheterotrophs are orchids; mycoheterotrophy has evolved >30 times independently in orchids. Heterotrophy in plants is often associated with increased DNA substitution rates, i.e. an increase in the number of DNA mutations over time. Despite the increasing number of plastid genomes being sequenced, there is still little known about mutational processes in the mitochondrial genome. Yet, these genomes hold potential to serve as models for understanding of mutational processes and genome evolution in plants. The 4 main objectives are to: 1) use mitochondrial DNA to compare substitution rates between partial mycoheterotrophic and holomycoheterotrophic species in two North American orchid genera containing both nutritional strategies, Hexalectris and Corallorhiza ; 2) determine relationships based on mitochondrial and plastid DNA; 3) compare substitution rates in Hexalectris (~30 million years old) to a relatively younger Corallorhiza (~3 million yrs old); 4) work towards constructing the first nearly complete mitogenome of any orchid or mycoheterotrophic plant. We observe higher rates of genetic change in holomycotrophic species of Hexalectris in mtDNA, and in part in Corallorhiza. As we hypothesized, Hexalectris showed higher overall mitochondrial substitution rates compared to the younger Corallorhiza. This study helped us to get closer to constructing a mitogenome for MH, and have a better understanding on the effects of altered natural selection regimes associated with drastic shift in nutritional mode on genome evolution. 

Characterization of the role of arrestin proteins in response to cellulosic copper nanoparticles in Saccharomyces cerevisiae | Katelyn R. Perroz and Jen Gallagher 

Department of Biology, Eberly College of Arts and Sciences, West Virginia University, Morgantown, WV 26506 and Department of Biochemistry, Allegheny College, Meadville, PA 16335 

Copper antimicrobial materials generate reactive oxygen species which are effective against both bacteria and fungi, however copper is expensive and not biodegradable. Copper nanoparticles built on cellulose (c-CuNPs), are low-cost, biodegradable, and deliver copper in an efficient and controlled manner. Yeast, unlike bacteria, are eukaryotes with similar biochemical pathways to humans, making it difficult to design antifungal materials with low toxicity to humans. Previous studies found c-CuNPs decorating the outside of yeast cells and were believed to be too small to enter the cells through channels, but about 10% of the cells had c-CuNPs inside the cells. Large molecules and membrane bound proteins enter eukaryotic cells through the process of endocytosis. Endocytosis requires a class of proteins called arrestins to trigger the endocytosis of different cargos. To investigate how c-CuNPs are entering yeast, different arrestin mutants of yeast were treated with c-CuNPs. A subset of these arrestins do have a role in endocytosis of c-CuNPs into yeast cells. This research supports the use of nanotechnology to make copper antimicrobial compounds to improve public safety. 

Novel apigenin and genkwanin derivatives as potential cancer growth inhibitors | Samuel L. Gary and Nik Kovinich 

Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506 

The naturally occurring flavones apigenin and genkwanin have gained notoriety in recent years as effective antiproliferative compounds, highly selective to cancerous cells, but are not potent enough to provide a rational clinical use. In the present study, novel halogenated derivatives of apigenin and genkwanin were produced in attempt to increase the efficacy of anticancer activity of these compounds. N-Bromosuccinimide was used to brominate apigenin and genkwanin at two positions. The proliferation rates of HTC-116 and HT-29 human colorectal cancer cells were measured with MTT colorimetric assay after treatment with the derived and parent compounds. It is expected that significant inhibition by apigenin and genkwanin on both cell lines will occur, based on previous studies, and also an enhanced effect from the brominated derivatives, based on a study of chrysin derivatives. Further research into the molecular targets of these compounds could lead to the development of high-affinity drugs, which could serve as potent and cancer-selective treatments. 

Genetic analysis of yeast in response to 4-methylcyclohexane methanol from Elk River chemical spill | Noor Malik and Jennifer Gallagher 

Department of Biology, West Virginia University, Morgantown, WV 26506 

4-methylcyclohexane methanol (MCHM) spilled in the Elk River in 2014 contaminating drinking water for many West Virginians’. The cellular effects of MCHM are unknown. To identify possible targets, the expression of genes was measured in the model eukaryotic organism, S. cerevisiae, when exposed to MCHM. Many genes that changed in expression were regulated by Mediator, a multi-protein complex. This complex is found in all eukaryotes. Single mutants of different components of Mediator can make yeast more or less sensitive. Double mutants were generated to address how these mutations interacted. The double mutant med15/med2 was more sensitive compared to single med15 mutants. In parallel, yeast were evolved in lab to uncover pathways that may be important when responding to MCHM. After multiple generations, mutations that improved resistance in the cells will be passed on and identified using whole genome sequencing. Identifying these mutations will help us understand the long term effects of MCHM exposure as well as genomic regions responsible. 

Function of lipophorin receptors as they relate to the hedgehog pathway in horn development of beetles | Bennett A. Yunker, Logan P. Zeigler and Teiya Kijimoto 

Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506-6045 

The focus of this ongoing research is to understand how unrelated developmental genes interact to influence non-vital traits. To do so, horn growth in dung beetles was observed because they are influenced by a sex-determination gene and a cell differentiation gene. The lab’s previous research investigated the effects of knocking down one gene or the other, but where in these genes’ signaling pathways connect is unknown. Cholesterol modifies the protein early in the aforementioned cell differentiation pathway, called hedgehog. The goal was to express the hedgehog gene but not allow its pathway to be modified. That required a means of keeping lipids from entering the cells. Subsequently, beetles use lipophorins to transport lipids, like cholesterol, across membranes, but require receptors to do so. By isolating the gene for these receptors, cultivating it, then injecting it into larvae as a double-stranded RNA, the cells should “attack” the RNA and similar strands. This should inhibit lipid transportation into the cells, limiting the amount cholesterol being utilized so the effects of the unmodified protein observed. 

Bioelectrical impedance analysis of smallmouth bass | Jonathan Fisk, Kyle Hartman and Ed Olesh 

Davis College of Agriculture, Natural Resources, and Design,
West Virginia University, Morgantown, WV 26506 

One of the most popular sportfish in North America is the Smallmouth Bass (Micropterus dolomieu). Due to this popularity it is important have a healthy population for stakeholder’s satisfaction. Previously measurements of a fish’s condition were done by simple length weight measurements. However, these measurements do not tell the whole store as a fish can change its percent water weight by 30% in a day. However a technology known as Bioelectrical Impedance Analysis (BIA) is a non-lethal method that can be used to calculate the percent dry weight (%DW), a metric that could only be measured previously by sacrificing a fish, after an equation has been discovered. To do this we needed to collect wild Smallmouth Bass from several waterbodies in the Ohio drainage area and collect reactants and resistant measurements along with lengths and weights. These fish will them be sacrificed to obtain dry weights. With these measurements it is our objective to discover the equation for %DW, using the reactants and resistant measurements and thus no longer needing to sacrifice fish. 

Functionalization of silica nanoparticles with recombinant streptococcal collagen-like proteins for cancer research | Brittany T. Keller, Dudley McNitt and Slawomir Lukomski 

Department of Microbiology, Immunology and Cell Biology,
West Virginia University School of Medicine, Morgantown, WV, 26506 

Extra cellular matrix surrounding tumors differ considerably from healthy tissue by the inclusion of cellular fibronectin isoforms containing extra domain A (EDA/cFn). These distinct tumor microenvironments that are rich with EDA/cFn are recognized by the streptococcal collagen-like protein 1, Scl1. We hypothesize that immobilizing recombinant Scl1 (rScl1) protein onto silica nanoparticles will aid these nanoparticles in targeting specific tumor sites. We designed in silico rScl1 constructs, cloned and expressed them in Escherichia coli, and then purified through affinity chromatography on a Streptactin sepharose. SDS-PAGE analysis was used to confirm the purity and integrity of the rScl1 polypeptides, before assessing their collagen-like structure by circular dichroism spectroscopy. Purified rScl1 proteins were immobilized onto red-fluorescent streptavidin-coated silica nanoparticles via C-terminal strep-tag II, successful immobilization was confirmed by enzyme linked immunosorbent assay using anti-rScl1-specific antibody. rScl1- immobilized nanoparticles were tested for binding to EDA/cFn-coated surfaces. Our long-term objectives are to use these rScl1-functionalized silica nanoparticles for cancer diagnostic purposes, and as a vehicle for drug delivery to specific tumor sites for the eradication of cancer cells. 

Oncogenic MET kinase signaling impacts lung cancer immune checkpoint pathways | Emmanuel Chan, Xiaoliang Wu and Patrick C. Ma 

University of California at Berkeley, CA; WVU Cancer Institute,
West Virginia University, Morgantown, WV, 26506 

Immune checkpoint inhibition has become standard treatment in advanced lung cancer. It reactivates a patient’s own immune systems to combat cancer by blocking the tumor’s immune escape via the PD-1/PD-L1 checkpoint interaction. MET kinase is a cancer target recognized in the Hallmarks of Cancer for its promotion of cancer invasion and metastases. We hypothesized that MET signaling/inhibition would dynamically impact the immune checkpoint pathways. We utilized NCI-H596 adenosquamous lung cancer cell line expressing the oncogenic MET exon-14 juxtamembrane splicing mutation as a model. Expression of immune signature genes including PD-L1 and IDO1 in NCI-H596 cells were studied under both MET ligand hepatocyte growth factor (HGF) induction and targeted inhibition. In a RT2 Profiler PCR Array Gene Expression analysis, H596 cells were found to have a reprogrammed gene expression signature during the 1-3 days under HGF stimulation including upregulated PD-L1 and downregulated IDO1, also validated by Western Blot analysis. Therapeutic cellular inhibition using crizotinib and CM-1 downregulated both PD-L1 and IDO1 expression. RNA-sequencing of HGF-stimulated H596 cells is underway for further comprehensive transcriptomics signature bioinformatics analysis. 

Crowders affect protein aggregation in Huntington’s disease | Caleb Fan, Albert W. Pilkington, IV and Justin Legleiter 

C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, WV 26506-6045 

Huntington’s disease is an amyloid disease where an inherently disordered protein is misfolded and is correlated to symptoms. The misfolded Huntingtin protein, Htt, aggregates together to form a fibrillar structure which is observed in mouse models and across other amyloid diseases. How these aggregates form and interact in the cell is important to understanding toxicity development in Huntington’s disease. Evidence suggests that aggregation is decreased in the presence of lipids possibly due to a crowding effect lowering favorable orientations of Htt for aggregation. To test the crowding effect, Htt is incubated with various unreactive crowding agents and lipids. The amount of fibrils at various time points are compared to observe its effects on rate of protein aggregation. Preliminary results suggest that crowders increase the rate at which Htt aggregates. There is a higher concentration of fibrils when incubated with crowders than in the control. The crowders possibly force Htt to interact more as they reduce the amount of empty space. This suggests that slower aggregation rates with lipids are due to unknown interactions between Htt and lipids, not the crowding effect. 

Increase in mutant Huntingtin protein aggregation leads to decrease in protein-lipid interaction | John Bard, Albert W. Pilkington, IV and Justin Legleiter 

C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, WV 26506-6045 

Huntington’s disease is an expanded polyglutamine neurodegenerative disease characterized by the aggregation of mutant Huntingtin protein into protein plaques known broadly as amyloids. The process by which these amyloids are formed goes through a variety of intermediates such as oligomers (multimeric species ranging in size) and fibrils (long protein aggregates developing from oligomers) ending with amyloids. These amyloids are a hallmark of the disease, but current knowledge of the subject has many questioning the toxic mechanism and role of the amyloids in said mechanism. We aimed to show how differing aggregation levels of Huntingtin affected the interaction between Huntingtin and lipid membranes. This was done by incubating Huntingtin Exon 1(model system) to differing stages of aggregation (monomer, oligomer, and fibril) and running these different samples in a polydiacetylene assay to quantify the interaction of each with cell membranes. Preliminary results show that Huntingtin had less interactions with cell membranes in samples containing higher fibril numbers indicating that it is likely monomeric or oligomeric intermediates responsible for the degradation of membranes in cells.