bluuebunny/crossref_metadata_2025 huggingface dataset:
Integrating civic scientific literacy skills in a Biochemistry course
Authors: Taylor Ann TS
Published: June 2021

Individuals and organizations have to make regular decisions about biochemical data in their everyday lives. At many higher education institutions, all students are required to take science courses with the goal of developing scientific literacy. Scientific literacy has several distinct meanings, but this project emphasizes civic scientific literacy, the understanding of contemporary science‐related issues necessary for informed community engagement. The hypothesis is that consumer and citizenship skills for evaluating data sources can be integrated into Biochemistry courses, specifically a one semester survey course of biochemistry for non‐science (liberal arts) majors. Parallel strategies were integrated to teach students to access biochemical and health related scientific information, analyze arguments, and decide on personal or community action. Students were taught how research studies are conducted and the limits of each model through a website called Study Design 101 (https://himmelfarb.gwu.edu/tutorials/studydesign101/) and to evaluate arguments with the Claim‐Evidence‐Reasoning model. These approaches were integrated into classroom activities and homework assignments. Personal and community action opportunities were provided through outside of class activities that students chose for themselves. The impact of these activities was assessed through performance on the activities, open ended exam questions, and an end of course survey. Students gained both biochemical content and skills in evaluating claims with evidence, and reported the course helped them better understand what scientists do and how to make decisions based upon scientific evidence.

Support or Funding Information

Thanks to the Haines Fund for the Study of Biochemistry at Wabash College.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of DNA damage suppression from the tardigrade (Ramazzottius varieornatus) gene Dsup in E. coli through ultraviolet radiation and hydrogen peroxide exposure
Authors: Pough Jason D , Wolyniak Michael J
Published: June 2021

Tardigrades (genus Ramazzottius), also known as water bears, are microscopic aquatic multicellular organisms that can be found in just about any environment. The creatures are known to be extremotolerant, which is an oddity since most extremotolerant organisms are single‐celled bacteria or archaea. With well over 1000 different species, these stoic creatures can withstand dehydration, freezing, high pressures, and even the vacuum of outer space (Hashimoto 2017). Interestingly, while most mammals would find roughly 5–10 grays of ionizing radiation fatal, and some prokaryotes such as E. coli could survive roughly 50 grays, tardigrades can survive up to 5,000 grays of radiation. What allows tardigrades to survive radiation that would be fatal to most other organisms?

In this research, we have chosen to focus on the Ramazzottius varieornatus Damage suppressor gene (Dsup). The gene produces proteins that contribute to an organism's tolerance to harsh environmental stresses by suppressing both single strand and double strand breaks in DNA and therefore improving overall radiotolerance (Hashimoto 2017). What makes the gene even more intriguing, however, is that it has been cultured in human and animal cells and found to improve radiotolerance and viability of irradiated cells. With the information known, there holds some potential for having this gene produce a practical method for fixing DNA breaks and preventing mutation from ionizing radiation (i.e. UV rays from the sun).

To confirm the original findings as seem in Hashimoto et al. (2017) as well as refine them for the context of our laboratory, we transformed E. coli with a plasmid containing Dsup and subjected the resulting transformants to either ultraviolet light or hydrogen peroxide with the goal of challenging the DNA repair machinery as well as the ability of Dsup to prevent DNA breaks. DNA damage was assessed through the comet assay, a single‐cell gel electrophoresis technique for the detection of damage through silver nitrate staining. Overall, E. coli cells with the Dsup plasmid showed significantly lower levels of DNA damage when insulted with ultraviolet radiation. Ongoing research is assessing the effectiveness of Dsup in response to hydrogen peroxide damage. Going forward, we are working towards site‐directed mutagenesis experiments with the goal of better characterizing Dsup's role in suppressing DNA damage.

Support or Funding Information

Hampden‐Sydney College Office of Undergraduate Research

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
New approach for the DNA damage/repair analysis: vertical comet assay
Authors: Williams Robert , McCoy Daniel , Chen Chenf‐fu , Podlutsky Andrej
Published: June 2021

Ina traditional Comet Assay, the fluorescence of the “head” and “tail” regions of the comet is used to assess the level of DNA damage. The traditional Comet Assay technique issensitive and wildly used, however, also time‐consuming and results are inherently variable between different Las. The Vertical Comet Assay (V‐Comet, patent pending) aims to quantify the extent of DNA damage by collecting and analyzing the tail‐portions of comets. This is achieved through the collection of damaged DNA pieces into abuffer solution via electrophoresis, which can then be used to quantify the concentration of DNA or be used as a template for the qPCR. The collection of damaged DNA in this manner would allow for the improvement of bioanalytical and clinical assessments of genotoxic agents. Due to the capability to analyze the DNA fragments with qPCR, a gene‐specific damage profile can be established for a wide variety of genes, not just a whole‐genome assessment like in the traditional Comet Assay. Presently we use our technique for the comparison of isolated DNA from HeLa and HDFa cell lines following the X‐ray exposure conducted with/without the repair time. For the first time we could answer the question: do cells discriminate between genome regions to perform DNA repair? Our results are based on a comparison between DNA repair in the following regions: “junk” DNA, housekeeping genes and cancer‐related genes.

Support or Funding Information

1) NASA EPSCoR, NNH15ZHA003C 2) Research reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Numbers UL1GM118991, TL4GM118992, or RL5GM118990. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. UA is an AA/EO employer and educational institution and prohibits illegal discrimination against any individual: www.alaska.edu/titleIXcompliance/nondiscrimination.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hookah and Vaping: Safe Nicotine Delivery Systems?
Authors: Dunham Rolaun , Bethishou Britiel , Skochko Alexander , Stone Koni K
Published: June 2021

Hookah and vaping have recently been popularized as alternative to smoking tobacco. Hookah heats tobacco with a very hot piece of charcoal and then the air is pulled through the hot tobacco, this smoke is then pulled through water before it reaches the user's mouth and lungs. Vaping uses electricity to vaporize a propylene glycol/glycerine matrix that contains nicotine and flavoring agents. This vapor is then pulled directly into the mouth and lungs. The mainstream vapors from both hookah and vaping were collected on Cambridge filters, the filters were soaked in water and then the aqueous solutions were dried via vacuum centrifugation. After weighing the dried mass, the samples were reconstituted with water and then analyzed by UV spectroscopy; the Hookah samples had absorbance at 275 nm. Our original hypothesis was that the water soluble tar components would be trapped in the water that the hookah smoke is pulled through. Thus, there should have been no absorbance at 275nm. The vaping samples did not absorb at 275; since vaping does not burn or heat tobacco, we did not expect to find the same absorbance as the cigarette tar radical. Gel electrophoresis was used to analyze the nicking of plasmid (pUC18) DNA. Aqueous solutions of Hookah and vaping solutions were tested in our DNA nicking assay. Briefly, we use supercoiled pUC18 DNA, this DNA unwinds after a single nick. The supercoiled and nicked (open circular) DNA migrate as two distinct bands in the agarose gel. The density of each band is then quantitated to determine the amount of DNA nicking. The amount of nicking by both vaping and hookah solutions is orders of magnitude less that the nicking we have detected with cigarette smoke. However, we have detected aldehydes in both vaping and hookah solutions. Aldehydes are known to be toxic, primarily by their facile reaction with primary amines and thus their ability to wreck havoc with protein structure. We have implemented an assay to detect aldehydes in both hookah and vaping solutions. The reagent, Purpald, 4‐Amino‐3‐hydrazino‐5‐mercapto‐1,2,4‐triazole readily reacts with aldehydes and then the oxidized product of the reaction produces purple solutions. We then analyzed the purple solutions by UV/VIS. Based on the UV spectra, we have detected acetaldehydes in both vaping and hookah solutions.

Support or Funding Information

R. Dunham is supported by the CSU, Stanislaus McNair Scholars program.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Roles of High Mobility Group Box Proteins in Nucleotide Excision Repair‐associated Processing of DNA Interstrand Crosslinks
Authors: Gerberich Jillian , Mukherjee Anirban , Vasquez Karen
Published: June 2021

Many traditional anti‐cancer chemotherapeutic agents induce DNA interstrand crosslinks (ICLs), lesions that form covalent bonds between the DNA strands. ICLs are highly cytotoxic if not properly repaired because they can block basic cellular processes such as transcription and replication. Nucleotide excision repair (NER) is a critical repair mechanism in processing ICLs in human cells. In addition to NER, ICLs are also processed through DNA double‐strand break repair pathways (e.g. homologous recombination) in S‐phase. We have previously demonstrated that the high mobility group box protein (HMGB1), a non‐histone architectural protein, can bind to site‐directed ICLs with high affinity and modulate error‐free repair processing of such lesions. In addition, we have found that HMGB1 associates with the NER damage recognition protein XPA in human cells and facilitates the recruitment of XPA to the damaged site. Further, we have demonstrated that HMGB1 can architecturally modify the damaged substrate by inducing negative supercoils, which may facilitate DNA damage processing by NER. We hypothesize that HMGB1 can modulate NER processing of ICLs by aiding NER damage‐recognition factors in locating damaged DNA. To test this hypothesis we are using a mutation‐reporter system to measure mutation frequencies and spectra generated by site‐directed ICLs in human cells. There are two NER subpathways that recognize DNA damage via different mechanisms. I am investigating in which pathway(s) HMGB1 is involved by utilizing various cell lines that are proficient or deficient for an essential recognition protein in each pathway. Additionally, we are investigating if HMGB2 and HMGB3, protein family members that are highly similar in structure to HMGB1, are also co‐factors in NER. Preliminary evidence suggests that HMGB1 may be involved in transcription coupled‐NER of site‐directed ICLs. If future evidence supports these data, this would indicate that HMGB1 could associate with transcription machinery and aid in recruiting other NER co‐factors, such as XPA. Future genetic, molecular biological, and biochemical assays will provide additional insight as to the roles of the HMGB proteins in ICL repair. The processing and repair of ICLs in human cells is not completely understood, and therefore a detailed study to define the molecular mechanisms of ICL processing is warranted. Identifying new proteins involved in ICL processing may lead to new pharmacological targets, which may help us to improve the outcome of cancer treatment.

Support or Funding Information

NIH/NCI to K.V. (CA093729) and NSF GRFP to J.G.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cruciform DNA structure formed at short inverted repeats: A source of genetic instability in vivo
Authors: Mandke Pooja P , Kompella Pallavi , Lu Steve , Wang Guliang , Vasquez Karen
Published: June 2021

Genomic instability is a major driving force for many human diseases including cancer. Certain mutation “hotspot” regions of the human genome are susceptible to cancer‐associated instability. These regions often co‐localize with repetitive DNA sequences that can adopt alternative structures (i.e. non‐B DNA). One such structure is cruciform DNA which forms at inverted repeat sequences. Short inverted repeats (<100bp) are abundant in the human genome and our laboratory has shown that they are substantially enriched at translocation breakpoints in human cancer genomes. Also, they are mutagenic and stimulate the formation of DNA double strand breaks (DSBs) leading to deletions in mammalian and yeast cells. The DSBs can be caused by fork stalling (i.e. a replication‐dependent mechanism) or by cleavage of cruciform structures by the repair enzyme XPF (i.e. a replication‐independent mechanism). Together, these studies have provided a mechanistic insight into the role of short inverted repeats in disease etiology. To further delineate the biological significance of these sequences, we have developed a transgenic mouse model containing human inverted repeats. We hypothesize that short inverted repeats capable of forming cruciform structures increase genetic instability in vivo. To test this, we have introduced a recoverable mutation‐reporter shuttle vector, carrying either a GC‐rich short inverted repeat (GC mice) or B‐DNA‐forming sequence (control mice) into the mouse genome. Both male and female GC‐containing mice (n=7) and control mice (n=9) were aged for 2 years and the shuttle‐vector was subsequently recovered from the genomic DNA of spleen tissue. Transformation into competent cells was performed to obtain blue (wild‐type) and white (mutant) colonies. Mutation frequencies were calculated and mutation spectra were obtained by direct DNA sequencing. Preliminary data reveals that GC rich cruciform‐forming sequences induce a significantly higher mutation frequency than the control B‐DNA‐forming sequences. These preliminary results suggest that cruciform‐forming sequences contribute to genetic instability in vivo. The contribution of this study to cancer etiology is significant, as the results provide insights into the molecular changes induced by cruciform structures.

Support or Funding Information

NIH/NCI support to KMV (CA193124 and CA093729)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Post‐transcriptional processing at the promoter proximal RNA polymerase II pausing. A possible mechanism for premature termination
Authors: Koney Nii Koney‐Kwaku , Perley Danielle , Guo Kai , Hur Junguk , Nechaev Sergei
Published: June 2021

Promoter‐proximal RNA Polymerase II pausing, which occurs within the first 100 nucleotides of a gene, refers to a property of the Polymerase II to halt transcription temporarily but remain transcriptionally competent. Knowledge of proximally paused Polymerase II dynamics is important for understanding the regulation of mRNA production.

At the paused site, Polymerase II can either elongate to resume mRNA synthesis or terminate to release a short RNA. We hypothesize that both events occur on genes and, furthermore, suggest that the equilibrium between elongation rate and termination rate at the pause site reflects regulatory inputs at the promoter.

We adopted the small RNA sequencing approach to detect short RNA generated by paused Pol II genome‐wide on chromatin. Analysis of short RNA in subcellular fractions revealed genes that display RNA species with a 5′ monophosphate ends downstream of transcription start sites. The 3′ ends of these monophosphate RNAs reside within the location of the 3′ ends of RNAs from paused Polymerase II demonstrating they may be products of paused Polymerase II. Short RNAs generated by Paused Pol II may represent a novel class of small RNAs derived from promoter‐proximal transcription termination. Future experiments will focus on identity and functional roles of these transcripts.

Support or Funding Information

NIH COBRE P20GM104360

NIH R21CA217751

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CpG Methylation Inhibits Prolidase Promoter Driven Transcription
Authors: Lanaghan Zeljka Miletic , Eni‐aganga Ireti , Balasubramaniam Muthukumar , Dash Chandravanu , Pandhare Jui
Published: June 2021

Prolidase, a cytosolic exopeptidase, and a member of the matrix metalloproteinase (MMP) family, is encoded by the PEPD gene in humans. Since Prolidase is the only enzyme which can cleave dipeptides containing C‐terminal proline, or hydroxyproline, it plays an essential role in protein metabolism, collagen turnover, and matrix remodeling. Prolidase is essential in several physiological and pathological processes such as wound healing, inflammation, angiogenesis, cell proliferation, and carcinogenesis. Surprisingly, little is known about the molecular and cellular regulation of PEPD gene expression. In initial studies, we observed a dense CG‐rich portion around the transcription start site, suggesting a possible role of promoter methylation in the transcriptional regulation of PEPD gene. To study the transcriptional regulation of PEPD gene, we cloned and characterized the human PEPD promoter. The PEPD promoter was amplified from the human genome via PCR and inserted into a luciferase reporter construct. As demonstrated in our results, increase in luciferase activity was obtained after transfection of the PEPD luciferase reporter in HEK293T, indicating the successful construction of the luciferase reporter. Next, we performed in vitro methylation of the PEPD promoter using SssI methyltransferase which methylates DNA CpG islands. Methylation was confirmed by BstUI digestion that cleaves at unmethylated CpG dinucleotides but not at methylated CpG. The CpG methylation significantly inhibited PEPD promoter activity in comparison to the unmethylated construct. Taken together, we believe, these results will generate new knowledge on the molecular regulation of prolidase and provide better insight in our understanding of regulation of prolidase expression in various physiological and pathological conditions.

Support or Funding Information

Supported by National Institutes of Health (NIH) Grants DA037779 and MD007586 (to Jui Pandhare) and DA024558, DA30896, DA033892, DA021471, AI22960 and MD007586 (to Chandravanu Dash). Ireti Eni‐Aganga is supported by RISE Grant. The work is also supported by the RCMI Grant G12MD007586, the Vanderbilt CTSA Grant UL1RR024975, the Meharry Translational Research Center (MeTRC) CTSA grant (U54 RR026140 from NCRR/NIH, the U54 Grant MD007593 from NIMHD/NIH, and the Tennessee Center for AIDS Research (P30 AI110527).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dissecting the gene regulation of Proteus vulgaris Rts1 HigB‐HigA toxin‐antitoxin system
Authors: Zaldana Kimberly Silvia , Pavelich Ian , Moller Abraham , Dunham Christine M.
Published: June 2021

Bacterial toxin‐antitoxin systems are abundant genes that modulate a beneficial phenotypic switch to shift cells' focus towards survival in times of stress, including antibiotic exposure. During normal growth, toxin‐antitoxin complexes transcriptionally repress their own expression by binding at DNA operator regions that overlap with their own promoters. Toxin binding to the antitoxin‐DNA complex is important because it can result in increased repression. Upon stress, including nutrient limitation and antibiotic exposure, antitoxins are proteolyzed, releasing the toxin component. Released toxin prevents further growth by inhibiting replication, translation or cell wall synthesis. Given these important roles, it is critical to understand how toxin‐antitoxin systems regulate this phenotypic switch between a growth and dormant state that is antibiotic tolerant. The HigB‐HigA protein‐protein complex was first identified on the multi‐drug resistant Rts1 plasmid from Proteus vulgaris(Pv) associated with a post‐operative urinary tract infection. This toxin‐antitoxin complex is not responsive to antibiotics suggesting this aids in tolerance to antibiotics. HigA binding to HigB neutralizes the toxic effects of HigB and forms a heterotetramer. Most toxin‐antitoxin systems encode the antitoxin first and the toxin gene second, resulting in higher antitoxin expression to suppress toxin. However, the higB‐higA gene is unique in that its gene order is reversed with the higB toxin encoded first. Additionally, there are two putative promoters and, although it is clear that the main promoter is active (Phig), the activity of the second internal promoter (PhigA) that precedes the higA antitoxin is unknown although the antitoxin should be expressed to high levels. I hypothesize that PhigA is a strong promoter that results in high expression of the HigA antitoxin. I also hypothesize that the Phig promoter is weak, resulting in lower toxin and antitoxin expression. Here, I determine that unexpectedly, mutation of both the −35 and −10 sites, the −35 site or the −10 site causes an increase in beta‐galactosidase activity as compared to the wild‐type. These data suggest that the weak internal promotor (PhigA) is more active in regulating HigA antitoxin expression. Future work will explore how modulating the operator sites influences expression and will determine how transcriptional control may control HigB‐HigA expression.

Support or Funding Information

This research is funded by a Burroughs Wellcome Fund Investigator in the Pathogenesis of Infectious Disease award 1015487 (to CMD).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of histone chaperone Nucleophosmin‐mediated transcriptional regulation in oral tumorigenesis
Authors: Dey Suchismita , Senapati Parijat , Bhattacharya Aditya , Sudarshan Deepthi , Das Sadhan , Kumar Manoj , Kodaganur Gopinath S. , Maleikal Tessy Thomas , Kundu Tapas K.
Published: June 2021

Histone chaperones regulate the histone traffic in the cell and are involved in different cellular processes including transcription regulation. Nucleophosmin (NPM1), a histone chaperone, enhances acetylation‐dependent chromatin transcription. Acetylation of NPM1 by lysine acetyltransferase (KAT) p300 further enhances its histone chaperone activity and transcription activation potential. Moreover, the acetylated pool of NPM1 (AcNPM1) colocalizes with RNA Polymerase II foci in the nucleus, indicating its role in transcription. NPM1 is also a potent inducer of autoacetylation of p300 thereby enhancing its KAT activity. In the context of cancers, NPM1 is overexpressed in several solid tumors including oral squamous cell carcinoma and regulates genes implicated in oral cancer. AcNPM1 levels are also elevated with increasing grades of oral cancer. However, the precise role of AcNPM1 and the mechanism of transcription regulation by NPM1 are unclear. We performed chromatin immunoprecipitation followed by sequencing (ChIP‐seq) to identify the genome‐wide localization of AcNPM1 on chromatin. Interestingly we find that AcNPM1 is enriched on promoters of genes that are co‐occupied by RNA Polymerase II and other histone modifications related to active transcription. RNA‐sequencing after NPM1 knockdown in an oral cancer cell line revealed that NPM1 regulates the expression of genes involved in oral tumorigenesis. Inducible knockdown of NPM1 in an orthotopic mouse model also validates our finding that NPM1 indeed is involved in oral cancer manifestation. These results provide insights into the gene network regulated by NPM1 and AcNPM1 and the possible role of its overexpression and hyperacetylation in oral cancer. In the same context, the underlying mechanisms of transcriptional activation by NPM1/AcNPM1 and the role of NPM1 in the process of tumorigenesis are also currently under investigation.

Support or Funding Information

Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Sir JC Bose Fellowship, Department of Science and Technology (DST), India (Grant No. SR/S2/JCB‐28/2010), Department of Biotechnology (DBT), India (Programme Support on ‘Chromatin and Disease’, Grant No. BT/01/CEIB/10/III/01 and Virtual National Oral Cancer Institute, Grant No. BT/PR17576/MED/30/1690/2016)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of mitochondrial DNA transcription by protein post‐translational modifications
Authors: Dittenhafer‐Reed Kristin E
Published: June 2021

Mammalian cells contain genetic information in two compartments, the nucleus and the mitochondria. Mitochondrial DNA (mtDNA) encodes thirteen protein subunits required for oxidative phosphorylation. The remaining mitochondrial proteome, including additional oxidative phosphorylation subunits, and the proteins necessary for mtDNA replication, expression, and stability, is encoded by the nuclear genome. Therefore, to respond to metabolic changes, mitochondrial gene expression must be coordinated with nuclear gene expression. While the control of nuclear gene expression is widely studied, there is a need to understand the regulation of mtDNA transcription. Our central hypothesis is that reversible post‐translational modification of the mtDNA transcriptional machinery is a means to tune mtDNA transcription in response to changes in cellular metabolism. Here, using in vitro biochemical and cell biology approaches, we describe the effects of lysine acetylation and serine/threonine/tyrosine phosphorylation on the mtDNA binding ability and transcriptional activity of three proteins, mitochondrial transcription factor B2 (TFB2M), the mitochondrial polymerase (POLRMT), and mitochondrial ribosomal protein L12 (MRPL12). Our in vitro biochemical results indicate that phosphorylation of TFB2M may impede mtDNA binding and subsequently alter transcription initiation. Modification of MRPL12 may alter the ability of MRPL12 to bind and stabilize POLRMT. We also report a temporal response in the transcript level of mitochondrial and nuclear genes in response to cell growth conditions used to drive a dependence on mitochondrial metabolism. Importantly, mtDNA content is unchanged in these conditions, providing evidence for a regulated transcriptional response of the mitochondrial genome. It is expected that these findings will lead to a more complete understanding of the dynamics and coordination of mitochondrial and nuclear gene transcription, as well as adding to the understanding of the importance of protein post‐translational modifications in regulating aspects of mitochondrial function.

Support or Funding Information

This material is based upon work supported by the National Science Foundation Division of Molecular and Cellular Biosciences under Grant No. 1814845.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functional Analysis of RNA Exosome Mutants Linked to Disease Using a Saccharomyces cerevisiae Model System
Authors: Enyenihi Liz Eno , Corbett Anita H , Joshi Samika , Leung Sara , Hess Laurie , Sterrett Maria , Basrai Munira , van Hoof Ambro , Fasken Milo B
Published: June 2021

The RNA exosome complex is a key component of RNA processing and quality control that both degrades and processes many classes of RNA. This complex is highly conserved among eukaryotes and was first identified and studied in budding yeast (S. cerevisiae). Mutations in the human EXOSC2 gene, which encodes a cap subunit of the RNA exosome, have been linked to a novel syndrome characterized by retinitis pigmentosa, progressive hearing loss, premature aging, short stature, mild intellectual disability and distinctive gestalt. While the amino acid substitutions in EXOSC2 that cause this syndrome are known, how these amino acid changes impact RNA exosome function is not. The goal of my project is to analyze the functional consequences of retinitis pigmentosa‐linked amino acid substitutions modeled in the budding yeast ortholog of EXOSC2, Rrp4.

The two variants I have analyzed, rrp4‐G58V and rrp4‐G226D, correspond to patient mutations G30V and G198D, respectively. I first assessed growth of the mutant strains compared to wildtype yeast cells, which revealed that rrp4‐G226D mutant cells exhibit a growth defect at 37°C, whereas the rrp4‐G58V mutant cells grow normally. To assess whether these amino acid substitutions affect Rrp4 protein levels, I used immunoblotting. Results of this analysis reveal that the rrp4‐G58V and rrp4‐G226D proteins are expressed, but at somewhat reduced level compared to wildtype Rrp4. In the future, I will continue characterization of the mutants by using biochemical approaches to study the assembly of the RNA exosome complex and genetic analysis of rrp4 mutant interactions with RNA exosome cofactors.

Support or Funding Information

EMORY INITIATIVE TO MAXIMIZE STUDENT DEVELOPMENT

Emory Initiative to Maximize Student Development: R25 GM125598

Neurodevelopmental Role of an RNA Binding Protein Required for Cognitive Function: R01 MH107305

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of Mutations in the DEXD‐motif of the Yeast RNA‐dependent ATPase Sub2
Authors: McCann Jack Thomas , Hansen Sarah , Hoskins Aaron
Published: June 2021

Pre‐mRNA splicing by the spliceosome is an essential part of eukaryotic gene expression. Errors in splicing can be detrimental to an organism, which is why it is extremely important to understand the spliceosome's mechanisms of assembly and action. Key features of splicing include the numerous RNA‐protein remodeling and RNA unwinding reactions that aid in spliceosome assembly. Some of these reactions require one of three, well‐conserved DEAD‐motif proteins that are part of the splicing machinery (Prp5/DDX46, Prp28/DDX23, and Sub2/UAP56). While Prp5 and Prp28 contain the canonical DEAD tetrapeptide motif within their active site, Sub2 is unusual in that it instead contains a conserved DECD motif. The goal of my research is to understand the function of the cysteine within Sub2's DECD motif in vitro and in vivo. Contrary to previous reports, I have shown that yeast containing a DECD to DEAD mutation in Sub2 are viable with little impact on yeast proliferation. To explain the conservation of Sub2's DECD sequence, we looked for changes in yeast phenotypes under stress conditions. I exposed yeast containing either DEAD‐Sub2 or DECD‐Sub2 to hydrogen peroxide and found that yeast containing DECD‐Sub2 died at lower oxidative stress conditions than the DEAD mutant. This suggests that Sub2's DECD‐motif cysteine is capable of being oxidized and that this results in a disruption in gene expression. Since one cysteine oxidation product (sulfinic acid) is isosteric with aspartate, I am now testing a DEDD mutant of Sub2 for function as well as attempting to chemically characterize the amino acid modifications resulting from Sub2 oxidation. Together my results will provide insight into a potential function for Sub2's conserved DECD‐motif cysteine.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bioconjugates of Protein A to Gold Nanoparticles
Authors: Lee Christina , Naing Tiffany , Herrera Daisy , Herrera Natalie , Hoang Audrey , Marin Melinda
Published: June 2021

Gold nanoparticles (GNPs) possess rich surface properties with low toxicity that allow it to interact with and participate in conjugation of various biomolecules. As proof of principle, gold nanoparticles were synthesized to a spherical shape with a diameter between 20–30 nm and conjugated to protein A of Staphylococcus aureus bacteria. Conjugation protocols were optimized whereas any size or shaped nanoparticle can be conjugated to any biomolecule. Applications of this technique can be utilized in biomedical imaging techniques to offer more optical contrast and visibility of tagged molecules such as in Magnetic Resonance Imaging (MRI), Chronic Traumatic Encephalopathy (CTE) or to create gold Nano shells in the detection and treatment of cancer cells. GNPs can be synthesized into Nano shells, conjugated to an antibody using the conjugation protocols reported and infused into cancer cells. The gold nanoshells are able to absorb near infrared light, which excites the electrons and, in an attempt, to return to a lower energy, release heat to the external environment. As a result, cancer cells that are also attached receives this heat and are destroyed. The optimization of the nanoparticles to biomolecules will allow for nanomedicines described to become reality.

Support or Funding Information

NIH BUILD PODER Grant Award Number RL5GM118975

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Regulation of Cellular Proliferation by VACM‐1/CUL5 is Dependent on its Posttranslational Modifications by NEDD8
Authors: Sundquist Skylar Paige , Lee Si Eun , Burnatowska‐Hledin Maria
Published: June 2021

VACM‐1/CUL5 acts as the scaffold protein in the E3 ligase complex in the ubiquitin‐dependent protein degradation pathway. The overexpression of VACM‐1/CUL5 is known to inhibit proliferation, whereas inhibition of VACM‐1/CUL5 expression induces cellular proliferation. Thus, VACM‐1/CUL5 is implicated in cancer pathways. The effect of VACM‐1/CUL5 on cellular proliferation is dependent on its post‐translational modification (PTM) by NEDD8 protein (neddylation). The relationship between NEDD8 and VACM‐1/CUL5 is important for cell cycle regulation and offers a target for cancer therapy. This work explores the structure‐function relationship between VACM‐1/CUL5 and its neddylation. VACM‐1/CUL5 was mutated at the putative neddylation site Lysine (K) 724 and at three potential neddylation sites, K724, K727, and K728 (3K mutant). Our work suggests that the expression of the K724 mutant induces growth whereas expression of the 3K mutant does not affect cell growth. Interestingly, Western Blot analysis indicates that the 3K VACM‐1/CUL5 mutant is still neddylated, which suggests that VACM‐1/CUL5 may be neddylated at additional lysine sites. Our current work focuses on characterizing cells transfected with the mutated VACM‐1/CUL5 cDNAs to elucidate how site‐specific neddylation may control alternative signaling pathways.

Support or Funding Information

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Neurodegeneration‐related amyloids interact with the prion protein
Authors: De Cecco Elena , Narkiewicz Joanna , Grandolfo Micaela , Parisse Pietro , Legname Giuseppe
Published: June 2021

Tau protein is the major component of intracellular aggregates that characterize a number of neurodegenerative disorders collectively called tauopathies. Tau amyloids propagate from cell to cell through different routes, such as dynamin‐dependent endocytosis, actin‐dependent macropinocytosis, tunneling nanotubes. Here, we evaluate the role of the prion protein PrPC in promoting the internalization of synthetic tau aggregates in cultured cells. As previously found by our group, the prion protein facilitates the uptake of synuclein fibrils in neuroblastoma cell lines, in primary neurons and in mice. Using confocal microscopy, we show that also tau fibrils take advantage of the presence of the prion protein on the plasma membrane to gain entrance to the cell, and that antibodies directed against the binding site of the fibrils on PrPC can decrease the internalization of the aggregates. Moreover, tau fibrils interact with both the cellular form PrPC and the pathological conformer PrPSc, inducing clearance of the latter through a combination of lysosomal activation and inhibition of prion conversion. Our results indicate that the prion protein can act as a receptor for aggregated proteins (synuclein, tau), and that targeting PrPC could be a therapeutic strategy to slow down the spreading of tau pathology.

Support or Funding Information

This work was supported by the Program FIRB (Prot. RBAP11FRE9_001).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Proteomic analysis of the poly‐ubiquitin profiles in Alzheimer's Disease
Authors: Zhang Tian T , Dammer Eric B , Abreha Measho , Duong Duc M , Lah James J , Levey Allan I , Seyfried Nicholas T
Published: June 2021

Several neurodegenerative diseases including Alzheimer's Disease (AD), have characteristic ubiquitin positive pathological protein aggregates. Currently, the specific ubiquitylation protein substrates associated with these aggregates in AD brain is poorly understood. We analyzed control and AD postmortem brain tissues to examine the changes in the proteome and ubiquitylation substrates associated with AD pathology. Affinity capture using tandem ubiquitin binding entities (TUBEs) was utilized to enrich poly‐ubiquitylation substrates from AD and age‐matched control postmortem brain tissues (n=3 each group). Label‐free mass spectrometry (MS) based proteomic analysis identified a total of 2112 unique proteins across all samples and bead controls. After controlling for non‐specific binding, we identified 197 proteins that were at least two‐fold enriched in AD compared to control cases. Some of these targets include ubiquitin, Tau, p62 sequestosome and 14‐3‐3 proteins, which are known to associate with AD neuropathology. A cross comparison with previous proteomic studies revealed that 108 of those 197 proteins have increased site‐specific levels of ubiquitylation in AD brain. Tau poly‐ubiquitylation from AD brain homogenates was confirmed by affinity capture using either pan or linkage‐specific (Lys48 and Lys63) poly‐ubiquitin TUBEs and immunoblotting. These findings show the utility of MS to map ubiquitylated substrates in the human brain and provide a better understanding of the mechanisms underlying post‐translational modifications of pathological proteins in AD.

Support or Funding Information

National Institute on Aging (U01AG046161‐02, R21AG054206, 5R01AG053960, RF1AG057470, and RF1AG057471) and the NINDS Emory Neuroscience Core (P30NS055077).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Polyphenolic Content and Anti‐oxidation activity in Annona fruit and its Effects on Lymphoma Cancer Cells Proliferation
Authors: AlShaya Huda , Li Haiwen , Hamama Anwar , Siddiqui Rafat A
Published: June 2021

Introduction

Lymphoma is a cancer of the lymph nodes and lymphatic system. The two main types are Hodgkin and non‐Hodgkin. Non‐Hodgkin lymphoma is the most common. The lymphomas can be effectively treated with chemotherapy; however, the treatments have severe side effects that can be permanent. Studies have shown a relationship between a high intake of vegetables/fruits and reduced risk of cancer development, including lymphomas.

Hypothesis

We hypothesize that Annona fruit, which is rich in fibers and phytochemicals, can be very effective in inhibiting lymphoma cancer growth.

Experimental

The skin, pulp and seeds fractions of Annona fruits were extracted into water, methanol or chloroform and then assayed for their total phenolic contents and anti‐oxidation activities and then tested for cell proliferation inhibitory activity using Ramos‐1 lymphoma cell line.

Results

The highest concentration of total phenols was present in the skin fraction; however, the pulp has the highest total amounts. The methanol fractions of skin and pulp and water extract of seeds contained the highest anti‐oxidation activity. The methanol extracts of skin, pulp and seeds have moderate whereas chloroform extracts of pulp and seeds have strong inhibition of Ramos‐1 lymphoma cell proliferation.

Conclusions

The edible pulp of Annona fruits can provide phenolic anti‐oxidants which can also be effective in preventing/treating lymphoma. The skin and seeds are non‐edible but can be used to isolate potent compounds with potential anti‐cancer activity.

Future Directions

Studies are required to isolate and identify the active compounds from Annona fruit and to test them in an animal model.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Binding of Telomeric DNA G‐Quadruplexes by Abietane Diterpene Natural Products
Authors: Kanlong Joseph , McKnight Ruel
Published: June 2021

We have carried out studies on a homologous series of abietane diterpenes binding to a human telomeric G‐quadruplex (G4) DNA sequence [5′G3(T2AG3)3]. These G4 DNA structures are found in G‐rich telomeric regions at the ends of chromosomes and near promoter regions of oncogenes. Targeted stabilization of G‐quadruplex DNA by drugs has been implicated in telomerase inhibition and downregulation of oncogenes, both of which should result in an inhibition of cancer growth. The abietane diterpenes are a class of natural products isolated from certain plants (e.g., Hyptis verticillata) known in many cultures for their anti‐microbial, anti‐inflammatory, and anti‐cancer medicinal benefits. Binding characteristics were evaluated using circular dichroism (CD) spectroscopy, UV‐thermal denaturation, and fluorescence displacement studies. Overall, our studies show that the abietane diterpenes of this study bind to human telomeric G4 DNA, with selected compounds binding strongly. CD spectra indicate that these compounds interact with G4 DNA, either via the G4 DNA groove or by base stacking. For one compound in particular, we observed a pair of strong induced CD signals (300 – 400 nm) associated with changes in the drug environment as it binds DNA. We also observe strong positive signals around the 250 and 285 nm absorption bands of the diterpenes. These suggest the involvement of stacking interactions with the DNA bases or within the DNA grooves. Several of the abietane diterpenes bind to DNA, resulting in increased thermal stability suggesting that binding of our compounds results in G4 stabilization (ΔTm = 7°C). Thioflavin‐T (a fluorescent dye known to bind to G4 DNA) was used for fluorescent displacement studies and indicated that some diterpenes were unable to displace it from its binding site on the G4 DNA, suggesting the diterpenes occupied distinct binding sites. The binding of these abietane diterpenes to G4 DNA suggests promising therapeutic potential as lead compounds and warrants further investigation.

Support or Funding Information

This study was lead by Dr Ruel Mcknight, professor of Chemistry and Biochemistry at SUNY Geneseo.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sphingomyelin synthase inhibition in sarcomas results in ceramide accumulation and apoptosis
Authors: Pavek Adriene , Afrin Farjana , Meldrum Trevor , Pashikanti Srinath , Barrott Jared
Published: June 2021

Sphingolipid metabolism is critical to cellular homeostasis especially as cells undergo mitosis. It is, therefore, unsurprising that the sphingolipid pathway is upregulated in a number of a cancers. Sphingoid bases such as sphingosine and ceramide are rapidly synthesized to provide the building blocks of other sphingolipids. Inhibition of downstream enzymes that convert sphingosine and ceramide into sphingolipids can lead to an increase of ceramide, which results in cellular toxicity and initiation of apoptosis. Using the marine natural product Jaspine B and chemically synthesized analogues, we investigated the potency of inhibiting sphingomyelin synthase in sarcoma cell lines. Sphingomyelin synthase is a downstream enzyme that converts ceramide into sphingomyelin by adding the polar head group phosphorylcholine to ceramide. Jaspine B and its analogues exhibited submicromolar potency against several human sarcoma cell lines. It was determined that the inhibition of sphingomyelin synthase resulted in an increase in ceramide and sphingosine levels and that sphingomyelinase enzymatic activity increased. While the mitochondria mass remained the same, the membrane potential of the mitochondria was significantly affected by the presence of the inhibitors. In conclusion, inhibitors of sphingolipid metabolism are viable therapeutic options with high untapped potential.

Support or Funding Information

Idaho State University Start Up

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.