bluuebunny/crossref_metadata_2025_split huggingface dataset:
Adding Spice to the Lab with BASIL: Combining Wet‐Lab Protein Biochemistry with Computational Analysis to Analyze Proteins of Unknown Function
Authors: Pikaart Michael 
Published: June 2021

Most undergraduates studying biochemistry and molecular biology get their broadest exposure to wet‐lab techniques in protein and nucleic acid chemistry (and, increasingly, computer/visualization) in their upper‐level laboratory courses. Students in these classes are juniors and seniors with well‐defined career goals. Many will have already had a research background in a traditional one‐to‐one (or one‐to‐few) research mentoring setting, for example a summer research program – an experience known to increase student learning and persistence in the sciences. At the same time, extended full‐time PI‐directed research is limited in the number of students served, and can even present a barrier. To broaden the impact of teaching through research, many practitioners have adopted the CURE, or Course‐based Undergraduate Research Experience, approach.This presentation reports on BASIL (Biochemical Authentic Scientific Inquiry Laboratory), the result of a team of faculty who have worked to bring computational and wet‐lab protein science to the biochemistry teaching lab. Together, we have developed a protein biochemistry CURE to determine enzymatic function of proteins of unknown activity. With eleven modules available online (basiliuse.blogspot.com), the BASIL curriculum represents a one‐semester upper level biochemistry laboratory course; five of the modules are computational/structural, while six are wet‐lab protein expression, isolation, and analysis experiments. Because the computational tools may be more challenging for biochemistry faculty and students to adopt than typical bench methods, we have developed a suite of analysis tools available on a USB stick. This presentation summarizes the status of BASIL on multiple campuses and seeks to promote further adaptation of this CURE.Support or Funding InformationNSF IUSE Awards 1709278, 1503710This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Extensive DNA Repeats Residing in Orthologs of PKD1 Correspond With Inactivating Mutagenesis and Polycystic Kidney Disease
Authors: Larson Erik , Zebolsky Aaron , Dewey John , Swayze Emma , Alvarado Gloria , Ellis Monica , Heuvel Gregory Vanden 
Published: June 2021

Bi‐allelic inactivation of the PKD1 gene results in Autosomal Dominant Polycystic Kidney Disease (ADPKD), a common and lethal disorder. ADPKD is thought to follow a two‐hit pathway, where one inherited mutation in PKD1 is followed later by somatic inactivation of the other allele. Loss of PKD1 expression results in cysts, which are clonal in nature and can number in the thousands. The mechanisms are unclear, but human PKD1 appears prone to mutagenesis and inactivation. Interestingly, naturally occurring ADPKD is observed in humans, cats, and pigs, but not mice. We have therefore compared PKD1 gene sequences across species to determine if there are shared sequence or structural characteristics that could explain PKD1 instability. Despite conservation of PKD1 organization and coding across species, we find an over‐representation of G/C content and tandem repeats in PKD1 from species that are naturally prone to ADPKD. It is already established that guanine‐rich DNA triggers site‐specific genetic instability via the formation of G‐quadruplex (G4) DNA structures. Cytosine repeats also support structures called, i‐motifs, which have the potential to induce site‐specific genetic instability. We find that the human, feline, and porcine PKD1 genes are rich in G and C tandem repeats and G4 sequences, while murine Pkd1 is not. Circular dichroism spectroscopy demonstrates that structure formation occurs under physiological conditions. The possibility that G4 DNA contributes to PKD1 instability is further supported by the fact that cataloged somatic mutations are clustered near predicted G4 forming motifs. Our results identify G4 DNA and i‐motifs as structures that may form on both strands of the PKD1 gene, uncovering a molecular rationale to explain the instability of the gene and the emergence of cysts in ADPKD carriers.Support or Funding InformationWMed Pilot Grant ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of thermal manipulation during embryogenesis and post‐hatch acute heat stress on the mRNA expression dynamics of cytokines in broiler chickens
Authors: Al‐Zghoul Moh'd Borhan 
Published: June 2021

Heat stress significantly impacts chicken immunity and cytokine expression, but, the effects of embryonic thermal manipulation (TM) on cytokine expression in broiler chickens is unclear. The aim of the present study was to evaluate the effects of TM on the splenic mRNA expression dynamics of certain cytokines (IFN‐α, IFN‐β, IFN‐γ, IL‐4, IL‐8, IL‐15, IL‐16, IL‐17 and IL‐18) in broilers during subsequent exposure to acute heat stress (AHS). TM was performed by elevating the incubation temperature to 39°C at 65% relative humidity (RH) for 18 h daily during embryonic days (ED) 10 to 18. AHS was carried out by raising the temperature to 40°C for 7 h on post‐hatch day 28. At 0, 1, 3, 5, and 7 h of AHS, the spleen was collected from all groups to evaluate mRNA expression by relative‐quantitative real‐time RT‐PCR, and blood was collected to measure plasma levels of IL‐4, IL‐8, and IFN‐γ. At 0 h, TM led to a significant reduction in the basal mRNA level of IFN‐β and the plasma level of IFN‐γ and IL‐8. Moreover, AHS significantly decreased IFN‐β in control chicks, decreased IL‐4 in both TM and control chicks, and increased IFN‐γ and IL‐16 in TM chicks. IL‐8, IL‐15, IL‐17, IFN‐α, and IL‐18 expression significantly increased during AHS in both TM and control chicks, but expression dynamics was improved in TM chicks for all cytokines except IL‐17. AHS resulted in increased plasma IFN‐γ levels in TM chicks only, and it increased IL‐8 levels at 3 and 5 h of AHS in TM chicks but at 7 h in control chicks. Lastly, 3 h of AHS increased IL‐4 plasma levels in control chicks. The results of this study may indicate that TM has a long‐term effect on the expression dynamics of cytokines, especially during AHS, suggesting that TM affects post‐hatch immunity. Furthermore, this study may suggest that TM improves heat tolerance by increasing the expression of signaling proteins important to the wound‐healing process.Support or Funding InformationDeanship of Research/Jordan University of Science & Technology (Grant#: 128/2017).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Quantitative Analysis of p73 Gene Promoter Methylation
Authors: Nicolaci Angelo A , Carastro L. Michael 
Published: June 2021

The p73 gene is a member of the p53 tumor suppressor gene family. The p73 gene contains a distal P1 promoter and a proximal P2 promoter. The P1 promoter is responsible for expression of the TAp73 protein isoform containing a transactivation domain (TA). The ΔNp73 protein isoform is expressed from the P2 promoter and lacks the N‐terminal TA domain. In this study, we analyzed six CpG islands in these p73 promoter regions. The purpose of this project was to quantitatively assess the degree of methylation at each CpG site in the six CpG islands. Three prostate cancer (PCa) cell lines (DU145, PC‐3, LNCaP) and non‐cancerous primary prostate cells were cultured, and cell pellets were collected. Cellular genomic DNAs were subjected to pyrosequencing analyses of five CpG islands (CpG islands #1–5) located in the P1 promoter region and one CpG island (CpG island #6) in the P2 promoter region. In total, the degree of methylation was determined for 241 individual CpG sites. Pyrosequencing analysis of CpG island #1 indicated 15 of 23 CpG sites were methylated >90% in all four cell types. Seven CpG sites had a methylation frequency range of 71.1% to 96.5%. The remaining CpG site methylation ranged between 60% to 68.8% across the four cell types studied. In CpG island #2, 9 of the 14 CpG sites were methylated >91.9% in all four cell types studied, and the 5 remaining CpG sites ranged from 70.3% to 95.6% methylation. In CpG island #3, the 37 CpG sites were methylated <6.4% in all cell types studied. In CpG island #4, none of the 112 CpG sites were methylated >64.7% in primary cells. Differential methylation was observed in the 112 CpG sites among the three PCa cell lines, ranging from 0.0% up to 91.8%. In CpG island #5, the 45 CpG sites in primary cells were methylated <43.19 %, but PCa cells displayed differential methylation values from 0.0% up to 92.7%. In CpG island #6, the 11 CpG sites were methylated <9.0% in primary cells, whereas, PCa cell lines were differentially methylated from 0.1% up to 98.1%. Of the six CpG islands analyzed, CpG island #3 had the lowest degree of methylation in all samples analyzed. CpG islands #1 and #2 had high degrees of CpG methylation in all four cell types. Differential methylation was observed in CpG island #4, CpG island #5, and CpG island #6. Generally, CpG sites were methylated to higher degrees in PCa cell lines compared to primary cells. Furthermore, the overall trend in high frequency CpG methylation decreased from DU145 to PC‐3 to LNCaP. We are currently performing chromatin immunoprecipitation (ChIP) assays in the differentially methylated regions in order to identify potentially important transcription factors.Support or Funding InformationUniversity of Tampa Department of Chemistry, Biochemistry & Physics Summer Research Fellowship ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bacterial Expression of Chimeric Escherichia coli and Trypanosoma brucei DNA Methyltransferases
Authors: Taber Cassandra Carol , Militello Kevin 
Published: June 2021

Our laboratory is interested in DNA and RNA methylation in E. coli and T. brucei as little is known about this form of epigenetic regulation in microorganisms. One methyltransferase being studied at this time is a putative DNA methyltransferase (TbDmt) from Trypanosoma brucei. The exact function of TbDmt is unknown but the protein strongly resembles bacterial DNA methyltransferases such as DNA cytosine methyltransferase (EcDcm) from E. coli. To test our hypothesis that TbDmt is a DNA methyltransferase, we expressed TbDmt in bacteria and created two chimeric protein sequences switching the DNA binding domain and enzymatic domain of EcDcm and TbDmt. Exchanging the DNA binding domain and enzymatic domain of TbDmt with a known methyltransferase may help us discover the function of the enzyme and, if it is a methyltransferase, what DNA sequence is targeted for methylation. Plasmids were made containing the sequences for EcDcm, TbDmt, and both chimeric proteins where the genes are adjacent to the T5 promoter and lac operator. E. coli lacking a cytosine DNA methylation pathway were transformed with the plasmids and expression was induced with IPTG. All four proteins were produced at 20°C, but the proteins with the TbDmt DNA binding domain were less soluble than the other two. The plasmids were re‐isolated after three hours of growth in the presence of IPTG. The plasmids were then digested with eight restriction enzymes blocked by methylation. Each digestion was run on an agarose gel with DNA from an uninduced cell and unmethylated phage lambda DNA as controls. EcDcm methylated at its expected site, 5′CCWGG3′, as determined by resistance to PspGI. In addition, there is partial resistance to HpaII in cells containing EcDcm indicating methylation at 5′CCGG3′ or related sequences. We are currently testing restriction enzyme sensitivity in the samples containing full length TbDmt and chimeric TbDMTs. Further work will be done to improve the conditions in which the chimeric proteins are produced to enhance folding of the potential DNA methyltransferases. In summary, this work contributes to our limited knowledge of DNA methylation enzymes and targets in bacteria and protists.Support or Funding InformationNIH, 1R15AI133428‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Importance of Genomic RNA1 3′ CITE in Blackcurrant Reversion Nepovirus Protein Synthesis
Authors: Baquero Galvis Laura D. , Shields Elizabeth , Morrison Evan J. , Filbin‐Wong Megan E. 
Published: June 2021

Canonical translation initiation and regulation involve end‐to‐end interactions between initiation factors bound to a 7‐methyl guanosine triphosphate cap at the 5′ end and at a poly(A) tail, at the 3′ end of the messenger RNA. This characteristic is also prevalent in many RNA viruses such as the Blackcurrant Reversion Nepovirus (BRV), which has two bipartite genomic RNAs (RNA1 and RNA2) that contain the 3′ poly(A) tail but lack the 5′ cap. The mechanism implemented by BRV during translation initiation is not clearly defined; however, in other cap‐less, positive‐sense RNA plant viruses, structured RNA in the 3′ UTR binds translation initiation factors and delivers the machinery to the 5′ end via proposed RNA‐RNA kissing‐loop interactions. This non‐canonical (cap‐independent) mechanism is directly related to the primary, secondary and higher‐order RNA structure called a cap‐independent translation enhancer (CITE) in the 3′ UTR. Considering the end‐to‐end communication and recruitment of translation factors is dependent upon RNA structure, our goal is to determine the secondary and putative tertiary structure of the 3′ CITE found in the genomic RNA1 in BRV, and correlate the structure to translation initiation function. With this in mind, we identified a set of functionally‐deficient mutants using a luciferase reporter translation assay. We then probed our purified wild‐type and mutant RNAs with a series of covalent nucleobase and sugar backbone modifiers to identify the secondary (and potential tertiary) structure of each RNA. Linking the location of specific modifications to the efficiency of the translation process, our findings show that mutation of a conserved uracil within a highly stable G+C‐rich stem‐loop decreases translation by approximately 40%, but does not significantly alter the structure of the CITE RNA. However, mutating half of the stem to its Watson‐Crick complement significantly alters the RNA1 CITE structure and decreases translation by approximately 80%. From these results, we propose a model whereby the structure and stability of the stem is required to present the loop sequence (including the conserved U) for translation factor recruitment to the viral RNA. Additionally, this data indicates the locations and RNA structures within the CITE directly involved in translation machinery recruitment, allowing for a better understanding and comparison of the tendencies displayed by BRV as well as other positive‐sense RNA plant viruses.Support or Funding InformationColorado‐Wyoming Alliance for Minority Participation in STEM Research Grant MSU Denver College of Letters, Arts and Sciences Dean's Seed Grant MSU Denver Undergraduate Research Conference Student GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Thermodynamic examination of magnesium ion binding in the M‐box core 2 4×4 internal loop
Authors: Hall Simone , Gilbertson Ellen , Grover Neena 
Published: June 2021

RNA binds to magnesium ions through diffuse and site‐specific interactions. Diffuse binding of magnesium ions to the backbone occurs in all RNA and is critical for the stabilization of RNA structure. Magnesium ions also bind to specific sites in particular RNA structures, often with direct Mg2+‐RNA bond formation. In this study, Isothermal Titration Calorimetry (ITC) was used to experimentally measure the enthalpy, binding constants, and Gibbs free energy of diffuse and site‐specific binding of magnesium ions to the core 2 of the Salmonella Typhimurium M‐box riboswitch. This Mg2+ binding bacterial riboswitch upregulates magnesium transporting proteins when intracellular ion concentrations are above an acceptable level. Crystal structure of the M‐box riboswitch RNA shows eight magnesium ion binding sites clustered in three cores. The core 2 of the M‐box riboswitch is made up of a 4×4 adenine‐rich internal loop that binds magnesium ions at residues A63 and A80. RNA constructs were designed to mimic the wild type core 2 and were also modified at positions A63 and A80. A helix construct, with the internal loop deleted, was also utilized. A statistical model for RNA‐Mg2+ binding was used to fit the ITC data to determine the thermodynamic parameters of diffuse and site‐specific magnesium ion binding to the RNA. The models used to fit ITC for ion binding will be presented. In general, we find that the diffuse binding is spontaneous, endothermic and entropically driven with binding constants in the low millimolar range. Site‐specific binding showed micromolar binding constants.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanistic and Transcriptome‐wide interrogation of eukaryotic translation initiation factor 3 (eIF3)
Authors: Aitken Colin Echeverría , DiNardo Acadia , Yoo Yoon , Stanciu Andrei 
Published: June 2021

Translation initiation is the rate‐limiting and most‐regulated phase of translation, and its misregulation has been implicated in myriad maladies, including cancer and neurodegenerative disease. In eukaryotes, initiation is a complex pathway requiring the participation of at least twelve initiation factors (eIFs). Eukaryotic translation initiation factor 3 (eIF3) is the largest and most complex of these factors and appears to participate in events throughout the pathway.In particular, eIF3 is a pivotal player in the recruitment of mRNA to the translation pre‐initiation complex (PIC). Biochemical and genetic studies have identified interactions of eIF3 with the PIC near the mRNA entry and exit channels of the ribosome; structural studies have revealed that eIF3 binds the solvent face of the small (40S) ribosomal subunit, projecting arms into both mRNA channels and ultimately encircling the PIC.We recently probed the effects of a library of S. cerevisiae eIF3 functional variants using an in‐vitro reconstituted system. These experiments demonstrated that mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment of a native yeast mRNA. Using a series of model mRNAs to control the amount of nucleotide sequence in either the mRNA entry or exit channel, we further revealed a critical role for eIF3 in stabilizing mRNA at the exit channel. Together, these experiments are consistent with the entry‐ and exit‐channel arms of eIF3 playing distinct roles during PIC formation, mRNA recruitment, and scanning.We have also investigated the collaboration of eIF3 with Rps3/uS3 in stabilizing mRNA within the mRNA entry channel. We found that Rps3/uS3 residues previously implicated in the helicase activity of the elongating ribosome stabilize mRNA binding within the ribosomal entry channel and enhance the effects of eIF3 on mRNA binding in both channels. More recently, we have observed that eIF3i and eIF3g contribute to the PIC‐dependent enhancement of eIF4A ATPase activity, which recent work suggests may function to remodel the mRNA entry channel.Our current work is focused on dissecting the interactions between eIF3, the PIC, and other initiation factors in both the mRNA entry and exit channels, as well as on the inter‐subunit face of the PIC, using both in‐vitro and next‐generation sequencing approaches. In particular, we are exploring the genome‐wide translational effects of depleting or disrupting the eIF3 complex, using ribosome profiling. In parallel, we are investigating the effects of mutations to the 40S protein Rps2 on the PIC:mRNA interaction, as well as how mutations to Rps2 and Rps3 affect the sensitivity of this interaction to mutations within the eIF3 complex.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Dissection of the Mechanism of Eukaryotic Initiation Factor 3 (eIF3)
Authors: Yoo Kyungyoon , Aitken Colin Echeverría 
Published: June 2021

Eukaryotic translation initiation involves a complex series of events culminating in the assembly of a ribosome containing the methionyl initiator tRNA (Met‐tRNA) positioned over the AUG start codon of an mRNA. This process is the rate‐limiting and most regulated step of translation. It begins with the formation of a pre‐initiation complex (PIC) on the small (40S) ribosomal subunit. This PIC is then recruited to the 5′ end of an mRNA, from which it scans in the 5′ to 3′ direction to identify the start (AUG) codon. This convoluted pathway is mediated by multiple protein factors called eukaryotic initiation factors (eIFs). Previous genetic and biochemical studies have shown that eIF3, the largest of these protein factors, participates in events throughout the pathway. However, characterization of eIF3's role has been challenged by this pervasive participation and its complicated architecture: eIF3 is a multisubunit complex containing at least five essential subunits, complicating mutational analysis. To circumvent this problem, we are reconstituting yeast eIF3 by recombinant expression from bacterial strains. Previous structural and biochemical studies have demonstrated the feasibility of this approach. We aim to couple it with an easily manipulatable in vitro reconstituted system to enable systematic dissection of the entire complex. Our initial studies will focus on a set of mutations targeted at understanding the role of eIF3 in mRNA recruitment: truncation of the eIF3c Nterminal domain, which has been seen to promote accurate start‐codon selection through interaction with eIF1; isolation of the eIF3a/c heterodimer that stabilizes mRNA at the ribosomal exit channel; truncation of the eIF3b C‐terminal domain, which promotes the formation of the eIF3b‐g‐i module found near the mRNA entry channel; and truncation of the eIF3b RRM domain. The effects of these mutations on component events during translation initiation will shine new light on the role of eIF3 and its subunits.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.