bluuebunny/crossref_metadata_2025_split huggingface dataset:
TRANSLATIONAL REGULATION OF HYPOXIA‐INDUCIBLE FACTOR 2α IN RESPONSE TO IRON AND CELLULAR STRESSORS
Authors: Lin Willey , Deck Kathryn , Eisenstein Richard 
Published: November 2023

Hypoxia‐inducible factor 2α (HIF‐2α) is a heterodimeric helix‐loop‐helix transcription factor that plays an integral role in the regulation of erythropoiesis and iron metabolism and is a key player in the oxygen‐sensing and signaling pathways. HIF‐2α also stimulates lipid synthesis and storage in liver suggesting a role in controlling fuel metabolism in hypoxia and iron deficiency. HIF‐2α's regulatory role in iron metabolism makes it a subject of interest in relation to blood disorders, in particular anemia induced by iron deficiency or inflammatory disorders. HIF‐2α expression has been thought to be primarily controlled via oxygen‐ and iron‐dependent degradation by the action of prolyl hydroxylases and the von Hippel Lindau (VHL) E3 ubiquitin ligase. However, our lab and others have shown that dysregulation of HIF‐2α mRNA translation leads to hematological disorders. We are interested in examining the controls of HIF‐2α mRNA translation in response to iron and the presence of different stressors including the inflammation associated with diet‐induced obesity. Our research currently focuses on the 5′ untranslated region (UTR) of the mRNA of HIF‐2α. In the 5′ UTR of HIF‐2α mRNA is an inhibitory regulatory element called the iron responsive element (IRE). Iron regulatory proteins (IRP) bind to the IRE and are important regulators of HIF‐2α mRNA translation. The IRE impedes translation when bound to IRP as during iron deficiency. When iron levels are elevated, the IRP is inactivated and HIF‐2α mRNA is translated.To study translational regulation of HIF‐2α we created a reporter construct with the HIF‐2α 5′ UTR linked to GFP. Our plasmid, obtained from R. Tsien, also encodes RFP to control for transfection efficiency. Constructs containing the full length and truncated versions (all lacking the IRE) of the HIF‐2α 5′ UTR were used to investigate the role of the IRE and possibly other translational regulatory elements. Plasmids were transfected into HEK 293 cells and subjected to iron in the form of hemin (H), or an iron chelator, desferal (D). The wildtype HIF‐2α 5′UTR‐GFP construct was induced by hemin and repressed by desferal with a H/D ratio of 2.6. The deletion mutant with the shortest 5′ UTR (80% deletion) directed more than a 2‐fold higher level of GFP activity compared the wildtype and other truncation mutants indicating the presence of a negative regulatory element that is 3′ of the IRE. Surprisingly, all deletion mutants exhibited modest (~1.4 fold H/D ratio) response to iron suggesting the presence of additional regulatory elements responsive to iron that are independent of the IRE. Additional studies are underway to examine the translational regulation of HIF‐2α in animals.Support or Funding InformationSupport NIH DK66600
Achieving Tenure as a Clinical Researcher
Authors: Foreman K. Bo 
Published: November 2023

On average, a tenure track position at a four‐year college typically consists of a seven‐year probationary period. In a recent survey, approximately 20% of faculty are denied tenure resulting in loss of employment. To achieve tenure, faculty are typically required to meet specific benchmarks in research, teaching, and service. Meeting these benchmarks is often challenging. Therefore, the purpose of this talk is to address some of these challenges, specifically related to benchmarks in research. This talk will be structured around implementing a focused line of research, discussing the importance of infrastructure, and understanding the expectations of the institution.A seven‐year probation period may sound like a considerable amount of time to implement a focused line of research; however, this time passes quickly. Even with a focused line of research from a postdoctoral fellowship or doctoral research experience, new tenure track faculty members may face the challenge of implementing their research in a new institution and acquiring funding to support their research. To overcome this obstacle, developing strong collaborations with clinical faculty and/or clinical environments who share similar research goals can lead to subject recruitment, referrals, research ideas, and funding.Appropriate infrastructure is also critical and should incorporate both the physical structure and institutional support necessary to be successful. Depending on the line of research, typically the physical structure includes space, equipment, and associated startup funding. Physical structure is critical to have in place to be able to focus on research and scholarly productivity without having to worry about procuring space and equipment. In addition to the physical structure, institutional support must also be considered which includes items such as structured mentorship, educational opportunities, grant resources, statistical resources, and accounting support to name a few. Both physical structure and institutional support are critical to acquiring funding to keep a research program viable and productive.Lastly, understanding the expectations of the institution is important and often incorporates multiple levels of evaluation. Evaluation typically is performed at the department, college, and university levels. Because of this, faculty members need to make sure their focus is in line with the mission of their department, college, and university and firmly valued by the administration. This can be accomplished through initial interviews, yearly formal and/or informal reviews, peer evaluations, and frequent discussions with colleagues, mentors, and supervisors.In summary, this presentation will provide insight into some of the successes and pitfalls faced in achieving tenure as a clinical researcher.
Computational Design of Helical Peptides Targeting Transmembrane Domain of Cellular Receptors
Authors: Hu Hailin , Zhang Shao‐Qing , DeGrado William Frank 
Published: November 2023

Computed helical anti‐membrane protein (CHAMP) design has been shown to be a successful strategy to generate specific binding partners of αIIb and αv integrin transmembrane (TM) domains. In this research, we attempt to extend this method to new transmembrane receptor targets. Our first focus is the design of an antiparallel CHAMP for the mouse Erythropoietin Receptor (EpoR) transmembrane domain, which displays a typical sequence pattern of Ala Coil (Small‐X6‐Small motif). Searching this motif in the sequence space in our previously generated transmembrane helix dimer database results in several templates with optimal geometry as a starting point for design. Thiol exchange and Flash‐EDT assays demonstrate that the designed EpoR‐CHAMP interaction has a comparable affinity as in EpoR TM domain homodimerization. Our results demonstrate the strength of CHAMP design strategy and will probably generate a potent EpoR antagonist for biological research. Furthermore, we are also exploring to target other growth factors, like EGFR family receptors and other integrin members using this method.Support or Funding InformationThis work is supported by NIH grant GM54616. Hailin Hu is supported by China Scholarship Council.
Juncture of the cytoskeleton and innate immune signaling pathways: the role of Suppressor of IKK epsilon (SIKE)
Authors: Dohleman Emerald L , Steele Hannah J , Knoblauch Jilan B , Sonnenschein Halie A , Lawrence Kenneth F , Bell Jessica K 
Published: November 2023

The innate immune system rapidly responds to challenges by pathogens via activation of an inflammatory response. As a convergence point for multiple inflammatory and anti‐viral signaling pathways, TANK binding kinase 1 (TBK1) serves as a catalytic hub to initiate host defenses. Suppressor of IKK epsilon (SIKE) is a newly identified TBK1 substrate. The goal of this project was to identify the function of SIKE within the host's anti‐viral response. Co‐immunoprecipitation – tandem MS/MS analyses of the SIKE interaction network identified several interactions with cytoskeletal components. Immunofluorescence assays of endogenous SIKE with cytoskeletal markers and colocalization analyses indicated that SIKE colocalized with tubulin, actin, and a‐actinin. Reciprocal immunoprecipitation (RcIPs) studies confirmed the SIKE:tubulin and SIKE:a‐actinin interaction that appeared enhanced following dsRNA stimulation, a mimic of viral infection. ELISA assays were used to characterize the binding affinity between SIKE and the cytoskeletal proteins under unstimulated and simulated viral‐stimulated conditions. The SIKE:actin interaction was not detected by RcIP. In vitro immunoprecipitation assays mapped direct interactions between SIKE:tubulin and SIKE:a‐actinin and were used to further probe for a weak SIKE:actin interaction. Assays employed purified cytoskeletal proteins and full‐length (residues 1–207), N‐terminal (residues 1–112), C – terminal (residues 113–207) or a phosphomimetic (S6E) of a 6x‐His‐tagged SIKE construct. Together these studies establish an interaction between SIKE and cytoskeletal proteins that may provide a direct link between innate immune signaling and cytoskeletal components.Support or Funding InformationThis work was supported by NIH grant R21AI107447 and the University of San Diego SURE program.
Noncanonical PDZ Interaction between PKA RIα R368X Acrodysostosis Mutant and P‐Rex1 PDZ Domain
Authors: Del Rio Jason C , Bruystens Jessica G.H. , Chávez‐Vargas Lydia , Ma Yuliang , Kornev Alexandr P. , Vázquez‐Prado José , Taylor Susan S. 
Published: November 2023

Protein Kinase A (PKA), a tetrameric enzyme comprised of two catalytic (C) and two regulatory (R) subunits, is the prototypical kinase that responds to intracellular cAMP. Hindering the capacity of the RIα subunit isoform to bind cAMP, results in overregulation of the C subunit, a rare condition called Acrodysostosis (ACRDYS). Patients with ACRDYS typically have mutations in either RIα or PDE4D genes, and display stunted growth, mental retardation, and in some cases, hormonal resistance. Recently, a new interaction between RIα and the tandem PDZ domains of Phosphatidylinositol 3,4,5‐Triphosphate‐Dependent Rac Exchanger 1 (P‐Rex1) was discovered. P‐Rex1, a multi‐domain scaffolding protein with Rac specific guanine nucleotide exchange factor (GEF) activity, is highly expressed in certain leukocyte populations, and is intimately involved with regulation of cell migration. Using peptide blots, we scanned the Tandem PDZ domains of P‐Rex1 (residues 607–783) for interactions with RIα. We found that RIα bound to the α2 helix and the linker region between the tandem PDZ domains. Pulldown assays from HEK293 cells further confiirmed the interaction of RIα and P‐Rex1 PDZ domains. Interestingly, an ACRDYS mutation of RIα (R368X), deleting the last 14 amino acids from the C‐terminus of RIα, bound stronger to the PDZ domains of P‐Rex1 in contrast to the canonically defined mechanism of PDZ domain interactions, which bind to and recognize the C‐terminus via a β‐strand addition mechanism. Having solved the structure of this R368X ACRDYS mutant allowed us to model this noncanonical complex in silico using ZDock. We found that the PDZ domain may bind in the pocket of the second cyclic nucleotide binding domain (CNB‐B) of RIα, in agreement with our peptide blot data. This suggest that Acrodysostosis may not only alter regulation of the C subunit, but also change the PKA signalosome. We hope to validate this interaction by X‐ray crystallography.Support or Funding InformationThis Work was supported by NIH Grant GM34921 to Susan S. Taylor.Jason C. Del Rio was supported in part by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology through an institutional training grant from the National Institute of General Medical Sciences, T32 GM007752.
In Vivo Assessment of a Tissue‐Engineered Vascular Graft in a Rat Model
Authors: MacIsaac Corina , Stringa Pablo , Lausada Natalia , Nieponice Alejandro , Geest Jonathan Vande 
Published: November 2023

Heart disease is the leading cause of death in the United States, of which coronary artery disease (CAD) is one of the primary types. The current treatment of CAD involves bypassing the blocked coronary artery with an auxiliary blood vessel obtained from elsewhere in the patient's body. As an alternative to using this type of bypass graft, much research has been done on the fabrication of a biocompatible tissue engineered vascular graft (TEVG). The development of this TEVG is hypothesized to eliminate problems of bypass graft failure and compliance mismatch present in the current treatment. The basis of the TEVG is a scaffold which must provide (1) mechanical support of the TEVG and (2) a mesh for cells to proliferate and migrate through. This is sought to be achieved by electrospinning non‐synthetic biopolymers (gelatin, fibrinogen, collagen and tropoelastin) into a tubular construct. This procedure generates a thin fibrous mesh which allows for cell growth within the construct. The use of non‐synthetic biopolymers allows the scaffold to better compliance match a native coronary artery, as well as favor cell attachment. Preliminary experiments have been performed with different combinations and layering of the biopolymers, various speeds at which the construct was electrospun, and different amounts of glutaraldehyde cross‐linking time. Success has resulted from electrospinning constructs from a 10% by volume solution 80:20 gelatin/fibrinogen (w/w) in 1,1,1,3,3,3‐Hexafluoro‐2‐propanol (HFP). By analyzing the mechanical properties of these constructs with a microbiaxial optomechanical testing apparatus, the constructs have shown stress and strain relationships comparable to previously tested porcine left descending coronary arteries. Fluorescence imaging has also shown that porcine aortic smooth muscle cells will migrate and proliferate within this TEVG.In addition to mechanical and in vitro cell culture testing, in vivo assessment of the acellular TEVGs has also been performed. This was achieved by performing infrarenal aortic interpositional graft placement surgery to implant the construct within a rat's aorta. This is a survival surgery and the rats remained alive for up to a month before sacrificing for analysis. Preliminary results of the in vivo assessments necessitated the use of synthetic polymer incorporated into the construct for added strength to be sutured at the anastomoses. However, results of these preliminary assessments show the effectiveness of using this surgery for analysis of graft function in vivo. Future research involves increasing the strength of the construct without the addition of a synthetic polymer, as well as seeding endothelial cells onto the constructs before implantation.Support or Funding InformationFunding for this research is provided by: NIH 1R2HL111990 to JPVG (Co‐I Doetschman and Slepian) and HHMI 52006942 to the Biomedical Research Abroad: Vistas Open! program.
Calorimetic Study of Calcium Binding Proteins with their Interacting Peptides
Authors: Wei Chin‐Chuan , Schaffer Kyle , Jensen Drake , Ngo Rebecca 
Published: November 2023

Several Ca2+‐binding proteins, including Calmodulin (CaM), NADPH Oxidase 5 (NOX5), and dual oxidase (Duox), were employed in calorimetry to understand their structural change upon ion and peptide ligand binding. A general mechanism for these proteins is that Ca2+ binding triggers a conformational change, forming a hydrophobic patch that is important for target protein recognition. Previously, we have characterized chimera proteins constructed by exchanging EF‐hands of CaM with those of Troponin C (TnC) to be used as an informative probe to better understand the functionality of each EF‐hand. Our results revealed that Ca2+ binding to chimeras impairs their hydrophobicity, and such conformational changes altered their interaction with a CaM‐binding domain (CMBD) peptide of Orai. 3TnC and 4TnC (3rd and 4th EF‐hand exchange position, respectively) exhibited a lower ANS binding and solvent accessible surface area change (ΔASA) based on structural homology modeling, but binding to Orai‐CMBD remained similar to CaM, suggesting that the exchanged EF‐hands in the C‐terminal lobe are capable of dynamically accommodating peptide binding. Our isothermal titration calorimetry (ITC) data indicates that approximately 0.3–0.7 protons are released to the buffer when CaM and chimeras are titrated with saturating Ca2+, suggesting that key residue(s) involved in Ca2+ binding are not altered significantly by the exchanged EF‐hands. The heat capacity change of Ca2+ binding (ΔCp) for CaM was determined to be ~ −0.66 kCal K−1 mol−1. The observed large negative value is probably due to 1) the increase in nonpolar ASA and decrease in polar ASA, which results in a positive ΔCp and a higher binding entropy change (ΔSb) and 2) the reorganization of hydrogen bonds including water molecules (i.e. solvation), contributing a relatively larger negative ΔCp. Interestingly, the ΔCp values of 1TnC and 2TnC are similar to CaM. On the other hand, 3TnC and 4TnC have very small ΔCp values ranging +0.004 to −0.011 kcal K−1 mol−1, possibly due the decrease of helical content determined by Circular Dichroism. The EF‐hand domain (EFD) of NOX5 has been suggested to be structurally similar to CaM. However, the proton release and ΔCP are ~0.07 and −0.0026 kCal mol−1K−1, respectively, indicating a fundamental structural change in CaM that is supported by modeling. We are currently investigating the heat capacity change upon peptide binding for these three Ca2+‐binding proteins, as well as their chimeras and mutants, to give insight about the binding modes and to rationalize apolar/polar surface changes to their ΔCp values. Differential scanning calorimetry (DSC) was also used to gather information about non‐covalent interactions in protein folding.Support or Funding InformationThis work is supported in part by National Science Foundation CCLI grant and Cottrell College Science Awards (CCSA) from Research Corporation.
Database for Storage and Rapid Retrieval of ProMOL Results
Authors: Baker Cameron D. , Mills Jeffrey L. , Bernstein Herbert J. , Craig Paul A. 
Published: November 2023

ProMOL is an extension to PyMOL, a widely used molecular visualization program, developed by students at RIT and Dowling College to help determine the function of proteins. The process is computationally intensive. This is exacerbated by the fact that we have a large number of motifs to search against. A database was created to store the results for fast and easy retrieval. The database is stored on a standalone server and results are transferred by TCP/IP. The implementation itself is very simple and provides a base for future development. Results show a runtime improvement from over 30 minutes to just under a minute for alignment results for a single protein. The speed gain comes from the retrieval of prior results. This greatly improves search time for users looking for matches that have been encountered previously, either by themselves or by other users. Future improvements may include a NoSQL database on top of MySQL to increase the speed of data retrieval.Support or Funding InformationThis work was supported in part by NIH GM078077.
Identification of the Recognition Sequence in Gluten by the Prolyl Endoprotease of Eurygaster integriceps
Authors: Melton Stephanie 
Published: November 2023

Eurygaster integriceps Puton, Sunn pest, is the most globally destructive insect pest of bread wheat and durum wheat infecting 15 million hectares (HA) in Western and Central Asia and in Eastern Europe. To discontinue the harmful effects that current pesticide treatments have, environmentally safe biopesticides, inhibitors of the Sunn pest's digestive enzyme, are being sought. The objective of this study is to advance knowledge and understanding of the recognition sequences of the Sunn pest prolyl endoprotease (spPEP) within its natural substrate, gluten, as well as provide information towards the development of peptide inhibitors. The first method to the study is to separate gluten into glutenins (high molecular weight) and gliadins (low molecular weight). Gliadins were separated according to pI with BioRad's Rotofor Cell system and then by molecular weight with 12% SDS gels into their α/β, γ, and ω isoforms, Current results from the SDS gels indicate several gliadins have similar pI's between 7.22 pH and 8.72 pH consistent with the theoretical pI and molecular weight generated using the ExPASy Bioinformatics Resource Portal. The information from the NCBI database for the subdivisions of gliadins allowed identification of the proteins for spPEP in‐gel digestion. To date, γ gliadin has been identified with the predicted characteristics. Once digested, the fragments will be analyzed using MALDI‐Mass spectrometry to determine the mass and amino acid sequence of each fragment. Future experiments will include sending fragments to the Molecular Structure Facility at UC Davis, Davis CA for N‐terminal sequencing. When all the analysis is gathered, the data will be compared with the complete collection of gliadin sequences from NCBI to identify the cut and noncutting sites of the spPep for future design of inhibitors.Support or Funding InformationFunded by Stephen F. Austin State University Graduate Research Assistant (GRA) Program
Noninvasive Biomarker Candidates for Cadmium‐induced Nephrotoxicity by 2DE/MALDI‐TOF MS and SILAC/LC‐MS Proteomic Analyses
Authors: Moon Aree , Kim Sun Young , Lee Hye Min , Ko Eun‐yi , Kim Kyeong Seok , Kim Hyung Sik 
Published: November 2023

Cadmium (Cd2+) is a major environmental pollutant that induces cytotoxicity by heavy‐metal accumulation. Prolonged Cd2+ exposure leads to cell damage by oxidative stress mainly in the kidneys, a critical organ for detoxification. In order to identify reliable on invasive protein biomarkers for Cd2+‐induced nephrotoxicity, we performed 2DE/MALDI‐TOF MS and SILAC/LC‐MS analyses using conditioned media (CM) of HK‐2 human kidney epithelial cells treated with CdCl2. Here, we identified heat shock cognate 71 kDa protein isoform1 (HSPA8) and α‐enolase (ENO1) as potential biomarker candidates for the evaluation of Cd2+‐induced nephrotoxicity. Treatment with CdCl2 increased the protein level of HSPA8 in CM and lysates of HK‐2 cells. The mRNA level of HSPA8 was also increased by CdCl2 treatment, indicating transcriptional regulation. The level of ENO1 was increased in CM, but not in lysates of CdCl2‐treated HK‐2 cells. CdCl2 did not affect the mRNA level of ENO1. We provide evidence that the increases of HSPA8 and ENO1 in CM were due to Cd2+‐induced cell death through oxidative stress. The increases of HSPA8 and ENO1 levels were also detected in CM of HK‐2 cells treated with other nephrotoxic agents such as HgCl2, NaAsO2, cisplatin, amphotericin B, and cyclosporine A. Urine and kidney tissues of CdCl2‐treated rats showed increased levels of HSPA8. Taken together, this study identified HSPA8 and ENO1as non‐invasive biomarker candidates by two comparative proteomic analyses. These new biomarker candidates may have potential as alternatives to traditional biomarkers for the efficient and sensitive assessment of nephrotoxicity.Support or Funding InformationThis work was supported by a grant from Korea Food & Drug Administration [10182KFDA992‐3202] and by the National Research Foundation of Korea (NRF) grants [2013R1A2A2A04013379].