bluuebunny/crossref_metadata_2025 huggingface dataset:
Choice of Size Parameter Alters Interpretation of Fossil Hominin Distal Humeral Morphology
Authors: Zelazny Kaya G , Sylvester Adam D , Ruff Christopher B
Published: June 2021

Allometric trends in human distal humeral morphology may account for a substantial portion of morphological variation observed among fossil hominins, affecting both taxonomic assessments and behavioral reconstructions. However, previous allometric studies have focused on the relationship of humeral morphology to size parameters of the distal humerus itself (i.e., centroid size), which is potentially confounded by interspecific differences in scaling of humeral dimensions to body mass, a factor that creates direct biomechanical demands on the forelimb in all non‐human primates.

In this study, we compared the relationship between distal humeral centroid size and body mass in extant hominids (N=246 modern humans, 47 Pan, 44 Gorilla, 22 Pongo) and three fossil hominins (A.L. 288‐1, StW 431, KNM‐ER 1503/1504) where body mass could be estimated from femoral head size. We then scaled principal components extracted from geometric morphometric (GM) analyses of three regions of the distal humerus (articular, periarticular, and distal diaphyseal) by both body mass and centroid size in order to evaluate differences resulting from choice of size parameter. Finally, we analyzed the relationship of nine fossil hominins to humans of equivalent body mass (A.L. 288‐1, StW 431, KNM‐ER 1503/1504) and centroid size (KNM‐KP 271, A.L. 288‐1, StW 431, KNM‐ER 1504, KNM‐ER 739, SKX 10924, SK 24600, TM 1517, Gombore IB‐7594).

While centroid size was highly correlated with body mass in all taxa (r=0.88–0.97), humans were highly positively allometric and had significantly smaller centroid sizes relative to body mass than other hominids, as did A.L. 288‐1 and KNM‐ER 1504. StW 431, however, showed a relationship between distal humeral size and body mass more similar to that seen in non‐human great apes. There was significant allometry in the traits differentiating modern humans and other hominids that resulted in large‐bodied humans showing more great ape‐like distal humeral characteristics than smaller humans in the articular and distal diaphyseal but not the periarticular region. This increased differences in size‐scaled morphology between modern humans and fossil hominins in several cases, most prominently A.L. 288‐1. The differences between humans and other hominids in the relationship of centroid size to body mass create the appearance of a distinct fossil hominin morphological group composed of A.L. 288‐1 and other small fossil humeri (SKX 10924, SK 24600) when principal components extracted from the GM analyses are regressed on centroid size. When body mass is used as the independent variable for regression, however, this distinct fossil grouping disappears, and it becomes clear that this morphology is also seen in small‐bodied chimpanzees and orangutans.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
How do Mysticete (Baleen) Whales Make Two Sounds Simultaneously?
Authors: Reidenberg Joy S. , Laitman Jeffrey T.
Published: June 2021

Mysticete (baleen) whales use the larynx to generate sounds. Air flowing past the U‐shaped vocal folds causes vibrations that are transferred through overlying throat tissues to water. Sounds can be made repetitively without surfacing for a breath due to air recycling between lungs and larynx. We hypothesize that tissue movements direct airflow between sound generating and air recycling regions of the larynx. Methods involved examination of twenty mysticete specimens found beached along the northeast US coastline. Larynges were recovered post mortem from carcasses of minke (Balaenoptera acutorostrata), fin (B. physalus), sei (B. borealis), blue (B. musculus), humpback (Megaptera novaeangliae), and right (Eubalaena glacialis) whales. All specimens were collected under the auspices of the Marine Mammal Health and Stranding Response Program, and possession was permitted via a letter of authorization from NOAA‐NMFS. Results show that the larynx has two valves. One valve is located between the vocal folds. This valve is aided by a newly discovered cushion of tissue on the ventral surface of the cricoid cartilage. It appears to function like a plug, apparently hermetically sealing the gap between the vocal folds. Air flowing around this interlock is channeled superiorly between the corniculate cartilages. A second valve is found between the paired corniculate cartilages. Closure is aided by raising the epiglottis to plug the gap between the corniculates. This seals the rostral larynx, preventing air from escaping towards the nose. Air is instead diverted ventrally between the vocal folds and into the laryngeal sac. These two valves direct airflow towards vocal folds or corniculate flaps. Flow between the vocal folds may generate song, and allow extended vocalizations as air is captured and recycled between the laryngeal sac and the lungs. Flow between the corniculate flaps may generate pulses, but these sounds would continue only until the nasal region becomes pressurized.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A rare bilateral occurrence of extensor medii proprius in a female cadaver
Authors: Nierstedt Ryan , Friedman Brett , Desai Maya , Kneen Kassy , Barlow Sara , Waheed Muhammad , Barremkala Malli
Published: June 2021

During a routine anatomical dissection of 48 upper limbs from 24 fixed cadavers, variants of extrinsic extensor hand muscles, including extensor medii proprius (EMP) and extensor indicis medii communis (EIMC), were observed. EMP was found bilaterally in 1 cadaver (4.2%; 95 year old female) and unilaterally in 2 cadavers (8.3%; 74 year old male, 78 year old female), while EIMC was only seen unilaterally in 3 cadavers (12.5%). Both EMP and EIMC demonstrated a tendinous insertion deep to extensor digitorum muscle into the extensor hood of the 3rd digit. All EIMC variants shared a muscle belly with extensor indicis (EI), while EMP variants maintained individual muscle bellies. The muscle belly for all EMP variants showed proximal attachments at the middle‐ or distal‐third of the ulna. All EMP and EIMC variants received innervation from the posterior interosseous nerve. In addition to contributing to prevalence reports on anatomical variations within the dorsum of the hand, our findings of a bilateral EMP in a female cadaver represent a rare variation not previously published. These EI variants may aid in clinical settings, such as identification, surgical planning, and muscle or tendon repair.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Unilateral Rectus Capitis Posterior Major Muscle Duplication: Clinical Implications
Authors: Snyder Sean B , Petrone Ashley Brooke , Maslowski Douglas J , Zdilla Matthew J , Lambert H. Wayne
Published: June 2021

Here, we present an anomaly of the rectus capitis posterior major (RCPM) muscle, found during routine cadaver dissection in a gross anatomy course. The RCPM is one of four muscles in the suboccipital region, the superior portion of the posterior neck, inferior to the occipital region of the head. The suboccipital muscles, including the RCPM, maintains posture of the neck to support the head and facilitate flexion, extension and lateral bending of the neck and rotation of the head at the atlanto‐occipital joint. We observed a unilateral, right‐sided duplication of the RCPM. This anomaly has several clinical implications: (1) Contraction of this duplicate muscle may cause postural instability or shift toward the right side, leading to improper weight bearing of the head. This may also cause loss of range of motion in the head of neck. This may lead to chronic neck pain and cervicogenic headache. (2) The greater occipital nerve passes through the suboccipital triangle, as it relays cutaneous innervation from the occipital scalp. We observed the greater occipital nerve passing between the two RCPM muscles, suggesting a potential for compression and sensory deficits. (3) The vertebral artery also passes inferior to the suboccipital muscles on its course to the brain stem, and compression of the vertebral artery by the RCPM may reduce blood supply to the brain stem, leading to dizziness or other neurovascular symptoms. Overall, this anomalous muscle, though likely rare, may be identified as a potential cause of the aforementioned symptoms, and surgical intervention may be warranted in severe cases.

Support or Funding Information

N/A

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Biochemistry and the Opioid Crisis: Using the Primary Literature to Teach Core Concepts and Underscore the Societal Relevance of Biochemical Research
Authors: LOCHNER JANIS
Published: June 2021

Macromolecular structure, interactions and dynamics are essential underpinnings of an education in biochemistry. Biochemists often convey these core concepts by systematically probing the structure function relationship of archetypal proteins. G‐protein–coupled receptors are the largest class of signaling receptors in the genome and the target of over 30% of marketed drugs. The mu opioid receptor (MOR) is a member of this influential receptor family and constitutes the main opioid target for the management of pain. Insight into opioid action has been facilitated by acquiring structures of the MOR in inactive and agonist‐induced active states. Importantly, these structures enabled the application of structure‐based approaches to drug discovery in a quest to find an ideal opioid that elicits analgesia but fails to trigger the respiratory depression associated with the epidemic of opioid‐induced deaths.

To provide an authentic learning experience, a primary literature module that explores the structure function of the MOR, and the ongoing work on developing optimized opioids as well as non‐opioid analgesics was integrated into our undergraduate biochemistry class. Students in the class undertook a deep reading of the primary papers detailing MOR structure and then addressed questions on the mode of ligand engagement and signal transduction in a think‐pair‐share method during class time. In a subsequent class session, teams of students critically assessed findings from published studies on designing ‘biased’ opioids that activate signaling pathways relevant to analgesia, but not those that produce unwanted effects. To complement this focus on the biochemistry of opioid analgesics, primary papers that detail the development of non‐opioid alternatives for the pharmacological treatment of pain were probed. Since humans with loss‐of‐function mutations in the gene that codes for the sodium channel subtype Nav1.7 exhibit complete insensitivity to pain, a literature review session focused on the diverse biochemical approaches being used to develop potent, selective inhibitors of Nav1.7. This literature module succeeded in illuminating the contemporary relevance of structural biochemistry by delving into the molecular interactions relevant to the escalating opioid crisis

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Developing a 3D Physical Model of 16S rRNA m1A1408 Methyltransferase, NpmA to Enhance Student Understanding of the Mechanisms of Resistance to Aminoglycosides
Authors: Toledo Silvia , Renn Jaxon , Rosenthal Caleb , Robinson Ashley , Bygd Madison , Ademola‐Green Dami , Martinez‐Vaz Betsy
Published: June 2021

Aminoglycosides are broad‐spectrum antibiotics common in clinical, veterinary, and agricultural settings and are often reserved for treating severe bacterial infections. With antibiotic resistance becoming a global crisis, understanding the mechanisms by which bacteria attain such resistance is more urgent than ever. One such mechanism specific to aminoglycosides is ribosomal modification by methyltransferases (RMTases). RMTases pose a significant threat as they grant simultaneous resistance to various aminoglycosides and are transmitted between species. The purpose of this project was to develop a 3D physical model of NpmA, an RMTase that confers blanket resistance to aminoglycoside antibiotics by transferring a methyl group to the A1408 nucleotide in helix 44 of the 30S ribosomal subunit. Methylation of A1408 makes helix 44 unrecognizable to these antibiotics. Database searches and sequence alignments were performed to identify conserved amino acids and structural features important in the catalytic mechanism of NpmA. Details of the protein structure and its interaction with helix 44 were obtained by analyzing the Protein Databank File 4OX9. To construct the physical model, the structure file (4OX9) was imported into Jmol and modified into a format suitable for 3D printing using scripts created by undergraduate researchers. The 3D model features NpmA interacting with rRNA and S‐adenosyl‐L‐methionine, the methyl group donor for the methylation reaction. The physical model also highlights key amino acids such as Arg207, E146, Trp107 and Trp197, which are critical to flip A1408 from helix 44 and position it into the enzyme's active site prior to methylation. A Jmol tutorial was created to complement the 3D model and assess students' learning of the structure and function of NpmA. Initial assessment of this activity showed improvement of students' protein visualization abilities, and computational skills. Future work will focus on field‐testing the complete exercise in a microbiology course to evaluate the impact of the 3D model on students' understanding of the mechanisms of antimicrobial resistance.

Support or Funding Information

This project was supported by NSF‐DUE 1725940 awarded to the CREST (Connecting Researchers, Educators, and Students) Project

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Managing course embedded research projects of any size using the Open Science Framework
Authors: Berndsen Christopher
Published: June 2021

The large biochemistry lecture section is not an environment where course‐embedded research projects are found. However, large lecture sections offer the opportunity to expose a large number of students with diverse backgrounds to an open, collaborative research experience. CUREs, while relatively common in biochemistry and molecular biology laboratory settings, are seen infrequently in lecture settings because of constraints on instructor time, access to projects, and having resources to manage a large research project. I will describe the implementation of a lecture‐based research activity that has been used in Biochemistry sections of over 100 students and was managed within the Open Science Framework (OSF). The research project reinforces concepts of biomolecule structure and mechanism through analyzing and modeling understudied areas of the biochemical literature. While performing this project over the course of the semester, students learn to search through scientific databases, analyze biomolecule structure, effectively convey scientific information in visual/text formats, and perform modeling to predict and explore the biomolecule structure‐function connection. OSF facilitates project management by serving as a central repository for the storage of data and hosts resources for analyzing results, collaboration between participants, describing conclusions, and dissemination of final project. This latter feature helps students develop public portfolios of their work for gaining employment or admission to graduate/professional school. Thus using the Open Science Framework to manage a course‐based research project aids students in learning the entire research cycle and developing professional materials to benefit their careers. In eight semesters of executing this project in my classroom, the activity has involved nearly 500 students and the data generated have been included in three peer‐reviewed publications.

All software and guides are free to use and are available through the internet, thereby reducing the cost burden and opening access to classrooms of all levels and resources. Instructor produced materials for this project including guides for using software/servers and OSF are available for use or adaptation through the Open Science Framework and the presenter's website. Moreover, new contributions and collaborations are welcome.

Support or Funding Information

The Thomas F. and Kate Miller Jeffress Memorial Trust, Bank of America, Trustee

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Integrating a Research‐Based Experience in the Biochemistry I Laboratory: From Separation of Colored Compounds to Protein Purification
Authors: Sherrer Shanen M. , Mertz Pamela S.
Published: June 2021

As part of our CUR Transformations Project work within the Department of Chemistry and Biochemistry, we aimed to create an additional research‐based experience for the Biochemistry I laboratory course. We designed a series of chromatography experiments that focus on careful observation, experimental design, data interpretation, and presentation skills. Students first work with three different types of columns during a two‐week chromatography lab. The first week they work with two mixtures of colored compounds, each mixture contains 3 out of the 5 compounds. These two mixtures are run through gel filtration, anion exchange, and cation exchange columns. Students analyze their observations in week 1 to design a purification scheme for separation of all 5 colored compounds together in one mixture in week 2. After finishing the two‐week lab, students write a paper based on their results in the format of Nature Methods. Following this experiment, students start a 3‐week lab where they design a scheme to separate two unknown colorless proteins in a mixture. They are given the following information: isoelectric points for both proteins, one of the two proteins has a 6x histidine‐tag, and the 260 nm extinction coefficient of the tagged protein. Students then choose a cation exchange, anion exchange, or an affinity column to use for protein separation. Students analyze their chromatography procedure by using the Bradford reagent to detect protein in column fractions, denaturing gel electrophoresis to assess purification and determine molecular weights of the two proteins, and a western blot to verify identity of one of the proteins using an antibody to 6x histidine‐tags. Lastly, students present their results to their peers in week 4.

Support or Funding Information

We would like to acknowledge support for this CUR Transformations project through NSF DUE IUSE grant # 16‐25354.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ataxia telangiectasia and Rad3‐related kinase (ATR) may prevent replication stress in planarian during regeneration
Authors: Sawyer Nathaniel , Tirgar Reyhaneh , Shamoon Ushna , Akpati Lois , Nguyen Lillian , Nam Edward A
Published: June 2021

S. mediterranea is a planarian with extensive regenerative ability which requires substantial cell division. However, DNA replication stress occurs every cell division and must be resolved. From yeast to man, an evolutionary conserved DNA replication stress response (RSR) is essential for life. The RSR is a signal transduction pathway regulated by Ataxia telangiectasia and Rad3‐related kinase (ATR) and Checkpoint 1 kinase (CHK1) to respond to replication stress. However, the RSR has not been established in planarian nor do we know if it functions in regeneration. We hypothesize that ATR may be essential for regeneration. Preliminary results show reduced regenerative ability in the presence of hydroxyurea (HU), a replication stress inducing agent. However, low concentrations (2mM) did not attenuate regeneration, indicating that ATR and the RSR may be present and able to overcome replication stress. To test this, we treated amputated tails with HU, ATR inhibitor (ATRi), and a combination of both. In 2mM HU, tails regenerated similarly to control. We observed only a one day delay in regeneration with ATRi alone. However, ATRi and 2mM HU in combination prevented planarian regeneration completely. This synergistic effect suggests that ATR is significant in the response to replication stress during regeneration. To obtain genetic evidence, we are in the process of knocking down ATR as well as other RSR genes like CHK1, WRN, etc. Further determining the importance of RSR genes in planarian will elucidate the conserved components and allow us to understand how ATR and the RSR contributes to the incredible regenerative abilities in planarian.

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.
Modulation of gene expression by Salvia hispanica seed extract in MC3T3 cells
Authors: Mendez Daisy May , Rey Maximo , Hernandez Tomas , Vel Jaysonn , Sanchez Diego , Nair Saraswathy , Banu Jameela
Published: June 2021

Postmenopausal bone loss weakens bones and make them fragile and easily susceptible to fractures. Available therapies have severe side effects increasing the need for new therapeutic agents that can be effective in protecting bone loss and have less‐side effects. Recently, Salvia hispanica (SH) is reported to have several health benefits and high calcium levels. Our studies using a rodent model for postmenopausal bone loss showed bone protective properties, we decided to determine the influence of SH seed extract on osteogenic gene expression.

We cultured MC3T3 cells, differentiated them into osteoblast and treated them with Salvia hispanica (SH) seed extract at optimal concentration. Cells were harvested, for gene expression studies, RNA was isolated using Purezol RNA isolation reagent (BioRad). cDNA was synthesized using standard methods. The cDNA was assessed for the expression of genes related to osteogenesis in real‐time RT‐PCR using gene specific primers and Taqman assays. Alkaline phosphatase, BMPr2, osteocalcin, osteopontin, DCAT (inhibitor of beta catenin – wnt signaling pathway) and Type 1 collagenase.

Alkaline phosphatase increased (58%) when treated with SH seed extract while type 1 collagen (18%), Bgl (19%), BMP2r (80%) decreased in the SH group. The expression of DCAT1, an antagonist of b‐catenin, also significantly decreased (75%) after treatment with SH. This suggests that SH may act via the Wnt signaling pathway to modulate bone metabolism.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Decoding Melanoma Antigen Gene (MAGE) Expression
Authors: Colemon Ashley , Ramanathan Saumya
Published: June 2021

Melanoma Antigen Genes (MAGEs) are a family of tumor‐associated genes whose protein products share a MAGE‐homology domain (MHD). Type I MAGEs are physiologically restricted in expression to the germline, specifically to the testis, however, they are aberrantly re‐expressed in a wide variety of cancers. Their expression often correlates with poor patient prognosis. There is a significant gap in knowledge regarding the regulatory mechanisms that cause this aberrant expression of Type I MAGEs. The overall goal of this project is to understand these regulatory mechanisms. We have tested the hypothesis that like many germline genes, the expression of the MAGE‐A gene family is regulated by promoter or DNA Methylation. Using DNA Methyltransferase inhibitor 5‐azacytidine, we determined that MAGE‐A gene expression can be controlled by CpG methylation. This data was validated by complementary bioinformatics analysis using the UCSC Genome browser methylome datasets showing conserved differentially methylated positions within the respective promoter regions of target MAGE‐As. In order to test the effect of the aberrant expression, we then introduced MAGE‐A genes into “normal” cells and tested deviations from known phenotypes. We discovered that when expressed several MAGE‐A genes can act as single‐gene drivers of cell proliferation and anchorage‐independent growth. Taken together our data indicate that MAGE‐A genes are expressed by epigenetic mechanisms, specifically CpG methylation and their aberrant expression in cancer is not happenstance but results in providing cells with a proliferative advantage.

Support or Funding Information

R25 Bridge to the Doctorate Grant‐1R25GM107754

NSF Implementation Award to L.L. # NSF HRD 1332284 Fisk University Implementation Award

NSF Research Initiation Award to S.R. #HRD1764201

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Conserved mechanisms of transcriptional pausing regulate diverse RNA polymerases
Authors: Palo Michael Z. , Mishanina Tatiana V. , Landick Robert
Published: June 2021

Transcription of DNA into RNA requires multi‐subunit RNA polymerases (RNAPs) in all forms of life. Pausing is an important regulatory feature of RNAPs that facilitates formation of RNA structures and the process of termination in bacteria. The elemental pause is an offline state of RNAP caused by conformational rearrangements and may be a precursor to longer‐lived backtracked and RNA hairpin‐stabilized pausing. While the backtrack pause has been well‐characterized in eukaryotes, biochemical data describing the elemental pause are lacking and hairpin‐stabilized pausing has not been described. As these last two types of pausing are important for the proper regulation of transcription in prokaryotes, it is important to know whether they are conserved in eukaryotes. Using in vitro transcription assays on synthetic oligonucleotide scaffolds, we investigated the response of Saccharomyces cerevisiae RNA polymerase II (Pol II) to the consensus elemental pause and his hairpin‐stabilized pause sequences from Escherichia coli. We demonstrate that RNA duplexes can form within the Pol II elongation complex and modulate pausing behavior. These duplexes can also halt transcription elongation in a manner analogous to intrinsic terminator hairpins found in prokaryotes. We also characterize the previously‐described HIV‐1 TAR pause as a consensus elemental pause sequence, confirming that features of the elemental pause are conserved from bacteria to mammals. These data show that aspects of pausing as a form of transcriptional regulation are conserved from bacteria to mammals, and guide the search for novel examples of elemental and hairpin‐modulated pauses in eukaryotes.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Methods of Mutation Efficiency Analysis for CRISPR/Cas9 in Fathead Minnow
Authors: Mielke Marissa J. , Blatz Donovan J. , Petros Mera T. , Kittelson Ashley , Via Zachary W. , Cavallin Jenna E. , Villeneuve Daniel L. , Ankley Gerald T. , Maki Jennifer A.
Published: June 2021

CRISPR/Cas9 mutation of fathead minnow genes allows for assessment of the key event relationships highlighted in Adverse Outcome Pathways (AOPs), diagrammatic representations of biological data from chemical exposure experiments. An AOP developed to hypothesize how mercaptobenzothiazole (MBT) exposure results in decreased anterior swim bladder inflation in fathead minnow reasons that MBT inhibits thyroid peroxidase (TPO), leading to a decrease in T4 hormone synthesis and the swim bladder phenotype.

In vitro fertilization and microinjection methods for fathead minnow were previously developed in collaboration with the Environmental Protection Agency research labs, and verified by successful CRISPR/Cas9 tyrosinase mutation. Tyrosinase mutant fish were identified by altered pigmentation and genotypically confirmed by Sanger sequencing and TIDE analysis (Tracking of Indels by Decomposition; tide.nki.nl). In a continued collaborative effort, mutation of TPO and additional AOP‐related targets have been attempted and assessment of possible mutant fish is ongoing.

In this work, methods of mutation analysis were assessed, including T7 endonuclease I (T7EI) kit, TIDE analysis and assessment via DNA Bioanalyzer. Results of the T7EI protocol with TPO experimental fish provided evidence of mutations, however, both the TIDE and Bioanalyzer data showed that mutation efficiencies were low to nonexistent in these fish. This is consistent with the lack of observable phenotype. Since the CRISPR/Cas9 attempt was not successful in generating mutations, the validity of the TPO AOP can neither be supported nor refuted until guide strand design is optimized to induce a higher mutation efficiency.

Current efforts are focused on guide strand redesign for a second attempt at TPO mutation. Additional targets are simultaneously being pursued for mutation with the in vitro fertilization and CRISPR/Cas9 system. Based on the data obtained here, TIDE analysis will be the primary technique utilized for assessment of mutation efficiency in subsequent studies. Though time‐consuming, TIDE analysis has proven reliable, provides clear results and is cost‐effective.

Support or Funding Information

U.S. Environmental Protection Agency, Office of Research and Development, Mid‐Continent Ecology Division, Duluth, MN. The College of St. Scholastica Faculty Development Committee Research Grant.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the role of ComFA ATPase in powering DNA uptake during bacterial transformation
Authors: Lin Xiaoxuan , Foster Hannah , Burton Briana
Published: June 2021

Horizontal gene transfer (HGT) is a process that largely promotes bacterial diversity through lateral exchange of genetic materials. As one of the major forms of HGT, natural transformation has been observed in over 80 species. The process of transformation involves a membrane‐associated protein machinery that mediates the binding, uptake, and recombination of exogenous DNA. Despite its large impact, little is known about the mechanism, and particularly, the power source for DNA uptake across membranes. One of the most likely sources powering DNA uptake is a motor protein. It has been postulated that ComFA, a member of DEAD‐box helicase family and a ssDNA‐dependent ATPase, contributes to the force needed for DNA import. In this study, we made a series of mutants to analyze the role of highly conserved residues in multiple regions of Streptococcus pneumoniae ComFA. First, we show that several mutants exhibit a defect in transformability in vivo, with a 10‐ to 100‐fold decrease transformation efficiency. Second, we show that ATP hydrolysis is not required for ComFA‐DNA binding but may stimulate DNA release. Moreover, we present in vitro data suggesting the coupling mechanism between ATP hydrolysis and DNA binding of ComFA. Our results suggest the mechanism of ComFA in transformation and its possible DNA‐binding surfaces. Our current work focuses on examining the ComFA‐DNA interactions using p‐benzoyl‐L‐phenylalanine (pBpa)‐crosslinking. Overall results of this study support the hypothetical molecular structure of ComFA and its role in powering DNA uptake in natural transformation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Specific Ionic Interactions Contribute to Substrate Binding of Lysine Deacetylases
Authors: Toro Tasha B , Broussard Christian G , Swanier Jordan S , Watt Terry J
Published: June 2021

Analysis of the human proteome has identified thousands of unique protein sequences that contain acetylated lysine residues in vivo. Lysine deacetylases (KDACs) are enzymes that reverse this post‐translational modification, by catalyzing the hydrolysis of ɛ‐N‐acetyllysine residues in proteins via a conserved mechanism. Deacetylation is important for many cellular processes and aberrant KDAC activity has been linked to numerous diseases. While proper deacetylation is known to be critical for proper cellular function, details regarding substrates of particular KDACs and how substrate specificity is determined are lacking. We have observed that the activity of KDAC8 with charged substrates in vitro is sensitive to the ionic strength of the reaction buffer. This observation led us to hypothesize that charged residues near the acetylated lysine may be important for the interaction of KDAC8 with its substrates. To test this hypothesis, we have employed an in vitro assay which relies on the reaction of fluorescamine with lysine residues to produce a quantitative fluorescent signal upon deacetylation of a peptide substrate. Using this method, KDAC8 activity was measured with several substrates under conditions of varied ionic strength. Interestingly, for several positively charged peptide substrates, KDAC8 activity increases as ionic strength is decreased, but the opposite is often observed when negatively charged peptide substrates are present. Preliminary results with derivatives of a known peptide substrate suggest that a positively charged side chain adjacent to the acetylated lysine may make a specific contact with KDAC8, as the ionic strength effect varies with the position of the charge in the substrate sequence. Screening a larger set of peptides with charged residues at specific positions relative to the acetylated lysine for activity with KDAC8 provides insight into the contributions of charge to substrate specificity. Comparison of these data with molecular docking data of the same peptides with the active site of KDAC8 can predict specific interactions between charged residues in the KDAC8‐substrate pair. Together, these results will lead to a better understanding of KDAC8 substrate specificity. Extension of this approach to additional KDACs and substrate behavior in vivo will provide insight into substrate specificity of the KDAC family and the biological targets of each KDAC.

Support or Funding Information

Funding was provided by NIH 5G12MD007595, R15GM129682, TL4GM118968, 5RL5GM118966, and the Louisiana Cancer Research Center.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating HSF1‐Mediated Interactions of Human Angiogenin with Chromatin
Authors: Vinopal Claire Grace , Dickson Kimberly A.
Published: June 2021

Angiogenin (ANG) is a pancreatic‐type secretory ribonuclease that was identified for its ability to promote new blood vessel growth. More recently, ANG has been implicated in neurodegeneration, inflammation, and reproduction, revealing its broad biological roles. The goal of our work is to understand the molecular activities of human ANG. During sustained growth of cultured human cells, ANG interacts with chromatin to upregulate rDNA transcription. Recent work in our lab demonstrates that ANG interacts with heat shock transcription factor 1 (HSF1). Our work is aimed at expanding our knowledge of ANG interactions with the human genome and determining if ANG works cooperatively with HSF1 to regulate stress‐response genes. Using chromatin immunoprecipitation (ChIP) sequencing, we have identified genomic targets of ANG biding and characterized the co‐regulation of heat shock genes by ANG and HSF1.

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 Protein‐Ligand Interactions in MsrA from E. coli
Authors: Winkie Mitchell L , So Joanne , Schlessman Jamie L , Smith Virginia F
Published: June 2021

The amino acid methionine is highly susceptible to oxidation via reactive oxygen species, resulting in loss of function in proteins containing oxidized methionines. This loss of protein function has been implicated in numerous neurodegenerative and age‐related diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (Lou Gehrig's Disease), emphysema, and arthritis. The enzyme methionine sulfoxide reductase (MsrA) catalyzes the stereospecific reduction of oxidized methionine in free amino acids, peptides, and proteins, restoring function in the latter. The catalytic mechanism of MsrA has been studied extensively, but less is known about how MsrA is able to recognize its large variety of substrates, from single amino acids or small molecules to oxidized methionines in the context of a protein. Earlier work from our lab using intrinsic and extrinsic fluorescence spectroscopy suggests that the presence of sulfoxide group on a methionine in a target protein enhances existing weak hydrophobic interactions with MsrA. The current study builds on these findings using fluorescence spectroscopy to characterize how MsrA of E. coli recognizes its substrates and repairs specific oxidized methionine residues in an intrinsically‐disordered form of staphylococcal nuclease and a nonapeptide derived from it.

Support or Funding Information

This work was supported by a Research Corporation award (grant number CC5482) to VFS. The Office of Naval Research supported Trident Scholar research by JS. MJW was supported by a Garrigues Program scholarship award.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nitrite and NO Processing by CblC: A Human B12 Trafficking Chaperone
Authors: Mascarenhas Romila N , Gherasim Carmen , Banerjee Ruma
Published: June 2021

Cobalamin (Cbl) or vitamin B12 derivatives are processed and escorted to the two known B12‐dependent enzymes: cytosolic methionine synthase (MS) and mitochondrial methylmalonyl CoA mutase (MCM). Mutations in the B12 trafficking genes lead to inborn errors of cobalamin metabolism belonging to the cblA‐G, J and mut complementation groups. Of these, cblC is the most common locus for mutations and patients present with both homocystinuria and methylmalonic aciduria. Functionally, CblC is involved in the early steps of the cytosolic B12 trafficking pathway and catalyzes divergent chemical reactions. Cobalamins entering the cytoplasm with various upper ligands are processed to a common cob(II)alamin intermediate by the CblC protein before being allocated to the methycobalamin or 5′‐deoxyadenosylcobalamin branches. Alkylcobalamins are cleaved via a nucleophilic displacement mechanism in the presence of glutathione (GSH) producing GSR and cob(I)alamin, which is subsequently oxidized to cob(II)alamin, and cyanocobalamin is cleaved via reductive elimination forming cob(II)alamin and cyanide. Herein, we report the discovery of a new activity catalyzed by CblC, i.e. nitrite reductase. The interactions between nitrite and NO with cobalamins have been characterized in solution, but the biological relevance of these reactions is largely unknown. It was previously shown that nitrosylcobalamin (NOCbl) has antitumor activity that preferentially targets cancer cells in which cobalamin receptor expression (TCII) is upregulated. The mechanism of action of NOCbl has been ascribed to its ability to release nitric oxide (NO•). However, the ready oxidation of NOCbl to nitritocobalamin (NO2Cbl) was not taken into consideration. In this study, we have investigated the reactivity of human CblC towards NOCbl and NO2Cbl. We show that CblC binds NOCbl with a konof 0.07 ± 0.01 mM−1s−1. The cobalamin is bound in a base‐off conformation as are the natural B12 derivatives and this is likely facilitated by the strongly σ‐donating NO‐ligand that promotes the dissociation of the coordinating dimethylbenzimidazole group. Surprisingly, neither thiols nor reductants remove the nitrosylgroup from CblC‐bound NOCbl. Addition of thiols to CblC‐bound NO2Cbl generates nitrite and cob(II)alamin (anaerobically) or aquocobalamin (aerobically). The nitrite that is release can potentially serve as an NO• source via the activity of cellular nitrite reductases. Collectively, our results show that the promiscuity of CblC towards cobalamins can be exploited to deliver small molecules or to inhibit intracellular B12 metabolism.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidating the Heme Domain's Regulatory Role on Human Cystathionine β‐Synthase (hCBS) Product Formation
Authors: Ruiz Donovan
Published: June 2021

Human cystathionine β‐synthase (hCBS) is a vital enzyme found within the human transulfuration pathway and is responsible for the biotransformation of serine and homocysteine into cystathionine. hCBS at high cysteine concentrations has been shown to release hydrogen sulfide as a byproduct via an alternate reaction, suggesting hCBS to be closely involved with regulating metabolic sulfur. Catalysis in both the primary and alternate reactions proceeds via a PLP dependent β‐replacement ping‐pong mechanism, with the last step before product dissociation from the internal aldimine (Schiff base) intermediate being the condensation of H2O or H2S, respectively. Consequently, mis‐regulation of substrate levels or loss of CBS activity has been causally linked to hyperhomocysteinemia, a biological condition signifying abnormally high levels of homocysteine within the circulatory system; this condition being one of many causes leading to cardiovascular disease. A highly conserved PLP catalytic and SAM regulatory domain can be seen when comparing yeast analogue (yCBS) to mammalian variants. However, the mammalian variants, specifically the human analog hCBS, differs by containing a unique heme binding domain not found in yCBS. Given that the heme prosthetic group is not required for catalytic activity, the role that this additional domain plays in regulating cystathionine production in hCBS is not yet clearly understood. Current methods for measuring CBS catalytic activity involve using radioactively labeled substrates and typically have focused only on yeast and truncated hCBS variants. We have adapted a previously established colorimetric coupled enzyme assay to measure hCBS kinetics, coupling CBS with cystathionine β‐lyase (CBL), and lactate dehydrogenase (LDH); where the stoichiometric oxidation of NADH, monitored spectrophotometrically at 340 nm, is correlated directly to the formation of cystathionine. Preliminary results suggest this coupled assay is a viable method for measuring product formation kinetics of full length wild type hCBS. Current and future work aims to 1) asses catalytic rate activity for wild type hCBS and 2) asses the activity of single point mutant variants with mutations localized within the heme binding domain. Through these experiments, we hope to elucidate a deeper understanding of how the heme binding domain affects cystathionine product formation.

Support or Funding Information

NIH MARC: T34‐GM008574

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determining the Cause of Peak Invertase Activity Delay using Isothermal Titration Calorimetry
Authors: Chan Dindi , Noh Jihoon , Mason Marco , Kenealey Jason
Published: June 2021

Invertase catalyzes the hydrolysis of sucrose producing glucose and fructose. However, its mechanism of action is not well understood as other studies have shown that there is an initial delay before the enzyme reaches peak activity. This indicates that there may be another step for the active form of invertase to be activated. To better understand this mechanism, isothermal titration calorimetry (ITC) was used to measure the enzymatic activity of invertase. In this study, varying concentrations of invertase between 0.0125 to 0.125mg/mL were used to react with 1M and 2M sucrose solutions. Results show an exponential relationship between varying concentrations of invertase and the time to reach peak height. Increasing 0.0125mg/mL of invertase by tenfold, decreases the time required for the enzyme to reach peak activity by 85%. This suggests at increased invertase concentrations there is more active invertase, which may be because invertase forms a type of multimer for full activity. The effect of temperature was also explored, 1.2M sucrose was titrated into 0.016mg/mL invertase at temperatures from 25 to 55 °C. By comparing 25 and 45°C, we observed that the time required to reach peak activity decreased by 62%. However, at 55°C, the time increased by 24%. Also contributing to the delay is the viscosity of sucrose. Concentrations of sucrose ranging from 0.5 to 2M were titrated into 0.0125mg/mL of invertase. The time for the enzyme to reach peak activity increases by 23% when increasing sucrose concentration from 0.5M to 2M. However, this increase in time was also observed in the control (0.5 to 2M sucrose concentration titrated into 0.1M sodium acetate buffer). Therefore, the viscosity of sucrose does not completely account for the delay. In conclusion, the delay in peak maximal activity is in part due to increased sucrose concentration, probably due to inefficient mixing. Additionally, the delay in maximal activity is decreased in the presence of increasing invertase concentration indicating that there are inter‐invertase interactions that are required for maximal invertase activity.

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