bluuebunny/crossref_metadata_2025 huggingface dataset:
Axial Image Orientation in Anatomy Disciplines: Clinical View not Anatomical View
Authors: Champney Thomas
Published: June 2021

Historically, horizontal (axial) images in the anatomical disciplines have been presented with posterior (dorsal) at the top of the field and anterior (ventral) at the bottom of the field. With the development and clinical use of axial imaging techniques (computed tomography (CT) and magnetic resonance imaging (MRI)), the clinical axial presentation has been standardized with posterior at the bottom of the field and anterior at the top of the field. This situation has required students and faculty to switch between two different orientations depending on the type of information presented. When learning embryology, neuroanatomy or any other subject, students should be able to assume that all axial images are in the same orientation – with posterior (dorsal) at the bottom of the field and anterior (ventral) at the top of the field. This makes an easier and smoother transition from foundational basic science instruction to clinical imaging. For the past three years, the anatomy, embryology and neuroanatomy instruction at the University of Miami has made a concerted effort to only use the clinical view when teaching first year medical students. This has required faculty to develop new images in the proper orientation. The students appreciate having one consistent orientation throughout their medical education. All stakeholders in the field should adopt this standard approach to displaying horizontal images. Educators, journal editors, book publishers and assessment organizations should insist that all figures be in the “clinical” orientation. This will provide a standardized view throughout medical education and will prevent confusion in students who have to switch from an anatomical view (during their basic science education) to a clinical view (during the remainder of their career).
An Exploration of Learner‐Centered Teaching and the Impact on Physical Therapy Students Teaching a High School Anatomy Summer Program
Authors: Callier Dylan , Chambless Taylor , Bruzzini Kristen
Published: June 2021

An evaluation of a student‐faculty collaborative study investigating the efficacy of graduate level physical therapy students conducting a student‐centered program entitled Body Basics: Exploring Anatomy to high school students. The study examined the impact of student‐centered learning on physical therapy students and whether the experience may influence their ability to articulate knowledge of anatomy to individuals with little or no anatomical background. This study presents the case that assuming a teaching role requires students to explain concepts in anatomy and kinesiology to a “lay audience” helping them attain clinical skills in communicating difficult concepts to patients they will eventually encounter.
Student experience and impact of participation in the Samuel Merritt University Community Service Honor Society
Authors: Allen Megan , Peck Lacey Beth , Elliott Craig , Puder Barb
Published: June 2021

The Samuel Merritt University Community Service Honor Society was established to acknowledge students who demonstrate excellence in community service, outreach, and volunteerism. An anonymous survey was developed and administered to evaluate student participation in community service events, assess the quantity of hours served, and study the impact that serving the community has on the participating students' education. Information regarding student demographics, student cohorts, age, gender, and ethnicity was obtained and evaluated. Additional information was evaluated regarding the type of outreach service events, volunteering challenges, student personal accomplishments, and educational benefits of being an active member of the Honor Society.
Encouraging Puerto Rican High School Students to Pursue a STEM Career Through an Anatomy & Neurobiology Outreach
Authors: González‐Solá Maryví , Rosario‐Canales Martín
Published: June 2021

Often Puerto Rican high school students are unaware of the opportunities in sciences. Furthermore, many students abandon school and encounter dangerous environments. Therefore, our objective was to create an anatomy and sciences outreach activity, entitled “What should I do after I graduate? Guidelines for success after high School”, for 84 high school students from a private school in Caguas, Puerto Rico to motivate them to study a STEM career. We created 8 stations with specific topics (cardiovascular system, respiratory system, neurosciences and drugs, muscle and skeletal system, reproductive system, environmental pollution and sciences orientation) and divided the 84 students into groups of approximately ±10 after giving them the pre‐test with 8 questions related to each station. Then the groups rotated through each station to learn about that topic. Later, they took a post‐test (same questions) in order for us to assess their learning experience. Each question worth 1 point with a maximum score of 8 points. Finally, they filled evaluation forms on each station (4‐excellent to 1‐poor) and made comments. A paired‐samples t‐test was conducted to compare PRE and POST tests (only 54 students completed). There was a significant difference in the scores for pre test (M=5.3, SD=1.18) and post test (M=6.77, SD=.90) conditions; t (53)=‐7.9, p 蠄 0.05. The results suggest the outreach activity had a positive impact in these students. The highest scored station in the evaluation form was the cardiovascular system and the sciences orientation station. It appears that our outreach activity had an impact on 54 students confirmed by our results, but we are confident we had a positive impact on the entire 84 students based on the evaluation form comments.
Embryology in the Medical Curriculum: The Perceptions and Opinions of CurrentAnatomy Faculty
Authors: Cassidy Keely
Published: June 2021

The anatomical sciences have seen a decrease in hours within U.S. medical education in recent years, most notably in the subject of embryology. With more changes to come, it is prudent to ask embryology educators about content placement, pedagogical methods, and personal experiences. The researchers distributed an online mixed methods survey to current anatomy and embryology faculty. Eighty‐eight percent of the 42 respondents teach at allopathic medical schools, 31% with at least 16 years of experience teaching embryology and 31% with less than 5 years. They primarily teach gross anatomy content, with a smaller percentage of embryology or various anatomy topics within an integrated curriculum. The integration of embryology into other courses is typical, remaining true in this study with 70% integrated and 30% stand‐alone courses. Sixty‐three percent of these individuals teach embryology because they volunteered, 25% were assigned, and 12% cited other reasons. When asked to rate their confidence level teaching embryology on a scale of 1 (low) to 10 (high), 79% reported at least a 7, with a mode of 7.5. Analysis of Likert items show that in general, they find embryology interesting and moderately difficult as a subject but find it to be important and applicable to the education of future physicians. Respondents answered an open‐ended question on how to improve the teaching and learning of embryology with several major themes: better integration and curricular placement, improved animations, and greater focus on clinical application. In this time of reform and integration, anatomy educators' perceptions and opinions of the current status of embryology in medical education must be gathered in order to assess its efficacy and to formulate inquiry‐guided recommendations for the future.
Is Carnivoran Skull Morphology Dependent on Predatory Style?
Authors: Dewar Eric , Kallamata Enian , Murray Brett , Hartnett Magen , Sood Ankit
Published: June 2021

Living carnivorans are diverse both in skull morphologies and in feeding style. Because predation as a feeding style is highly reliant on vision, we wished to determine if carnivoran skull morphology—particularly the morphology of the orbit—were related to feeding style. We photographed the skulls of museum specimens of 48 extant carnivore species (n = 225) and three Eocene‐age carnivorans (n = 7). From this sample, we measured the relative size and completeness of the orbit as well as the relative proportions of the face, orbital region, and cranium. We compared these measurements with taxonomic affinity, diel activity pattern, and feeding style of these species.

The longest orbital rims were found among the felids and the grey seal. The felids and phocids also had orbits with the largest relative diameter (measured from the postorbital process–dacryon). Diurnal hunters and more omnivorous species such as ursids and herpestids tended to have smaller orbital diameters. Nocturnal hunters such as felids and genets had the shortest faces and longest crania overall, in contrast to Canis and Ursus, which had the longest faces but shortest crania. Herpestids, hyaenids, and mustelids were intermediate in these proportions. Using these baseline data on Eocene carnivorans, we found that the small canid Hesperocyon overlapped with both canids and herpestids, while the Eocene felids Hoplophoneus and Dinictis demonstrated early establishment of the typical “cat‐like” skull proportions and feeding behaviors.
Phenotypic Analysis of Long Bones in Pannexin 3 Knockout Mice Using a Geometric Morphometric Approach
Authors: Caskenette Deidre , Laird Dale , Penuela Silvia , Wilson Tim , Willmore Katherine
Published: June 2021

Pannexin 3 (Panx3) is a channel‐forming glycoprotein that is expressed in skin, mammary gland and skeletal tissues. In vitro studies have shown that Panx3 is induced at the growth plate where it is thought to promote cell differentiation, a key role in bone formation. However, whether or not the ablation of Panx3 in mice causes phenotypic anomalies is unknown.

The study objective is to analyze the phenotype of long bones in a new Panx3 knockout (KO) mouse to assess the role of Panx3 in bone formation.

Ten KO and 10 wild‐type (WT) adult mice were scanned using high resolution in vivo micro‐CT. Each long bone studied was digitized using homologous landmarks. Data analysis using geometric morphometrics (multivariate statistical methods) allowed for a quantitative comparison of shape, size and variation of long bones between KO and WT mice.

KO mice demonstrated distinct long bone shape differences that were greatest at the joint surfaces. As well, long bones in KO mice were 5% smaller. A significant portion of the shape difference (20%) can be attributed to the size component of shape. KO mice demonstrated greater shape variation that also appeared to be driven by different patterns of growth.

Our findings suggest that Panx3 affects long bone shape through its effect on size. Importantly, KO mice appear to have altered and less predictable patterns of long bone growth. Thus, Panx3 may have a role in maintaining optimum bone growth.
Collagen XXIV Null Mouse Bones Are Not Osteoporotic Due to an Insufficiency of Osteoblastogenesis Factors
Authors: Gordon Marion , Lu Ziyao Eric , Zhou Peihong , Jeon Jae Yoon Louis , Hahn Rita , Gerecke Donald , Ramirez Francesco , Svoboda Kathy
Published: June 2021

Collagen XXIV is a non‐traditional member of the fibrillar collagens expressed in bone. The density of wild type (WT) and knock out (KO) mouse femurs was analyzed by micro CT. Femurs of 3 and 5 month old null mice were found to be osteoporotic as compared to those of WT mice. As a first avenue of investigation to understand this, the expression levels of mRNAs involved in the regulation of osteoblastogenesis were examined. cDNA was made from 7 days, 1 month, 2 months, 3 months, 6 months, 1 year and 1.5 year old femur RNA extracted from WT and KO mice. qPCR was used to evaluate the mRNA levels. GAPDH mRNA levels determined from the qPCR were used to normalize the results. Factors examined were RANK ligand (RANKL), Runx2, Lrp5, Indian Hedge Hog, FGF18, BMP4, Hey1 and Notch1. These mRNAs encoding osteoblastogenesis factors generally exhibited a greater expression level in the KO mice than in the WT mice. This was true for almost every time point. Since collagen XXIV null mice have osteoporotic bones, yet the bone cells express higher levels of osteoblastogenesis differentiation signals, we suggest the KO bones attempt to compensate for the cells' inability to make good mineralized bone. Future work will examine whether steps in bone remodeling is affected in KO mice. This work was supported by NEI EY009056, NIEHS ES005022 and NIAMSD AR055073
Covariation Among Elements of the Bony Torso in Anthropoid Primates
Authors: Ward Carol , Peacock Sarah , Winkler Zachariah , Maddux Scott
Published: June 2021

A cornerstone assumption for reconstructing ape evolution is that the hominoid torso differs from that of monkeys, reflecting adaptation to below‐branch arboreality. The fossil record leaves only sporadic bony remains, however, forcing paleontologists to rely on hypothesized covariation among bones to infer torso structure. Recent data suggesting more variation within the torso than previously appreciated challenges long‐held assumptions used to make these inferences. To explore these associations, linear and landmark data were collected from vertebrae, sterna, ribs, pelves, clavicles, scapulae and humeri of 256 anthropoids. Also, intact torsos of 57 cadavers were CT scanned. Data reveal significant correlations among lumbar vertebral, lower rib, and iliac morphology. Correlations between upper and lower portions of the rib cage are weak, as are relations among ribs, pectoral girdle and humerus. Patterns of allometry also vary among regions. These results reflect differing selection pressures and structural constraints on parts of the torso, and provide data with which to more accurately infer aspects of torso form, and locomotor adaptation, in fossil anthropoids.
The Effects of Clinical, and Sub‐Clinical mTBI on Cognitive Function in Varsity Athletes
Authors: Brewer Danielle , Wilson Timothy , Owen Adrian
Published: June 2021

Emerging research suggests that head trauma, causing clinical (medically diagnosed, ie. concussion), or sub‐clinical minor Traumatic Brain Injury (mTBI) is cumulative and correlates negatively with cognitive function. Given the high prevalence of head trauma in contact athletics, better understanding the relationship between contact‐athletic activity, mTBI, and independent aspects of cognitive function is imperative.

Sixty‐eight varsity football athletes were recruited to examine how cumulative head trauma and self‐reported mTBI affect cognitive function. Participants completed a 12‐test online cognitive battery (www2.cbstrials.com) at time points prior to, bi‐weekly throughout, and after the season. With each cognitive test, participants also completed a personal history questionnaire to assess their ongoing athletic activity and mTBI status. Through assessing three distinct aspects of cognitive function (verbal ability, short‐term memory, and reasoning ability) via the cognitive battery in a longitudinal fashion, we will comprehensively examine the effects of contact athletic participation on independent aspects of cognitive function. Results look to better characterize the relationship between mTBI and cognitive function as well as inform sport decisions made by athletes, recreationalists and athletic governance councils to guide participation and safe play decisions.
Expression of Tyrosine Hydroxylase in the Anterior Lobe of the Human Pituitary Gland: A Possible Local Autocrine/Paracrine Role in the Regulation of Pituitary Hormone Secretion
Authors: D'Assumpcao Carlos , Carranza Claudia , Banno Joseph , Nagy György
Published: June 2021

The main source of dopamine (DA) regulating anterior pituitary function is derived from the hypothalamic tuberoinfundibular (TIDA) neurons. Recent studies performed on mice have indicated the presence of a local source of DA because tyrosine hydroxylase (TH), the rate‐limiting enzyme of DA biosynthesis, has been found within the AL. Here, we provide evidence for the presence of TH in human cadaveric pituitary glands. The presence of TH is a good indicator of an intrinsic or local production of DA. Serial axial sections of pituitary glands obtained from two (70 and 87 year old) male cadavers have been histologically prepared and stained with hematoxylin‐eosin (HE) and immunohistochemistry has been performed using specific antibodies for TH. TH immunoreactivity has been found in the AL of both pituitaries. Similar to the AL of the mouse, TH positive cells have been localized bilaterally, at the border of the intermediate lobe (IL) and the AL. Incidentally, a distinct area in the left side of the AL of one of the pituitary glands has been found avoiding HE staining, which indicates a proliferation of cells forming an adenoma called “incidentaloma”. Curiously, an increased number of TH immunoreactive cells have been found in the area of the incidentaloma compared to the normal AL tissue of the same gland. The increased number of TH positive cells within the incidentaloma and the asymptomatic nature of it may suggest a compensatory elevation of local production of DA for being able to inhibit hormone secretion of the adenoma in an autocrine/paracrine manner. Therefore, a new mechanism of DAergic control of pituitary hormone secretion locating within the AL of the human pituitary gland can be hypothesized.
Cellular Identification of Doublecortin Positive Immature Neurons in Monkey Cortex
Authors: Heyworth Nadine , Calderazzo Samantha , Kyada Margee , Rosene Douglas
Published: June 2021

Doublecortin (DCX) is a microtubule‐associated protein expressed transiently during the maturation and migration of newborn neurons. In the adult brain, two neurogenic regions including the Subventricular Zone (SVZ) and the Subgranular Zone (SGZ) continue to produce adult generated neurons (aGN) throughout adulthood. The transient expression of DCX has also been found in various neocortical and allocortical regions in the adult brain of the rodent, rabbit, cat, monkey and human. These regions include the piriform cortex, medial prefrontal cortex, entorhinal cortex, as well as neocortical areas of the temporal lobe and insula. DCX+ cells are reported to localize mainly in superficial layers of cortex (II/III) with scattered DCX cells in deeper cortical layers. Some studies have reported these cells differentiate into excitatory neurons while others report a GABAergic phenotype. In addition to cellular identity, the origin of these neurons remains unresolved with one possibility that these cells are derived postnatally from the SVZ while others contend that they are generated prenatally.

To address these issues, tissue was obtained from rhesus monkeys injected with BrdU to label aGN. Sections were processed with immunohistochemistry and confocal microscopy to determine if DCX cells in the temporal lobe co‐localize with BrdU and/or with markers of inhibitory interneurons including Calbindin, Parvalbumin, and Calretinin, or with the excitatory neuron marker CaMKII. Results show no DCX+ cells co‐localized with BrdU, indicating that DCX+ immature neurons are not adult generated and hence are leftover from development. Preliminary analysis of co‐localization with DCX shows that at least some co‐localize with GABergic markers of interneurons.
Multiple Pathways Mediate Thyroid Hormone Receptor Nuclear Import
Authors: Allison Lizabeth , Roggero Vincent , Parente Laura , Zhang Jibo , Dziedzic Rose
Published: June 2021

The thyroid hormone receptor α1 (TRα1) is a nuclear receptor for thyroid hormone. While TRα1 carries out its function as a transcription factor in the nucleus, the receptor also shuttles rapidly between the nucleus and cytoplasm. Our prior studies show that nuclear import of TRα1 is directed by two nuclear localization signal (NLS) motifs, one in the N‐terminal A/B domain and the other in the hinge domain. Here, we investigated which importins mediate nuclear import of TRα1, using a combined approach of in vitro nuclear import assays and small RNA interference. Nuclear localization of TRα1 in digitonin‐permeabilized HeLa cells was temperature and energy‐dependent and could be reconstituted by the addition of cytosol or recombinant importins α1 and β1. In transient transfection assays, knockdown of importin 7, importin β1, and importin α1 markedly reduced nuclear localization of TRα1, while importin α3 had a lesser effect. Taken together, data suggest that TRα1 utilizes multiple import pathways, with nuclear entry facilitated primarily by importin 7, as well as by importin β1 in association with importin α1.
Population Health of Horseshoe Crabs (Limulus polyphemus) at Harvested and Unharvested Beaches in South Carolina
Authors: Walsh Rachel , Darden Tanya , Robinson John
Published: June 2021

The horseshoe crab (Limulus polyphemus) is a valuable species environmentally, commercially, and biomedically. The population genetic health of horseshoe crab populations in South Carolina has been relatively unstudied. This project is a part of a greater ongoing study examining the genetic health of horseshoe crab populations along the southern coast of South Carolina. A suite of 11 microsatellites was used to analyze the Coffin Point and Harbor Island horseshoe crab spawning sites. Genetic structure was analyzed along with various genetic health metric tests. Results indicated the two sites represent a single population (Jost's D=0.0019, p=0.2679; FST=0.0058, p=0.2385). The population analyses showed moderately high diversity (HE= 0.762), no inbreeding (FIS= ‐0.0057), and a high Ne value based on the lower 95% confidence interval obtained from linkage disequilibrium‐based estimates of Ne (Ne > 2240.7). These are all positive indicators for the adaptive potential of the population. The results of this study serve as an important baseline for future conservation and management of horseshoe crab populations.
Investigating the Role of Threonylcarbamoyl Adenosine in HIV‐1 Replication in vivo
Authors: Kirkland Marina , Venketaraman Vishwanath , Swairjo Manal
Published: June 2021

In all retroviruses, reverse transcription of viral genes is primed by a host‐cell tRNA whose 3′‐end 18 nucleotides are complementary to the viral primer binding site. For lentiviruses, including HIV, the natural primer is tRNALys3 which harbors several modified bases in its anticodon stem‐loop (ASL). Interaction of the fully modified ASL with the HIV RNA template is required for efficient reverse transcription of viral RNA, and continuation of viral life cycle. One of the modified nucleosides of the ASL of tRNALys3 is N6‐threonylcarbamoyl adenosine (t6A37), a universally conserved modification found at position 37 in all tRNAs decoding ANN codons. t6A37 stabilizes the ASL structure and prevents ribosomal frameshifting in the course of translation. Eukaryotic t6A37 biosynthesis is catalyzed by the enzyme Sua5 and the KEOPS complex, comprised of the four subunits Kae1, Pcc1, Bud32 and Cgi121.

We hypothesize that t6A37 plays an important role in viral replication in vivo, by stabilizing ASL structure or by direct interaction with viral RNA. To test this hypothesis, we set out to suppress the biosynthesis of t6A37 in CD4+ cells by targeting its biosynthesis genes with siRNA and testing viral load upon HIV‐1 infection. Background expression levels of the core human t6A37 biosynthesis gene Osgep (Kae1 ortholog) were determined by qRT‐PCR. Treatment with anti‐OSGEP siRNA resulted in knockdown of Osgep expression by 60% 48 hrs post‐transfection and sustained for 72 hrs, 4 times longer than the reported half‐life of OSGEP protein in human cells.
Structural insights into ribosome structure and translation regulation
Authors: Korostelev Andrei , Bah Eugene , Koh Cha San , Madireddy Rohini , Svidritskiy Egor , Zhang Ying
Published: June 2021

Protein synthesis (translation) is accomplished by ribosomes in all living cells. Ribosomes decode genetic information encoded in messenger RNA and catalyze peptide‐bond formation. We study the mechanisms of translation translation regulation, using structural and biochemical methods. Here, we will present new structural insights into regulation of translation on the ribosome under cellular stress and/or disease.
Characterization of the Small Splicing Protein Dib1, a Component of the U4/U6‐U5 snRNP
Authors: Hernandez Cody , Lucas Amber , Maeder Corina
Published: June 2021

The spliceosome is a highly dynamic molecular machine composed of small nuclear ribonucleoprotein particles (snRNP). The snRNP facilitates the joining of exons via two transesterification reactions that remove the non‐coding intronic regions. Once the introns are removed, the resulting mature RNA (mRNA) transcript can then be translated into functional protein. The fidelity of this process is crucial, and mistakes in splicing can lead to diseases such as Retinitis pigmentosa. One of the critical steps in splicing is the activation of the U4/U6‐U5 triple snRNP upon joining the spliceosomal complex.­­ In this study, we focus on the small, essential splicing protein Dib1, whose function in splicing remains unknown. Dib1 is 15kDa protein with a thioredoxin‐like fold. Previous data suggests that Dib1 interacts with splicing proteins that bridge the U4/U6 and U5 snRNPs. We characterized the role of Dib1 by rationally designing dib1 mutants and assaying for effects on Saccharomyces cerevisiae growth. We purified these Dib1 mutant proteins and examined their structures by circular dichroism in order to determine the effects these mutants have on the structure of Dib1 protein. In addition, splicing assays were conducted in order to determine the effects the mutant may have on mediating splicing. These assays will help further characterize Dib1 and its role in splicing as well as lend insight into further understanding the splicing machinery.
Structural Characterization of nc886, a Cellular Non‐coding RNA Regulator of the Antiviral Kinase PKR
Authors: Calderon Brenda , Conn Graeme
Published: June 2021

The double‐stranded (ds)RNA‐activated protein kinase (PKR) senses dsRNA produced during viral replication and acts as the first line of defense in the innate immune response to viral infection. Activated PKR phosphorylates the eukaryotic initiation factor 2 (eIF2), halting protein synthesis and viral replication. How PKR basal activity is regulated in the absence of viral infection is unknown. Recently, the cellular non‐coding RNA 886 (nc886) was discovered and proposed to act as a novel, endogenous regulator of PKR. We hypothesize that nc886 regulates PKR by binding to the monomeric form and preventing the dimerization step of activation. This maintains PKR in an inactive state that is poised for a rapid response to viral infection. Our goal is to define the molecular mechanism of nc886‐mediated PKR inhibition using RNA structure probing and mutational analyses combined with binding and activity assays. We show that nc886 RNA exists as two stable, structurally and functionally distinct conformations. Ribonuclease digestion reveals that the two conformers differ in their apical region, while the terminal stem portion of the RNA structure is conserved. The two conformers have strikingly different binding affinities for PKR, but both are functional inhibitors of PKR in in vitro kinase assays. By delineating the structural basis for PKR interaction with the different nc886 conformers, we will uncover the structural motifs that control PKR inhibition and determine how PKR is poised to act in the antiviral response.

Source of Research Support: NIH R21‐AI097803 and 5T32GM008367
Diversification of a Bacterial ADP‐Ribosyltransferase
Authors: Sung Vicky , Kallert Stephanie
Published: June 2021

OBJECTIVE

To accelerate the enzyme evolution, we diversified a bacterial ADP‐ribosyltransferase, cholix toxin catalytic fragment, to modify different substrates.

METHODS

Previously, we identified a diffusible intermediate that could modify target residues in a proximity dependent manner (submitted in the same meeting, Topic #2278‐ASBMB‐Enzyme Mechanism, Number 540). We utilized a phage display library screening strategy to select for variants to bind to different substrates of interests, followed by enzymatic screening for the selected variants retaining enzymatic activity. Site‐directed mutagenesis, enzymatic assays and 2‐D‐electrophoresis analysis were used to detect the in vitro modification of the substrates by the engineered enzymes.

RESULTS

We showed several examples of engineered cholix toxin catalytic fragment variants that can modify various substrates, including enhanced green fluorescent protein (EGFP) and mouse immunoglobulins.

CONCLUSION

We anticipate our findings to be a starting point for more sophisticated engineering. Such enzymes can be useful tools for basic research or therapeutic applications.
Investigating the Structural Basis of the CTD Code
Authors: Gibbs Eric , Showalter Scott
Published: June 2021

The goal of this project is to study the structure and function of the C‐terminal domain of RNA Polymerase II (CTD). The CTD, which contains tandem repeats of the consensus heptad sequence YSPTSPS, orchestrates transcription by recruiting trans‐acting regulatory factors, directed by a set of post‐translational modifications known as the CTD code. Since the discovery of the CTD code, much effort has gone into the identification of novel modifications, the determination of their effects on specific CTD interactions, and the structural characterization of transcription factors in complex with the CTD. Much less is known about the CTD in its unbound state and of the possible structural effects of the post‐translational modifications that constitute the code. Recently, we have begun biophysical characterization of the unmodified and hyperphosphorylated CTD using a combination of techniques, including 13C Direct‐Detect Nuclear Magnetic Resonance (NMR) spectroscopy and Mass Spectrometry. Preliminary results suggest that the CTD exists in an extended conformation that cannot be adequately described by the previously proposed beta–spiral model. The models we have developed serve as novel structural hypothesis, relevant for determination of the structural basis for the CTD code and the elucidation of the molecular mechanisms of eukaryotic transcription.

This work is funded by NSF CAREER award (MCB‐0953918) to Scott A. Showalter.
HSF4 Promotes Human Lens Epithelial Cells G1/S Arrest through Stabilizing p53
Authors: mi huang , meng gao
Published: June 2021

The differentiation from constantly dividing epithelial cells into fiber cells is the key step during lens development. Failure in lens fiber cells differentiation, which requires cell proliferation inhibition and cell cycle exit, causes cataract formation. It has been discovered that HSF4 (Heat Shock Factor 4) gene mutations led to congenital and senile cataracts. And the lens of HSF4 knockout mice showed increased proliferation of epithelial cells and premature differentiation of fiber cells. However, the molecular roles that HSF4 mutations play in cataract formation remain obscure. In present study, we found that wild type HSF4 suppressed human lens epithelial cells (HLECs) proliferation by promoting G1/S arrest. We further identified that HSF4 could interact with p53 and stabilize p53 protein by inhibiting its ubiquitin degradation.The p53 transcriptional activity was also increased by HSF4. In contrast, HSF4 cataract mutants had no effects on either HLECs proliferation or p53 protein stabilization. On the other hand, the wild type HSF4 lost its ability in cell proliferation inhibition and G1/S cell cycle arrest in p53‐null H1299 cell line. Our data reveals that HSF4 may work as a switch between lens epithelial cell proliferation and fiber cell differentiation, and p53 is required in this process.
Disulfide‐Mediated Stabilization of the IκB Kinase Binding Domain of NF‐kB Essential Modulator (NEMO)
Authors: Zhou Li , Yeo Alan , Ballarano Carmine , Weber Urs , Allen Karen , Gilmore Thomas , Whitty Adrian
Published: June 2021

Human NEMO is a 419 residue protein that, in complex with the catalytic subunits IKKα and IKKβ, forms the IκB kinase (IKK) complex, a key regulator of NF‐κB signaling. NEMO is a rod‐like homodimer comprising mostly α helix. It has been shown that a NEMO fragment spanning residues 44‐111, which contains the IKKα/β binding site, is structurally disordered in the absence of bound IKKβ. Here we show that enforcing dimerization of NEMO1‐120 or NEMO44‐111 constructs through introduction of one or two inter‐chain disulfide bonds – through oxidation of the endogenous Cys54 residue and/or at position 107 through a Leu107Cys mutation – induces a stable α‐helical coiled‐coil structure that is pre‐organized to bind IKKβ with high affinity. Chemical and thermal denaturation studies showed that, in the context of a covalent dimer, the ordered structure was stabilized relative to denatured state by up to 3 kcal/mol. A full‐length NEMO‐L107C protein formed covalent dimers upon treatment of mammalian cells with H2O2. Furthermore, NEMO‐L107C bound endogenous IKKβ in cells and reconstituted NF‐κB signaling in NEMO‐deficient cells. Our results indicate that IKKβ binding domain of NEMO possesses an ordered structure in unbound state, provided that it is constrained within a dimer as is the case in the constitutively dimeric full‐length NEMO protein. The stability of the NEMO coiled coil is maintained by strong inter‐helix interactions in the region centered on residue 54. The disulfide‐linked constructs we describe herein may be useful for crystallization of NEMO's IKKβ binding domain in the absence of bound IKKβ, thereby facilitating the structural characterization of small‐molecule inhibitors.
Molecular Dynamics of DNA Mechanical Contributions of Various Promoter Sequences in p53 Complexes Motivated by a Novel Additive Binding Energy Model
Authors: Han In Sub , Thayer Kelly M.
Published: June 2021

The tumor suppressor protein p53 binds genomic DNA as a cell cycle regulator that ensures DNA gets repaired before replication. When excessive damage renders cells non‐salvageable, the protein initiates apoptosis; however, disruption of this process may lead to tumor growth. An additive binding‐energy model was developed as a means to categorize known DNA binding sequences based on their hydrogen bonding patterns and DNA bending mechanics. The model incorporates reported key discriminatory hydrogen bonding interactions and the mechanical DNA bending caused by the “TG step” that occurs in the 1TUP wild type p53 crystal structure. We have found that binding categorically occurs in five groups hypothesized also differ in binding mechanisms. The high frequency of poly(A) and poly(G) tract regions in the non‐consensus p53 binding sites has prompted us to pay special attention to the DNA mechanics and its role in DNA‐protein interactions specifically in the p53 binding interface. To further investigate this hypothesis, sequences representing these features taken from known biological promoters were simulated with molecular dynamics using the AMBER suite programs. The results can iteratively refine the additive binding energy model as more detailed mechanistic properties are elucidated.
Structural Point Mutations of p53 Protein and Their Effects on the Zinc Coordination Complex
Authors: Quinn Taylor , Thayer Kelly
Published: June 2021

The p53 protein is an important transcription factor in the cell cycle. As such, mutations of this protein are found in at least 50% of all cancers, most of which are in the DNA‐binding domain containing residues 102‐292. This research is focusing on the molecular dynamics of p53 with point mutations that affect the structural stability of the protein, namely, R175H, R282W, G245S, and R249S. The system will be evaluated in reference to the Zn2+ cation that is tetrahedrally coordinated to Cys176, Cys238, Cys242, and His179. Changes in the stability of the coordination complex have been shown to have a greater effect on the stability of the overall protein, as it restricts the movement of the L2 and L3 loops. These systems and their changes are evaluated using molecular dynamic simulations with the AMBER12 suite, cpptraj, VMD and PyMOL. The objective of this study is to investigate how point mutations change the structural stability of the protein in relation to the zinc complex. Structural metrics including distance and surface measures shed light of the effects of these mutations on the dynamics of the protein. This informs future research initiatives for rational drug design.
Construction of p53 Protein Tetramer Structure
Authors: Carpenter Cassidy
Published: June 2021

The p53 protein controls the cell cycle checkpoint responsible for maintaining the integrity of the genome and inactivation of the p53 tumor suppressor is a common development in many different cancers. The monomer structure of p53 supports the hypothesis that DNA binding is critical for the biological activity of p53 and provides a framework for understanding how mutations inactivate it. However, the full tetramer structure, which has not been solved crystallographically in its entirety, is necessary to contextualize the findings from the monomer. Tetramer construction of the p53 protein was carried out using pymol, protein folding, and energy minimization. Additionally, the p53 tetramer interface was characterized by comparing how the tetramer structure interacted with DNA and other monomers contrasted to just the monomer structure. Lastly, high frequency mutations from the literature were mapped in order to identify significant mutations that could disrupt the tetramer formation. The exploration of these mutations on the constructed p53 tetramer provide the ability to understand how mutations to p53 are situated with respect to the tetramerization interfaces.
Unique Sensitivity to Extracellular Proteases and Peptide Protection of the HERG Potassium Channel
Authors: Zhang Shetuan , Guo Jun , Lamothe Shawn , Hogan‐Cann Andrew , Kang Yudi , Yang Tonghua
Published: June 2021

The human ether‐a‐go‐go–related gene (hERG) encodes the pore‐forming subunit of the rapidly activating delayed rectifier potassium channel (IKr), which is important for cardiac repolarization. Dysfunction of hERG causes long QT syndrome, arrhythmias and sudden death, which occur in patients with cardiac ischemia. Cardiac ischemia is also associated with activation of various proteases. We characterized the effects of proteases on hERG/IKr with the aim to develop novel strategies to prevent protease‐mediated damage. Using whole‐cell patch clamp and Western blot analysis we demonstrated that the hERG/IKr channel is selectively cleaved by the serine proteases, proteinase K and protease XXIV. Using molecular biology including making chimerical channels between protease K‐sensitive hERG and insensitive hEAG, we identified that the S5‐pore linker of hERG is the target domain for proteinase K. We discovered that the scorpion toxin BeKm‐1, which binds to the S5‐pore linker of hERG, can effectively protect hERG from proteinase K‐mediated damage. The reduction in mature ERG expression was observed in a rabbit cardiac ischemia model and in hERG‐HEK cells cultured in hypoxic conditions. BeKm‐1 effectively prevented the hypoxia‐induced reduction of the mature hERG expression. In conclusion, hERG is uniquely susceptible to proteases, which may contribute to arrhythmias under conditions such as cardiac ischemia. Protecting hERG channels from proteolytic damage using specific peptides may represent a novel strategy to prevent and treat arrhythmias associated with ischemic heart disease.

(Supported by the Canadian Institutes of Health Research & the Heart and Stroke Foundation of Ontario)
An Investigation of the Impact of NHE1 Phosphorylation on Steady State Intracellular pH
Authors: Hasle Daniel , Provost Joseph , Wallert Mark
Published: June 2021

The Sodium‐Hydrogen Exchanger 1 (NHE1) is involved in cell scaffolding, mobility and intracellular pH (pHi) homeostasis in most cells. The C‐terminus of NHE1 is phosphorylated by several protein kinases, each contributing to NHE1 function. While investigation of one or two sites have been conducted with various agonists, we do not have an understanding of how these sites coordinate or if there is a hierarchical phosphorylation regulation of NHE1. To determine the potential phosphorylation‐dependent regulation of NHE1 on pHi, we focus on four protein kinases with known sites on NHE1; ERK 1⁄2, protein kinase B (AKT), RhoA kinase (Rock) and ribosomal s6 kinase (RSK). To measure the impact of these four kinases we will treat cells with three different agonist; lysophophatidic acid (LPA), platelet‐derived growth factor (PDGF), and phenylephrine (PE). We first determined the dose response for each agonist in fibroblasts deficient in NHE1 expression (PS120 cells) and in stable expressing human NHE1 in PS120 cells (PSN cells). To determine the role of each kinase on NHE1 basal activity, cells were treated with each agonist and teady state pHi was determined 15‐20 min after addition of agonist. Increases in pHi of 0.18 to 0.2 were observed for agonists. The role of each kinase was determined by treating cells prior to agonist with a specific cell permeable kinase inhibitor (1uM MK‐2206, 10uM BI‐D1870, 10uM Y27632 and 0.5uM SCH772984). The impact of direct phosphorylation was determined for each agonist in cells expressing human NHE1 with Ser/Thr to Ala mutations for each kinase site. This work begins to identify the coordinated and role of multiple phosphorylation events on NHE1.
Differing Functions of Liver and Intestinal‐Fatty Acid Binding Proteins: Examination of the LFABP/IFABP Double Knockout Mouse.
Authors: Gajda Angela , Zhou Yin Xiu , Kodukula Sarala , Lackey Atreju , Pappas Arete , Agellon Luis , Storch Judith
Published: June 2021

The intestinal enterocyte expresses two fatty acid‐binding proteins (FABP): intestinal‐ (IFABP; FABP2) and liver‐FABP (LFABP; FABP1). These proteins have similar tertiary structures, but display different mechanisms of long‐chain fatty acid (LCFA) transport, and some differences in ligand binding specificities. The individual functions of IFABP and LFABP in intestinal lipid absorption and metabolism are incompletely understood. We previously reported that ablation of these proteins in mice results in divergent phenotypes in response to high‐fat feeding; LFABP‐/‐ mice gain more weight and fat mass than WT mice, while IFABP‐/‐ mice are lean. In the present studies, we generated LFABP and IFABP double knockout mice (DKO) to study the effects of simultaneous ablation of these proteins. Male C57BL/6 (WT), IFABP‐/‐, LFABP‐/‐ and DKO mice were fed 10 or 45 kcal% fat diets containing LCFA for 12 weeks. Interestingly, the phenotype of the DKO mice was integrated between those of the LFABP‐/‐ and IFABP‐/‐ mice, with body weight and fat mass that were between those found for the single knockouts. Remarkably, DKO mice display no evidence of lipid malabsorption, indicating that these proteins are not required for bulk uptake of FA into the intestine. Overall, simultaneous ablation of LFABP and IFABP does not reveal a dominant effect of either protein, but rather an additive effect of ablation of each of the proteins. The results suggest that IFABP and LFABP have different functions in intestinal lipid homeostasis, resulting in differential downstream alterations in systemic energy metabolism.
Oral administration of a Ketone Ester regulates blood cholesterol in rats and humans
Authors: Kemper Martin , Pawlosky Robert , King M Todd , Wainwright Camilla , Clarke Kieran , Veech Richard
Published: June 2021

Control of cholesterol in Type 2 Diabetics and Pre‐diabetics has been problematic as statins increase blood glucose. We propose that a unique β‐hydroxybutyrate (βHB) Ester can provide non‐lipogenic calories and decrease cholesterol synthesis without increasing glucose. The ketone bodies, βHB and Acetoacetate, can be metabolized by most tissues, but not liver, limiting cholesterol precursors. In humans, a single meal containing this ketone ester (KE) dose dependently decreased plasma mevalonate, a marker for cholesterol synthesis, without directly inhibiting HMG‐CoA Reductase. After two days of KE, rats had 31% lower plasma cholesterol and 26% lower mevalonate than controls. In a 30 day study, KE fed rats had lower plasma total cholesterol (‐40%) and mevalonate (‐27%) than controls. Levels of mevalonate precursors, Acetoacetyl‐CoA and HMG‐CoA were lower by 33% and 54% respectively, but Acetyl‐CoA was unaffected. Low liver cholesterol increases absorption of cholesterol through the Low Density Lipoprotein receptor (LDL‐R), KE feeding increased LDL‐R levels in whole cell (+24%) and membrane preparations (+67%). KE fed animals had increased protein levels of SREBP‐2, a key transcriptional activator of LDL‐R, both full length and transcriptionally active forms increased, by 91% and 140%. A 5 day study of KE effect on cholesterol in human Type 2 Diabetics showed not only lower total cholesterol (‐10%) but also lower fasting glucose (‐17%) and Triglycerides (‐18%). We report preliminary findings that KE decreases cholesterol and its synthetic precursors while decreasing fasting blood glucose and triglycerides.
Phosphoinositide Binding to ErbB3‐Binding Protein 1 Contributes to Its Nucleolar Localisation
Authors: Lewis Aurelia , Karlsson Thomas , Altankhuyag Altanchimeg
Published: June 2021

Phosphoinositides (PIs) play essential functions as signalling molecules, either directly or by acting as precursors to other signalling molecules. Although their functions have been mostly elucidated in the cytoplasm, PIs are also intra‐nuclear where they contribute to chromatin remodelling and mRNA processing. In addition, PI‐specific kinases, phosphatases as well as phospholipases regulate PI levels within the nucleus. In a previous study we identified ErbB3 binding protein 1 (Ebp1) as a nuclear PI‐binding protein by lipid pull‐down and mass spectrometry. Ebp1 is a conserved protein located in the cytoplasm and nucleus and its differential localisation is correlated to its role in cell proliferation. EBP1 is known to have different cellular activities such as proliferation, differentiation and apoptosis depending on the expression of its long form p48 or the shorter form p42. In this study, we have validated that recombinant p48‐Ebp1 binds to different sets of PIs via two distinct PI‐binding sites located at the N‐ and C‐termini and site‐directed mutagenesis revealed that these PI‐binding sites consist of patches of basic residues. The C‐terminal PI interaction motif was shown to contribute to the sub‐cellular targeting of EBP1 to the nucleolus. We have hence also determined that endogenous EBP1 colocalises with a marker of the nucleolus, nucleophosmin, as well as the class I PI3K catalytic subunit p110β and its product phosphatidylinositol(3,4,5)triphosphate in the nucleolus. Mutations in the same PI‐interaction motif have been identified in different carcinomas and one of these mutations prevents nucleolar targeting. Taken together, these results suggest that PIs may retain Ebp1 in different sub‐cellular compartments to affect its functions.