bluuebunny/crossref_metadata_2025 huggingface dataset:
Perceived relevance of anatomy to clerkship: opinions of an inaugural class of final year MD undergraduate students
Authors: Oyedele Olusegun
Published: June 2021

This poster outlines results of a pilot survey undertaken to evaluate the perceptions of a graduating class of medical students about the relevance of the anatomy instruction received to subsequent training during clerkship years. A questionnaire was completed by Year 4 students at the University of British Columbia's Southern Medical Program (n=32). This group of students formed the inaugural intake into the MD undergraduate distributed program at Kelowna, BC. Students received their anatomy instruction in Years 1 & 2 of a PBL‐based curriculum in which regional anatomy was taught through didactic sessions and cadaveric dissections, supplemented by prosections and use of anatomical models. The aim of the survey was to assess how much students considered the anatomy instruction that they received relevant to their subsequent clinical clerkship. Furthermore, we aimed to ascertain the degree of confidence felt by the students in their current knowledge of anatomy and in what ways factors such as the method of instruction, teaching styles of particular professors or location of their campus (hence access to a diversity of resources) affected this perception. Data analysis is ongoing and results will be presented.
Teaching the Extraocular Muscles of the Eye: Construction and Evaluation of a Low‐Cost, High‐Fidelity Ophthalmotrope for Medical Trainees
Authors: Maini Arjun , Welke Laura , Nagy Gyorgy
Published: June 2021

Examining extraocular eye movements is important in assessing nerve health and possible injuries as they pertain to the anatomy of the eye. The anatomy of the orbit and the function of the six extraocular muscles of the eye is a challenging and frequently misunderstood concept amongst medical trainees. In gross anatomy, educators have employed simple teaching tools such as balloons and static models to represent the relevant anatomy and help explain these difficult concepts. Absent from this education, however, is a movable, interactive and accurate model of the eye that demonstrates activation of the muscles along with the resultant movement of the globe of the eye. The goal of this project is thus to create a functional and high‐fidelity model of the extraocular muscular movements of the human eye, or simply an “ophthalmotrope”, to assist medical students with their anatomical understanding of this concept. A further goal is to ensure that this ophthalmotrope is fabricated from low‐cost and accessible materials as to ensure its reproducibility and use at schools where costs of pre‐fabricated plastic models is prohibitive. The final goal of this project is to investigate learning outcomes and student perceptions of this new teaching tool compared with those that have been employed previously at Ross University. Such outcomes will be measured through surveys and by a comparison of grades between two student groups, one with the teaching resource and one without the resource. We hypothesize that the addition of this new eye model will result in an increase in student performance on orbit anatomy related questions in both practical and written tests along with a qualitatively reported increase in understanding and confidence with the relevant anatomy.
An Untold Story: The Important Contributions of Muslim Scholars for the Discovery of Human Anatomy and the History of Evolutionary Thinking
Authors: Alghamdi Malak , Malik Aamina , Ziermann Janine , Diogo Rui
Published: June 2021

Evolutionary and anatomical textbooks tend to completely neglect, or include just a few pages about, the contributions of Muslim scholars for the discovery of human anatomy and particularly for the history of evolutionary thinking. This negligence is the result of complex historical biases, has profound implications, creates several misconceptions, and does not allow the general public and teachers, students and researchers to fully understand the true history of anatomical and evolutionary discovery. In an original project done at one of the most historical US universities ‐ Howard ‐ involving graduate students, faculty members, and their colleagues from other institutions, we did an extensive literature review of ancient texts written in various languages, particularly before Vesalius (about human anatomy) and Darwin (about evolutionary thinking), as well as of the few more recent reviews on the subject. Here we present, for the first time, the outcome of this review and discuss some of the Muslim scholars who made relevant contributions to these fields of science, which were unfortunately often forgotten in time, particularly in the West. This American Association of Anatomists meeting is the ideal place to unfold this untold story, both symbolically and pragmatically, due to the relevance of this story to students and professionals involved in evolutionary biology and anatomy, and to the broader public.
Comparative Study on the Effectiveness of Integrated vs. Discipline‐Based Digital Learning Resources
Authors: Mitchell Katherine , Lee Lisa
Published: June 2021

The continuing trend of integration and the consequent decrease in student‐instructor contact hours in basic science curricula in professional schools makes digital ancillary resources increasingly important. Majority of learning resources for basic sciences however are varied in content, accuracy and are presented in a discipline‐specific manner. Few integrated educational resources exist and even fewer studies report the effectiveness of an integrated presentation of the material in the digital environment. The objective of the study was to investigate educational effectiveness of a digital learning resource presented in an integrated manner compared to the traditional, discipline‐specific presentation. Two digital resources covering female pelvic anatomy, embryology and histology were created, both presenting the same content but one presenting each discipline separately while the other presents the material in an integrated manner. First‐year graduate students enrolled in a gross anatomy course at the University of Colorado Modern Human Anatomy Program were recruited and randomly assigned to view one of the two learning modules. The learning outcome was quantitated by pre‐ and post‐test score comparison as well as a retention study 30‐days after viewing the resource. A survey was conducted to analyze students' preference and perception of the effectiveness of the digital resources. The results of the study have important implications in designing and presenting educational resources in the digital age.
Assessing Medical Student's Perspective on Mobile Devices and Apps in an Integrated Curriculum
Authors: Harmon Derek , Burgoon Jennifer
Published: June 2021

INTRODUCTION

Since 2007, 590 million iPhones and 237 million iPads have been sold, while 85 billion mobile apps have been downloaded from Apple's iTunes store. From accessing patient medical records and CAT scans to utilizing a digital dissection guide and pharmacological references, the medical field has been at the forefront of technology utilization within practice and education. Since the fall of 2013, The Ohio State University College of Medicine has provided Apple iPads to incoming first year students. The students use the iPads to take practical examinations, watch e‐module lectures and download mobile apps they determine to be beneficial.

OBJECTIVE

Conduct focus groups with second year medical students to assess mobile device and mobile app usage during the first two years of an integrated medical curriculum.

METHODS

Focus group sessions ranging from single student groups, to groups of ten students were conducted. Medical students were asked to answer seven questions and discuss their answers related to personal use of mobile devices and mobile apps.

RESULTS

Although analyses of the results are ongoing, initial indications are that medical students are highly comfortable with mobile device usage; continually use mobile apps within each medical learning block and students demand integrated educational mobile apps.

CONCLUSIONS

Based on the focus group responses, medical students consider interactive, intuitive and high quality educational mobile apps to be the most beneficial. Creation of mobile educational resources must be a high priority for future medical education.
Medical Gross Anatomy for Large Classes in a ‘Flipped’ Classroom.
Authors: Meredith M Alex , Dupree Jeffrey , Clemo H Ruth , McGinn Melissa , Simpson David , Massey Hugh , Haar Peter , Digiovanni Susan
Published: June 2021

Traditional Medical Gross Anatomy courses are under considerable pressure to incorporate active learning tools while delivering clinically‐relevant material to large classes in a truncated schedule. Toward those goals, we have devised a course for 215 medical students that examines the basic regions of the body in 24 dissection sessions in a ‘flipped’ classroom format while involving longitudinal exercises and directly incorporating basic Radiology and Pathology. Prior to each laboratory session, students are required to view a ~30 minute custom‐made video introducing the basic anatomy of the exercise that also demonstrates the actual dissection steps. Following the 3‐hour dissection exercise, a faculty presentation extends the anatomical and functional information as well as introduces the clinical relevance of structures or region just examined. Each exercise culminates with a non‐graded self‐assessment quiz. In addition, a CT scan of each cadaver (n=32) and standard path reports for submitted tissue samples provide the students with additional structural and clinical insights into their cadaver. Observations obtained in these sequential exercises are archived in a daily dissection‐group log. A final, grand‐rounds style group presentation synthesizes the longitudinally‐obtained observations into a plausible clinical scenario consistent with a significant health deficit and/or cause of death. The effectiveness of the new curriculum will be evaluated by comparing board scores with those from classes prior to the curriculum change (data not yet available).
Developing Core Competencies in First Year Medical Students: Peer Presentations of Case Studies in Neurobiology
Authors: Salm Adrienne , Culberson James
Published: June 2021

In recent years the American Association of Medical Colleges and the Accreditation Council for Graduate Medical Education have placed increased emphasis on developing core competencies in trainees. Such competencies are meant to reflect characteristics that, ideally, will be displayed by every graduate upon completion of training. Here we describe a classroom innovation consisting of student‐led peer presentations of neurologic case studies that occurs during weeks 5 and 6 of a seven‐week medical neurobiology course for first year medical students. After completing study of major sensory/motor pathways comprising CNS functional systems, students are divided into small groups and randomly assigned a classic neurology case problem. After brief basic instruction in neurology case study problem solving, groups are given class time to work on presentations that they will give to one another. Our experience is that students bring a great deal of creative energy to this endeavor and that the exercise has been favorably received by most. In addition, we note that developing core competencies such as strong peer‐communication and teamwork, problem‐solving and the ability to apply a knowledge base to medical problems are strengthened by student participation in this exercise. Thus, in addition to facilitating the learning of challenging material, this classroom innovation is also consistent with the shift towards competency‐based education.
IL‐10 Improves Cardiomyocyte Survival via Downstream MyD88‐dependent TLR4 Innate Signaling of Jak/Stat3 Pathway
Authors: Bagchi Ashim , Akolkar Gauri , Mandal Soma , Singal Pawan
Published: June 2021

Recent evidence indicates that changes in the balance between anti‐inflammatory (IL‐10) and pro‐inflammatory (TNF‐α) cytokines is crucial in heart failure. IL‐10 has been shown to antagonize the pro‐apoptotic effect of TNF‐α via the activation of Jak/Stat3 pathway. In parallel, IL‐10‐mediated cardiac cell survival is regulated by the Toll‐like receptor 4 (TLR4) innate signaling where myeloid differentiation gene factor 88 (MyD88) plays a key role. However, interplay between these two signaling cascades is unknown. Our objective was to determine the role of MyD88‐dependent TLR4 innate signaling in acquiring adaptive response to IL‐10 via the Jak/Stat3 pathway. We found an increased TLR4 activity in isolated cardiomyocytes overexpressing MyD88 in presence or absence of IL‐10 treatment. Furthermore, inhibition of TLR4 activity by MyD88‐shRNA suggested that TLR4 activation is dependent on MyD88 which also abrogates downstream AktSer86/Jak/Stat3 pathway. On the contrary, Stat3 inhibition by DPP 5, 15 also suppresses IL‐10‐induced activation of TLR4 by IRAK M (An inhibitor of TLR4 signaling) inhibition; this suggests that Jak/Stat3 via a feedback mechanism also regulates TLR4. In addition, IL‐10 helps in the dissociation of interleukin‐1 receptor activated kinase 4 (IRAK4) into IRAK1 instead of IRAK2 and there was down‐regulation of TNF‐α receptor associated inducible protein (TRAIP) and its associated adaptor molecules TRADD and FADD. Data suggest that MyD88/IRAK1/IRAK4 recruitment, blocks TNF‐αR associated cellular changes and suppresses apoptosis. Up‐regulation of TLR4 signaling by IL‐10 involving Akt‐Jak/Stat3 pathway could be a potential therapeutic strategy in cardiovascular diseases (Supported by CIHR).
Differential Regulation of CCN1 During Valve Remodeling in TGFβ isoform Activation, Collagen production through Integrin Signaling.
Authors: Ghatak Shibnath , Misra Suniti , Moreno Rodriguez Ricardo , Sugi Yukiko , Hascall Vincent , Markwald Roger
Published: June 2021

Introduction

The failure of CCN1 null mice to form an atrioventricular (AV) septum has provided us with a unique opportunity to test mechanistic hypotheses to explain the role of CCN1 in valvular cushion development.

Methods and Results

We tested the hypothesis that CCN1 is a secreted signaling molecule that activates integrin dependent intracellular kinases generally associated with such phenomena as cell adhesion, DNA synthesis, and/or secretion of extracellular matrix, including hyaluronan (HA). Next, we found that, CCN1 does not induce cell proliferation on its own but enhances DNA synthesis induced by other mitogens, including bFGF and PDGF, and also induced by low MW HA (LMWHA; 150 kD, and 500 kD). Further, we found that CCN1 regulates HA synthase2 and hence stimulates synthesis of HA . Further, CCN1 binding to β‐3 integrin activated p‐Erk and p‐Fak kinases that were required to increase activated MMP‐1 (collagenase), indicating that CCN1 and HA induced by CCN1 combine to activate MMP‐1 through Erk pathway. In contrast to LMWHA, CCN1 with native HA (HMWHA) induced TGFβ3, not TGFβ1, and collagen III, not collagen I, expression.

Conclusion

In summary,this study provides direct evidence that endocardially secreted CCN1 can stimulate cushion development and maturation into valve leaflets and that effect of CCN1 can be influenced by HA

Research Support:2R01 HL033756‐24A2, 1R03 CA167722‐01.
Neural Crest‐Derived Valve Interstitial Cells Calcify In The Klotho‐Deficient Mouse Model Of Aortic Valve Disease
Authors: Wirrig Elaine , Yutzey Katherine
Published: June 2021

Calcific aortic valve disease (CAVD) is characterized by calcification and stenosis of the valve. Cell lineage studies were performed to determine if the developmental origin of a valve interstitial cell (VIC) predetermines its calcific potential. Neural crest (NC)‐derived cells in the skull calcify during normal bone formation, therefore, it is hypothesized that NC‐derived VICs are primed to calcify in CAVD. In this study, klotho‐deficient (Kl‐/‐) mice were used as a model of CAVD as they develop robust aortic valve (AoV) calcification. Parallel lineage‐tracing experiments in wild type and Kl‐/‐ mice from crosses with Tie2‐Cre, Wnt1‐Cre, and Mef2cAHF‐Cre were performed to follow endothelial‐, NC‐, and second heart field (SHF)‐ derived VICs respectively. In normal AoVs, endothelial‐ and SHF‐ derived VICs contribute broadly to all leaflets, whereas NC‐derived VICs are found primarily in the right and left coronary leaflets and in the hinge region of all three leaflets. In the calcific lesions of Kl‐/‐ mice, there was a noted absence of endothelial‐derived VICs, which constitute the majority of the AoV. In contrast, NC‐derived VICs were abundant in calcific lesions in the hinge region of the leaflets. Thus, osteogenic potential may depend on the embryonic origin of the VICs. Understanding the capacity of VICs to calcify will elucidate the cellular mechanisms underlying CAVD. Funding is provided by NIH F32HL110390.
The Timing of In Utero Caffeine Exposure Influences the Morphological, Functional, and Trans‐generational Effects on Adult Hearts
Authors: Wendler Christopher , Poulsen Ryan , Fang Xiefan
Published: June 2021

It is recognized that the disruption of the intrauterine environment by nutritional or chemical factors may influence the developing fetus, resulting in long‐term adverse effects in adulthood and subsequent generations. Previously, we demonstrated that in utero caffeine exposure in mice leads to altered cardiac function and morphology in adult offspring, which is accompanied by changes in DNA methylation and cardiac gene expression. We next hypothesized that the timing of in utero caffeine exposure influences which generation is affected, either the exposed embryo (F1) or subsequent generations (F2, exposed gametes, or F3 unexposed). To test this hypothesis, we treated pregnant CD‐1 dams with 20 mg/kg of caffeine daily from E6.5‐9.5, to target DNA re‐methylation in the embryo, or from E10.5‐13.5, to target DNA de‐methylation occurring during gametogenesis. In the F1 generation, early caffeine exposure from E6.5‐9.5 increased LV internal diameter (LVID) and volume, decreased LV posterior wall (LVPW) thickness and decreased % fractional shortening (FS) at 1 year of age, which is consistent with dilated cardiomyopathy. Adult cardiac gene expression was also affected including increased Myh7 expression. For late caffeine exposure from E10.5‐13.5, no effects on cardiac morphology or function were observed at either 12 weeks or 1 year in the F1 generation. However, in the late caffeine exposed F2 generation, we observed increased LVPW thickness, decreased LV volume and increased %FS, which is consistent with concentric cardiac hypertrophy.

Conclusion

In utero caffeine exposure leads to changes in adult cardiac morphology and function. The timing of caffeine exposure not only influence which generation is affected but also the cardiac phenotype.
Follistatin‐BMP Signaling Regulated by Isl2/CoupTFIb is Critical for Vascular Development
Authors: Li Ru‐Fang , Chang‐Yi Wu
Published: June 2021

While many regulators and signaling molecules controlling the process of vascular development are in zebrfish, genetic regulating the vein identity, intersegmental vessels (ISV) and cardinal vein plexus (CVP) patterning is still not fully understood. We previously identified the transcription factors isl2/coupTFIb required for specification of the vein and ISV growth mediated by Notch pathway in zebrafish. Microarray analysis showed highly overlapping downstream targets controlled by isl2/coupTFIb, suggesting that cooperatively regulate vascular development. In this study, we analyze the functional relevance of one potential target from array hits, follistatin 1a (fst1a). Fst1a is a bone morphogenetic protein (BMP) antagonist related to axis formation during the development and shown involved in venous angiogenesis. However, no description of fst1a functioning in vascular so far. The interaction of notch and BMP signalings to regulate vasculization is unknown.

We first examine amino acid sequence comparison and show fst1a is highly conserved among the vertebrates. Our in‐situ hybridization results showed fst1a is expressed in the lateral plate mesoderm and vessels. Overexpressing fst1a driven by fli1 promoter in zebrafish embryos, we observe the vascular defects in the growth of ISV and CVP. We further address the genetic interaction between fst1a and isl2, we found fst1 expression is increased in isl2 morphants by in‐situ hybridization. In addition, knockdown of fst1a can partial rescue the vascular defects in isl2 morphants. We suggest fst1a likely regulated by isl2. Since fst1 involved in BMP and isl2 functions downstream of notch, we conclude fst1a‐BMP signaling play an important role to regulate vascular patterning and interacts with isl2/coupTFIb‐Notch signaling.
Inhibition of DNA Methyltransferase 1 Promotes Proliferation and Alters Gene Expression in Mouse Embryonic Cardiomyocytes
Authors: Fang Xiefan , Rivkees Scott , Wendler Christopher
Published: June 2021

Introduction

Our previous research demonstrated that in utero caffeine exposure from embryonic day (E) 6.5‐10.5 down‐regulates the expression of DNA methyltransferases (Dnmts) and enhances cardiac gene expression in embryonic ventricles.

Hypothesis

We hypothesize that inhibition or down‐regulation of Dnmts promotes proliferation and differentiation of embryonic cardiomyocytes.

Method

Ventricles were dissociated from mouse E13.5 embryos and treated with 0.5‐1 mM 5‐azacytidine, a non‐specific DNMT inhibitor, or Dnmt1 siRNA. Immunofluorescence staining was performed to identify cardiomyocytes and illustrate morphological changes. Total RNA was isolated and used for quantitative real‐time PCR (qPCR).

Results

Knock down of Dnmt1 significantly decreased global DNA methylation in cultured cells. Immunofluorescence staining showed that the percentage of α‐actinin and connexin 43 positive cells was increased in cultures treated with Dnmt1 siRNA compared to control. Dnmt1 knocked down cardiomyocytes formed larger beating units compared with control cells. 5‐azacytidine treatment increased the expression of cardiac structural genes (Myh7 and Myh7b), hormonal genes (Bnp), and transcription factors (Gata4 and Nfatc1) in HL‐1 cardiomyocytes. Dnmt1 knock down by siRNA increased the expression of Myh6, Tnni3, Tnnt2, Tnnc1, Bnp, Gata4, Mef2c, Nfatc1, Camta1, and Camta2 in primary embryonic cardiomyocytes. Ongoing studies will evaluate cardiomyocyte function after Dnmt1 knock down and identify CpG sites that regulate cardiac gene expression.

Conclusion

Reduced Dnmt1 expression increases cell proliferation and cardiac gene expression of embryonic cardiomyocytes in culture.
The role of non‐muscle myosin II in early chick morphogenesis
Authors: Hoang My , Chu‐LaGraff Quynh
Published: June 2021

Cardiac looping, a highly conserved feature of vertebrate embryonic heart development, comprises of two phases: c‐looping and s‐looping. This process involves the transformation of the relatively straight heart tube into a distinct c‐shaped, then an s‐shaped loop. A previous study indicated that contraction involving muscle myosin and non‐muscle myosin II plays a minor role in the bending component of the first phase of cardiac looping, c‐looping. Here, we investigated whether myosin‐based contraction was necessary for the second phase of cardiac looping, s‐looping. We treated whole, cultured chick embryos with Verapamil, an L‐type channel inhibitor, to block actin‐myosin binding. Verapamil treatment induced heartbeat cessation, cardiac swelling, abnormal actin distribution, and unsuccessful s‐looping. Using Y‐27632, a ROCK inhibitor, to directly inhibit myosin‐based contractions, we showed that cardiac swelling was not a physical constraint that hindered s‐looping. Y‐27632 treated embryos experienced sustained heartbeat, growth retardation, and arrested s‐looping. We will further quantify the levels of non‐myosin and other proteins in the Rho‐ROCK signaling pathway to determine the mechanism that mediates s‐looping.
FRET biosensors reveal AKAP‐mediated shaping of subcellular PKA activity and a novel mode of Ca2+/PKA crosstalk
Authors: Schott Micah , Grove Bryon
Published: June 2021

A‐Kinase Anchoring Proteins (AKAPs) serve as scaffolds for cAMP‐dependent Protein Kinase (PKA) and other signaling enzymes within specific subcellular compartments. However, some AKAPs are not static anchors, but form dynamic signaling complexes that can traffic to different subcellular compartments when triggered. Gravin (AKAP12) is a multivalent AKAP that anchors PKA and other enzymes to the plasma membrane but redistributes to the cytosol upon intracellular Ca2+ elevation. Through the redistribution of gravin, we postulate that Ca2+ may regulate the interaction of PKA with downstream substrates, thus representing a novel crosstalk mechanism between Ca2+ and PKA‐dependent pathways. To assess this, we measured the impact of gravin‐V5/His expression on compartmentalized PKA activity using the PKA FRET biosensor AKAR3. In cultured AN3 CA cells, which lack endogenous gravin, expression of gravin‐V5/His increased forskolin‐stimulated PKA activity in AKAR3 constructs targeted to the plasma membrane compared to control cells lacking gravin or expressing a ΔPKA‐gravin construct missing the PKA‐binding domain. In contrast, gravin‐V5/His expression decreased forskolin‐stimulated PKA activity in cytosolic AKAR3 constructs. A mutant gravin construct with inhibited membrane localization had no effect on membrane and cytosolic PKA activity. Finally, pretreatment with the calcium‐elevating agent thapsigargin caused the redistribution of gravin and prevented the gravin‐mediated increase in plasma membrane PKA activity following forskolin or isoproterenol treatment. These results reveal that Ca2+ alters receptor‐mediated PKA activity through gravin redistribution and supports the hypothesis that gravin mediates crosstalk between Ca2+ and PKA‐dependent pathways.
A New Model of Tubulobulbar Complex Budding in the Seminiferous Epithelium of the Testis
Authors: Lyon Kevin , Bosseboeuf Emy , Vogl Wayne
Published: June 2021

Endocytic structures known as tubulobulbar complexes internalize intercellular attachments during sperm release. They are elongate structures capped with a clathrin‐coated pit. Their membranous core is a double‐membrane tube which is surrounded by a network of actin filaments. A bulbous region develops in the distal region which lacks the associated actin network but has a close association with cisternae of endoplasmic reticulum. The bulb eventually buds from the complex and enters endocytic compartments of the Sertoli cell. When rat testes were perfused for one hour ex vivo with oxygenated Krebs Henseleit buffer (with BSA) at 33°C containing either cytochalasin D (10, 20 or 40 µM) or the drug carrier (DMSO), apical TBCs at stage VII of spermatogenesis were associated with lower levels of actin in treated epithelia relative to controls. When evaluated at the ultrastructural level, the actin networks appeared patchy. In addition, ectopic bulbs and swollen tubular regions occurred. When thin sections of normal untreated samples were analyzed, large elongate bulbs and short tubular sections occasionally were observed. Together, these results suggest a new model for TBC vesiculation in which the actin network begins to disassemble and the tubular region begins to swell into a bulb. As the actin network continues to disassemble, the coated pit and most of the tubular region are incorporated into the enlarging bulb. Then the remaining short ‘neck’ of the bulb undergoes scission. Supported by a NSERC Discovery Grant to AWV.
A Recombinant DNA Lab Exercise Based On Racing Endonuclease and Ligase Enzymes
Authors: Hill Rachel , Junker Matthew
Published: June 2021

Recombinant DNA methods underlie much of modern biochemical and molecular biology research and industrial and pharmaceutical manufacturing. This laboratory exercise introduces these methods using a novel competition experiment in which restriction endonuclease and DNA ligase enzymes are “raced” against each other. The exercise is based on a common protein expression plasmid and the relatively inexpensive restriction enzymes EcoR V and Hind III. The digestions and ligations produce DNA fragments that are well‐resolved by agarose gel electrophoresis. In addition, ATP and DNA ligase concentrations were optimized to enable reproducible control of the number of different types of ligation products formed. This facilitates the identification of the composition of all products formed in the race experiment, including which DNA fragments have been covalently joined to form each ligation product. The distribution of products is further quantified by analysis of gel images using freely available image analysis software. Successful determination of the “winner” of the race experiment requires student conceptual mastery of both the DNA digestion and the ligation processes. The experiment is highly efficient in that the basic methods of DNA digestion and ligation can be covered within a single 3 hour laboratory period. In addition, the direct visualization of ligation products by gel electrophoresis and the quantification of product formation give the exercise a distinct biochemical flavor. This distinguishes it from many molecular biology exercises in which DNA ligation is detected indirectly by changes in the phenotype of transformed bacteria.
Using Computational Modeling to Teach Experimental Design
Authors: Wright Amanda , Gammack David
Published: June 2021

In many laboratories during an undergraduate science education, students are given a set of procedures to follow to obtain a pre‐determined outcome. Often students give little thought to the underlying scientific principles and lack a true understanding of the experimental protocol. Not only does this laboratory format not represent ‘real’ science, it leaves students unprepared for the often unpredictable and intimidating nature of experimental design. Here we have chosen classic biochemical experiments paired with computational modeling approaches to help students navigate the process of designing an experiment, from initial planning to hypothesis development to data analysis. Students can use the modeling mechanisms to help predict experimental outcomes and gain a better understanding of scientific concepts.
SUNY/NYAS STEM Mentoring After School Program Statewide Scale Up Project, Year Three
Authors: Ortiz Phillip , Breton Kristian , Duncan‐Poitier Johanna , Elphick Gwendolyn , Groome Meghan , Lansing Jill , Moore Gaylen
Published: June 2021

The United States is facing a crisis: Not enough students are being trained in science, technology, engineering and mathematics (STEM) to support and foster economic growth. To increase exposure to, experience in, and knowledge of STEM in younger students, the State University of New York (SUNY) and the New York Academy of Sciences (NYAS) have partnered to deliver informal, after school STEM experiences to underserved middle school students. Now in year three, our program trains SUNY graduate students and post‐doctoral fellows as mentors at six SUNY campuses throughout New York State. SUNY mentors learn pedagogy and content via streaming videos, on‐campus instruction and a credit‐bearing online course, and are placed as mentoring pairs in an informal afterschool setting. For Spring 2014, 25 classes were taught with 6 different curricula; 46 mentors and 371 students participated. Findings demonstrate that, as in years one and two, mentoring has multiple positive outcomes for the students and, just as strikingly, for the mentors: (1) After‐school mentoring improves the confidence of both mentor and mentee; (2) Students participating in the STEM Mentoring Program become more positive about STEM disciplines; (3) Students participating had an increase in STEM content knowledge; (4) Mentors develop more confidence in their abilities to teach across multiple situations; (5) Online course work is effective and efficient in training mentors. Since year one the program has impacted over 1,000 students and 100 mentors.
Transforming Undergraduate Education in the Molecular Life Sciences‐A Special Summer Symposium
Authors: Vega Quinn , Huff Mary
Published: June 2021

From July 30th to August 2nd at Missouri Western University, a special ASBMB small meeting will bring together both new and experienced educators to share, synthesize, and generate effective classroom strategies in undergraduate molecular life science courses. Through a series of presentations, workshops, and open discussions led by successful educators in the field, the most recent strategies identified to improve student learning and success in undergraduate biochemistry and molecular biology courses will be highlighted. Presentations and workshops will demonstrate how these strategies have been implemented in first year courses as well as upper division lecture and specialized laboratory sessions, providing a comprehensive view on how to invigorate the undergraduate curriculum. In addition, discussions focused on evaluating these strategies for publication or grant submission will be included. Throughout the symposium, there will also be extensive opportunities to discuss educational strategies with speakers and other attendees as well as opportunities to gain assistance with future course design.
Fueling the Interdisciplinary Flame: Exploring Plant‐Based Alternative Fuels in the Undergraduate Laboratory
Authors: Gregorzak Gabriel , Fajardo Jacqueline , Baillie Mark , Hlousek‐Radojcic Alenka
Published: June 2021

A laboratory module intended for an integrated course was developed to highlight relevant and interrelated concepts described in both introductory biology and general chemistry with an enrollment of nearly 500 students. The ever‐present depletion of petroleum energy reserves is an ongoing societal concern. Concomitantly, there is growing interest in identifying natural sources of alternative fuels and optimizing the efficiency of their use. To enhance student awareness of alternative fuel availability from natural resources, we have developed a lab centered on the production of biofuels produced from seed‐oil extracts. Oil was physically extracted from a variety of seed types including pumpkin, sunflower, walnut, & flax, and utilized an efficient, small‐scale, and green approach. The physical extraction technique replaced commonly used organic solvent extraction methods used in many academic and educational labs. Extracted oil was then subject to transesterification to yield the biofuel product. The energy content of this biofuel was measured and compared to ethanol using calorimetric analysis. This distinctive laboratory experience will allow large numbers of freshmen students to recognize the broader implications of their curriculum beyond the boundaries of the classroom.

This project was made possible through funding by the Howard Hughes Medical Institute.
Expression of Neuronal Nitric Oxide Synthase in the Extratesticular Pathway and Its Role in Murine Sperm Maturation
Authors: Pollak Rebecca , DeLeon Patricia
Published: June 2021

Neuronal nitric oxide synthase (nNOS) is one of two constitutive enzyme variants responsible for the production of nitric oxide (NO) from L‐arginine in mammalian cells. In somatic cells, this membrane‐associated protein has been shown to be activated by Ca2+ and to interact with Plasma Membrane Calcium ATPase 4 (PMCA4), the major calcium efflux pump in murine sperm (Wennemuth et. al. 2003), which negatively regulates it. NO is an important second messenger, and is required for a variety of sperm functions, including motility and fertilizing ability (Ramya et. al. 2011). Recently, the DeLeon lab has shown the expression of PMCA4 in the murine epididymis. However, no work has been done on the expression of nNOS in the extratesticular pathway of any mammalian species. Therefore, we set out to investigate the expression pattern of nNOS in the post‐testicular pathway and its role in sperm maturation. Here, we show the presence of nNOS in all three regions of the murine epididymis (caput, corpus, and cauda), including the basal and apical regions of the cells, via immunofluorescence for the first time. Western blotting results confirmed the expression of nNOS throughout the epididymis, and showed differential expression of nNOS, with highest expression in the corpus. Furthermore, preliminary immunofluorescence results suggest that the process of capacitation, in which the sperm experience a major influx of calcium, may influence the localization of nNOS. Experiments to isolate epididymosomes and determine if they are able to deliver nNOS to sperm are in progress.
Use of Social Media to Manage Out‐of‐Class Discussion
Authors: Tansey John
Published: June 2021

Discussion is potentially the least used of the science educator's tools. The cause of this is multifaceted, but the reality remains that some topics, such as ethics, or current events, are often best approached through discussion. Unfortunately, discussions consume valuable class time and many science educators are uncomfortable directing or managing classroom discussions. Social media provides a forum through which it is simple to generate a discussion group for a class or combination of sections, and manage discussions outside of the classroom. Advantages of using social media include flexibility and ease of use for students, ability to return to a conversation at any point in the future, ability to manage conversations and quantitate contributions, and provide students that would otherwise not speak up in class a voice. This technique has been used with positive results in both general chemistry and an honors non‐majors course.
Selective DNA Damage Slowed Histone Turnover
Authors: Kellow Rowaid , Neidigh Jonathan
Published: June 2021

DNA repair requires remodeling of the histones bound to DNA in eukaryotic cells. The remodeling of histone proteins to allow DNA repair enzymes access to the damaged DNA requires histone post‐translational modifications. After repair is complete, it is likely that the modifications are reversed or the modified histone is replaced by a newly synthesized histone. If histones at DNA damage sites is degraded, the turnover of histone proteins during repair will increase. We used stable isotope‐enriched amino acids to label histone proteins prior to DNA damage. The cellular DNA was selectively damaged using 5‐hydroxymethyl‐2′deoxyuridine (HmdU). After metabolism to its triphosphate and incorporation of HmdU during replication, HmdU is recognized as an oxidized thymine base and DNA repair is initiated. To control for the effect of DNA damage on the replication rate, the DNA of cells was also labeled by adding stable‐isotope enriched uridine to the cell culture media. We used LC‐MS and GC‐MS methods to measure the turnover kinetics of histones and DNA, respectively, during DNA damage and repair. Contrary to our expectation of unchanged or accelerated histone turnover, we observed that the turnover of histone H4 slowed when the DNA was damaged. These results imply that replication‐independent histone turnover is slowed, likely because DNA damage inhibits transcription.
The Q Motif is Involved in DNA Binding in ChlR1 (DDX11) Helicase
Authors: Ding Hao , Wu Yuliang
Published: June 2021

Helicases are molecular motors that couple the energy of nucleoside triphosphate (NTP) hydrolysis to the unwinding and remodeling of structured DNA or RNA. The conversion of energy derived from NTP hydrolysis into unwinding of double‐stranded nucleic acids is coordinated by seven sequence motifs (I, Ia, II, III, IV, V, and VI). The Q motif, consisting of an invariant glutamine (Q) residue, has been identified in some, but not all helicases. Previous studies have suggested that the Q motif is involved in ATP binding. Mutations in the human ChlR1 (DDX11) gene are associated with Warsaw Breakage Syndrome characterized by cellular defects in genome maintenance. ChlR1 is known to play essential roles to preserve genomic stability, particularly in sister chromatid cohesion; however, its molecular mechanism remains unknown. To examine the roles of the Q motif in ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Q motif of ChlR1. ChlR1 recombinant protein was overexpressed and purified from HEK293T cells. The ChlR1‐Q23A mutant abolished helicase activity of ChlR1, and further DNA binding assays suggested the Q motif affected the DNA binding ability of ChlR1. ATP hydrolysis assays revealed that the ChlR1‐Q23A had impaired ATP hydrolysis activity. However, further ATP binding assays showed that the Q motif is not essential for ATP binding. The Q motif in FANCJ helicase, a ChlR1 sequence homology protein, regulates FANCJ's dimerization, while our size exclusion chromatography indicated that ChlR1 protein functions as monomer. Taken together, our results suggest that the Q motif in ChlR1 helicase is involved in DNA binding but not ATP binding.

This work was supported in part by the Natural Sciences and Engineering Research Council of Canada (YW)
Investigating the Importance of DinB‐RecA Interactions to the Regulation of DinB's Activity and the Fidelity of the DNA Damage Response
Authors: Tashjian Tommy , Lin Ida , Cafarelli Tiziana , Godoy Veronica
Published: June 2021

The error prone DNA Polymerase IV, DinB, specializes in synthesizing DNA from a lesion‐containing DNA template, an activity that allows cell survival during DNA‐damaging conditions at a mutagenic cost. We have shown that there is direct physical interaction between DinB and Escherichia coli's main recombinase, RecA. We also have in vitro evidence suggesting that DinB•RecA interaction is conserved across many bacterial species, including human pathogens. Circular dichroism spectroscopy experiments show that the DinB•RecA complex undergoes a drastic conformational change compared to the single proteins alone. This is supported by the fact that DinB(C66A), a derivative with tight binding to RecA, and RecA copurify by size exclusion chromatography at a size that is much smaller than the size of the individual proteins, suggesting that compaction and tertiary structure changes are likely involved upon complex formation. Alteration of the physical interaction between DinB and RecA leads to changes in the mutagenic outcome in E. coli cells that are exposed to DNA damaging agents. Our current model is that the DinB•RecA complex not only alters the activity of DinB, but results in the sequestration of RecA from the lowest fidelity polymerase, DNA Polymerase V. The DinB•RecA complex, therefore, is central in controlling the fidelity of the DNA damage response.

Source of Funding: R01 and its supplement from the National Institute of Health (NIH) Institute of General Medicine to V. Godoy
TGF‐beta Regulates Alternative Splicing of CD44 by Inducing Smad3 Binding to CD44 pre‐mRNA
Authors: Zhang Ying , Tripathi Veenu , Sixt Katherine , Heller Mary
Published: June 2021

During late stage of breast cancer, TGF‐beta signaling promotes tumor progression by stimulating EMT, cell invasion and disseminations to distant sites. Previous studies have showed that TGF‐beta supports tumor progression through Smad‐dependent transcription regulation and Smad‐independent translational regulation. A few reports also suggested a role of alternative splicing, which represents another important mechanism of gene regulation, in aggressive cellular behavior induced by TGF‐beta during cancer progression. However, it is still unknown how TGF‐beta regulates alternative splicing. We found that TGF‐beta regulate alternative splicing of CD44 by inducing binding of Smad3 on CD44 pre‐mRNA surrounding regions encoding variant exons to inhibit recruitment of the splicing machinery to variant exons with suboptimal splice sites, thereby promotes a switch in CD44 expression from epithelial variant isoforms to the standard isoform. This isoform switch is essential for breast cancer progression. We show that the interaction of Smad3 with CD44 pre‐mRNA requires both input from TGF‐beta signaling and activation of oncogenic signaling, which occurs selectively in the tumor cells. Thus, Smad3 enables the tumor promoting function of TGF‐beta by serving as a regulator in alternative splicing under oncogenic stimulation in tumor cells.
Small Molecule Disruption of T‐box Regulated Transcription
Authors: Todd Gabrielle , Lu Kun , Frohlich Kyla , Cantara Will , Kitchen Douglas , Kloos Zachary , McDonough Kathleen , Agris Paul
Published: June 2021

T‐boxes are gram positive specific riboswitches that sense the charged status of tRNAs to regulate­ transcription of operons containing corresponding tRNA synthetase, amino acid metabolism and amino acid uptake genes. An uncharged tRNA spans two structural elements of the T‐box. The 5′ specifier loop nucleotides are complementary to the anticodon of a specific tRNA, and an antiterminator domain binds to the uncharged 3′ CCA arm. The latter interaction cannot form when a tRNA is charged, leading to the formation of an alternate transcription termination hairpin. T‐boxes can be found in most gram positive species, often regulating multiple operons (up to 19) with different tRNAs. Redundant use of T‐boxes and specificity of this element to gram positive bacteria make T‐boxes an excellent target for the development of small molecule, broad spectrum antibiotics with a low probability for antibiotic resistance development. Our lab has identified a small molecule compound that interacts with the specifier loop in silico, selectively kills gram positive bacteria and is non‐toxic to human cells. We have also characterized the Mg2+‐dependent interaction between tRNAGly and the B. subtilis glyQS 5′ UTR T‐box element using gel shift assays and isothermal titration calorimetry. These assays will be extended to elucidate the mechanism of small molecule inhibition of T‐box regulation in vitro.
RTT105 in Ty1 Retrotransposition: a Possible Role in Virus‐like Particle (VLP) Assembly
Authors: Scales Jessica , Peterson Mark , Keeney Jill
Published: June 2021

Retrovirus‐like transposon mRNA encodes envelope and replication proteins (gag and pol), and serves as genomic material for virus‐like particles (VLPs), a cytosolic structure in which the element's genome is reverse transcribed. In Saccharomyces cerevisiae, there are several families of such retrotransposons, including the abundant Ty1 element. Previously, genome‐wide screens have identified candidate genes that mediate Ty1 mobility. One candidate, RTT105, was identified as a negative regulator of Ty1 mobility. Because RTT105 contains no conserved domains, the nature of RTT105's role in Ty1 retrotransposition remains unknown. Rtt105p localizes at Ty1 RNA and Gag foci, suggesting a role in ribonucleoprotein complexes. Here, we use RNA‐Seq analysis of isogenic WT and RTT105 knockout strains to determine changes in gene expression that may explain RTT105's regulation of Ty1. In rtt105Δ, we identified significant down regulation of signal recognition particle (SRP), a conserved chaperone that binds ribosome‐nascent chain complexes for post‐transcriptional processing. SRP aids in translocating Gag to the ER where it encapsulates Ty1 RNA in trans at presumptive VLP assembly sites. Because endogenous Ty1 mobility is maintained at low levels by the host cell, our data suggest that RTT105 may support SRP‐dependent processing of Ty1. Deletion of RTT105 may induce down regulation of SRP‐dependent processing of Ty1, thereby decreasing genome‐wide mobility of the element. This result illuminates an important step in retrotransposon processing and regulation, and suggests at least one route by which Ty1 activity is actively maintained by the cell.

This research was funded by HHMI, NIH, and the Huck Institutes of the Life Sciences.