bluuebunny/crossref_metadata_2025 huggingface dataset:
Student Learning and Retention in Introductory Anatomy Flipped Class Models Compared To Traditional Lecture Format
Authors: McCullough Monica J. , McCullough Matthew G.
Published: November 2023

To improve student understanding and retention in an introductory anatomy class, instruction was shifted from a traditional lecture format to a ‘flipped class’ model at a small liberal arts college. Class members varied in the health sciences from physical education to pre‐med. The first cohort of students were taught via traditional lecture format and were exposed to PowerPoint instruction which lacked discussion. Assessments were typically multiple‐choice and model labeling. The second cohort of students were taught via flipped‐style instruction where lecture was recorded and assigned as homework while class time consisted of collaborative case studies, augmented reality and regular discussion/student interaction. This cohort was assessed similarly to that of the first cohort of students. The third cohort of students were taught via flipped‐style instruction, similar to that of the second cohort, but were assessed on application and synthesis of content. The first cohort had 41.7% D and F range grades for their final exam and 19.5% D and F range grades overall for the course. The second cohort had 19.1% D and F range grades for their final exam and 27.6% D range grades overall for the course (with 0% failures). The third cohort had 7.4% D range grades for their final exam (with 0% failures) and 31% D range for their final grade (with 0% failures). While the mind‐shift for students to a more active format of engagement was difficult for some, the retention of information as well as the enriched relationships among classmates and with instructor improved the academic performance in these introductory anatomy courses.
Can a cranial nerve animation enhance Dental student learning? Employing an innovative teaching aid in the classroom
Authors: Lone Mutahira , McKenna Joe P. , Cryan John F. , Vagg Tamara , Toulouse André , Downer Eric J.
Published: November 2023

The structure and function of the cranial nerves is a core curriculum topic for Dental students. However, due to the perceived complexity of the subject, it is often difficult for students to develop a comprehensive understanding using textbooks and models. It is generally accepted that the acquisition of anatomical knowledge can be facilitated by visualization of structures [1]. The aim of this study is to assess a new animation of the cranial nerves as a learning aid for first‐year Dental students. A cartoon‐type animation detailing the names, roles and anatomy of the cranial nerves, with particular emphasis on a life scenario, was developed based on Mayer's theory of multimedia learning [2]. Questionnaires were designed to assess the participants’ attitude towards the animation and their knowledge of the cranial nerves before and after visualization of the animation. Following consent, the participants completed the pre‐animation questionnaire and viewed the animation in a lecture theatre (n=49). The participants had continued access to the animation for 2 weeks after which they completed the post‐animation questionnaire (n=55).

Results from the pre‐animation questionnaire demonstrate that although more than half the respondents (57.1%; n=28) recognised the concept of cranial nerves, the majority (87.7%, n=43) had little knowledge of cranial nerves and indeed could not associate the nerves with their function. Data indicates that animations are recognised as helpful tools in understanding anatomy (87.7%; n=43) and that animations can be beneficial in teaching difficult topics (94%; n=46). Results from the post‐animation questionnaire indicate that the majority of the respondents viewed the animation after the initial presentation (83.6%; n=46) and also indicate that the animation assisted in developing knowledge of the cranial nerves (81.8%; n=45). While only a small number of respondents (9%; n=5) determined that cranial nerves are an easy topic, the majority (67.2%; n=37) indicated that the animation assisted study and improved knowledge of cranial nerve anatomy (72.7%; n=40). The findings of this study suggest that this new animation facilitated understanding of the cranial nerves and their functions.

Social Research Ethics Committee, UCC (SREC‐UCC) approval was obtained for the study.

Support or Funding Information

This study was funded by a teaching grant from the Dental School and Hospital, UCC.
QTD Effects on Veterinary Anatomy Learning
Authors: Ahn Dongchoon , Sim Jeoungha
Published: November 2023

Veterinary anatomy is interesting subject to veterinary medical students. However, the learning in laboratory of veterinary anatomy is hard and energetic work to students to meet in first grade because they have to learn a lot of new words or concepts under long time consuming after the professor's demonstration. Team based learning (TBL) was modified and introduced to veterinary anatomy education to help students in improvement of cooperation, team work, motivation for study, and to relief the teacher's task since 2006. Although the demonstration video files aided the student's work in laboratory, they were stayed in the room long time than expected and received annoyance in dissection the cadaver. We introduced the Qualifying Test for Dissection (QTD), a quiz, before entering the dissection room in last year, and it resulted in good effect. Here we report the effects of QTD with surveyed data. QTD was consisted of some easy questions, for example, translation English words to Korean, or, conversely, and the terms of veterinary anatomy. The students were qualified to entering the dissection room if get score over 80 % on the test. These quizzes were taken by individuals or team except two times, and then observed the finish time of dissection. At the end of semester, several questions were surveyed on the sheet by 5‐point scale. They finished the dissection tasks within 5 hours. All except one students answered that the QTD was helpful in their learning, and, except three students, thought that the level of the quiz was suitable. Two third of students prepared the QTD for 2 to 6 hours and felt burden moderately. They liked the test by individual than by team. They answered the QTD merits as helpful, first, in identification and dissection the he structures, second, the understanding professor's demonstration, and third reducing the dissection time. It also brought positive attitude to prepare the written examinations for knowledge. This QTD method is very useful to improvement of the study activities including shortening the dissection time and understanding a lot of concepts or structure in dissection room without burdening.
A Comparison of Three Neuroscience Curriculum Models in an Osteopathic Medical School
Authors: Sabry Hassan Sherif M. , Thomann Charity , Bhatia Shalini , Theobald Robert
Published: November 2023

Objective

Neuroscience has enjoyed tremendous growth over the past 20 years, including a substantial increase in the number of neuroscience departments, programs, and courses at the undergraduate level. To meet the need of new neuroscience courses, there has also been growth in the number of introductory neuroscience textbooks designed for undergraduates. However, textbooks typically trail current knowledge by five to ten years, especially in neuroscience where our understanding is increasing rapidly. Consequently, it is often important to supplement neuroscience and textbooks with information about recent findings in neuroscience. The purpose of this educational study is to compare 3 different models of teaching and evaluate their effectiveness as they are applied to get a better idea of how neuroscience education is being delivered.

Methods

3 Models of teaching were compared in this study; The Longitudinal model where the neuroscience course was taught separately, irrespective of the rest of the gross anatomy course, the Interdisciplinary model where the neuroscience course was taught as a single unit of different disciplines, irrespective of the other different courses and systems, and finally, the Interdisciplinary/system‐based model where the neuroscience course was taught as a single unit of different disciplines, following the system‐based curriculum adopted by the school. A retrospective study of the students’ grades and student's evaluation in surveys that were presented to postgraduate neuroscience students was conducted. These surveys were presented to students at the end of each neuroscience course in an Osteopathic Medical School between the years 2009 to 2015, regarding how the neuroscience education is being delivered.

Results

Students have scored highest scores in the longitudinal model of teaching followed by the Interdisciplinary model and lastly by Interdisciplinary/system‐based model. With scores data, there was statistically significant differences in the scores between the three models. The mean score of students’ evaluations showed that the students were more content with the Interdisciplinary/system‐based model of teaching, followed by the Interdisciplinary model and lastly by the Longitudinal model.

Conclusion

The results of this study provide data that can be used to determine future directions and priorities for neuroscience education. Data will hopefully help faculty who teach neuroscience at medical institutions to better define their activities.

Support or Funding Information

The authors declare no funding to report.
TEACHERS PERCEPTION ON THE INTEGRATION OF MORPHOLOGICAL SCIENCES DISCIPLINE IN INTEGRATED CLINICAL CURRICULUM IN SANTA CASA DE SÃO PAULO SCHOOL OF MEDICAL SCIENCES
Authors: Barros Mirna Duarte , Silva Vivian Alessandra , Pinto Antonio Cardoso , Pereira Celina Siqueira Barbosa , Liquidato Bianca Maria
Published: November 2023

Introduction

In the modern medical education, several curriculum options can be adopted. The integration of basic and clinical science is a challenging goal, encouraging early medical practice with theoretical basis of excellence. The Santa Casa de São Paulo School of Medical Sciences proceeded a curricular reform adopting the integration of basic and clinical sciences with a vertical integration of content in order to improve student learning.

Objectives

Report the perception of the basic science teachers in an integrative joint discipline with the clinic.

Experience Report

Clinical disciplines were subdivided by Organic Systems, with the inclusion of basic science content linked to the needs of the clinic practice. The Respiratory System was held in the third year of the medical course, with 15% of the total hours of discipline. The contents of anatomy, histology and embryology were taken to level the brought knowledge of the basic cycle (first two years), but has also been reconfigured with contextualized approaches through clinical cases, radiological images, procedure videos, in addition to traditional tools such as study on histological slides and prodissection anatomical specimens. Five professors from the Department of Morphology engaged in vertical approach to content, teaching and coordinating practical activities.

Results

Teachers reported that the use of clinical context raised in deepening students learning the morphological content, since it had connected in future clinical practice. The use of clinical cases created a favorable emotional context, making the learning of morphology more meaningful. Students had the opportunity to analyze problems and apply their knowledge to solving and develop criticism. The approchement between the contents showed students the importance of morphological knowledge in patient care.

Conclusion

Teachers evaluated positively the curriculum change and considered that the learning of basic sciences had become more significant when associated with a specific clinical context, favoring the retention of knowledge.
What to Teach or Not to Teach, that is the Question: Anatomy Objectives in a Modern Medical Curriculum
Authors: Metti Nada , Anwar Fadi , Lyons Virginia , Bee Mary
Published: November 2023

With the decline of contact hours over the last decade and recent emphasis on competency‐based medical education, a significant reduction has occurred in the amount of material that faculty are able to cover in the anatomical sciences. As a result, many faculty struggle with the decision of what to teach within a limited amount of time. A need exists for faculty to have guidelines or recommendations of defined objectives in the anatomical sciences. In response to that need, a task force has been formed over the past year to tap into the collective wisdom of a group of anatomy faculty and clinicians. This team is working together to develop a core subset of anatomical information that is essential for all future health professionals to know. The organization, timeline, and development of this Anatomical Content Task Force and its quest to determine essential content for histology, gross anatomy and neuroscience will be presented.
Utilization of the Point of Care Ultrasound (POCUS) Training in Patients Management by Medical Residents: A Place in the Anatomy Curriculum
Authors: Jirjis Rami , Hussein Inaya Hajj , Jurjus Abdo , Zakaria Khalid
Published: November 2023

Point of Care Ultrasound (POCUS) has proven through multiple studies its usefulness and effectiveness in Emergency Department (ED), intensive Care Unit (ICU), and certain procedures. POCUS also showed usefulness in rapidly verifying diagnosis in patients who require immediate surgical intervention. In addition, ultrasound has been introduced in schools during the anatomy courses.

Assessing the perception of residents, who have done POCUS training via workshops or as part of their curriculum prior to their residency training, will provide constructive feedback whether the training was useful, retained, or further integration of POCUS training workshops into medical school curriculum is needed. We will also explore the role of potential barriers faced by the residents that might help improve the usage of such vital technology to achieve better outcomes clinically.

A cross‐sectional survey will be administered via email using Qualtrics to Wayne State School of Medicine 2013 and 2014 Alumni, regarding POCUS retaining, utilization, access, training and preference. Data will be analyzed using multiple logistic regression to identify independent variables associated with POCUS usage.

This study will serve as a pilot study and provide us with a better understanding of the effectiveness early POCUS training, in Anatomy or other settings, and will lead us through further research to propose redirection of resources and curricular content to enhance the medical experience regarding the utilization of POCUS techniques.
How to use anatomical dissections and prossections to contribute in avoiding erros in clinical practice
Authors: Barros Mirna Duarte , Mendes Carlos Lazzarini
Published: November 2023

Introduction

In a country of social and technological inequalities like Brazil, radiological features image as portable ultrasound are not always available. The challenge teaching performance of procedures that use the transfer of knowledge that add skills, especially those who need the anatomic three‐dimensional view, it is still a cause for constant quest for technical perfection of this task. Basic technical procedures merit special attention on education, which, despite commonplace, lack of effective warning mechanism for technical and tactical errors, due to anatomical traps, can result in difficult complications treatment, often specialized, not to mention the temporary or permanent sequelae, and may lead to death.

Objective

Present anatomical landmarks to the correct procedure of central venous access via subclavian, and chest access by water seal drainage in dissected anatomical specimens for students from pre‐clinical disciplines of the Santa Casa de São Paulo School of Medical Sciences, SP, Brazil.

Experience report

Students of third and fourth years of medical course attended disciplines about morphology, physiology, pathology and clinic in an integrative way for every Body System. They were presented to a prossected cadaver model of the right infraclavicular region through muscular windows, aiming to restitutio ad integrum of anatomical structures. The topography of the subclavian vessels was valued to subsidize important knowledge for the correct procedure of placements of central catheters. Dissection of the side wall of the thorax, in which myo‐aponeurotic layers, costal framework, parietal pleura, diaphragm muscle, thoracic organs and abdominal intraperitoneal and parietal peritoneum, were exposed to exalting thoraco‐abdominal transition, in order to promote identification of possible errors in access to pleural cavity at the time of pleural drainage in water seal.

Results

The teaching method allowed the identification of key anatomical landmarks, with visualization and manipulation of anatomical structures, repetitively, allowing for training procedure central venous access through the subclavian and thoracic drainage in in water seal.

Conclusion

Anatomical dissections and prossections applied to clinical practice, preserving structures and topography, offer unlimited training interventions, minimizing error to perform procedures which are not available imaging technology as support.
Utility of 3 Dimensional Printing of Hearts with Complex Congenital Heart Disease in the Education of Pediatric Trainees and Sonographers
Authors: Prasad Deepa , Ashwath Ravi
Published: November 2023

Background

Complex congenital heart defects (CCHD) are often hard to describe and understand. Visualization of the CCHD in 3 dimensional (3‐D) views based on the two dimensional data obtained by echocardiograms and computed tomography (CT)/magnetic resonance imaging (MRI) is a daunting task. However, the interventions have to be based on and carried out in a 3‐D environment. These kind of disconnect between 2 D imaging and interventions in the 3D space pose a challenge to communicate effectively to the novice trainees and sonographers. With recent technological advancements in cardiac CT and MRI, it is possible to obtain excellent 3D reconstructions of the heart that can be used to rapidly and economically create 3D models of the heart. This can add value and improve understanding of complex heart defects especially of the CCHD. There is no data at present regarding its utility in using it as an educational tool. We aim to assess if 3‐D printing of the heart models would lead to improved understanding of congenital heart disease by trainees.

Methods

Pediatric residents, sonographers and cardiology fellows were randomly recruited and provided with a pretest questionnaire to assess their baseline understanding of normal and abnormal cardiac anatomy. CT and MRI scans were shown to the trainees initially prior to completion of pretest questionnaire. The trainees were then allowed to hold and examine the 3‐D heart models, after which a post test questionnaire is given to them. Data were analyzed to assess the improvement in understanding of CCHD, based on a scoring system.

Preliminary results

A total of 10 subjects (5 pediatric residents, 2 sonographers and 2 cardiology fellows) were included in the pilot study. 80% of the residents, 100% of the sonographers and fellows felt that their understanding of congenital heart disease improved with 3‐D heart models and 100% of the study subjects felt that 3‐D models should be absolutely utilized in the education of pediatric trainees.

Future directions

We will print more heart models and recruit atleast 100 subjects to achieve statistical power.

Conclusion

3‐D heart models can prove to be an effective educational tool for pediatric trainees to improve understanding of normal and abnormal cardiac anatomy.

Support or Funding Information

FRAP (Fellowship Research Award Program) grant
The Anatomy Training Program: a Participant's Perspective
Authors: Umorin Mikhail
Published: November 2023

The Anatomy Training Program (ATP, http://www.anatomy.org/anatomy‐training‐program.html) is a joint undertaking between the American Association of Anatomists and the Anatomical Society (UK). It aims at training anatomists so that they can teach Gross Anatomy at a graduate/professional school level. I participated in the ATP during the 2013 – 2015 academic years. I had no prior anatomical education, and the dental school at which I am a postdoctoral fellow sponsored me to participate in the program. My objective for this presentation is to present the positive and negative aspects of the ATP program by describing and reflecting on my experiences in the ATP. As a result of a significant time commitment in ATP I learnt Anatomy to the level that I currently teach it at our dental school. During the ATP program I learnt didactic and dissecting skills. I have a foundation for treating teaching as a science and view teaching as a complex student‐teacher interaction. I have learnt an important self‐improvement tool: reflection. I started using this tool as a method of improving my anatomical knowledge and teaching methods. I have recently interviewed at and received a job offer from a US medical school. In conclusion, ATP has been vital to advancing my career in anatomical education. In addition, reflection has become a life skill that I use to improve my performance in all sorts of endeavors ranging from cooking to home improvement to job related activities.

Support or Funding Information

American Association of Anatomists
Student Demographics of Samuel Merritt University Community Service Honor Society Volunteers
Authors: Allen Megan A , Peck Lacey Beth A , Elliott Craig A , Puder Barb
Published: November 2023

The Samuel Merritt University Community Service Honor Society was established to acknowledge students who demonstrate excellence in community service, outreach, and volunteerism. A 3‐year longitudinal study examining specific participant data was collected from consented and enrolled students. Demographic information over 3 years was gathered including age, gender and program cohort of the students. Additionally, trends regarding number and frequency of volunteer hours, and types of outreach services completed were examined. Finally, a comparison of enrolled students vs. inducted students was completed.
Breaking the fourth wall: Quaternary organizations forge a link to the novel non‐enzymatic function of RNR‐α
Authors: Aye Yimon
Published: November 2023

The classical textbook description of the enzyme ribonucleotide reductase (RNR) has been the rate‐limiting catalytic role in the de novo biosynthesis of dNTPs required for DNA replication and repair. The nucleotide‐regulated α subunit of RNR in unison with the metalloprotein parter subunit b, or its p53‐regulated isoform, p53b, constitutes the active enzyme that catalyzes NDP reduction to dNDPs in humans. RNR activity is positively correlated with cell proliferation and is a known target of nucleoside anticancer agents. However, seemingly at odds with the major role of RNR in cancer proliferation, selective overexpression of α alone confers anti‐metastasis activity in transformed cells and in transgenic mice, and is correlated with longer lifespan in cancer patients. Indeed, α‐specific gene therapy approaches had been in clinical trials. The basic biology underlying this enigmatic α‐specific tumor suppression is unknown and is long postulated to involve non‐reductase functions. Our most recent serendipitous discovery of a new nucleotide‐driven α‐specific signaling role has shed the first light on the long proposed non‐canonical function of α. We present the detailed mechanistic deconvolution of the newly discovered function of α underpinning the clinically relevant tumor suppressor phenotype.

Support or Funding Information

NIH Director's New Innovator award (1DP2GM114850‐01)
Acetylation of Replication Protein A (RPA) Improves its DNA Binding Property
Authors: Surendran Sneha , Ononye Onyeka Ebelechukwu , Balakrishnan Lata
Published: November 2023

Genome maintenance is critical for cellular survival and growth. Replication Protein A (RPA), a single‐strand DNA (ssDNA) binding protein, is vital for various aspects of genome maintenance such as replication, recombination, repair and checkpoint activation. RPA binding to ssDNA protects it from degradation by cellular nucleases, prevents secondary structure formation and from illegitimate recombination. Within the cell, RPA is subject to many post‐translational modifications including phosphorylation, SUMOylation and ribosylation. These modifications regulate the activity of RPA with DNA and other binding partners.

RPA has been reported to be also modified by acetylation. We found that human RPA (hRPA) can be in vitro acetylated by p300, an acetyl transferase (AT). To study the effect of this modification on its ssDNA binding function, we made use of electro‐mobility gel shift assay (EMSA) and bio‐layer interferometry (BLI) technology. Using various length oligos, we tested the binding property of unmodified and acetylated RPA. Our results showed that acetylation of RPA increased its binding affinity compared to unmodified RPA. Interestingly, the acetylated form was also able to bind more stably to shorter length oligos compared to the unmodified form. This suggests that the acetylation of RPA improves its ssDNA binding function. This alteration in its enzymatic activity would have significant implications in maintenance of genome fidelity since improved DNA binding function of RPA will protect the genome from both endogenous and exogenous stresses. Additionally, using mass spectrometry analysis we have identified the lysine residues that get modified by the acetyl group both in vitro and in vivo. We are currently studying the factors that trigger this post‐translational modification in the cell.

Support or Funding Information

This work was supported by a grant from the National Institutes of Health GM098328 (to LB).
Defining the cut site specificity of Ref – a RecA dependent endonuclease
Authors: Wan Yui Chun Serena , Ronayne Erin A , Cox Michael M
Published: November 2023

Recombination enhancement function (Ref) is a RecA‐dependent endonuclease that creates double strand breaks within in a displacement loop of the dsDNA substrate. DNA cleavage is not random, and we are currently deciphering the precise cleavage specificity of the Ref nuclease. Cleavage occurs throughout the D‐loop, although some locations are clearly preferred. The overall cleavage patterns of Ref are highly repeatable, indicating that the process is likely influenced by sequence and/or protein interactions. When reactions are carried out with ATPase deficient RecA or with non‐hydrolyzable ATP analog so that RecA filaments are static, patterns with preferred cleavage peaks recurring every three nucleotides are observed. As the binding site of a RecA subunit is three nucleotides or base pairs, these results suggest that cleavage may be directed by, or occur at, the subunit‐subunit interface of a RecA filament. This DNA cleaving system has the targeting potential of the CRISPR system in genome editing, although the complexity of the Ref system currently limits its application. This project is aimed at a further understanding of the nuclease mechanism of Ref, protein‐protein interactions between Ref and RecA, and will be useful for future applications of the Ref‐RecA system.
Understanding the Efficacy of Inhibitor TRDL‐551 on Replication Protein A (RPA) Binding Affinity
Authors: Kaur Jasmeet , Balakrishnan Lata
Published: November 2023

Replication protein A (RPA), a single stranded DNA (ssDNA) binding protein, prevents ssDNA from being a target of cellular nucleases, recombining with other complementary sequences, or folding‐back on itself to form hairpin structures. Each of these scenarios can cause high rates of genome instability. Due to the protective function of RPA it now used as a therapeutic target for cancer cells, wherein DNA replication and repair often occur at high frequencies. For the current study, we utilized a chemical inhibitor of RPA, TDRL‐551, which has been previously characterized as a potent inhibitor of RPA binding to substrate. In this study, we chose to further analyze the TDRL‐551 inhibitor by testing different binding conditions either by the unmodified or acetylated form of RPA. Results from our laboratory have shown that acetylated RPA binds with higher affinity to ssDNA compared to the unmodified form. We tested RPA (unmodified and acetylated) binding under two different conditions; (i) RPA was pre‐bound to substrate and then incubated with inhibitor or (ii) RPA was pre‐bound to inhibitor and then exposed to substrate. Our results showed that in the first case, there was no effect of exposure to inhibitor on both unmodified and acetylated RPA binding. However, when RPA was first incubated with the inhibitor, both the unmodified and acetylated RPA showed reduced binding affinity to the ssDNA substrate. In conclusion, our studies show that the length of the DNA substrate, the post‐translational status of RPA and the time of exposure of the inhibitor to the RPA all play a significant role in determining the potency of this inhibitor. Further studies are being done to ascertain the efficacy of this inhibitor before using it in translational research.

Support or Funding Information

This work was supported by a grant from the National Institutes of Health GM098328 (to LB)
Identifying the role of genomic uracil in folate deficiency
Authors: Chon James , Field Martha , Gondokusumo Jabez , Stover Patrick
Published: November 2023

Folate deficiency is implicated in deleterious health outcomes including neural tube defects (NTDs), neurodegeneration and colon cancer. Folate is a cofactor for the de novo synthesis of thymidylate (dTMP), and folate deficiency increases DNA damage, alters histone methylation, and increases uracil misincorporation into the genome during DNA synthesis. Excision of uracil in DNA is shown to cause DNA damage, but paradoxically, knockdown of uracil excision enzyme uracil DNA glycosylase (UNG) increases rates of DNA damage. The biological significance of uracil incorporation into DNA in folate deficiency is unknown, but shows tissue‐specific differences. It is not known whether uracil in DNA has functional or regulatory consequences, but uracil incorporation in DNA interferes with transcription factor binding in vitro. Uracil levels in nuclear DNA are postulated to be governed by the cellular dTTP/dUTP ratio. Cellular dUTP levels are regulated by the enzyme dUTP diphosphatase (dUTPase), which cleaves two phosphate groups from dUTP, yielding dUMP, which can be used as the substrate for synthesis of dTMP. Cellular dTTP levels are determined by rates of de novo dTMP biosynthesis, which requires folate cofactors and functions as a multi‐enzyme complex in the nucleus. In this study, we sought to determine the contribution of these two activities to uracil accumulation in DNA.

The multienzyme complex that synthesizes dTMP is comprised of serine hydroxymethyltransferase (SHMT1 and SHMT2α), thymidylate synthase (TYMS), dihydrofolate reductase (DHFR), and methylenetetrahydrofolate dehydrogenase (MTHFD1). Both SHMT and MTHFD1 synthesize 5,10‐methylenetetrahydrofolate, the cofactor required to synthesize dTMP. SHMT also acts as a scaffold for the multienzyme complex and is critical for recruitment of these proteins at sites of DNA replication and repair. CRISPR knockout of SHMT1 in HeLa cells leads to a 2‐fold increase in uracil in DNA, compared to control cells. Treatment of HeLa cells with dUTPase siRNA leads to a 2.5‐fold increase in uracil in DNA and a 9‐fold increase in gamma‐H2A.X, a marker of DNA damage.

These data suggest that dUTPase prevents genomic uracil incorporation by its enzymatic activity, keeping dUTP concentrations low, and that SHMT1 prevents uracil incorporation by its scaffolding activity, indicating that the uracil incorporation into DNA is regulated beyond the nuclear dTTP/dUTP ratio.

Support or Funding Information

National Institutes of Health Grants DK58144 and HD059120
Neural Crest‐Placode Cell Interactions during Cranial Ganglia Assembly
Authors: Taneyhill Lisa A.
Published: November 2023

The cranial sensory ganglia are created from two motile cell populations, neural crest cells and placode cell‐derived neurons. Defects in the migration and interaction of these cells can cause abnormalities in craniofacial development and in the formation of the sensory nervous system, leading to conditions such as cleft lip/palate, neuralgia, and peripheral neuropathies. Although prior studies have shown that neural crest cells and placode cell‐derived neurons intermingle and coalesce during cranial ganglia assembly, the mechanisms underlying these processes are still elusive. To this end, we are using the cranial ganglia in the developing chick embryo as a model to elucidate the molecules and signaling pathways that mediate these intercellular interactions. Using gene perturbation assays, we have delineated important roles for cellular adherens junction components in controlling cranial ganglia formation throughout multiple stages of gangliogenesis. Furthermore, we have identified a member of the Annexin protein super‐family that functions during the neurogenesis of cranial placode cells and possibly other non‐placodal cell types. These findings provide additional insight into the mechanisms that orchestrate the cellular movements essential for cranial gangliogenesis, and, collectively, will impact our understanding of the normal formation of, and diseases associated with, the sensory nervous system. Importantly, our results may be directly translatable to other tissues generated from heterotypic cell types and will therefore have broad implications for human health.

Support or Funding Information

This work is supported by NIH R01DE024217 to L.A.T.
Enhancer H3K4 methyltransferase MLL4 controls cell fate transition
Authors: Ge Kai
Published: November 2023

Enhancers control cell type‐specific gene expression and direct cell fate transition. Enhancers are marked by H3K4me1/2. We have shown previously that MLL4 (KMT2D) is a major enhancer H3K4me1/2 methyltransferase with partial functional redundancy with MLL3 (KMT2C). However, the role of MLL4 in enhancer regulation and cell fate transition is poorly understood. Here we show in embryonic stem cells (ESCs), MLL4 associates with, but is dispensable for the maintenance of, active enhancers of ESC identity genes. As a result, MLL4 is dispensable for cell identity gene expression and self‐renewal in ESCs. In contrast, MLL4 is essential for activation of de novo enhancers and induction of cell identity genes during ESC differentiation. Similarly, MLL4 is dispensable for maintaining fibroblast cell identity but is essential for reprogramming into induced pluripotent stem cells. Together with our findings in adipocytes and naïve CD4+ T cells, these results indicate that whileMLL4 is dispensable for maintaining cell identity, it controls cell fatetransitions by orchestrating de novoenhancer activation.

Support or Funding Information

Supported by the intramural research program of NIDDK, NIH to K.G.
Dissection of Gene Regulatory Networks that control retinal cell fate specification
Authors: Wang Sui , Cepko Connie
Published: November 2023

Gene regulatory networks (GRNs) regulate critical events during development. In complex tissues, such as the mammalian central nervous system (CNS), networks likely provide the complex regulatory interactions needed to direct the specification of the many CNS cell types. To understand these GRNs, we started by dissecting a GRN that regulates a binary fate decision between two siblings in the murine retina, the rod photoreceptor and bipolar interneuron. A simple strategy was employed and developed to dissect the rod‐bipolar GRN. First, we identified enhancers of key transcription factors (TFs) that regulate the specification of rod and bipolar cells. Second, by analyzing enhancers, we uncovered upstream regulatory TFs that regulate these enhancers. Third, we established the relationships among different TFs, and mapped TFs into GRNs via performing gene perturbation assays. Specifically, the rod‐bipolar GRN centers on Blimp1, one of the TFs that regulates the rod versus bipolar cell fate decision. We identified a cis‐regulatory enhancer, B108, which mimics Blimp1 expression. Deletion of genomic B108 by CRISPR/Cas9 in vivo using electroporation abolished the function of Blimp1. Otx2 and RORβ were found to regulate Blimp1 expression via B108, and Blimp1 and Otx2 were shown to form a negative feedback loop that regulates the level of Otx2, which regulates the production of the correct ratio of rods and bipolar cells. Furthermore, based on this study, we developed novel enhancer screen assays and TF pull‐down assays to identify enhancers and regulatory TFs in a more profound and large‐scale fashion to facilitate GRN studies.

Support or Funding Information

This work was supported by HHMI (to C.L.C. and S.W.).
Regulation of Hepatic Mouse Major Urinary Protein Genes by the Zinc Finger and Hemeoboxes 2 and Alpha‐fetoprotein Regulator 2
Authors: Jiang Jieyun , Creasy Kate Townsend , Peterson Martha L , Spear Brett
Published: November 2023

Objectives

We have previously found that AFP, H19, Glypican 3 (Gpc3) and Lipoprotein lipase (Lpl) are targets of Zinc Finger and Hemeoboxes (Zhx) 2 based on their elevated expression in the adult liver of BALB/cJ mice compared to other strains. Since mouse major urinary proteins (MUP) mRNA are expressed at lower levels in the adult liver of BALB/cJ mice compared to other mouse strains, we tested whether this trait is due to Zhx2.

Methods

At first, by using Real‐Time Polymerase Chain Reaction (PCR) techniques, we tested hepatic MUP mRNA levels in different mouse strains including BALB/c, BALB/cJ mice, BALB/cJ mice with Zhx2 liver transgene, and recently developed Zhx2 knock‐out mice. Next we performed liver regeneration and Afr2 analysis, C3H/HeJ (Afr2a) and C57BL/6 (Afr2b) mice (2–3 months old) were given intraperitoneal injections of 50 mL of mineral oil (MO) containing 10% (vol/vol) carbon tetrachloride (CCl4); control animals were given intraperitoneal injections of 50 mL of MO alone (22). Three days later, the animals were killed, and their livers were removed for RNA preparation and analysis. Lastly, we studied how Zhx2 regulates MUP using in‐vitro cell based assays.

Results

Our data indicate that MUP genes are target of Zhx2 regulation. In contrast to previous Zhx2 targets, which are elevated when Zhx2 levels are reduced, MUP expression decreases when Zhx2 levels are lower. AFP, H19, Gpc3 and Lpl are reactivated in regenerating liver and HCC. With all four genes, the level of induction is significantly less in C57BL/6 mice than in other mouse strains; this trait is governed by a single locus on mouse chromosome 2 called alpha‐fetoprotein regulator 2 (Afr2). The gene for the Afr2 phenotype has not yet been identified. Since all known Zhx2 targets are also regulated by Afr2, we tested whether MUP expression was influenced by this locus in regenerating liver. We found that MUP mRNA levels decreased in regenerating liver (in contrast to AFP, H19, Gpc3 and Lpl, which are increased in regenerating liver) and that this decrease is influenced by Afr2. Additionally, data from in vitro cell‐based assays demonstrated that Zhx2 could activate MUP24 promoter and increase MUP expression in AML cells.

Conclusion

Taken together, MUP is a new target of Zhx2 and it is positively regulated by Zhx2.
Regulatory Characteristics of Vibrio vulnificus gbpA Encoding a Mucin‐Binding Protein Essential for Pathogenesis
Authors: Choi Sang Ho , Jang Kyung Ku
Published: November 2023

Background

Adhesion to mucus layer is a prerequisite for pathogenesis of enteropathogens.

Results

GbpA is a mucin‐binding protein essential for pathogenesis of Vibrio vulnificus, and IscR, CRP, and SmcR cooperatively regulate gbpA in a growth‐phase dependent manner.

Conclusion

Cooperation of IscR, CRP, and SmcR precisely tunes gbpA expression during pathogenesis.

Significance

This study elucidated the regulatory mechanism of gbpA transcription for the first time.

Binding to mucin is the initial step for enteropathogens to establish pathogenesis. An open reading frame, gbpA, of Vibrio vulnificus was identified and characterized in this study. Compared to the wild type, the gbpA mutant was impaired in binding to mucin‐agar and the mucin‐secreting HT29‐MTX cells, and the impaired mucin binding was restored by the purified GbpA provided exogenously. The gbpA mutant was defective in lethality and intestinal colonization in a mouse model, indicating that GbpA is a mucin‐binding protein and essential for pathogenesis of V. vulnificus. The gbpA transcription was growth‐phase dependent, reaching a maximum during the exponential phase. The Fe‐S cluster regulator (IscR) and the cyclic AMP receptor protein (CRP) coactivated whereas SmcR, a LuxR homologue, repressed gbpA. The cellular levels of IscR, CRP, and SmcR were not significantly affected by one another, indicating that the regulator proteins function cooperatively to regulate gbpA rather than sequentially in a regulatory cascade. The regulatory proteins directly bind to the upstream of the gbpA promoter PgbpA. DNase I protection assays, together with the deletion analyses of PgbpA, demonstrated that IscR binds to two specific sequences centered at −164.5 and −106, and CRP and SmcR bind specifically to the sequences centered at −68 and −45, respectively. Furthermore, gbpA was induced by exposure to H2O2, and the induction appeared to be mediated by elevated intracellular levels of IscR. Consequently, the combined results indicated that IscR, CRP, and SmcR cooperate for precise regulation of gbpA during the V. vulnificus pathogenesis.

Support or Funding Information

This work was supported by Mid‐career Researcher Program Grants 2015R1A2A1A13001654 through National Research Foundation funded by Ministry of Science, ICT, and Future planning; the R&D Convergence Center Support Program of the Ministry of Agriculture, Food and Rural Affairs; and a grant (14162MFDS972) from Ministry of Food and Drug Safety, Republic of Korea.
Structural basis for histone H2B deubiquitination by the SAGA DUB module
Authors: Morgan Michael Thomas , Haj‐Yahya Mahmood , Ringel Alison E. , Bandi Prasanthi , Brik Ashraf , Wolberger Cynthia
Published: November 2023

Monoubiquitinated histone H2B plays multiple roles in transcription activation. H2B is deubiquitinated by the Spt‐Ada‐Gcn5 acetyltransferase (SAGA) coactivator, which contains a four‐protein subcomplex known as the deubiquitinating (DUB) module. The crystal structure of the Ubp8/Sgf11/Sus1/Sgf73 DUB module bound to a ubiquitinated nucleosome reveals that the DUB module primarily contacts H2A/H2B, with an arginine cluster on the Sgf11 zinc finger domain docking on the conserved H2A/H2B acidic patch. The Ubp8 catalytic domain mediates additional contacts with H2B as well as with the conjugated ubiquitin. We find that the DUB module deubiquitinates H2B both in the context of the nucleosome and in H2A/H2B dimers complexed with the histone chaperone, FACT, suggesting that SAGA could target H2B at multiple stages of nucleosome disassembly and reassembly during transcription.

Support or Funding Information

Supported by grant GM‐095822 from the National Institute of General Medical Sciences (C.W.) and by a Ruth L. Kirschstein National Research Service Award (M.M.). A. Brik is a Neubauer Professor. GM/CA @ APS has been funded by the National Cancer Institute (Y1‐CO‐1020) and the National Institute of General Medical Sciences (Y1‐GM‐1104).
Hematopoietic Cell Stress Response Gene Transcriptional Regulation By TFII‐I and E2F Family
Authors: Aryan Mahmoud
Published: November 2023

TFII‐I is a ubiquitously expressed, multifunctional transcription factor that has been shown to activate and repress transcription. TFII‐I is relatively large and contains a DNA binding domain, a nuclear localization domain, and protein‐protein interaction domains, including 6 R‐repeats that resemble helix‐loop‐helix motifs and a leucine zipper. Genome wide analysis of TFII‐I in human erythroleukemia K562 cells through biotinylation and ChIP sequencing revealed TFII‐I interacting with DNA segments near binding sites for E2F transcription factors. Co‐immunoprecipitation experiments provided data suggesting that E2F1, E2F4, and E2F6 interact with TFII‐I. The E2F family of transcription factors has been shown to regulate G1/S transition stages during the cell cycle. E2F1 works as a transcriptional activator, thereby promoting cell cycle progression, whereas E2F4 and E2F6 are transcriptional repressors and block cell cycle progression. TFII‐I has also been shown to be involved in cell cycle regulation. Chromatin immunoprecipitation (ChIP) experiments revealed binding of TFII‐I and E2F transcription factors at regulatory DNA elements in the Activating Transcription Factor 3 (ATF3)‐gene locus. ATF3 is a transcription factor that binds to the cAMP response element and is a critical component of the cellular stress response. Expression of ATF3 is induced in response to amino acid starvation and/or endoplasmic reticulum stress. Results from the Encode database suggests that transcription factors E2F4 and E2F6 co‐localize with RNA polymerase II at the ATF3 promoter just upstream of a TFII‐I binding site. By treating K562 cells with histidinol and thapsigargin to induce amino acid starvation or endoplasmic reticulum stress, respectively, the upregulation of ATF3 correlated with differential E2F binding patterns in the promoter region. Moreover, TFII‐I knockdown prompted reduced interactions of E2F4 and E2F6 at the ATF3 gene locus. These findings direct us to conclude that the associations between E2F4/6 and TFII‐I may have intricate roles in the configuration of the ATF3 gene locus thereby regulating mechanisms of cellular stress response.

Support or Funding Information

Mahmoud Aryan, Alex X. Fan, PhD1, Mir Hossain, MS1, and Jörg Bungert, PhD1 1Department of Biochemistry and Molecular Biology, Center for Epigenetics, Powell Gene Therapy Center NIDDK: RO1DK083389, RO1D0K52356