bluuebunny/crossref_metadata_2025 huggingface dataset:
Gaining Competencies During Early Medical Training: Medical Students As Teaching Assistants In Dissection‐Based Anatomy Course
Authors: Brown Martin Tucker , Zhang Guiyun
Published: November 2023

Competency based curricula have been widely implemented in medical schools. To strengthen medical students’ competency training during the early years of their medical education, we established an anatomy Teaching Assistant (TA) program. Ten second‐year medical students were recruited into the program. Their primary responsibility was to serve as TAs in an 8 week, dissection‐based anatomy course for Physician Assistant (PA) students. Additional dissection‐based projects, such as laminectomy, advance dissection of head & neck, and development of learning tools, were incorporated into the program. A questionnaire was conducted at the end of the program. The questions addressed five of six ACGME core competencies, namely development of professionalism, communication skills, lifelong learning, improvement of knowledge, and patient care. Nine of 10 students responded to the questionnaire. All of the responders agreed that they learned various aspects of professionalism, such as commitment to excellence, respect and compassion for learners, providing accurate scientific information, and commitment to timeliness. Interestingly, only 67% agreed that they must wear professional attire during professional activities. As expected, all agreed that their knowledge of anatomy was strengthened after participating in this program. Through interactions with PA students, peer TAs and faculty members, all students agreed that their interpersonal and communication skills were improved. Learning when to provide answers, and when to challenge students with questions, enhanced their leadership and decision making skills (89%). Most of the TAs acknowledged improvement of lifelong learning skills, including the ability to consult various learning resources, initiate advanced study to answer student questions, and create innovative learning tools. All agreed that abilities related to future patient care were enhanced, including problem solving and teaching. All of the TAs agreed that faculty members were strong role models for professionalism, indicating that faculty can significantly influence medical students in their professional growth. PA students highly evaluated the teaching efforts of the TAs. 100% of the PA students acknowledged the TAs were helpful resources for their dissection and the learning tool development by a TA was helpful. In summary, our anatomy TA program provided a unique opportunity for second year medical students to gain skills and behaviors expected of health professionals. Through this program, TAs strengthened anatomical knowledge, improved communication skills, gained a deeper understanding of professionalism, and demonstrated competencies related to independent study and lifelong learning.
Learning to Lead: Facilitating Leadership Skills through Peer and Faculty Feedback in a Team‐Based Anatomy Course
Authors: Halasz Sasha Renee’ , Ansari Mohammad Humza , Pawlina Wojciech , Lachman Nirusha
Published: November 2023

Leadership, among other non‐technical discipline‐independent skills, has been identified as an important parameter in promoting patient safety and quality improvement in healthcare. While literature suggests that early exposure to non‐technical skill development positively correlates with improved technical skill, incorporating this into medical school curricula remains challenging. For first‐year medical students, team‐based learning environments provide an opportunity to demonstrate and reflect on leadership roles when assessment of non‐technical skills is incorporated as a formal component of their final grade. At Mayo Medical School, 20% (subjective faculty (10%) and peer (10%)) of the final anatomy and histology grade is awarded for performance of non‐technical skills. As part of our quality improvement initiative, we analyzed five consecutive years of data regarding leadership feedback in order to better understand: (1) student interpretation of leadership (2) student level of confidence in displaying leadership skills (3) student ability to critically assess leadership skills. Quantitative data from self and peer assessment were compared with qualitative analysis of leader and peer reflections. We found that leader assertiveness or lack thereof was a common theme in written feedback to team leaders, being mentioned at least once in 69% of all groups. We also discovered that 71% of leaders gave themselves a score lower than the aggregate team member score on leadership metrics. Despite this, score assignment on several individual components of leadership including respect, integrity, responsibility, compassion, problem‐solving, commitment to excellence, and overall professionalism was highly congruent between leaders and team members; leader selfscores matched aggregate team scores within two points 90% of the time. These results suggest that first‐year medical students strongly associate a proper level of assertiveness with leadership skills, are less confident in their own leadership abilities than the peers they are leading, and tend to agree with peers on relative areas of leadership strengths and weaknesses. Better understanding of medical student perception of leadership will help to direct leadership skill discussion during faculty‐driven direct feedback sessions.
Use of iPad and ExamSoft&[trade] in administering morphological sciences practical examination in an integrated curriculum
Authors: Vasan Cheryl , Goldman Evan , Gentile Matthew , Perlis Susan , Ferrarie Joseph , Ferretti Colin , Greenfield Pete , Vasan Nagaswami
Published: November 2023

At Cooper Medical School of Rowan University, morphological sciences are taught in an integrated organ system based curriculum. In the first year students in the last 8 weeks completed the skin, muscle and skeleton module. In the second year, the remainder of the organ systems is studied. Each practical exam includes cadaver, histology, pathology, radiology and embryology questions. As the class size has increased and with a limited number of faculty, manual grading of the practical exam had become onerous. Furthermore, manual grading challenged the validity and reliability of the assessment instrument. Therefore migration to an automated testing system was made. This move has proven to increase the validity, made the grading process more efficient and students are able to receive their practical exam scores faster with greater qualitative feedback than previous years. Recognizing the deficiency, the authors of the abstract met and operationalized the innovative system and purchased the required number of iPads through the Office of Medical Education. Student and faculty development using a mock exam was conducted. On the day of the practical exam, unique exam specific password was distributed to each student prior to the start of the practical. In addition, instructions were given on how to navigate, submit answers, and close out of the exam. IT personnel were on‐hand to solve technical problems. This poster will describe the step‐by‐step process of the approach, as well as present student and faculty perceptions.

Support or Funding Information

This study was supported by the Office of Medical Education.
Teaching Ultrasound Of The Musculoskeletal System During Anatomy Using the 7D Interactive Software Program
Authors: Blandford Ian , Benninger Brion
Published: November 2023

INTRODUCTION

Ultrasound (US) is increasingly being applied to thoracic and abdominal cavities with incredible momentum. However, there is a demand for musculoskeletal (MSK) US imaging as well. Although life‐threatening diagnosis can be made via US of the cavities, there are enormous numbers of everyday MSK complaints presented to the healthcare system. The objective of this study was to investigate if the MSK US educational software could develop MSK ultrasound users.

METHODS

Literature search was conducted to find MSK US educational software use while teaching anatomy. The 7D Imaging interactive educational software (mskNAV) was provided to 20 students to navigate through the proximal upper limb (shoulder to elbow) section of the software during a one‐week period while dissecting the shoulder to elbow region. The students were evaluated on US machine use, probe skill, structure identification and clinical application on a standardized patient and didactic knowledge via a written exam. Fukuda Denshi UF‐760AG ultrasound machine with SonicEye finger probes was used.

Literature search revealed no known studies with interactive MSK US software. Students scored an average of 86% when evaluated on all aspects of the US examination of shoulder structures (machine & probe use, structure identification, and clinical relevance). Given the number of patients that present with MSK complaints, MSK US is underused and under taught. Medical students and residents are keen to learn MSK US techniques, however, there is insufficient qualified users to instruct them. There is a great need for interactive US education software that allows comprehensive self‐teaching. This study used the mskNAV iPad app to assess whether students can acquire ultrasound skills and knowledge during an anatomy course.

CONCLUSION

This study revealed the interactive mskNAV module was successful in integrating US skills with relevant structures and clinical applications while learning anatomy.
Self‐ and Peer Assessment of Professional Speaking Skills Using a Classroom Response System: Three “Clicker” Systems and Ten Years of Data Collection
Authors: Sprunger Leslie K
Published: November 2023

A gross anatomy lab offers many opportunities for professional skills development. We have used an exercise in a gross anatomy laboratory for first year veterinary medical students which is focused on delivery of short educational presentations using new vocabulary to a small audience. At the end of a lab session, students present a brief overview of the day's work to 2–3 incoming students from another section. Each presenter is required to submit a self assessment using a rubric and a classroom response device. Similarly, their classmates are required to submit a peer assessment at the end of the presentation using the same rubric. The rubric incorporates five questions with quantitative scale responses, two optional questions with a menu of qualitative feedback (developed by students) and two questions to match presenters to their audience in subsequent data processing. Presenters receive an email within 24–48 hrs with their own self assessment data aligned with the peer feedback (names removed). Each student presents five to eight times over the course of the semester.

Three different commercial classroom response systems have been used for this activity. All have served the purpose of the exercise but the most recent one (Top Hat, a bring your own device system) has met with the the most favorable response from the students and the smallest number of problems with data entry. A macro‐enabled template spreadsheet was developed in Microsoft Excel to facilitate processing of data prior to emailing feedback to presenters. Data has been collected to assess whether the exercise facilitates development of students’ professional speaking skills as reported by themselves as well as by their peers. In addition, students have been surveyed to measure their perception of the value of the exercise itself and also the value of the self‐ and peer assessments that are part of the exercise. Peer vs. self assessment scores from the qualitative responses are compared in the aggregate, as well as for individual students. Likewise, scores are tracked over the semester to identify, for example, trends in developing vocabulary skills or changes in the ability to organize a 5–10 minute informative presentation. There is often a tendency for students to give each other somewhat inflated scores, leaving little room to indicate improvement over time. Coaching students on the value of realistic feedback and the lack of value of hyperbole helps in this regard.

Feedback about the value and effect of the presentations has also been collected during most years. Overall, students have indicated that the process is very valuable in helping keep them on task during the lab sessions (in anticipation of having to summarize it for their peers later), in increasing their confidence in the use of medical vocabulary, and that they appreciate having the opportunity to provide similar feedback for their peers.
Effectiveness of BlueLink Cadaveric Multimedia Resources in a Pre‐professional Course
Authors: Dudgeon Sarah Nixon , Alsup B. Kathleen , Fox Glenn M
Published: November 2023

Self‐directed study is a skill necessary in successful learning. One method of self‐directed study is usage of multimedia resources. We have chosen to assess the effectiveness and usage of BlueLink multimedia resources, including course‐specific labeled cadaveric visuals, in illustrating learning objectives for two semesters of a pre‐professional 400‐level anatomy course (n=400). Resources typically supplied to these students, including textbooks, Netter atlases, and lecture slides were held constant in the study. On a five‐point Likert scale, students self‐reported usage of BlueLink resources and increased comprehension of course material. Data showed a strong, positive correlation between usage of BlueLink resources and increased comprehension of course material.

Support or Funding Information

Funded by the University of Michigan Medical School Division of Anatomical Sciences, Department of Surgery.
Implementing the Sony Action Cam® and Camtasia® Software to Supplement Dissection‐Based Anatomy in DPT Programs
Authors: Immonen Jessica , Siefring Chris , Chung‐Hoon Kaiwi , McWhorter J. Wesley
Published: November 2023

The average length of doctor of physical therapy (DPT) programs is three years; however, some programs are shorter in duration and may experience unique demands. A dissection‐based course can improve student understanding and retention of information, but this approach may be time consuming (1–4). One possible consideration may be the implementation of the Sony Action Cam® and Camtasia® video editing software to capture real‐time anatomical dissection to meet accelerated curriculum demands. Thus, the objective of this report is to explore whether instruction utilizing this technology is effective in the delivery of anatomical curriculum and relieving time constraints in the lab.

The Sony® camera and editing software was used to create video tutorials of dissections for DPT students’ cadaver lab. During labs, the Sony® camera was also employed to live‐stream dissections to a HD t.v., allowing students to see real‐time manipulations of structure. Videos created using this technology were high resolution and provided students with more than narration by including tracers, mnemonics and notes. First‐year DPT students (N=54) were surveyed with SurveyMonkey® regarding the efficacy of the technology in the delivery of anatomy curriculum and correlating didactic curriculum to clinical work.

In the first year DPT class, 93% found the technology “beneficial” in correlating didactic material to their future clinical work. 71% of students surveyed declared that live streaming of the dissections provided better understanding of material when students learned in large groups (N=16). In conclusion, this report is significant to improving the delivery of DPT applied anatomy curriculum. For this purpose, the Sony® camera has greater capabilities than the GoPro® and editing the videos with Camtasia® was deemed more helpful than videos with unedited narration. Live streaming of dissections improved understanding of material at the time of presentation and video tutorials provided a concise review method after class hours.

Support or Funding Information

Internal Funding from Rocky Mountain University of Health Professions
Performance in an Anatomy Pre‐course Predicts Performance in a Professional Veterinary Anatomy Curriculum
Authors: McNulty Margaret A. , Stevens‐Sparks Cathryn , Taboada Joseph , Daniel Annie , Lazarus Michelle D.
Published: November 2023

Previous research, including work that has been performed by the co‐authors, has found minimal to no correlation between prior undergraduate coursework and performance in either a professional medical or professional veterinary curriculum. Our preceding work showed that a one‐week pre‐course in veterinary anatomy significantly improved performance within a professional veterinary anatomy curriculum. The purpose of the current study was to determine whether performance in this pre‐course indicated professional anatomy curriculum accomplishments. A one‐week anatomy pre‐course was offered before start of classes to two student cohorts at LSU's School of Veterinary Medicine, the classes of 2018 and 2019. This pre‐course focused on musculoskeletal anatomy of the canine thoracic limb. Immediately following pre‐course completion, students were evaluated via written & practical examinations. Grades in the subsequent fall professional veterinary anatomy course were collected and compared to the students’ performance on the aforementioned pre‐course examinations using Kendall's tau correlation analyses. In both student cohorts, there was a significant (p<0.05) positive correlation between performance on the written exam within the pre‐course and performance on written exams in the professional anatomy course, and this finding was irrespective of whether the material was covered in the pre‐course. Similarly, there was also significant (p<0.05) positive correlation between performance on the practical exam within the pre‐course and performance on practical exams in the professional anatomy course for all participants. The results from this study indicate that performance in a one‐week pre‐course in anatomy can predict student performance in a professional veterinary anatomy curriculum. Grounded theory qualitative analysis of open ended feedback following the pre‐course will be used to determine differences, if any, between those who performed well in the pre‐course compared to those who did not. Together, our results suggest that participation and performance in a one‐week pre‐course focused in anatomy is a reliable measure of performance within a professional veterinary anatomy curriculum. Our results also suggest that participation in an anatomy‐focused pre‐course may be a method of identifying students prior to the start of the semester who would struggle within the professional anatomy curriculum, and efforts can be directed to assist those students with improved study methods. Further studies will evaluate whether performance in the anatomy pre‐course influences and/or predicts performance in other aspects of professional schooling.
Cell differentiation in the murine intestine requires NADPH oxidase 1
Authors: Darby Trevor , Jones Rheinallt
Published: November 2023

It is increasingly documented that cells deliberately generate of reactive oxygen species (ROS) to function as signaling intermediates in the control cell proliferation. These comprise of regulatory networks that regulate progenitor cell differentiation in the murine intestine. However, how ROS mediates these responses remain to be fully defined. Recent reports have shown that the ROS‐generating enzyme NADPH oxidase 1 (Nox1) is highly expressed by colon epithelia. Indeed, a recent report identified Nox1 as a novel IBD susceptibility gene where mutations in Nox1 was discovered in patients presenting with severe pancolitis. Here, we report that ROS generation by Nox1 in the murine colon is necessary for normal intestinal stem cell proliferation and differentiation. Gut epithelial‐specific Nox1‐null mice exhibited increased gut permeability, skewed differentiation, and slowed recovery rates following gut injury. Together, these results implicate Nox1 as an essential protein in epithelial cell homeostasis and response to injury, and reveal a novel mechanism for the maintenance of intestinal tissue structure.
Hypoxia‐Regulated Autophagic Pathways Mediate Epithelial Defense during Mucosal Inflammation
Authors: Kitzenberg David , Saeedi Bejan , Kelly Caleb , Schwisow Kayla , Campbell Eric , Lanis Jordi , Curtis Valerie , Glover Louise
Published: November 2023

Intestinal epithelia provide the primary cellular interface between microbiota and host in the mammalian mucosal ecosystem. As such, epithelia actively participate as innate immune sensors of luminal bacteria that breach this barrier. Defects in epithelial autophagy have been identified in inflammatory bowel disease (IBD). Autophagy, a primordial degradation pathway, is implicated in diverse but interconnected immune responses such as mitochondrial signaling and elimination of invasive bacteria. Improved epithelial mitochondrial function proves protective in IBD, and a functional link between mitochondrial autophagy and bacterial autophagy has recently been established. However, crosstalk between these seemingly disparate processes is poorly characterized, and the regulatory pathways involved in epithelial cell‐intrinsic autophagy remain unknown. Recent work has defined a central role for the hypoxia‐inducible factor HIF in promoting epithelial bioenergetics and antimicrobial defense. Using a ChIP‐on‐chip approach, we identified autophagy as a novel pathway coordinately regulated by HIF, and highlighted a cohort of IBD‐implicated genes involved in the selective autophagy of mitochondria (mitophagy) and bacteria (xenophagy). Enteropathogen invasion was found to be an inherent driver of epithelial hypoxia and HIF‐depleted epithelia displayed dysregulated mitochondrial energetics and abrogated mitophagic and xenophagic responses. Conditional epithelial HIF‐deficient mice demonstrated increased bacterial dissemination and disease activity following acute invasive bacterial challenge. Moreover, augmentation of HIF‐regulated mitochondrial bioenergetics proved protective in mouse models of colitis. These findings place epithelial HIF as a central orchestrator of mitophagic and xenophagic responses during mucosal inflammation.

Support or Funding Information

This work was supported by a Crohn's and Colitis Foundation of America Career Development Award (LEG) and National Institute of Health NIDDK R01 grant DK103712 (LEG).
Differential Regulation of Macrophage Glucose Metabolism by M‐CSF and GM‐CSF: Implications for 18F‐FDG PET Imaging of Inflammation
Authors: Tavakoli Sina , Short John D , Downs Kevin , Nga Nguyen Huynh , Asmis Reto
Published: November 2023

Background

The high glycolytic capacity of macrophages allows for the utilization of 18F‐FDG in the detection of inflammation associated with various diseases. Microenvironmental signals (such as growth factors, cytokines and pathogen‐derived products) have profound effects on determining the activation state of macrophages, which in turn is delicately regulated through reprogramming of cell metabolism. Specifically, glycolysis has been suggested to enhance the pro‐inflammatory macrophage phenotype induced by pathogen‐derived stimuli. Here, we examined whether this phenomenon extends to endogenous microenvironmental activators by investigating the metabolic effects of two critical myeloid growth factors with opposing effects on the macrophage inflammatory state, i.e., macrophage colony‐stimulating factor (M‐CSF) and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF).

Methods

Murine peritoneal macrophages (Mϕ) were incubated ex vivo for two days in the absence (Mϕ0) or presence of M‐CSF (MϕM‐CSF) or GM‐CSF (MϕGM‐CSF). Glucose uptake was measured using 3H‐2‐deoxyglucose. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), markers of oxidative and glycolytic metabolism, as well as mitochondrial content of the cells and lactate production were quantified. Expression of activation markers, glucose transporters and metabolic enzymes was measured by high‐throughput quantitative reverse‐transcription PCR.

Results

M‐CSF and GM‐CSF stimulated comparable levels of 3H‐2‐deoxyglucose uptake and induced similar expression levels of glucose transporter‐1 and ‐6 in Mϕ. Similarly, mitochondrial biogenesis and oxidative metabolism were induced to the same levels in MϕM‐CSF and MϕGM‐CSF. Unexpectedly, activation of glycolysis was more pronounced in MϕM‐CSF compared to MϕGM‐CSF, as evidenced by higher basal ECAR and ECAR‐to‐OCR ratio, as well as lactate production. A number of critical glycolytic enzymes were also differentially upregulated in MϕM‐CSF compared to MϕGM‐CSF.

Conclusion

Despite their pro‐inflammatory activation state, MϕGM‐CSF showed the same level of glucose uptake and oxidative metabolism as MϕM‐CSF, which are associated with inflammation resolution. Furthermore, MϕM‐CSF, not the pro‐inflammatory MϕGM‐CSF, demonstrate the higher glycolytic activity. These findings strongly suggest that the reported association between a pro‐inflammatory macrophage phenotype and increased glucose uptake and glycolysis is limited to certain microenvironmental signals, and does not represent a universal phenomenon, calling for a more prudent approach in the interpretation of 18F‐FDG imaging of inflammation.

Support or Funding Information

This work was supported by grants from Radiological Society of North America Grant to S.T. (RR1131) and the National Institute of Health (HL115858) to R.A.
Control of CRISPR Priming and Interference Activities by a Cascade‐Activated Switch
Authors: Whitis Natalie Rose , Xue Chaoyou , Sashital Dipali G , Carver Roy J.
Published: November 2023

CRISPR‐Cas (Clustered Regularly Interspaced Short Palindromic Repeats ‐ CRISPR‐associated proteins) is an adaptive immune system in bacteria that scans DNA within the cell and integrates pieces of viral DNA into the bacterial genome (acquisition) in order to recognize and destroy the virus the next time it infects the cell (interference). The immune system differentiates the viral genome from its own CRISPR loci by recognizing PAM (protospacer‐adjacent motif) sequences that are present in viral targets but not in the host genomic locus. Mutations in the PAM trigger a more efficient method of integrating new spacers (priming), which requires both the acquisition and the interference machinery. Based on structural analysis of the target‐binding interference complex Cascade, we identified several amino acid residues that may have a role in PAM recognition and tested the effects of alanine mutations at these positions on Cascade function. Surprisingly, we identified several mutants with measurable defects in interference but no effects on priming. Our in vitro DNA‐binding assays have revealed that these Cascade mutants cause modest defects in target binding, but completely block target cleavage by the interference endonuclease Cas3, consistent with the observed loss of interference in vivo. Together, these results suggest that Cascade may bind DNA in two alternate conformations that switches activity from interference to priming, and that the PAM‐recognition region of Cascade controls this putative conformational change.
A Small Molecule Enhanced CRISPR/Cas9 Methodology for Genome Editing of Dog (Canis familiaris) induced Pluripotent Stem Cells (diPSC)
Authors: Federico Francesca , Federico Niksha , Sternik Gabriel , Federico Tessya , Esmaeli‐Azad Babak
Published: November 2023

Bacterial CRISPR‐Cas9 system has emerged as an effective tool for sequence‐specific gene knock‐out through non‐homologous end joining (NHEJ), but it remains inefficient for precise editing of genome sequences (gene knock‐in) via homology‐directed repair (HDR). Because of this a long and tedious screening process via cell sorting or selection, expansion, and sequencing is often required to identify correctly edited cells. Improving the efficiency of precise CRISPR gene editing remains a major challenge for many applications, such as generating sufficient numbers of genome‐edited animals or genome edited patient reprogrammed cell lines for disease modeling. Using our patented technology to develop Artificial 3D Microenvironment Niche (US Patent # 904737), we have developed a reporter based screening approach for high‐throughput identification of chemical compounds that can modulate precise genome editing through HDR. In preliminary experiments, we have identified small molecule classes that enhance CRISPR mediated HDR efficiency more than 9 fold. Additionally, we have found that small molecules that inhibit HDR, enhance insertion and deletion mutations via NHEJ and “vice versa”. We plan to further test this refined genome editing methodology for growth hormone gene modifications in dog (Canis familiaris) induced pluripotent stem cells (diPSC), attempting to correct genetically inherited disorders in dogs and for the development of “designer pets” of the future. Our preliminary findings with this experimental system will be presented.

Support or Funding Information

This project was supported by Internal R&D funds from DNAmicroarray, Inc.
Synthesis and Characterization of Nucleic Acid Aptamers Targeted at Aspergillus Surface Carbohydrates
Authors: Bush Jessica Anne , Rohlman Christopher E
Published: November 2023

Aspergillus is a common fungus that is found naturally throughout the world. Of the 200 known species of Aspergillus, 16 have been identified as being harmful to humans and other animals. These spores are inhaled on a daily basis and processed within the body without any negative consequences, however prolonged exposure to high quantities of Aspergillus can cause allergic symptoms, toxic symptoms, and infection (Hummel et al., 2006). People at highest risk are those with weakened immune systems such as surgery, HIV/AIDS, and cancer patients. Traditional methods for the detection of Aspergillus infection in the body are difficult and invasive including biopsies of cerebral lesions and extraction of cerebrospinal fluid, and frequently yield negative results (Hummel et al., 2006). These detection methods are often not feasible for immune‐compromised patients. Aptamers present a new potential detection method for Aspergillosis. Aptamers are single stranded DNA or RNA sequences, which bind to a target molecule with high affinity and specificity (Navani et al., 2009). These sequences can be selected bind to ligands associated with Aspergillosis. Because aptamers have the capacity to bind target molecules with high affinity and specificity they have become a common diagnostic and therapeutic tool. In research, aptamers can have several applications such as artificial gene synthesis, DNA sequencing, library construction, molecular probes, and polymerase chain reaction (McKeague and DeRose, 2012). Such application allows us to target and identify a wide variety of infectious diseases including fungal infections caused by the mold, Aspergillus. By selecting for specific nucleic acid aptamers we can develop a new detection method for Aspergillosis that is more sensitive and less invasive. Once selected, fluorescent tagging can be used to observe their interaction with the target molecule. The design of this experiment was to develop a method for selecting aptamers that bind to a variety of carbohydrates that can be found on the cell surface of Aspergillus. As a means for developing and evaluating the selection process several carbohydrate targets were studied. This selection process binds randomized nucleic acid cassettes to a target carbohydrate found on the Aspergillus surface. Once bound a washing process is used to eliminate DNA sequences that did not tightly bind to the carbohydrate. Nucleic acid sequences that do bind are then eluted and isolated to select successful aptamer candidates. Asymmetric PCR is then used to convert the double‐stranded DNA into the single‐stranded DNA that will be used as a pool for the next round of selection. Several rounds of selection were completed in which the nucleic acid pool was significantly refined. We are currently characterizing the aptamer binding interaction with Aspergillus.
Investigation of the DNA Damage Induced protein DinD, and its effect on DNA Synthesis
Authors: Romero Zachary Joseph , Lusetti Shelley
Published: November 2023

The integrity of DNA is constantly challenged by exogenous and endogenous factors. Many proteins are involved in this constant battle to maintain genomic stability. The protein RecA is one of many proteins up regulated during the bacterial DNA damage response. It serves a variety functions one of which is Homologous Recombination at the site of double stranded DNA breaks. RecA preforms this function by forming nucleoprotein filaments on the single stranded regions of DNA, and mediating strand invasion of another homologous DNA molecule. After DNA pairing RecA gets off the DNA and DNA synthesis occurs. Our lab is interested in another DNA damage induced protein DinD that has been shown to inhibit RecA's activity. Previous work done in our lab has shown when added to a reaction where RecA is preforming DNA pairing DinD freezes the reaction. Inhibition of RecA's function could be attributed to sequestering DNA binding sites from the recombinase. However, further analysis also showed that DinD was not just sequestering binding sites on DNA, but instead it was disassembling the nucleoprotein filament of RecA. The in vivo functions of this activity are not completely understood. However, it is reasonable to think that under heavy DNA damage it is important for RecA to get off the DNA and allow faster synthesis. Here we present experiments designed to investigate whether DinD acts this way under DNA stress to allow for efficient DNA synthesis.
Elucidating the Role of Transcriptional Mutagenesis in the Acquisition of Adaptive Mutations
Authors: Saxowsky Tina T. , Wilson Gracie , Nixon Gavin , Iverson Jessika , Angkanaworakul Pannapat , Brock Chance
Published: November 2023

For nearly a century, we have understood mutagenesis as a growth‐dependent process involving DNA replication. More recently, cells have been shown to accumulate adaptive mutations in response to selective pressure that induces prolonged growth arrest. While multiple mechanisms related to DNA damage and genome maintenance have been shown to contribute, the specific role of DNA base damage and base excision repair in the process has not been explored. Base damage can be bypassed by RNA polymerase, resulting in a pool of mutant mRNAs through transcriptional mutagenesis (TM), and potentially a population of mutant proteins that could alter the phenotype of the cell. In non‐dividing cells, such as those subject to drug pressure, the influence of persistent DNA damage on transcription could affect cellular outcomes. We hypothesize that TM could be an important first step in acquiring drug resistance, as the mutant protein generated could allow cells to resume DNA replication and convert this damage to a permanent advantageous mutation, a process we call retromutagenesis. Using Saccharomyces cerevisiae as a model organism, we have established an experimental system that examines adaptive mutagenesis in response to canavanine exposure. Within this system, we have demonstrated that prolonged exposure to canavanine yields an increase in intracellular reactive oxygen species, and that cells deficient in the repair of the oxidative lesion 8‐oxoguanine (ogg1 knockouts) show an increase in adaptive mutation frequency. Additionally, DNA sequencing of the target gene in adaptive mutants from this repair deficient cell line has revealed a strand bias for the DNA damage leading to canavanine resistance in the template strand related to transcription. These results support our hypothesis that mutagenic transcription past DNA damage in non‐dividing cells could contribute to the mechanism by which adaptive mutations arise.

Support or Funding Information

Cottrell College Science Award, Research Corporation for Science Advancement
The Yeast Genome Project: Characterizing APD1
Authors: Douds Catherine , Ben Jelloun Idriss Touimi , Keeney Jill B.
Published: November 2023

Yeast is considered to be “man's oldest industrial microorganism” and has been used by humans for over 5,000 years. Today, its use has expanded and Saccharomyces cerevisiae has become an ideal eukaryotic model due to its quick growth and easily manipulated genome. After its genome was sequenced in 1996, scientists came together to create a full encyclopedia of systemized gene functions characterized according to the Gene Ontology Consortium (GO). However, nearly twenty years later, the functions of more than 10% of these genes still remain unknown. The ORFan gene project aims to aid the characterization of the S. cerevisiae genome by exploring the function of specific open reading frames (ORFs). In order to classify these unknown ORFs, a putative pipeline has been constructed beginning with the identification of an ORFan of interest through the Saccharomyces Genome Database (SGD). This project focuses on YBR151W or APD1. The selected ORF was fluorescently tagged at the C and N terminals to verify cell localization. Next, a comparison between the wild type and target gene knockout strains will be analyzed through RNAseq. This comparative analysis uses programs such as FastQC, FastX, RSEM, Bowtie2 and R, and will provide information about the impact of gene deletion on global gene expression levels. Further exploration into the putative function of APD1 has led to studies involving actin patch localization, as well as the protein's potential role in oxidative stress mechanisms. We have observed the effects of actin patch localization under various forms of oxidative stress such as HU and H2O2 between wildtype and Δapd1 strains.

Support or Funding Information

Howard Hughes Medical Institute Undergraduate Grant Program, Funded by HHMI GCAT SEEK SNF grant #1248096, NIH Grant R15‐GM0587 to Jill B. Keeney
TFAM Mutants Support mtDNA Replication In Vivo
Authors: Alexeyev Mikhail , Kozhukhar Natalya
Published: November 2023

Objective

To evaluate ability of the transcription‐deficient TFAM mutants to support mtDNA replication in vivo.

Methods

We developed a GeneSwap approach that allows for the replacement of WT TFAM allele with a mutant in cultured cells. The approach consists of the three steps: 1) inactivation of the chromosomal TFAM alleles with CRISPR/Cas, 2) introduction of a floxed WT TFAM allele, 3) reintroduction of mtDNA and 4) co‐introduction of Cre recombinase and mutant TFAM allele.

Results

We were able, for the first time, to cultivate cells lacking TFAM and thus demonstrated that lethality of the TFAM knockout is conditional. TFAM knockout (KO) cells lack mitochondrial DNA (mtDNA) and are defective in respiration. We further demonstrate that TFAM mutants with severe defects in mtDNA transcription and respiration can support mtDNA replication.

Conclusion

even very low levels of mtDNA transcription may be sufficient to support mtDNA replication in vivo.

Support or Funding Information

HL66299 and OD010944.
Genome wide methylation of RASSFs associated genes in Kidney Cancer
Authors: ALOKAIL MAJED S , ALENAD AMAL M
Published: November 2023

The RASSF are tumor suppressor proteins that undergo loss of expression through promoter methylation in numerous types of cancers including kidney cancer. RASSF proteins have several functional domains that modulate associations with other proteins like Salvador‐RASSF‐Hippo (SARAH). RASSFs can associate via the SARAH domain with downstream kinases such as MST1 and MST2 and SAV1 in order to promote apoptosis. This study identifying differential methylation patterns that can distinguish between conventional clear cell renal cell carcinoma (ccRCC), chromophobe, oncocytoma and papillary RCC. Genome wide methylation using the Illumina Infinium Human Methylation 450 Bead Chip, RASSF1A was highly methylated in ccRCC, Papillary and Oncocytoma tested (95%, 100% and 45% respectively), while in chromophobe, methylation was lower at only 15% of samples. Analysis of the Hippo pathway genes indicates that methylation of this pathway may vary between the cancer types in particualar methylation of MST1, which was identified to be methylated in 80% ccRCC, 38.9% papillary RCC, 20% chromophobe and 13.3% oncocytomas. Methylation of the RASSF2 promoter was detected in 36.8% papillary RCC, 31.6% ccRCC, 16.7% chromophobe and 5.3% oncocytomas. In conclusion, the methylation levels varied across the promoters of Salvador‐RASSF‐Hippo genes associated with kidney cancer.

Support or Funding Information

King Saud University
Molecular Analysis of Adaptational Strategies under Heat Stress by Whole‐Genome Sequencing of Laboratory‐Evolved Saccharomyces cerevisiae
Authors: Satomura Atsushi , Miura Natsuko , Kuroda Kouichi , Ueda Mitsuyoshi
Published: November 2023

Experimental evolution is an effective method to connect genotypes to phenotypes. The outcomes obtained from the method potentially facilitate the rational engineering of useful strains for bioproduction.

In this study, we carried out experimental evolution of Saccharomyces cerevisiae under heat stress [1, 2]. In order to track the cell populations, intermediate strains obtained during the adaptational experiments have been preserved. Whole‐genome sequencing of 19 intermediate strains revealed that a total of 49 genetic mutations had emerged during the experimental evolution. A phylogenetic tree analysis showed that there were at least 5 events in which these strains had acquired mutations in a CDC25 gene. One of the CDC25 mutants successfully adapted to a 38°C‐environment. These mutations led to the downregulation of cAMP‐dependent protein kinase (PKA) signaling pathway, which controls variety of processes such as cell‐cycle progression and stress tolerance. In general, excessive downregulation of this pathway causes a trade‐off between induced thermotolerance and diminished growth rate. However, these strains have acquired thermotolerance without any growth defects. Our study demonstrates that experimental evolution leads to fine tuning of signaling pathways and engineering of beneficial cellular traits [3].

Support or Funding Information

This work was supported by a Grant‐in‐Aid for JSPS Fellows (No. 26‐1413) from Japan Society for the Promotion of Science, the commission for Development of Artificial Genes Synthesis Technology for Creating Innovative Biomaterial from the Ministry of Economy, Trade and Industry (METI), Japan, and, JST‐CREST.
The involvement of histones and post‐translational modifications during DNA repair in bdelloid rotifers
Authors: Jones Alexander C , Ebijoyeldhas Miracline , Boerma Marjan , Orr Lisa M , Byrum Stephanie D , Tackett Alan J , Schurko Andrew M
Published: November 2023

Histones are architectural proteins that package eukaryotic DNA into nucleosomes and regulate the expression and replication of the genome. Four canonical histones (H2A, H2B, H3, and H4) are present in duplicates to form an octamer within a nucleosome. Histone N‐and C‐terminal tails are often targets for post‐translational modifications (PTMs). For example, C‐terminal phosphorylation of the histone variant H2AX serves as an epigenetic marker for DNA damage and links damaged chromatin to DNA repair proteins. Bdelloid rotifers are aquatic microinvertebrates that have the ability to effectively repair DNA double‐strand breaks (DSBs) induced by high doses (>1000 Gray) of ionizing radiation. The bdelloid Adineta vaga possesses five histone H2A variants, three of which (H2Abd, H2Abd1, H2Abd2) have C‐terminal tails that are significantly longer than those in other eukaryotes. These tails have the potential to act as targets for PTMs. The objective of this project was to investigate the role of bdelloid H2A variants in DNA repair by characterizing PTMs associated with these proteins and to identify their binding partners. We induced DSBs by irradiating three species of bdelloids with 280 Gray of ionizing radiation. Histones were then isolated at different time points post‐irradiation and high‐resolution mass spectrometry was used to characterize the histones present. We identified each of the five bdelloid‐specific H2A variants along with histones H2B, H3 and H4. Several PTMs (acetylations, methylations and phosphorylations) were identified on the N‐and C‐terminal tails of all histones. Of the identified PTMs, many were only present during DNA repair; among these, several were located in the long C‐terminal tails of H2A variants. To determine the significance of the H2A variants and associated PTMS in bdelloids, we want to identify binding partners to these proteins during DNA repair. To accomplish this, we produced 6×histidine‐tagged proteins representing each of the five variants in A. vaga. Western blots with custom polyclonal antibodies specific for each variant were used to verify the identity of the tagged proteins. Recombinant proteins and peptides (+/− PTMs) will be used in pulldown assays to identify binding partners of histones during DNA repair. In sum, we have identified several novel histone PTMs associated with DNA repair in bdelloid rotifers. These findings will improve our understanding of the role of histones and epigenetics in the specialized DNA repair system in bdelloids.

Support or Funding Information

This work was supported by grants from the NCRR (5P20RR016460) & NIGMS (8P20GM103429) from the NIH and from the Arkansas Science and Technology Authority.
Identification and Characterization of a Minimal Functional Splicing Regulatory Protein
Authors: Ontiveros Robert J. , Doan Justin , Adams Eric S. , Hernandez A. L. , Black Douglas L. , Keppetipola Niroshika M.
Published: November 2023

In higher eukaryotes, alternative splicing of a single gene transcript into multiple final mRNA isoforms contributes significantly towards the diversity of cellular protein composition. The process of alternative splicing is regulated in part by RNA binding proteins which bind within and adjacent to regulated exons, influencing the location and assembly of a functional spliceosome. Splice variants have been identified in many neurodegenerative diseases and cancers, underscoring the importance of alternative splicing. Detailed mechanistic and atomic understanding of how splice variants are generated will provide new targets for therapeutic intervention. The Polypyrimidine Tract Binding Protein 1 (PTBP1) is a well characterized RNA binding protein with roles in alternative splicing regulation, mRNA localization, and IRES‐mediated translation initiation. Studies highlight that PTBP1 binds preferentially to either upstream or downstream sites of target exons to promote their exclusion or inclusion from the spliced transcript. PTBP1 contains four RNA Recognition Motifs (RRMs) joined via three linker regions that bind to pyrimidine rich sequences with varying affinity and structural preferences. How the complete structure of PTBP1 binds to its target RNA and the role of each RRM during exon inclusion/exclusion is unclear and hinges on obtaining atomic structures of full‐length PTBP1 bound to a target RNA molecule. Attempts to crystallize RNA‐bound PTBP1 have been hindered by the flexible linker regions between RRMs 1 and 2, (linker 1, 42 amino acids), and RRMs 2 and 3 (linker 2, 81 amino acids). The aim of this study is to identify and characterize a minimal functional PTBP1. A series of PTBP1 mutants were created with deletions in the linker 1 (Δ19, Δ29, Δ37, Δ40) as well as the linker 2 (Δ21, Δ44, Δ53, Δ68). Mutants were tested in cell culture for protein expression and splicing repression activity using an alternatively spliced reporter gene in mouse neuroblastoma cells (N2A). Western blots indicate that the mutants are well expressed and highlight that deletions in the linker regions do not interfere with the proper folding of the protein. Splicing assays reveal that the shortest construct (linker 1Δ40 and linker 2 Δ68) maintains splicing repression of the reporter gene cassette exon similar to full length PTBP1. We are currently constructing an in vitro expression plasmid of the mutant to express and purify recombinant protein to begin crystallization trials.