bluuebunny/crossref_metadata_2025 huggingface dataset:
Making Meaning: A qualitative inquiry into undergraduate meta‐emotional and sociocognitive development during cadaver dissection
Authors: Soneral Paula A.G. , Wyse Sara A. , Shaw Tim
Published: November 2023

Cadaver dissection has been a crucial part of training medical professionals for centuries, a learning experience often enriched with life lessons beyond the content of gross human anatomy. Relatively little data exists on the long‐term impact of cadaver dissection on medical professionals, and an even greater paucity of data exists on how undergraduate learners make meaning from this multidimensional experience. The objective of this study was to explore the perceived impact of cadaver dissection on the cognitive, relational, and emotional development of undergraduate students. How do undergraduate students make sense of their cadaver dissection experience in relation to their personal beliefs, prior knowledge and experiences? Undergraduate students enrolled in a 400‐level gross human anatomy course at a small liberal arts college worked in teams to dissect a human cadaver over the course of a four‐week intensive course. At the conclusion of the course, we administered a 15 item, open‐ended reflective survey and performed content analysis to code the responses for themes.

Several multifaceted themes emerged as the most salient perceived aspects of the cadaver dissection experience, spanning four domains of learning: (1) cognitive and metacognitive, (2) meta‐emotional and affective, (3) socio‐cognitive, and (4) psychomotor. Within these domains, students reported appreciation for the complexity and interconnectedness of the human body (cognitive 50%), increased awareness of the importance of compartmentalizing emotions in order to learn (affective and meta‐emotional 57%), concern for ethics (socio‐cognitive 50%), and visceral responsiveness to the odor, sounds, and tactile components of the lab (psychomotor 80%). These preliminary data seem to mirror those from retrospective studies of medical professionals (Skinner 2013), wherein participants reflected on the cadaver dissection experience as foundational for future learning in areas of patient care, self‐efficacy, collaboration, and content mastery. However, undergraduate students distinctly emphasize managing, coping, and relating as the most important aspects of their learning, compared to anatomy content learning gains. We propose a developmental framework to help instructors select appropriate interventions and pedagogies for learners of gross human anatomy at all stages of professional development.
Does Lecture Attendance Matter? A Study of Exam Performance in Medical Neurobiology
Authors: Meyer Edgar Rayner , Notebaert Andrew J , Perkins Eddie , Lynch James C , Simpson Kimberly L , Darling Ryan D
Published: November 2023

With the proliferation of the use of podcasts and online‐based learning, there is debate over lecture attendance and its value in students’ medical education. This study examined the relationship between medical students’ attendance at neurobiology lectures and their subsequent performance in the course. Attendance data from two consecutive classes of medical neurobiology (2014 and 2015) were collected using Turning Technologies Audience Response Solutions during each basic science and clinical correlation lecture. In the spring of 2014, attendance at clinically correlated lectures was mandatory, but not for basic science lectures. For the course in the spring of 2015, no lectures were mandatory, but quizzes were randomly administered at any of the 2015 course lectures as potential bonus points. Each student's percent attendance was correlated with various performance measures in the course including final grade, NBME subject exam score, and three course exams. Upon initial examination of the 2014 data, no significant relationship between students’ lecture attendance and their scores on related exams was noted (R2 = 0.0297). Survey data collected from the 2015 cohort of students revealed that 73.1% and 79.7% of respondents go to basic science or clinical correlation lectures >75% of the time or always, respectively. Also, 65.7% of respondents used the podcasts to supplement lecture while 46.3% used podcasts to substitute for lectures. A means comparison between student attendance at particular lectures and their resultant performance on exam questions specifically related to that lecture topic will be analyzed. Initial results question whether lectures are really important for medical students to attend and whether mandatory attendance is only addressing extrinsic motivation and therefore does not result in improved performance. Future studies should address the quality of lectures and whether there are other ways to increase intrinsic motivation for attending lectures.

Support or Funding Information

None
The Impact of Team‐based Learning (TBL) on Physician Assistant Student's Academic Performance in Gross Anatomy
Authors: Makeeva Valeria , Isbell Jonathan A , Caruthers Kara , Brooks William
Published: November 2023

A number of factors including the acquisition of sufficient numbers of cadavers, lack of appropriately trained educators, and increasing numbers of students enrolling in graduate health science programs have made complete dissection by students difficult at many institutions. Team‐based learning (TBL) is an alternative education method involving pre‐class independent learning, in‐class readiness assurance consisting of individual and team assessments, and in‐class application exercises used to augment traditional dissection. TBL was integrated into the 2014 physician assistant/nurse anesthetist gross anatomy laboratory at the University of Alabama at Birmingham. In 2013 all students attended didactic lectures, and each student participated in one‐half of dissections. The structure of the 2014 course was similar except that students who did not participate in a day's dissection instead participated in a TBL session that ran concurrently with the laboratory. At the end of each laboratory period, students who participated in the TBL session went to the anatomy laboratory, and those students who dissected peer‐taught that day's dissection. De‐identified data from the 2013 and 2014 courses were used to assess the impact of this addition on student academic performance by t‐tests. Students in the 2014 course who experienced TBL performed significantly better on 75% of written and laboratory practical examinations (p<0.05). These mean examination increases correlated with a positive shift in final grades. A greater benefit was noted for physician assistant students than nurse anesthetist students. Importantly, mean percentage TBL grades were not significantly higher than mean examination scores, indicating that the addition of TBL scores to final grade calculations was not responsible for grade inflation in the experimental group. Additionally, there were no significant differences in the mean baseline academic ability of the comparison and experimental PA cohorts as determined by similar undergraduate grade point averages and exam scores in other coursework. TBL is an effective adjunct to traditional cadaveric dissection for students in the health sciences by providing opportunities for students to apply anatomy to clinical practice.

Support or Funding Information

This project was funded by an Educational Development and Innovation Grant from the UAB Center for Teaching and Learning.
Performance statistics of structures: does the anatomical region or structure type impact item difficulty or discrimination on gross anatomy practical examinations?
Authors: Thompson Andrew R. , Cotter Courtney E. , Hanna Jandy B
Published: November 2023

Student examination performance is likely influenced by a number of factors, including (but not limited to) student learning, student approaches to studying, teacher emphasis, and clarity of objectives. Gross anatomy practical examinations are somewhat unique in that assessment has traditionally been achieved through free‐response identification questions. Variation in student examination performance on free‐response items, both within and between anatomical regions, is often accounted for by the casual observation that certain regions and/or structures are more “difficult” than others, but that explanation can be subjective and is of limited use from a pedagogical standpoint. The goal of this study was to directly assess the influence that anatomical region and structure type have on gross anatomy practical examination performance on free‐response items by coding and analyzing several years of question item data (i.e. difficulty and point biserial). This information can help with quality improvement of gross anatomy laboratory teaching and assessment.

Five years of examination data (N>500 items) were categorized by structure type (e.g. vessel, muscle, bone, nerve, organ, etc.) and anatomical region. Within‐structure patterns were also explored, such as branching level and physical size. Difficulty and point bi‐serial correlations (PBS) were calculated for each item and then compared across categories with Wilcoxon tests. Results indicate that, overall, vessels, nerves, and ligaments tend to be the most difficult structure types, while bones, muscles, and organs are the easiest. Evaluation of vessel branching revealed that main vessels are typically easier than subsequent branches (various p<0.05). The general size of a nerve did not have a significant influence on difficulty. These patterns were generally consistent within each anatomical region, although vessels in the head and neck region are significantly more difficult than nerves (p=0.0176). Limited differences in PBS were exhibited. These results provide insight into several factors that influence examination performance and are useful for improving assessments in the gross anatomy laboratory.
VARK LEARNING STYLES OF FIRST YEAR DENTAL STUDENTS
Authors: ALSHEHRY SAMI , Sedlmayr Jayc , Cork John
Published: November 2023

Introduction

Students have different modalities for learning. One of the protocols developed to classify learning styles is the VARK ( V:visual, A:audition, R:reading, K:kinetics) assessment that consists of 16 multiple choice questions. Detailed analysis of the questionnaire results is provided by the VARK institute in New Zealand. Students can prefer a single learning style over all the others or employ a combination of two, three or all four styles.

Aim

To survey the different learning style preferences of first year dental students?

Materials and methods

61 first year dental students from LSUHSC were involved in this study. They were provided with the paper form VARK questionnaire. After gathering all of the data, Excel sheet were emailed to VARK institute for analysis.

Results

The highest percent of students were K (22.5%). The following A, AK, V, VA, VARK, VK and VRK were between 5%–10%. The rest (AR, ARK, R, RK, VAK, VAR and VR) were below 5%.

Conclusion

Based on this analysis, we can conclude that the majority of students are V and K learners and less A and R.
Assessing Musculoskeletal Competency in Osteopathic Medical Students
Authors: McCarthy Sarah A. , Olson Luke , Chen Chun Cheng Andy , Kulesza Randy , Kalmey Jonathan K.
Published: November 2023

Musculoskeletal related conditions are the second most common reason patients seek medical attention in the primary care setting and the second most costly cause of morbidity behind cardiovascular disease. Furthermore, due to the changes that occur in the musculoskeletal system during the aging process, the frequency of musculoskeletal related office visits will increase as the number of elderly patients increases in the U.S. Therefore, it is very important that physicians are thoroughly educated, competent, and confident to diagnose and treat musculoskeletal conditions. However, previous research examining a cohort of graduating osteopathic medical students and a cohort of allopathic medical residents has demonstrated that musculoskeletal knowledge retention, based on a standardized competency exam, is less than optimal. Our study evaluated first‐year osteopathic medical students before and after they had taken a Musculoskeletal System course through administration of a 25‐question examination developed by internal medicine and orthopedic residency directors utilized in previous studies. The competency examination covered a broad area of topics that were considered important by surgical and nonsurgical criteria. We administered the examination before and after the course to two separate cohorts of first year medical students, the class of 2014 and the class of 2017. We compared these two cohorts to determine if changes within our Musculoskeletal course enhanced overall performance on the competency examination. The results show that the Musculoskeletal System course significantly improved examination performance by 27.8% for the class of 2014 and 28.4% for the class of 2017. But what was more interesting was that the average post‐course score of the class of 2017 was significantly higher than the average score of students in the class of 2014 showing 23% more students demonstrated competency. Our first‐year osteopathic medical students performed similarly to graduating osteopathic medical students (32% vs. 33%) and outperformed allopathic medical residents (32% vs. 22%) in the portion of students demonstrating competence. In summary, our Musculoskeletal System course improved musculoskeletal knowledge in students, and our first‐year osteopathic medical students did as well or better compared to graduating osteopathic students and allopathic residents, respectively. This study indicates that despite first‐year students comparing favorably with more experienced students in musculoskeletal knowledge there is room for improvement within our Musculoskeletal System course. Future changes to the course may include more case study scenarios and a focus on more high impact, high frequency musculoskeletal related presentations and management.

Support or Funding Information

This work was supported by an internal grant from Lake Erie College of Osteopathic Medicine.
Customizing Anatomy Videos to Curricula Maximizes Learning and Retention: Implications for First Year Medical Education
Authors: Mastriano Daniel , Kurtzman Jane , Paskin‐Flerlage Samuel , Brady Daniel , Eucalitto Patrick , Laurance Brett , Márquez Samuel
Published: November 2023

Medical students routinely view anatomy video resources found through Internet searches that are not tailored to their schools’ specific curriculum. These videos are long in duration as they include sequential dissection steps before the end product is revealed. As an alternative, a series of Downstate student driven videos were created to be concise with no video exceeding 110 seconds in duration. These videos skip the process of sequential dissection and instead offer the end result of each dissection activity. Videos are matched to each anatomy lab activity with narration and onscreen text captions that orient, identify and emphasize the contiguous anatomical structures within three‐dimensional space. The videos were offered as a supplemental learning resource to the Downstate class of 2019. Qualitative results showed 98% of students requested access to similar videos for future anatomy units and 97% recommended these videos to future classes. Quantitative results recorded by Panopto software showed 147 out of 186 unique users utilized this resource with 4720 views resulting in 5870 total minutes viewed within one week of access to the videos. Comparing all video media resources across all institutional courses over a calendar year the videos, in a one‐week period, resulted in approximately four times as many views as the next most utilized resource. These anatomy videos serve as an invaluable adjunct for acquiring, integrating, and applying anatomical knowledge here at Downstate. Other institutions may deem using customized videos a prudent investment in medical education.
Extracts of the tapeworm, Hymenolepis diminuta, recruit CCR2+PDL1+ Myeloid Cells with the capacity to inhibit dextran sodium sulphate induced‐colitis
Authors: Lopes Fernando , Reyes Jose Luiz , Leung Gabriella , Mancini Nicole , Wang Arthur , McKay Derek
Published: November 2023

Background

Awareness of the immunological basis of host‐parasite interactions can reveal new approaches to cure and/or treat inflammatory disease. We have shown that mice infected with the tapeworm Hymenolepis diminuta (Hd) or systemic delivery of worm antigen (HdAg) developed significantly less severe dinitrobenzene sulphonic acid (DNBS)‐induced murine colitis.

Aim

In ongoing analyses of this helminth‐mouse model system we assessed the effect of systemic delivery of HdAg and adoptive transfer of HdAg‐recruited peritoneal exudate cells (PECs) on the outcome of dextran sodium sulphate (DSS)‐induced colitis.

Methods

Colitis was induced by 5 days of DSS in drinking water and 3 days of normal water in wild‐type (WT) mice ± HdAg or adoptive transfer of HdAg‐recruited total PECs or F4/80+Ly6Glo sorted cells. Disease was assessed by colon length, weight loss, a cumulative disease activity score and histopathology. Cytokine levels were assessed by ELISA. T cell proliferation was assessed in anti‐CD3+ anti‐CD28‐treated splenic CD4+ T cells co‐cultured with PECs recruited by PBS or HdAg.

Results

HdAg suppressed DSS colitis reducing splenic IFNγ, IL‐17 and TNF α and increasing IL‐10 production. Treatment with HdAg resulted in a CCR2‐dependent recruitment of F4/80+Ly6Glo monocytic‐like myeloid‐derived suppressor cells (MDSCs) into the peritoneum. Adoptive transfer of these cells blocked DSS‐colitis. The mixed peritoneal cells recruited in response to HdAg, blocked anti‐CD3+anti‐CD28 antibody evoked T cell proliferation via nitric oxide.

Conclusion

Injection of an extract of adult H. diminuta inhibits DSS‐induced colitis and was accompanied by recruitment of immunosuppressive CCR2+PDL1+ monocytic‐type MDSCs. The adoptive transfer of the HdAg‐evoked MDSCs inhibits DSS‐induced colitis, demonstrating a novel aspect by which helminth‐derived molecules could suppress intestinal inflammation.

Support or Funding Information

CIHR

NSERC CREATE, Host‐Parasite Interactions Program
Entamoeba histolytica Stimulates the Release of the Alarmin Molecule HMGB1 upon Contact with Macrophages
Authors: Begum Sharmin , St‐Pierre Joëlle , Moreau France , Chadee Kris
Published: November 2023

Entamoeba histolytica (Eh) is the causative agent of amebiasis, which occurs in 10% of infected individuals when the parasite invades the underlying colonic mucosa. Parasite‐induced host pro‐inflammatory responses are critical in disease pathogenesis, but the mechanisms underlying this response is poorly illustrated. The contact between Eh and macrophages triggers a raging pro‐inflammatory response by activating NLRP3 inflammasome (IL‐1β) and other uncharacterized pathways (TNF‐α). High Mobility Group Box 1 protein (HMGB1) is a critical mediator of inflammation during infection and it shapes the magnitude and degree of the inflammatory response. HMGB1 is a non‐histone nuclear protein, which have overlapping binding side with histone H1 in the chromatin. It is released in the extracellular space during infection by activated or damaged immune cells and act as an alarmin molecule. In this study, we investigated the early release of HMGB1 upon Eh infection with macrophages and identification of the parasite virulent factors that initiated this response. Human monocytic cell line (THP‐1) and mouse bone marrow derived macrophages (BMDMs) were treated with live Eh, Eh components for different time point and western blotting was used to quantify HMGB1 secretion. Only stimulation with live Eh but not Eh components triggered robust time‐dependent (as early as 5 minutes) secretion of HMGB1. HMGB1 release from macrophage was contact‐dependent as inhibiting the Gallectin adhesion with exogenous galactose abrogated HMGB1 secretion. Another Eh surface virulent factor cysteine protease 5 (EhCP5) partially inhibited HMGB1 release, and the enzymatic activity of the CP5 was not required for the secretion. Moreover, HMGB1 protein secretion was independent of caspase‐1 activation, as secretion was not inhibited with the Pan caspase inhibitor, Z‐VAD‐fmk. Similar results were obtained using BMDMs from caspase‐1−/− mice treated with live Eh. Surprisingly, there was the simultaneous release of the nuclear protein histone H1 along with the HMGB1 upon Eh infection. These results demonstrate that the potent pro‐inflammatory mediator HMGB1 release is the earliest innate response upon Eh contact with macrophages. This mediator can act by itself or along with other molecules as a danger signal (alarmin response) to sense invasive infection that can attract bystander cells and/or promote extensive inflammation, which is critical in disease pathogenesis.

Support or Funding Information

Grant Support: NSERC
Spatial Abilities, Anatomy Knowledge and Technical Skills: A Systematic Review
Authors: Langlois Jean , Bellemare Christian , Toulouse Josée , Wells George A.
Published: November 2023

Objective

Spatial abilities consist of visualization, orientation and manipulation of structures in space. Spatial abilities have been correlated to anatomy knowledge and the performance in technical skills in systematic reviews. The objective was to conduct a systematic review of the relationship between anatomy knowledge and performance in technical skills as related to spatial abilities.

Methods

Search criteria included ‘spatial abilities’, ‘anatomy knowledge’ and ‘technical skills’. Keywords related to these criteria were defined. A literature search was done up to December 31, 2014 in Scopus (including Medline) and in several databases on OvidSP and EBSCOhost platforms. A bank of citations was obtained and was reviewed by two independent investigators. Citations related to abstracts, literature review, thesis and books were excluded. Articles related to retained citations were obtained and a final list of articles was done. Articles were assessed for quality using Scottish Intercollegiate Guidelines Network‐50 (SIGN‐50). Data were extracted from articles and methods relating spatial abilities, anatomy knowledge and technical skills were identified.

Results

A series of 106 articles was obtained. One additional article was identified through other source. A series of 53 articles was identified after duplicates were removed. Forty‐nine articles were then excluded. Four articles were retained, fully reviewed, and excluded with reasons, yielding no eligible articles.

Conclusion

No eligible articles were found in a systematic review on spatial abilities, anatomy knowledge and technical skills. Future studies will be required to assess the importance of anatomy knowledge before performing a technical skill as related to spatial abilities.

Support or Funding Information

None.
The Influence of Spatial Ability On Anatomy Examination Questions in an Integrated Medical Curriculum
Authors: Xiong Jennifer , Edmondson Anna C. , Martin Charys
Published: November 2023

Background

Students with high spatial visualization ability (Vz) have been found to outperform students with low Vz in anatomy. However, how Vz influences anatomy performance has not been established. Thus, this study aimed to assess the influence of Vz on medical student performance on different levels of anatomy questions categorized by Bloom's taxonomy levels and discrimination index (DI) and to observe the relationship between Vz and anatomy performance. We hypothesized that there would be a positive correlation between Vz and performance on more difficult exam questions categorized by DI and Bloom's taxonomy. We also hypothesized that there would be a positive correlation between Vz and anatomy written exam, anatomy lab exam, and overall anatomy performance.

Methods

First year medical students in a systems‐based integrated medical curriculum (n=61), completed the Mental Rotations Test (MRT) prior to the start of anatomy to establish Vz. All anatomy exam questions were categorized into four Bloom's taxonomy domains of increasing difficulty level (identification, comprehension, application, and analysis). These questions were also categorized into three tiers via DI.

Results

No significant relationship (p>0.05) was found between Vz and questions categorized by DI or Bloom's taxonomy. Data also indicated that although entrance Vz plays an insignificant role in medical student anatomy lab exam, anatomy written exam, and overall performance in the anatomy course, there is a correlation between entrance Vz and anatomy performance in the very first systems‐based module (r2=0.017, p≤0.05).

Discussion

These findings suggest that entrance Vz may influence anatomy performance at the beginning of the curriculum; however, students with lower Vz find ways to cope and increase anatomy performance throughout the curriculum. Due to the significant relationship between Vz and the first systems‐based module, further analysis was completed to assess the relationship between Vz and anatomy question difficulty. This analysis indicated that there was no significant interaction between Vz and questions categorized by DI or Bloom's taxonomy within that first systems‐based module (p>0.05), suggesting that Vz's effect on performance in anatomy may not have a relationship with question difficulty categorized by Bloom's taxonomy or DI. Further research is necessary to explore how Vz influences anatomy performance and how students’ ability to train Vz and change study strategies influences the effect of Vz on anatomy performance throughout the medical curriculum.
Stereoscopic (3D) Visualization Improves Medical Student Comprehension of Head and Neck Vascular Anatomy
Authors: Cui Dongmei , Wilson Timothy D , Rockhold Robin W , Lehman Michael N , Lynch James C
Published: November 2023

The use of 3D stereoscopic models (3DSM) of head and neck blood vessels in anatomy education has not been studied in detail. We investigated whether 3DSM created from a prior study (Cui et al. 2015) would improve student learning of head and neck vascular anatomy. Comparisons were made between the use of 3DSM and identical but 2D flat screen images extracted from the 3D models in a first year medical gross anatomy course. Anatomical knowledge was tested via pre‐ and post‐learning session anatomy knowledge tests. In addition student fluency with mental rotation (pre‐ and post‐session rotation tests.) Results were analyzed using a Wilcoxon rank‐sum test and linear regression analysis. A total of 39 first year medical students participated in the study. Baseline pre‐learning session test scores were equivalent, 5.85±2.37 and 5.03±1.93 for 3DSM and 2D groups respectively. Students who utilized the 3DSM (n=21) scored significantly higher on the post‐learning tests compared to those using 2D images (n = 18) (11.43±2.79 vs 8.75±2.81) (p =0.0033). There was no significant difference on the mental rotation test scores between 3DSM (17.29±5.36) and 2D groups (17.39±6.42) after learning sessions (p=0.6208). In summary, the use of virtual stereoscopic 3D models improved medical student performance on knowledge tests of head and neck vascular anatomy, suggest new avenues for the use of virtual models in medical education.

Support or Funding Information

Faculty Scholarship Exchange Award
Potential of “Living” Photography in an Online Anatomy Resource to Improve Students’ 3D Spatial Understanding
Authors: Pascoe Mike , Lee Lisa
Published: November 2023

BACKGROUND

Although numerous online resources for gross anatomy are available, most feature two‐dimensional photographs generated by traditional digital cameras. Demonstrating the complex, three‐dimensional relations of anatomical structures in 2D images is challenging and difficult for students to understand. To that end, we developed two online learning modules on the gross anatomy of the orbit featuring photos taken by either plenoptic (allowed adjustment of the focal point between different areas within a single “living” picture) or tradition photography.

OBJECTIVE

The project goal was to determine whether the incorporation of the plenoptic photographs into an online anatomy module would result in improved learning outcomes compared with a learning module containing traditional digital photographs.

METHODS

115 health professions students were randomly assigned into two groups and each group was allowed to view one of the two learning modules on gross anatomy of the orbit. The two modules were identical in content except for the type of photography (traditional = control; plenoptic = experimental) used to illustrate a dissected orbit. Pre‐and post‐quizzes composed of a mixture of low‐ and high‐degree of spatial difficulty were administered. A survey was also distributed to assess student evaluations of the module. Paired, parametric statistical tests were used to examine significant group differences in quiz score improvement between the two types of modules (control vs. experimental; α = 0.05).

RESULTS

Of the 115 students, 49 completed the study (43% participation rate) and of the participants, 26 were in control group and 23 in experimental group. All participants demonstrated improved performance in post‐quiz compared to the pre‐quiz but the increase was not significantly different between the two groups (control: 42.7%, experimental: 47.0%, P = 0.495). When quiz performance was analyzed based on degree of spatial difficulty, both groups demonstrated significant increases on questions with low‐ and high‐degrees of spatial difficulty (P<0.001). However, students in the experimental group demonstrated a larger increase (28.2%) in score for the questions with high degree of spatial difficulty compared to the control group (25.8%; P = 0.040). Qualitatively, all participants reported for both modules that they were well organized, easy to learn from, intellectually stimulating, and would recommend it to their peers.

CONCLUSIONS

The results of our study demonstrate that an online learning module on gross anatomy can positively impact student learning and that plenoptic photography has potential to enhance students’ understanding of challenging 3D anatomical relations.

Support or Funding Information

Rymer Small Grant, Academy of Medical Educators, University of Colorado Anschutz Medical Campus
Live Cell Interrogation of dCas9:sgRNA Dynamics Off and At the Target
Authors: Pederson Thoru
Published: November 2023

Although the CRISPR‐Cas9 system has been widely used for genome editing, little is known about its dynamics in live cells such as the kinetics of sgRNA:Cas9 assembly and DNA recognition. By labeling dCas9 (nuclease‐dead Cas9) using a fluorescent protein and sgRNAs with GFP‐like RNA aptamers (Broccoli), and employing a single genomic locus on human chromosome 3 as the target, we monitored the assembly of sgRNA:dCas9 and tracked its nuclear roaming and docking on the target. We generated stable cell lines with tunable dCas9 expression and constitutive sgRNA transcription. In the absence of dCas9 expression the sgRNA was so unstable as to not be detectable. In contrast, after dCas9 induction the sgRNA displayed two kinetic components, one with a half‐life of ca. 15 min. (likely sgRNA:dCas9 complexes roaming the nucleoplasm) and another more long‐lived (likely bound to both target and non‐target DNA). Fluorescence Recovery After Photobleaching (FRAP) revealed that sgRNA:dCas9 association with the target had an average residence time of 100 min. and that mismatches in the sgRNA seed significantly reduced the target residence time. A compelling correlation was observed between the target residence times of these sgRNA seed mutations and the cleavage activity observed for these same mutations in studies of others with nuclease‐active Cas9, suggesting that our tracking of dCa9 dynamics at the target reflects a basic thermodynamic feature of the loading step it shares with nuclease‐active Cas9. Our in vivo measurements, in combination with existing data from in vitro studies, should facilitate the design of efficient and precise CRISPR‐Cas9 applications in various cellular and therapeutic settings.
Optimization of CRISPR/Cas9 Platform‐Mediated Gene Editing of Swine Embryos for The Creation of Novel Biomedical Swine Models
Authors: Schomberg Dominic T. , Meudt Jennifer J. , Krentz Kathleen J. , Rubinstein C. Dustin , Zhang Aiying , Shanmuganayagam Dhanansayan
Published: November 2023

Swine are increasingly used to model complex human diseases for basic scientific discovery and for the preclinical development and validation of therapies and medical devices. The overwhelming genetic, anatomical, physiological and pathophysiological similarities to humans make swine an ideal platform for modeling the complex interplay of factors that influence human disease. The recent advances in gene editing technologies have been instrumental in opening the door to the creation of novel genetic swine models. However, in comparison to the creation of genetic rodent models, the process of creating swine models can be relatively costly, and time and labor intensive, if the techniques and experimental parameters are not optimized to allow high efficiency of edited embryos to be implanted into surrogate swine. The overarching objective of this study was to develop an optimized CRISPR/Cas9 platform for high efficiency of targeted gene editing via cytosolic microinjection of CRISPR/Cas9 components into presumptive swine embryos. We used the introduction of mutations in the neurofibromin 1 gene as the backbone for optimizing the platform. CRISPR guide RNAs (gRNA) were created for regions targeting sites both upstream and downstream of an exon within the gene for targeted cleavage by Cas9. Repair was mediated by a specific single‐stranded oligodeoxynucleotide (ssODN) template thus introducing a 2500 bp deletion. The functionality of the CRISPR/Cas9 system was validated using a plasmid‐mediated transfection of porcine kidney cells; the accuracy of editing in the target region was confirmed by Sanger sequencing. The optimization of the CRISPR/Cas9 platform was performed by the cytosolic microinjection of the editing elements into in vitro fertilized single‐celled presumptive zygotes. Varying parameters including the use of Cas9 as mRNA vs protein, Cas9 concentration, gRNA concentration, and ssoDN concentration were examined. The editing efficiencies were evaluated first with a customized multiplex nested PCR and then verified by Sanger sequencing. We found that each CRISPR concentration studied to be equally permissive of embryonic development through the blastocyst stage and that all conditions produced gene editing events at some frequency. However, the use of naked Cas9 mRNA and higher CRISPR concentrations produced greater rates of gene edit. We present our findings and optimization that allows for efficient creation of swine models.

Support or Funding Information

The research was supported by the Biomedical & Genomic Research Group Discretionary Fund (University of Wisconsin‐Madison), NF Network, NF Central Plains, NF Michigan, NF Northeast, and Washington State NF Family.
Phylogenetic Classification of Gongora Species (Orchidaceae) Based on DNA Barcode Regions matK, rpoB, rpoC1, and trn‐H‐psbA
Authors: Guardado Christopher , Rodriguez Olivia , Foss Keagan , Ludovic Daniela , Ribadeneyra Clara , Trivino Cesar , Baenziger Edward , Ribes‐Zamora Albert , Simmons Alexandra D
Published: November 2023

Objectives

Traditional species classification of orchids is based upon morphology, which is often hindered by inherent limitations such as species similarities. According to the American Orchid Society, the genus Gongora shows extensive morphological affinities, which result in mislabeling in the collections; however, advancements in gene sequencing technology enable the economic feasibility for species identification based on the orchid's genome. Specifically, DNA Barcoding is a molecular technique that uses “DNA barcodes”, which are short DNA regions, that can be used for species identification among other applications (Feng et al, 2015). Additionally, barcoding allows for the study of the evolutionary relationship between species examined and would therefore facilitate classification of unknown species (Singh, 2012). Our objective is to elucidate the classification of 38 Gongora species using four DNA barcode sequences.

Methods and Results

DNA was extracted from 82 individuals from 38 Gongora species. Then, PCR was employed to amplify the four DNA barcode regions of interest (matK, rpoB, rpoC1, and trn‐H‐psbA). The effectiveness of the PCR was analyzed by gel electrophoresis. Subsequently, amplicons were sent for DNA sequencing at an outside sequencing laboratory. The sequenced DNA was analyzed using MUSCLE in Mega 6.0 program. We developed a phylogenetic tree for about 32 species of orchids for each DNA barcode region. The rpoB and rpoC1 sequences provided similar phylogenetic trees thereby supporting the current classification of those Gongora as individual species. Furthermore, the phylogenetic trees also imply a possible evolutionary scheme. The highly variable trnH‐psbA provided a similar species identification but contradicting species evolution.

Conclusions

DNA barcodes provide unique insights into possible Gongora species identification; however, the need to combine all four DNA barcodes into one phylogenetic tree is necessary to provide conclusive results for species classification and evolutionary relationships. The current results suggest that the rpoB and rpoC1 are useful markers for evolutionary analysis and trnH‐psbA could elucidate the differences between closely related species.

Support or Funding Information

University of St. Thomas Research Committee and Biology Department
Using budding yeast to study properties and function of the human Replication Protein A2 N‐terminus in response to specific‐types of DNA damage
Authors: Haring Stuart J. , Senger Barbara L. , Wilson Timothy M.
Published: November 2023

Background

Replication Protein A (RPA) is a heterotrimeric complex that serves critical roles in eukaryotic genome duplication and maintenance. In human cells, the N‐terminus (NT) of the 32‐kDa subunit of RPA (Rpa2) is hyper‐phosphorylated by multiple kinases in response to DNA damage. We demonstrated previously in the budding yeast Saccharomyces cerevisiae that phosphorylation of the yeast Rpa2 NT does not occur under commonly assayed genotoxic conditions, yet this domain is vital for the damage response. Using a chimeric yeast Rpa2 protein containing the human Rpa2 N‐terminus, we have demonstrated that this human domain is phosphorylated in yeast cells a manner nearly indistinguishable from that in human cells.

Study Objective

We wish to determine properties of human Rpa2 NT phosphorylation, including kinases required, sites involved and their interdependence, and what conditions trigger this phosphorylation to identify whether properties of Rpa2 NT phosphorylation are conserved in yeast and human cells.

Methods

We utilized chimeric human‐yeast Rpa2 proteins (with WT and mutant NT), yeast Rpa2 antibodies, and human Rpa2 phospho‐specific antibodies to examine the potential for the human Rpa2 NT to be phosphorylated in response to varying DNA damaging agents.

Results and Conclusions

We determined that the same sites in the human Rpa2 NT are phosphorylated on the chimeric protein in yeast, and that these show similar dependence on DNA damage induction. Unlike that reported in human cells, phosphorylation of one site does not depend on phosphorylation of another site within the domain (i.e., no sequential/primed phosphorylation). We demonstrated that phosphorylation of the human Rpa2 NT in yeast only appears to occur when cells are treated with agents that produce double‐strand breaks (e.g., HO endonuclease, phleomycin, camptothecin) but not when replication is inhibited by hydroxyurea. Finally, we have shown that Mec1 (yeast ATR), but surprisingly not Tel1 (yeast ATM), is necessary to phosphorylate T21 and S33 (both containing TQ or SQ) on the human Rpa2 NT. This is in contrast to human cells where ATR has only been reported to phosphorylate S33, not T21. We conclude that yeast provides a simple system to identify kinases involved, conditions that trigger human Rpa2 phosphorylation, and its relation to other properties of damage responses. This also provides the opportunity to study Rpa2 NT modification in two different systems to gain an understanding of conserved properties and to potentially study the significance of differences in the damage response between the two systems.

Support or Funding Information

This research was supported by NSF‐CAREER 1253723 to SJH.
Protein‐protein interaction inhibitors of the Fanconi anemia DNA repair pathway
Authors: Voter Andrew F. , Manthei Kelly A. , Keck James L.
Published: November 2023

Upregulation of the Fanconi anemia DNA repair pathway is a frequent mechanism by which tumors evolve resistance to DNA crosslinking chemotherapies. Previous studies have determined that the interaction between FANCM and the RecQ mediated genome instability (RMI) complex is essential for crosslink repair in this pathway and that disruption of the interface causes sensitization to DNA crosslinking agents. To identify small molecules that disrupt this interaction, we developed a high‐throughput fluorescence polarization assay that was used to screen a library of 75,000 small molecules. We confirmed the activity of a selective inhibitor in an orthogonal proximity assay and detected its direct physical interaction with the RMI complex using surface plasmon resonance and isothermal titration calorimetry. More potent inhibitors have been identified by structure activity relationship studies. These potent small molecules will be tested for synergies with DNA crosslinking agents in vivo to assess for therapeutic potential.

Support or Funding Information

University of Wisconsin – Madison Medical Scientist Training Program (T32GM008692), Integrated Program in Biochemistry, ICTR Clinical and Translational Science award (UL1TR00427) and traineeship (TL1TR000429) and the NCI (R21CA178475‐01)
FORCE SPECTROSCOPY OF NUCLEOSOMES AT THE PROMOTERS OF THE LH GENES REVEALS TWO DISTINCT STRATEGIES FOR THEIR REGULATION
Authors: Kaplan Ariel , Rudnizky Sergei
Published: November 2023

Luteinizing hormone (LH) is a heterodimeric protein that regulates function of the gonads, and is composed of two subunits, which are encoded by two different genes: chorionic gonadotropin‐a (Cga) and Lhß. A sequence‐based algorithm designed to predict nucleosome positions on DNA suggests that the promoters of both genes, and in particular the binding regions for important transcription factors, are packed into nucleosomes. Since gene expression requires that the promoter be accessible to the cellular transcription machinery, we hypothesized that nucleosome positioning regulates basal expression of these genes. We are performing single‐molecule force spectroscopy with optical tweezers, complemented by MNase mapping, to probe the architecture of chromatin on both gene promoters. In gonadotrope cells, the promoter of Lhß is occupied by a nucleosome containing the variant H2A.Z, while the Cga promoter appears nucleosome‐depleted. Optical tweezers experiments show that a nucleosome reconstituted on the Cga promoter is weaker, and poorly localized, as compared to the one on the promoter of LHß. This may suggest that it is evicted, in vivo, by chromatin remodelers. In contrast, the first nucleosome downstream of the transcription start site contains H2A.Z in Cga but H2A in Lhß, and is well localized on both genes, although more weakly bound on the Lhß than the Cga gene. Hence, these two genes are apparently regulated by distinct strategies, tailored to obtain higher expression for Cga, which encodes a subunit shared also by other hormones, and lower for Lhß, which is unique to this hormone. Our results highlight how DNA sequence, the usage of alternative histone variants and, possibly, remodeling machinery act synergistically to modulate gene expression.
DNA Cytosine Methyltransferase Increases Stationary Phase Fitness under Temperature Stress in Escherichia coli
Authors: Huss Rebecca A , Davidson Samantha , Militello Kevin T
Published: November 2023

Escherichia coli contains two orphan DNA methyltransferases. DNA adenine methyltransferase (Dam) methylates 5′GATC3′ sequences, and DNA cytosine methyltransferase (Dcm) methylates the interior cytosine in 5′CCWGG3′ sequences. Numerous functions of the Dam enzyme have been elucidated, however the role of Dcm is less defined. Growth curve assays in our laboratory have shown no significant difference in growth rate between strains containing dcm and those without it. However, our laboratory's DNA microarray analyses have revealed numerous gene expression changes in dcm knockout cells in early stationary phase, suggesting dcm influences stationary phase biology. Thus, we tested the role of Dcm in stationary phase fitness. A dcm containing plasmid was constructed by amplifying the dcm gene as well as the putative promoter upstream of the gene by PCR, and inserting it into the pET‐28a+ plasmid. The pET‐28a+ plasmid with and without dcm were introduced into T7 express bacteria, which has a large chromosomal deletion that includes the dcm gene. We call the new strains T7E/pDcm and T7E/pET‐28a+. In order to demonstrate that the cells containing dcm methylated their DNA, a restriction enzyme isoschizomer assay was conducted with PspGI and BstnI. The results indicated that the cells containing T7E/pDcm methylated their DNA at 5′CCWGG3′ sites. The T7E/pET‐28a+ and T7E/pDcm strains were competed over long‐term stationary phase at 37°C. Colony PCR assays were used to determine which strain was outcompeting the other at the conclusion of one and three month intervals. At 37°C, T7E/pET‐28a+ outcompeted T7E/pDcm. As our laboratory has revealed in recent microarray studies, the cold‐shock protein B gene (cspb) was upregulated in bacteria treated with the DNA methyltransferase inhibitor, 5‐azacytidine. This suggests a possible biological role for Dcm at colder temperatures. We then competed T7E/pET‐28a+ and T7E/pDcm at 20°C over long‐term stationary phase. When competing at 20°C, T7E/pDcm outcompeted T7E/pET‐28a+. In order to verify the surviving T7E/pDcm colonies still methylated their DNA, restriction enzyme isoschizomer assays were performed, with the results indicating methylation occurs in only the T7E/pDcm colonies. Our work suggests a new and unexpected phenotype for Dcm. Dcm provides a fitness advantage during stationary phase at low temperatures. We aim to determine the biological mechanism for this phenotype.

Support or Funding Information

This work was supported by the Geneseo Foundation.