bluuebunny/crossref_metadata_2025 huggingface dataset:
Do Secretory‐Stage Ameloblasts Divide? Experimental Evidence
Authors: Hogg Russell , Smith Timothy , Ward Nikolai , Gografe Sylvia
Published: November 2023

Our objective is to test the dictum that secretory ameloblasts cannot divide. This idea is often mentioned in dental biology textbooks without citation or evidence; its source seems to be studies of rodent incisors from the 1960's. However, rodent incisors are ever‐growing, and not acceptable models for enamel epithelia function in other mammals. Also, there are two reasons to expect that mature ameloblasts divide. First, recent studies show that tensile forces induce mitosis in epithelia, and it is likely that the enamel epithelium undergoes tension as enamel is deposited and the surface area of the growing crown expands. Second, we now know that ameloblasts are subject to the same biorhythms that synchronize proliferation of osteoblasts. If ameloblast division is similarly coordinated by the nervous system and occurs at night, it is unlikely that it will have been observed since most experimental protocols are carried out during daylight hours.

To test the hypothesis, we employed two methods. First, 3–6 day old outbred ICR mouse pups (Crl:CD1) used for another research purpose were euthanized at 4 hour intervals that spanned the entire day/night cycle. Specimens euthanized during the day were utilized as controls, while those specimens euthanized at night were considered as experimental tissues for the purpose of the study. Whole heads were thin‐sectioned in a coronal plane and stained using standard H&E protocols to identify mitotic figures in secretory‐stage ameloblast cells. Second, we labeled juvenile primate specimens for immunoreactivity to Proliferating Cell Nuclear Antigen (PCNA), which is expressed in cells that are undergoing DNA repair and/or replication in preparation for mitosis. Animal protocols were approved by respective Institutional Animal Care and Use Committees (IACUC) at Slippery Rock University and Florida Atlantic University.

In our mouse specimens, we have not found signs of mitotic figures; however in one juvenile collared lemur we did see signs of strong PCNA expression in cervical loop ameloblasts that had just entered the secretory stage. This weakly supports our hypothesis, and warrants searches for other patterns of gene expression and mitosis.
Distribution of Neural/Stem Progenitor Cells in the Brain of the Leopard Gecko (Eublepharis macularius)
Authors: McDonald Rebecca Paula , Bradley Stefanie Simone , Vickaryous Matthew Kenneth
Published: November 2023

Endogenous neural stem/progenitor cells (NSPCs) have been identified across vertebrate species. Among adult mammals, NSPCs are spatially restricted to just two neurogenic regions: the subventricular zone of the lateral ventricle, and the subgranular zone of the hippocampal dentate gyrus. In other species – including teleost fish and urodeles – NSPCs are known to have a much broader distribution. For reptiles, less is known. Here we identify NSPCs along the rostral caudal axis of the brain of the leopard gecko (Eublepharis macularius; hereafter, ‘gecko’). The ventricular system of the gecko brain is bordered by a distinct neurogenic niche, the ventricular zone (VZ). All VZ cells (including those of the lateral, third and fourth ventricles) constitutively express the hallmark transcription factor of stemness, SOX 2. Additionally, VZ cells have a radial‐like morphology and pattern of protein expression (including immunostaining for the intermediate filaments Vimentin and glial fibrillary acid protein [GFAP]) closely comparable to mammalian embryonic radial glial cells. At the level of the cerebellum, putative NSPCs (SOX2+ and GFAP+) were also identified independent of the ventricular system. Proliferating cells (identified by phosphorylated histone H3 expression) are most abundant in the VZ of the lateral ventricle and cerebellar parenchyma. The VZ of the third ventricle demonstrates a comparatively modest level of proliferation, while the VZ of the fourth ventricle is largely quiescent. The presence of NSPCs, both dormant and constitutively active, across the lizard brain offers compelling evidence for its neurogenic and possibly regenerative capacity.

Support or Funding Information

Natural Sciences and Engineering Research Council Discovery Grant 400358
NEURON‐SPECIFIC TRANSGENE EXPRESSION OF IMP2 MEDIATED BY SYNAPSIN PROMOTER‐DRIVEN AAV9
Authors: Park Danielle , Blizard Sarah , Austin Scarlett , Harman Joshua , Haraszti Samantha , Xu Mei
Published: November 2023

Insulin‐like growth factor‐II (IGF‐II) mRNA‐binding protein‐2 (IMP2) is one of the three homologues (IMP1‐3) that play an important role in the post‐transcriptional regulation of gene expression during development in several tissues including the nervous system. An alternative splice product of IMP2 lacking exon 10 aberrantly expresses in human hepatocellular carcinoma and is identified as HCC. Several lines of studies have demonstrated that IMP1/ZBP1 (zipcode binding protein) is critical in axon guidance and regeneration by regulating localization and translation of specific mRNAs. However, a role of IMP2 in the neural tissue is largely unknown. Previously, we used a custom made IMP2‐specific antibody to locate the endogenous IMP2 expression in mouse neurons and glial cells at different developmental stages. Our pilot study shows that IMP2 expression is sustained throughout life and it may play a role to facilitate axon regeneration. In this study, we used the synapsin promoter‐driven adeno‐associated viral (AAV) 9 constructs to express YFP‐IMP2 and –HCC specifically in neurons both in vitro and in vivo. We applied AAV9.hSyn.YFP.PolyA, AAV9.hSyn.YFP‐hIMP2.PolyA and AAV9.hSyn.YFP‐hHCC.PolyA in the primary dorsal root ganglion mouse neuron culture as an in vitro model. We also injected the viral vectors into the crushed mouse sciatic nerve as an in vivo model. Our results demonstrate the moderate neuron‐specific transgene expression of IMP2/HCC in both models. The data indicate that the synapsin promoter‐driven AAV9 can be used as an important tool for further study of the role of IMP2 in the nervous system.

Support or Funding Information

This work is supported by the Department of Bio‐Medical Sciences, PCOM and Health Research Grant, Pennsylvania Department of Health.
(Z)‐Bisdehydrodoisynolic Acid (BDDA), a Non‐Steroidal Uterotrophic and Potentially Neuroprotective Compound in Brain Injury, Induces Proestrus‐like Luteinizing Hormone Secretion and Lordosis Behavior in Ovariectomized Female Rats
Authors: Clough Rich W. , Viscomi Natalie S. , Arbogast Lydia , Hou Yuqing , Banz William J.
Published: November 2023

Exploring therapeutic interventions in experimental Traumatic and Concussive Brain Injury (TBI, CBI) in rodents, we have previously reported that (Z)‐BDDA, a non‐steriodal but estrogenic compound of interest, significantly improved the rate and extent of behavioral improvement in rats subject to lateral fluid percussion brain injury (an experimental model of TBI). Z‐BDDA is a synthetic compound described as highly estrogenic based on uterotrophic activity in female rats. Estrogenicity in brain has not been established. Insofar as the effects of Z‐BDDA on TBI may be due to an “estrogenic” effect in brain, we sought to determine whether the compound does indeed have estrogenic effects on this organ. Accordingly, we explored the induction of Luteinizing Hormone (LH) secretion and lordosis behavior using a standardized priming model in ovariectomized (OVX) female rats. Female Sprague‐Dawley rats were OVXed under isofluorane anesthesia and assigned to various steroid treatment protocols intended to induce (preovulatory‐like) LH secretion sexual receptivity. Z‐BDDA (300 or 30 Ug/rat) or Estradiol Benzoate (EB, 10 ug/rat) was administered (sc) two weeks following OVX and some rats were also given progesterone (1mg/rat) 48 hours later. Lordosis behavior was assessed (at onset of dark‐phase) by placement in an arena containing a sexually mature male rat. Mount attempts by the male and lordotic responses in females were recorded to obtain a lordosis quotient. For endocrine studies, rats were treated as with the behavioral studies, but at onset of dark were euthanized and blood was collected immediately for LH measurement by radioimmunoassay. Our results confirmed a profound estrogenic effect of Z‐BDDA on both LH secretion and lordosis responding in female rats. Compared to control groups, Z‐BDDA was equipotent to EB in this priming model. Estrogenic compounds have shown high promise in the experimental brain injury literature but the use of estrogens per se, in the human context, is controversial. This warrants continued research with other compounds, such as Z‐BDDA, that are estrogenic and also have a variety of other attributes potentially beneficial in brain injury but that avoid the confounds of estrogen.

Support or Funding Information

Supported by SIU School of Medicine and the SIUC Neuroscience Research Center.
Vexin (Vxn) Regulates Neurogenesis and Cell Cycle Exit Downstream of Proneural Factors
Authors: Moore Kathryn , Logan Mary , Al Diri Issam , Steele Michael , Vetter Monica
Published: November 2023

Development of the retina is regulated by sets of proneural basic helix‐loop helix (bHLH) transcription factors that coordinate the generation of multiple cell types in a conserved sequence. These proneural bHLH factors promote the expression of target genes that regulate the timing of differentiation and maintain a balance between proliferation and differentiation. However, how these target genes mediate proneural effects remains incompletely understood. The objective of this study was to identify and characterize novel target genes of proneural bHLH factors. We identified vexin (vxn) as a novel target gene for Xenopus laevis proneural bHLH factors. In the developing Xenopus nervous system, Vxn is expressed by progenitors. Vxn encodes a novel protein with a predicted SH3‐family motif and is conserved across vertebrate species. To assess the function of vxn during retinal neurogenesis in Xenopus, we targeted retinal progenitors and performed RNA overexpression or knockdown using antisense vxn morpholinos (MO). We analyzed cell cycle exit, retinal neuron differentiation and changes in gene expression, and assessed subcellular location of Vxn protein.

Vxn is transiently expressed as retinal progenitors initiate differentiation. Vxn overexpression at neural plate stages inhibited cell proliferation and promoted increased expression of the cyclin‐dependent kinase inhibitor p27Xic1. Both exogenously expressed epitope‐tagged mouse or Xenopus Vxn protein, as well as endogenous Vxn protein, localized to the cell membrane and the nucleus in both the neural plate and retina. We demonstrated that Vxn must localize to the nucleus to promote neural differentiation and cell cycle exit by generating forms of Vxn tagged with either nuclear export or nuclear localization sequences. Overexpression of either Xenopus or mouse vxn in retinal progenitors promoted increased differentiation of early born retinal neurons, and also strongly cooperated with the proneural factor Atoh7 to enhance retinal ganglion cell differentiation. Overexpression of vxn caused reduced retinal progenitor expansion resulting in reduced clone size. Conversely, MO knockdown of vxn inhibited retinal neuron differentiation, preferentially maintaining cells as retinal progenitors and reducing expression of terminal differentiation genes. Thus vxn is expressed in progenitors as they initiate neuronal differentiation, and appears to function in the nucleus to regulate cell cycle exit downstream of proneural bHLH factors during neural development.

Support or Funding Information

Supported by NEI EY012274 to MLV. Thank you to Constance Dooley, Jianmin Zhang, Kaitllyn Phuong Le and Jacklyn Roberts for technical contributions.
Ronin (Thap11) is Implicated in a New Cobalamin Deficiency Syndrome Impacting the Central Nervous System
Authors: Achilleos Annita , Tong Xuefei , Poché Ross A
Published: November 2023

Combined methylmalonic acidemia and homocystinuria (cblC type), also known as hereditary Vitamin B12 deficiency, is a rare congenital metabolic and multi‐systemic disorder that is due to mutations in MMACHC. Babies born with cblC have severe neurodevelopmental defects including microcephaly, hydrocephaly and seizures as well as renal, cardiac and hematological defects. Mutations in MMACHC have been the sole known causative gene until recently when an X‐linked variant of the disease was described, termed cblX. This variant was found to be due to mutations in the transcription cofactor HCFC1, which is known to be an obligatory partner for the transcription factor RONIN (THAP11). Intriguingly, a single patient exhibiting cblX‐like defects has been found to carry a mutation in RONIN. These patients, with both HCFC1 and RONIN mutations, were shown to have a dramatic reduction in MMACHC transcription. Further, we have previously shown that the Hcfc1/Ronin transcriptional complex directly regulates Mmachc expression. These findings suggest that cblX comprises a novel family of rare and severe cblC‐like disorders that are transcriptional in nature. As a result, we have generated a mouse model that carries the human mutation in Ronin (RoninF80L), in an effort to better understand the cellular mechanisms underlying these devastating neurodevelopmental diseases. Here we report the generation of the RoninF80L mouse and its phenotypic and molecular characterization. RoninF80L homozygous mice die soon after birth due to respiratory failure and exhibit severe brain developmental defects including hydrocephalus and thinning of the neocortex. Molecular characterization of the RoninF80L protein in the brain has revealed instability of the protein, which led to a dramatic downregulation of Mmachc expression. In addition, ChIP‐PCR has revealed direct regulation of Mmachc expression by Ronin in the brain, which further supports the transcriptional nature of cblX and a common underlying cause of the disease. Together the phenotypic and molecular data recapitulate aspects of cblC and cblX, which confirms that the RoninF80L mouse model will serve as a powerful tool to further uncover the pathophysiology of this complex disease.
Effects of Dietary Isoflavones, Age, and Stroke on Estrogen Pathway Activation in Male Rats
Authors: Hart Muriel Lynn , Huber Kalene , Grisley Elizabeth Dawn , Cheatwood Joseph L
Published: November 2023

The effects of stroke can cause long lasting impairment on not only one's physical health but their quality of life and everyday function. Dietary modifications provide a potential solution to aid in neuroprotection and allow enhanced functional recovery post‐stroke. Previous investigation tested this, and found the addition of dietary soy compounds does show significantly improved functional recovery through behavioral tests in our rat models. Further studies aimed to identify specific effects on gene expression on the dietary inclusion of isoflavones alone, using Daidzein and Genistein, known neuroprotective estrogenic receptor activators. We not only examined the effect of isoflavone content on altered gene expression, but also age as another potential mediating factor either promoting or inhibiting anti‐inflammatory and neuroprotective pathways. In doing so, both adult and aged male Hooded Long Evan rats were separated into distinct dietary protein groups containing either Sodium Caseinate (CAS), or Sodium Caseinate with the isoflavones daidzein and genistein (CAS+ISO) two weeks before a permanent unilateral middle cerebral artery occlusion (MCAO). Tissue samples were collected from the injured hemisphere on Day +3 (n=6) and Day +0 (n=4; received no MCAO). Data were collected via a qPCR array after RNA extraction and cDNA synthesis, following the manufacturer's protocol guidelines (Qiagen). Expression of mRNA was compared between treatment groups based on age, isoflavone content, and stroke. Changes in mRNA expression greater than 2‐fold were considered significant. Detailed results will be presented on the poster.

Support or Funding Information

NIH NCCAM (1R15AT006593; JLC) and DuPont Nutrition and Health
Morphological comparison of the pudendal canal and its neurovascular elements in human and domestic mammals
Authors: Rodriguez‐Sosa Jose R. , Hall Margaret I , Plochocki Jeffrey H , Ruiz Saul A , Adrian Brent
Published: November 2023

The structural organization of the pelvis and perineum is complex and poorly understood, especially in non‐primate mammals. A relevant feature of these regions is the pudendal canal, which is the passage for the pudendal vessels and nerve at the caudal surface of the pelvic diaphragm (coccygeous and levator ani muscles). These elements are responsible for the blood supply and innervation of the perineum and involve extensive consideration in numerous clinical interventions. The pudendal canal is well described in humans, but not in other species. Therefore, with the aim of studying and comparing the pudendal canal and its neurovascular elements we dissected the pelvic diaphragm and traced the pudendal vessels and nerve in dogs (n=20), goats (n=10), pigs (n=10), horse (n=5), cats (n=3), and humans (n=20). We found that, unlike humans, the pudendal canal in domestic animals is a narrow passage that results from the extensive union of the pelvic diaphragm to the pelvic wall. In large animals, the pelvic diaphragm was a simple muscular layer with a prominent insertion in the external anal sphincter. In small animals, the diaphragm is formed by a group of muscular bundles with greater insertion at the caudal vertebrae. The presence of nerves were variable according to the specific conformation of the sacral plexus. However, the branching pattern of the pudendal vessels and nerve were similar. A notable feature in domestic animals was the presence of the caudal rectal nerve inside the pudendal canal, which is not a feature normally found in humans.
The Women in Science Undergraduate Organization at Otterbein University: Best Practices and Outreach Efforts
Authors: Hyatt Taylor B. , Bailey Hannah M. , Tansey John T.
Published: November 2023

Otterbein University's Women in Science chapter has been restructured to better help its members reach their potential. We have written a formal constitution, organized professional events, and expanded our community outreach efforts. The constitution explains the foundation of our program and our goal of promoting the following:

A greater understanding of the contributions of women in all fields of science.

Providing an open environment for members to share and explore interests and career paths within the sciences.

As an organization, we attended a presentation given Dr Angelika Nelson of Ohio State University. We had the opportunity to speak with her after the presentation, ask her questions about her career as well as ask her advice on our own career paths. She was able to share personal experiences with us and also expand upon those to make them applicable to an undergraduate audience. It is our hope that our members can apply what they have learned to their future experiences. Women in Science also hopes to help engage with the local community. Last year, we participated in Otterthon, a fundraiser that benefits the Children's Miracle Network, and plan to continue this event annually. Members of Women in Science participated in Girl Day at Otterbein University. This event occurs every year but this year's focus was Science, Technology, Engineering, Art, and Mathematics (STEAM). Our representatives spoke with the middle school aged girls and helped with a liquid nitrogen ice cream demonstration. For the coming year, we have planned a science outreach night with a local elementary school. This night will incorporate approximately ten stations that each feature a different interactive activity for the children. We are expecting several hundred people to attend this event. We plan to partner with our ASBMB‐SC chapter to participate in Starry Night, a local interest festival that promotes interdisciplinary learning in STEAM fields. Annually, over 1500 visitors attended the festival. Our hope is that Women in Science will continue to grow in membership and opportunities available for our members.
Use of Eyetracking Technology to Determine Biochemistry Expert‐Novice Differences in Reading Metabolic Pathways
Authors: Cortes Kimberly Linenberger
Published: November 2023

Being able to move between multiple representations is an important skill in biochemistry. Schematics are a type of representation commonly used in biochemistry instruction often combined with chemical structures to form a metabolic pathway. As little work has focused on schematics in biochemistry, this study uses the Tobii X2‐30 Compact Eye Tracker system to record experts and students enrolled in various biochemistry courses as they read various metabolic pathway images. This presentation will focus on differences in areas of interest (AOI), fixation, and scan path analysis.
Mentorship Training for Postdoctoral Researchers: Results from a Four Week Intervention Program
Authors: Pena Christopher George , Evans Teresa , Mustafa Shamim , Moreno Laura , McManus Linda
Published: November 2023

The acquisition of mentoring skills in biomedical research has traditionally been a passive process via apprenticeships. Moreover, mentorship quality directly affects scientific productivity. Consequently, the National Institutes of Health (NIH) has strongly encouraged formal mentorship training for academic researchers including postdoctoral trainees (postdocs). While the acquisition of knowledge and skills in mentoring is encouraged, it is unclear whether postdocs value these skills and how greatly their mentoring ability can be improved by formal training. Furthermore, outcomes assessment following formal mentor training is necessary to ensure effective instruction. The present program and programmatic assessment was designed to address these important questions.

The Offices of Postdoctoral Affairs and Career Development implemented a mentoring intervention program for postdoctoral trainees with the goals to increase awareness of trainee mentoring ability as well as improve trainee mentoring skills. This program, which consisted of four, two hour weekly sessions, included facilitated discussions and case studies centered around key competencies of mentoring: Elements of a mentoring relationship, effective communication, problem solving, and self‐evaluation. Program participants rated the importance and their development of 15 mentoring skills that aligned with these competencies in a pre and post intervention assessment via a Likert scale. Data were compiled across seven cohorts from 2012 to 2016 (n=34).

All postdocs highly valued each of the mentoring skills prior to participation in this intervention program. These views were unchanged after intervention. In contrast to unchanged valuation of mentoring skills upon completion of the program, postdocs reported a substantial positive gain in skill development in all mentoring skills. The greatest skill improvement was ‘setting reasonable goals for a mentee’. The lowest skill improvement was ‘keeping a mentee engaged’.

Postdoctoral research trainees highly value mentoring skills. Of importance, a formal process for mentorship training aids in trainee development of vital mentoring skills. Future studies are needed to address additional variables that may impact the outcomes of formal mentor training among postdoctoral trainees. Among these are trainee demographics including gender, area of research emphasis, duration of postdoctoral research training, trainee citizenship status, and the international location of predoctoral studies. Equally important will be consideration of the sustained impact of short term mentor training.

Support or Funding Information

Supported in part San Antonio Biomedical Education and Research (SABER) K12 GM11726 and Clinical Translational Science Award (CTSA) 1KL2RR025766‐01.
Flipped classroom approaches lead to no improvement in learning outcomes or student perceptions
Authors: Arnott John Albert , Planey Sonia Lobo
Published: November 2023

The active learning approach has been suggested to promote increased learning in science classes when compared to the traditional lecture‐based approach. The flipped classroom approach is one method that has recently gained popularity to implement active learning approaches within the classroom by encouraging student‐centered group learning and problem solving activities. While some studies report that flipped classroom experiences can improve student performance and produce favorable student perceptions, others report that these gains in performance are not consistent and that the positive effects from flipped pedagogy are short lived. In this study we compared learning outcomes and student perceptions between two, randomized, study arms that were exposed to either active learning or lecture style pedagogy before and after they were crossed over between the pedagogy styles. To ensure continuity of experience, demographics and GPA were similar in each group and they received the exact same material with the only difference being the pedagogy employed. Group A received flipped classroom pedagogy, while group B received traditional lecture pedagogy for four consecutive sessions at which time the groups were crossed over. After the cross over group A received traditional lecture pedagogy and group B received the flipped classroom pedagogy for four additional consecutive sessions. At the end of each session a five‐question evaluation quiz was given and no significant difference in quiz performance was observed between the flipped or lecture groups except that group B outperformed group A during the traditional arm of the study. No differences were observed when exam performance was compared. To assess students' attitudes about the flipped vs. lecture pedagogy, a previously validated Motivated Strategies for Learning Questionnaire (MSLQ) was utilized. A pre‐course survey, a midway‐survey and a post‐course survey was given. Findings suggest that while student confidence in their abilities was reduced initially, this recovered toward the end of the study. Taken together, flipped classroom approaches in this study did not have a significant difference on student performance or perception.
Proxies For Success–How Application Changes Correlate To PhD Path Pursuit For a Small Diversity Research Program
Authors: Shadding Cherilynn R. , Whittington Dawayne
Published: November 2023

STEM diversity research programs have shown progress in increasing the number of underrepresented students who pursue STEM at the highest degree levels. While many programs have delineated gains of program participants, fewer programs have outlined the path to their outcomes, namely the STEM Ph.D. Our program, Opportunities in Genomics Research (OGR) through two cycles of NIH funding showed significant increases in Ph.D. matriculation from cycle one to cycle two of funding. We explored typical measures such as GPA, institution classification, and graduate school ranking of participants and compared by cycles and found that these measures did not alone explain the improved outcomes. We examined changes we made to our application materials, where we increased the number of questions serving as proxies for gauging interest in pursuing a Ph.D. Our data show a significant correlation of pursuit of the Ph.D. path to increased proxies and proxy type (open‐ or close‐ended questions answered by applicant or referee). We believe that we are the first to statistically demonstrate that changes in application materials for diversity research programs correlates to improved program outcomes.

Support or Funding Information

2R25HG006687‐05
Design and Implementation of an Experiential Learning Workshop for Upper‐level Undergraduate Science Majors
Authors: Connelly Sandra J. , Johnson Rebecca , Mills Jeffrey L.
Published: November 2023

Historically, science courses designed for majors lack critical thinking, application of concepts beyond the field of study, and fall short of an interdisciplinary approach to learning and investigation. Instructors often struggle with implementing curricular designs for interdisciplinary students, even in light of obvious STEM educational reform efforts nationwide (e.g. “Science for All Americans,” “Benchmarks for Science Literacy,” and “Standards for Technological Literacy: Content for the Study of Technology”). Course redesigns that have centered on problem‐based learning for both majors and non‐majors are typically limited to single discipline classes and have reported moderate success, but are not without their shortcomings as well – specifically engagement of students in topics and the lack of multidisciplinary approaches. Our objective was to design an experiential learning course for biology and chemistry majors that aligns with graduation requirements and addresses some of the aforementioned shortcomings. The primary focus of this implementation was to assure that the course reflected the fundamental needs of science curricula: critical thinking, conceptual application, experiential learning, and interdisciplinary learning and investigation. Students worked in interdisciplinary groups to solve real world problems, including soil pollution remediation, algae as a source of biofuels, antibiotic resistance, and waste stream remediation for a local cidery. We have assessed the learning gains of the students using the “Student Assessment of their Learning Gains” (pre‐ and post‐course), weekly individual progress reviews, oral presentations of research proposals and final results, peer reviews of group work, ePortfolios, and evaluation of the course faculty by an in‐house Instructional Designer. Qualitative assessments of student professional and personal dynamics in the course have also been compiled and will be presented. We propose the development of similar courses in multiple overlapping disciplines to break down the silos of learning and better prepare students for their future endeavors.

Support or Funding Information

The course design and implementation presented is funded by a Provost's Learning Innovations Grant at Rochester Institute of Technology.
Making Connections: Impact of primary literature assignments on lecture and laboratory learning
Authors: Miller Keith R
Published: November 2023

One of the most daunting tasks for undergraduates is reading and writing about primary literature. To improve undergraduate skills for reading, writing, and critiquing primary literature, a curriculum scaffold was introduced in a two semester biochemistry lecture and laboratory setting. This curriculum moved students from using primary literature to address case studies and literature reviews to finally writing NIH‐style grants. Rubrics were developed for all assignments to assist students in understanding where improvement was needed. Rubrics were designed to provide enough detail to be informative but not so much that it was prescriptive. To assist students in engaging with the literature, during the first semester, students were expected to locate a primary peer‐reviewed research article that implemented that week's laboratory technique/lecture concept and write a summary on one of the figures. During the second semester, this assignment expanded to include critiques and presentations covering all the figures in an article. This primary literature curriculum was intended to develop student learning from passive to active engagement by applying their knowledge to reading and critiquing science. The curriculum was assessed using student evaluations, student writing samples, and research faculty evaluation of their students. Conclusions drawn demonstrated that students were better prepared in locating, reading, and assessing primary literature and had improved scientific communication skills.
Assessing Student Competencies Using Rubrics Associated with Figure Legends and Ability to Perform the Bradford Assay in a 400‐level Undergraduate Course on Proteomics
Authors: Shipman Richard , Grant Jennifer
Published: November 2023

The University of Wisconsin Stout is the polytechnic of the UW‐System. Aligned with our core values, Proteomics (BIO‐425) is a 3‐credit lecture and laboratory course that emphasizes the use of hands‐on techniques, particularly microchromatographic and protein digestion strategies. In addition to learning techniques like in‐gel digestion of proteins and protein chemistry techniques, students also developed and tested their own protein purification strategy in a mini‐project format. At the heart of this course, assessments revolve around completion of laboratory reports that follow the Abstract/Introduction/Methods/Results/Discussion (AIMRD) commonly followed by scientific articles.

One focus of our semester‐long course was to instruct students in the performance of the Bradford protein assay at the level of a professional in the field. R2 values for their standard curves, made using bovine serum albumin serial dilutions, were tracked over the semester. Data on student success with the Bradford assay, and particularly measures of student progress over the course of the semester, were evaluated. Time to competence appears to be the most effective measure of student success. On average it required three attempts for students to gain proficiency at the Bradford assay.

A second measure of student success is the time to proficiency in creating graphs within lab reports. Here we report the rubric used to evaluate graphs submitted by students, which measures appropriateness of axis labels, titles, and figure legends. While development of this rubric is ongoing, the present version provides insight into the intellectual development of the students taking this course.

In all, this presentation provides useful measures of student success that focus on data that can be harvested from lab reports submitted by the students. The authors are actively soliciting feedback on statistical measures that can be utilized to quantify learning outcomes in both formative and summative arenas.
Bypass and Misincorporation of DNA Polymerases at DNA‐Peptide Crosslinks
Authors: Sedgeman Carl Andrew , Guengerich F. Peter
Published: November 2023

The DNA of all living organisms is constantly exposed to chemicals that can cause diverse harmful lesions, one of which is DNA‐peptide cross‐links. The formation of DNA‐peptide cross‐links (DPCs) has been shown to occur from the modification to DNA bases by bis‐electrophiles and concomitantly nuclear proteins such as glutathione and O6‐alkylguanine DNA alkyltransferase (AGT). These conjugates have been discovered in cells and can induce both DNA mutations and cell death. Our hypothesis is that some translesion DNA polymerases are capable of replicating past DNA‐peptide cross‐links. A corollary of our central hypothesis is that AGT cross‐linked to DNA causes mutations after being partially degraded by proteases. For the investigation of the bypass and miscoding ability of DPCs by translesion DNA polymerases, DNA replication assays have been performed in duplex DNA substrates containing an N6‐dA‐DPC formed by glutathione and peptides from the active site of AGT (3–15 aa). Preliminary results indicate that human Pols kappa and iota have low processivity past these adducts compared to Pol eta and Sulfolobus solfataricus Dpo4. Bypass by Pol eta yielded higher levels of misincorporation. Steady‐state kinetics similarly show that Pol eta has a greater proclivity for misincorporation compared to Pols kappa and iota.

This work was supported in part by National Institutes of Health grants R01 ES010546 and T32 GM065086.
An evolutionary conserved DNA replication stress response in planarian
Authors: Tirgar Reyhaneh , Shamoon Ushna , Akpati Lois , Sawyer Nathaniel , Nam Edward
Published: November 2023

S. mediterranea is a planarian with extensive regenerative ability which requires substantial cell division. However, DNA replication stress occurs every cell division and must be resolved. From yeast to man, an evolutionary conserved DNA replication stress response (RSR) is essential for life. The RSR is a signal transduction pathway regulated by Ataxia‐telangiectasia and Rad3‐Related kinase (ATR) and Checkpoint 1 kinase (CHK1) to respond to replication stress. However, the RSR has not been established in planarian nor do we know if it functions in regeneration. We hypothesize that the RSR may be essential for regeneration. Preliminary results show reduced regenerative ability in the presence of hydroxyurea (HU), a replication stress inducing agent. However, low concentrations (2mM) did not attenuate regeneration, indicating that the RSR may be present and able to overcome replication stress. To test this, we treated amputated tails with HU, VE‐821 ATR inhibitor (ATRi), and a combination of both. In 2mM HU, tails regenerated similarly to control. We observed only a one day delay in regeneration with ATRi alone. However, ATRi and 2mM HU in combination prevented planarian regeneration completely. This synergistic effect suggests that ATR is significant in the response to replication stress during regeneration. To obtain genetic evidence, we are in the process of knocking down ATR as well as other RSR genes like CHK1, WRN, etc. Further determining the importance of RSR genes in planarian will elucidate the conserved components and allow us to understand how the RSR contributes to the incredible regenerative abilities in planarian.
A Novel Small Molecule Inhibitor of Human DNA Polymerase Eta Modulates the Efficacy of Cisplatin in Cancer Cells
Authors: Zafar Maroof Khan , Maddukuri Leena , Penthala Narsima R , Ketkar Amit , Eddy Sarah , Reed Megan R , Crooks Peter A , Eoff Robert L
Published: November 2023

Chemotherapeutic strategies involving platinum‐based drugs rely on DNA damage to limit tumor growth. Activation of DNA damage response pathways can modulate patient response to these types of therapies. For example, translesion DNA synthesis (TLS) by human DNA polymerase (pol) eta (η) can render platinum‐based chemotherapy ineffective through the direct bypass of DNA damage. Therefore, targeting hpol η with small‐molecule inhibitors is a promising strategy for combating chemoresistance in certain types of cancer, such as ovarian cancer, that rely on hpol η to survive genotoxic chemotherapy. The main objective of our study was to identify small molecule inhibitors of hpol η that modulate the efficacy of genotoxic anti‐cancer drugs in an effort to improve patient outcomes. Towards that end, the catalytic activity of hpol η was measured in the presence of eighty‐five novel indole thio‐barbituric acid (ITBA) and indole barbituric acid (IBA) derivatives. Several compounds that inhibit hpol η activity with low micromolar IC50s were identified from this initial screen. One of these compounds, PNR‐7‐02, an ITBA derivative with both N‐napthoyl moiety and 5‐chloro substituent on the indole ring inhibited hpol η activity with an IC50 value of 8 μM. PNR‐7‐02 also inhibited hRev1 and hpol lambda (λ) with low micromolar IC50, but inhibition by PNR‐7‐02 was approximately ten‐fold more potent against hpol η than B‐family pols. Michaelis‐Menten kinetic analysis revealed a partial competitive mechanism of inhibition of hpol η activity. Chemical labeling and molecular docking results further supported the kinetic mechanism of inhibition. A target‐dependent effect on cell viability was observed when comparing hpol η‐proficient and hpol η‐deficient HAP‐1 cells co‐treated with cisplatin and PNR‐7‐02. The calculated combination index values indicated a strong synergy between cisplatin and PNR‐7‐02 in hpol η‐proficient cells. This synergy was not evident in the hpol η knockout cells, supporting the idea that PNR‐7‐02 modulates cisplatin toxicity in a hpol η‐dependent manner. Results from cell sorting experiments were consistent with synergy observed between cisplatin and PNR‐7‐02. In summary, our lab has performed the first quantitative structure activity relationship for TLS pols to identify potent inhibitor of hpol η that sensitizes cancer cells to cisplatin treatment.

Support or Funding Information

This work was supported by NIH grants GM 084460 and CA 183895 (to R.L.E.).
Inhibition of Kynurenine Signaling Decreases Glioblastoma Multiforme Genomic Instability and Sensitizes Cells to Chemotherapeutic Treatment
Authors: Reed Megan R , Maddukuri Leena , Helm Emily , Bostian April C.L. , Zafar Maroof K , Eoff Robert L
Published: November 2023

Glioblastoma multiforme (GBM) is an aggressive grade IV astrocytoma that is commonly treated with genotoxic compounds. Aberrant activation of the aryl hydrocarbon receptor (AhR) through mis‐regulation of the kynurenine pathway (KP) in GBM cells contributes to the malignant properties of these tumors. Our group determined that AhR activation by the KP led to an increase in chromosomal damage and that this was due, in part, to increased expression of the translesion DNA polymerase hpol κ. Based on these results, we hypothesized that blocking the rate‐limiting step in kynurenine (KYN) production through inhibition of the enzyme tryptophan 2,3‐dioxygenase (TDO) would sensitize GBM cells to genotoxic treatment by reducing the basal levels of proteins and enzymes that facilitate tolerance/repair of DNA damage. GBM cells pre‐treated with the TDO inhibitor 680C91 were found to be more sensitive to the DNA damaging agents cis‐diamminedichloroplatinum (CDDP) and bis‐chloroethylnitrosourea (BCNU). Flow cytometry analysis of GBM cell lines treated with 680C91 in combination with CDDP or BCNU revealed an enrichment in cells with 4N (G2/M) and 1N (sub‐G1) DNA content. These results are consistent with the promotion of cell cycle arrest and death following DNA damage when KYN/AhR signaling is blocked. To study global changes in replication fork dynamics, replication‐associated proteins were enriched using affinity‐mediated isolation of proteins on nascent DNA (AMIPOND). Analysis of the resulting mass spectrometry data indicate that when GBM cells are treated with 680C91 there is a broad depletion of DNA damage response and repair proteins near sites of active DNA synthesis. Immunoblotting was used to support trends observed in the proteomics results. In conclusion, our study provides support for a model in which inhibition of TDO sensitizes GBM‐derived cells to genotoxic drugs through depletion of DNA repair/damage tolerance factors.

Support or Funding Information

Support or Funding Information: Supported by NIH grants CA183895 to RLE and CA140409 to PAC. This research was also supported by a grant from the Arkansas Breast Cancer Research Program, the UAMS CTSA and a Seeds of Science Award from the Winthrop P. Rockefeller Cancer Institute.
The Role of Noncoding Genetic Elements in the Transcriptional Regulation of the Voltage‐Gated Sodium Channels SCN1A and SCN8A
Authors: Inglis George Andrew Stanowski , Escayg Andrew
Published: November 2023

Epilepsy is a common neurological disorder that affects over 50 million people worldwide, and is characterized by recurrent, unprovoked seizures. Altered brain expression of the voltage‐gated sodium channel (VGSC) α subunit gene SCN1A has been shown to directly cause some forms of epilepsy. Heterozygous loss‐of‐function mutations in the VGSC gene SCN1A (encoding the protein Nav1.1) are responsible for 70–80% of cases of Dravet Syndrome (DS), a severe, treatment‐resistant form of childhood epilepsy. A significant decrease in SCN1A mRNA levels is also associated with temporal lobe epilepsy (TLE), the most common form of treatment‐resistant epilepsy in adults. Restoring SCN1A expression through increased gene expression is predicted to be therapeutic in these cases. Conversely, loss‐of‐function or reduced expression in the VGSC gene SCN8A (encoding the protein Nav1.6) has been demonstrated to increase seizure resistance and quality of life in mouse models of DS and TLE. However, despite the important role of the VGSC genes in epilepsy, and the clinical potential of modulating SCN1A and SCN8A expression as a therapy for patients with epilepsy, we have limited knowledge about the genetic elements or factors responsible for the transcriptional regulation of these two genes.

By analyzing several genomic markers of open or actively transcribed chromatin across neuronal cell types, we identified several genetic elements in both SCN1A and SCN8A with putative transcriptional regulatory ability. We have cloned these elements into a vector upstream of a human SCN1A promoter driving luciferase reporter expression. A number of these constructs resulted in altered luciferase activity relative to empty vectors or constructs with just the SCN1A promoter, suggesting the cloned elements have functional significance. Additional analyses, including identification of proteins responsible for transcription, are being conducted on a subset of these elements.

Support or Funding Information

This work was supported through funding from the NIH National Institute of Neurological Disorders and Stroke (NINDS) and the Emory University Genetics & Molecular Biology T32 Training Grant.
Rdl Expression Impacts Circadian Rhythm and Locomotion in Drosophila melanogaster
Authors: MacDonald Stephanie , Rogers Ryan P
Published: November 2023

The genes that modulate circadian rhythm in Drosophila melanogaster have been at the forefront of sleep research in an effort to characterize the impact of circadian rhythm on other biological processes and identify potential overlap in human sleep disorders. In flies, Resistant to Dieldrin (Rdl) encodes a subunit of the GABA receptor, which is upregulated by the circadian oscillator, WAKE. This interaction promotes RDL localization to the plasma membrane to modulate sleep; however, the specific function of RDL and its downstream interactions to facilitate sleep/wake switching have yet to be characterized. Prior research has shown that Rdl can suppress kazachoc (kcc), which in turn suppresses paralytic (para). para is known to control voltage‐gated sodium channels (VGSC's) in the same way that the human gene SCN1A does. This is noteworthy because mutations in SCN1A are linked to epilepsy in humans, and mutations in para result in seizure‐like behavior in Drosophila. The Drosophila Activity Monitor TriKinetics System and a custom‐built light controlling apparatus was employed to characterize locomotion in control and RdlMD‐RR mutant flies. Since circadian rhythm is largely influenced by light and dark patterns, the flies were monitored during 12‐hour light and dark cycles over a period of 5 days to determine any variation between genotypes. Analysis of the data show control flies exhibit consistent sleep and wake patterns with peaks of activity in daylight hours, and activity in darkness is significantly reduced. Conversely, RdlMD‐RR flies show less activity during daylight hours, and near paralysis in darkness. These data suggest that Rdl expression is related to restricting locomotion during sleep and that levels may be modulated by circadian oscillation. Future research will focus on characterizing the effect of Rdl on para expression using RT‐qPCR to elucidate the role of RDL in circadian regulation.

Support or Funding Information

This work was supported by funds from the Department of Sciences at Wentworth Institute of Technology.