bluuebunny/crossref_metadata_2025 huggingface dataset:
Structuring an Undergraduate Anatomy Teaching Experience
Authors: Orczykowski Mary E
Published: June 2021

The Division of Anatomical Sciences at the University of Michigan Medical School offers a team‐taught undergraduate anatomy course (Anatomy 403) to 170–200 students each semester. As a supplement to the lectures, the students review the anatomy in the laboratory through demonstration of our extensive plastinated anatomical specimen collection. Understanding of laboratory based anatomy is assessed through cadaveric image based practical exam questions. In the Winter 2017 semester, we created an additional undergraduate course (Anatomy 510) to give students who successfully completed Anatomy 403 an opportunity to teach future students as teaching assistants (TAs). Feedback from the first two semesters of Anatomy 510 suggested that course expectations and outcomes were unclear. We hypothesized that restructuring the teaching course would have a positive impact on TA satisfaction and student engagement in the laboratory. Under the revised structure, TAs create mini lab lesson proposals, develop these proposals, practice the lesson with peers and faculty, and execute the lesson in the Anatomy 403 laboratory. Further, students participated in an orientation at the beginning of the semester to discuss grading and expectations. Under the restructure, students spent more time reviewing with faculty and had increased faculty support for lesson development and setting up the laboratory. Lastly, to ensure a thorough review of the material, online readiness quizzes covering all laboratory structures were developed for the TAs. We are assessing the efficacy of the restructuring of the course through narrative feedback, anonymous survey data from both TAs and students, course evaluations for Anatomy 403 and 510, and 403 student outcomes on the practical exam. Data collection and analysis is ongoing. Preliminary results reveal that Anatomy 510 course expectations were clearer following the restructure of the course. Additionally, Anatomy 403 students felt that the presence of TAs and their mini‐lessons were helpful for their success in the course. These results will contribute to a better understanding of an effective undergraduate anatomy teaching experience.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of Bcl6 KO in Mouse Cochlear Hair Cells
Authors: Morrill Seth , He David
Published: June 2021

Although inner and outer hair cells (IHCs and OHCs) in the inner ear are both vulnerable to noise and ototoxic drugs, IHCs are less susceptible to those insults. The molecular mechanisms of such differential vulnerability are unknown. Hair cell‐specific transcriptome analysis shows that Bcl6, a known anti‐apoptotic gene, is differentially expressed in IHCs compared to OHC with a 3‐fold difference. We hypothesize that higher expression of Bcl6 in IHCs contributes to relative resilience of IHCs to ototoxic and mechanical assault. Here, we examine the role of Bcl6 on survival and differential vulnerability of IHCs and OHCs using a Bcl6 conditional knockout mouse model generated by mating the B6.Cg‐Tg(Atoh1‐cre)1Bfri/J mouse line with the B6.129S(FVB)‐Bcl6tm1.1Dent/J from Jackson Labs. Exons 7–9 were deleted from the Bcl6 gene. Auditory Brainstem Response (ABR)‐based thresholds were determined using tone pips with frequencies between 4–50 kHz in homozygous, heterozygous, and wild type littermate mice at postnatal ages of 30, 60, 90, and 120 days. Hair cell survival and morphology from the three cochlear locations (representing apical, middle and basal turns of the cochlea) were examined using confocal microscopy. A diminished hearing threshold and loss of hair cells, especially IHCs, would suggest that Bcl6 is necessary for hair cell maintenance and survival. Two lines of work are currently under way: 1) assessing changes in gene expression (transcriptomes) and identification of downstream targets of Bcl6 in hair cells using hair cells isolated and collected from three genotypes, and 2) applying mechanical and ototoxic insult to mice to determine whether loss of function due to Bcl6 deletion would make IHCs equally vulnerable to noise and ototoxic insults.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effect of Thoracic Kyphosis on the Midfacial Skeleton of Adults
Authors: Ceuninck Kristyna , Tran Tri , Engel Adam , Sensakovic William , Starbuck John
Published: June 2021

Kyphosis is a spinal deformity that manifests as forward rounding of the vertebral column, caused by vertebrae wedging of five degrees or more. Morphological integration (MI) refers to the interdependence of two or more phenotypic structures. The MI concept is based on the fact that parts of complex organisms do not vary randomly and instead display degrees of non‐independence that are thought to occur from shared genetic or developmental origins, and/or functional demands. Integrated traits may develop, evolve, and be inherited together. Due to the position of the skull resting atop of the vertebral column, posture may influence skull development and overall craniofacial anatomy and morphology, which can be assessed using an analysis of MI to explore correlation and covariation patterns between the spine and skull. We hypothesized that individuals with kyphosis (n=15) will exhibit statistically significant correlation differences relative to the control group (n=19) for T6 thoracic vertebral and midfacial linear distance measurements. Six anatomical landmarks were identified on the T6 thoracic vertebrae and the midfacial skeleton, and XYZ coordinates were recorded for analysis. A subset of 10 individuals (5 kyphosis, 5 controls) individuals were measured on two occasions to assess reliability and measurement error. A Euclidean Distance Matrix Analysis (EDMA) of MI was carried out on the entire sample by calculating correlation values for paired linear distance measurements (one vertebral and one midfacial) separately for the kyphosis and control samples (n = 225 for each sample). Next, EDMA calculated correlation differences and statistically assessed significance using a non‐parametric bootstrap (1,000 resamples) and confidence interval testing (α ≤ 0.10). Only 35 of the 225 (15.56%) correlation differences were statistically significant. Patterns of variation among these significant correlation differences were explored by examining sample directionality of differences, sign patterns, and strengths. The relevance of these results to clinical anatomy and biological anthropological studies are discussed.

Support or Funding Information

No funding information to disclose.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Comparative Study of Leg Musculature in Humans and Non‐human Primates and its Evolutionary Implications
Authors: Eriksen Amandine , Kenyon Brittany , Sirianni Joyce E.
Published: June 2021

In humans, the lateral compartment of the leg consists of the fibularis longus (FL) and fibularis brevis (FB) muscles, which originate from the lateral shaft of the fibula. These two muscles serve to plantar flex and evert the foot. While FL inserts on the first metatarsal, FB inserts on the fifth metatarsal. Occasionally, a third muscle is present, the fibularis digiti quinti (FDQ), which is described in previous studies as an accessory tendon arising from FB and inserting on variable parts of the fifth digit, which extends the fifth toe. The sparse literature for great apes indicates that FDQ presents similarly to humans as an accessory tendon. In monkeys, however, FDQ presents as a distinct muscle, with its own belly and a long, thin tendon that runs between FL and FB and inserts on the fifth distal phalanx. Previous studies of humans have found that 32–33% have a variant of the FDQ muscle, which typically manifests itself as an accessory tendon traveling from FB to the fifth digit.

In this study, we test the hypothesis that FDQ is present in a third of human legs and compare the variations of FDQ in humans to that of chimpanzees and macaque monkeys. Our sample includes cadavers of humans (n=25), chimpanzees (n=2), and macaque monkeys (n=90). All cadavers (n=117) were surveyed for the presence or absence of the FDQ muscle, noting the origin and insertion if present.

Since the FDQ is observed among different primates, it is viewed as an evolutionary primitive trait that is variably retained among the higher order of primates. While previous human research documents FDQ muscles as arising from the FB tendon, this study provides the first documentation of the FDQ in a single human with its own separate muscle belly and tendon, arising from the fibula and inserting on the fifth digit. Comparatively, this human FDQ is more similar to monkeys than previously documented human FDQ muscles. The variation observed in FDQ among humans and apes may reflect a reduced reliance on function – extension of the fifth toe. Although humans and apes appear to share a morphologically similar FDQ, their locomotion patterns are quite different. While the human foot is less dexterous, a necessary condition for walking bipedally, the ape foot is adept for an arboreal environment, which requires great dexterity for grasping while climbing. This suggests that while the foot of humans and apes has very different functional adaptations, they still share a reduced need to extend the fifth digit, leading to a less primitive condition as compared to monkeys.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sciatic Nerve Anatomical Relationship with the Piriformis Muscle and its Point of Division
Authors: Alimohammadi Majid , Mortazavi Dana , Doroudi Majid
Published: June 2021

The objective of this study is to examine the course of the Sciatic Nerve (SN) and identify anatomical variations 1) between SN and the piriformis muscle (PM), and 2) in SN's point of division into the common peroneal and tibial nerves. The anatomical relationship between SN and PM was classified based on Beaton and Anson's six types of classification, finding 96.3% of the 82 lower limbs followed a typical anatomical relationship (type I), and 3.7% of the lower limbs showed one branch of SN piercing through the PM with the other branch running posterior to PM (type II). No incidence of the other types was found. As for SN's point of division, the mean division distance is expressed ± confidence intervals for all lower limbs (10.54±1.47cm), right (11.52±2.44cm), left (9.57±1.60cm), male (10.27±2.17cm), and female lower limbs (10.89±3.86cm). The median division distance is calculated with an interquartile range (lower limit, upper limit) as 8.30cm (6.15,13.10) for all lower limbs, 8.55cm (6.30,15.17) for right and 8.20cm (6.10,10.91) for left lower limbs, 9.10cm (6.56,13.10) for male and 7.60cm (5.75,12.42) for female lower limbs. A significant difference was not found in SN's mean division distance between right vs. left lower limbs (p‐value = 0.19) and male vs. female (p‐value = 0.68).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Variation in Anatomic Textbook Illustrations of the Rami Communicantes and the Reality Upon Dissection
Authors: Titunick Michelle B. , Pagano Anthony S. , Metzger Keith A.
Published: June 2021

There is some variation among anatomy textbooks regarding the placement of the rami communicantes. While most authors (Morton, Foreman, and Albertine 2011; Moore, Agur, and Dalley 2018; Detton and Tank 2017; Standring 2016) describe the rami communicantes as connections between the ventral ramus and the sympathetic trunk, accompanying diagrams often do not illustrate this description. Review of textbook illustration and dissection of cadaveric specimens was conducted to determine the precise connections of the rami communicantes. Locations and connections of the rami communicantes, dorsal and ventral rami, and mixed spinal nerve were dissected and documented in eight cadavers. Photographs were taken using a Nikon COOLPIX L820 camera. In all eight cadavers dissected, both rami communicantes were connected to the ventral ramus.

These anatomical findings are consistent with what has been presented in many illustrations (Agur and Dalley 2017; Standring 2016; Drake, Vogl, and Mitchell 2015; Drake 2015; Moore, Agur, and Dalley 2018; Schünke et al. 2016). While the anatomical findings generally agree with textbook illustrations, some textbooks depict the gray rami communicantes connecting to the junction at which the ventral and dorsal rami branch (Netter et al. 2019; Agur and Dalley 2017; Drake 2015; Sobotta et al. 2013), depict variation within a textbook (Agur and Dalley 2017; Drake 2015), or variation within different parts of the same image (Drake 2015). This review suggests that textbook illustrations need to be more consistent and may need to be changed to better represent the reality of anatomical relationships.

Support or Funding Information

No external funding source.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mandibular Nerve and Maxillary Artery Anatomical Variations: Implications for Dentistry, Maxillofacial and Oral Surgery
Authors: Petrone Ashley Brooke , Shaffer Harry G , Zdilla Matthew J
Published: June 2021

BACKGROUND

The posterior division mandibular nerve (V3) gives rise to several branches in the infratemporal region, including the auriculotemporal (ATN), lingual (LN), and inferior alveolar (IAN) nerve. The IAN block is the most commonly performed local anesthetic technique used in dentistry, and atypical morphology of the IAN could lead to block failure or complications. Further, all of the V3 branches are located in close proximity to the maxillary artery, the main blood supply to the maxillofacial region, thus need to be considered and avoided during surgery.

OBJECTIVE

Here we present a case series derived from examination of n=12 cadavers as part of routine dissection in a gross anatomy course. We examined the infratemporal fossa of each cadaver for V3 and maxillary artery anatomical variations.

RESULTS

Variations in either V3 or the maxillary artery were clearly visible in 3 out of 12 cadavers. The potential clinical significance of two selected variations is summarized here:

Variant 1: Just inferior to the origin of the IAN and LN from V3, we observed IAN fenestration around the second part of the maxillary artery. This variant is clinically relevant from multiple standpoints, as the position of the artery is unexpected, and therefore may be unintentionally compromised.

Variant 2: Just inferior to the origin of the IAN and LN from V3, we observed an anomalous communicating branch between the IAN and LN. While we cannot ascertain the functional significance or origin of this branch, this coalescence of nerve fibers may limit the ability to “target” the nerves individually. For example, IAN block may also affect LN fibers, leading to alterations in taste and tongue sensation.

CONCLUSION

Variations in the branching of V3 or maxillary artery were clearly visible in 3/12 cadavers. As such, variations were found in 25 percent of specimens; however, several of the specimens were damaged during dissection, thus the true incidence of variations could likely be higher. Further, these variations may be more common in patients than expected and need to be carefully considered in surgical procedures. Lastly, several variations described here have been previously described in the literature, thus the incidence of these specific variants may be much higher than expected.

Support or Funding Information

N/A

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anatomic variant of the gracilis muscle: a case report
Authors: Kirera Francis , Wallace Doyle , Chalk‐Wilayto Janine
Published: June 2021

Gracilis is a medial thigh muscle that typically exhibits little anatomic variation. The muscle attaches proximally to the inferior pubic ramus. Unlike other adductors of the thigh, it inserts distally at the anteromedial aspect of the proximal tibia. The muscle and tendon are commonly used for reconstructive surgical procedures because of its ease of access as well as its consistent and abundant blood supply. Here we report a case of a unique unilateral anatomical variation in the gracilis muscle from an 84‐year‐old male cadaver. Variation in the distal attachment of the gracilis muscle was observed on the right side during routine dissection of the medial and posterior thigh compartments. Subsequent dissection determined innervation and muscular attachments. The skin and fascia lata were dissected to expose the medial thigh muscles, including the gracilis muscle. The following aspects were evaluated: (a) the origin and insertion of the muscle, (b) the measurements for the lengths and weights for the gracilis, semimembranosus, and semitendinosus muscles from both limbs, and c) the distance of the arterial pedicles from origin at the ischiopubic ramus for the gracilis muscles. Results showed the variant gracilis muscle inserted into the distal aspect of the semimembranosus and semitendinosus muscle bellies. As a result, the pes anserinus only contained tendons from the semitendinosus and sartorius muscles. The variant muscle was shorter and weighed less than the contralateral gracilis muscle. Two arterial pedicles inserted into the variant gracilis muscle belly as opposed to four on the contralateral side, suggesting reduced blood supply to the variant muscle. This report describes the details of the variation and its clinical significance in reconstructive surgery.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Relationships between the Cross‐sectional area of the Quadriceps Musculature and the Cross‐sectional area of the Femoral Diaphysis in Post‐menopausal Females: Balanced Muscle with Unbalanced Bone
Authors: Bolon Dalton S , French Evan D , Zdilla Matthew J
Published: June 2021

The structural integrity of the femur is of particular importance for post‐menopausal women and therefore warrants study. A history of force transmitted through the femur from, for example, weight bearing activity is associated with increased femoral bone mass. However, little study has assessed the relationships between the musculature of the thigh and the structure of the bone. Therefore, this study assessed the bilateral cross‐sectional areas of quadriceps muscles, the mid‐diaphyseal marrow cavity, and the mid‐diaphyseal cortical bone via axial MRI in post‐menopausal women (Mean age = 63 years). Paired t‐test revealed no significant difference in vastus medialis, vastus intermedius, vastus lateralis, and rectus femoris cross‐sectional areas between thighs (p=0.06; p=0.62; p=0.12; p=0.06, respectively). Also, a paired t‐test did not reveal significance between the cross sectional area of the medullary cavity between sides (p=0.91). However, the outer diameter of the diaphysis was significantly different between sides (p=0.02). Accordingly, there was significant different in the cross‐sectional area of the cortical bone between sides (p=0.02). The right‐sided cortical bone cross‐sectional area was 393.2±87.9mm2 (Mean±SD) whereas the left‐sided cortical bone cross‐sectional area was 374.4±83.4mm2. The results of this study suggest that factors outside of musculature play an important role in cortical bone mass. Cortical bone, but not the marrow cavity, differs between femurs. Though speculative, the difference in bone mass is likely due to uneven loads between legs, suggesting a general favoring of load to the right side.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Distance Discrepancies of Vater, Santorini and Pylorus Relationships
Authors: Bee Mary Tracy , Fouad Michael , Zakhary Paul , Skinner Alexandra
Published: June 2021

Surgical exploration of the duodenum requires injection into the major duodenal papilla or observation of nearby anatomical structures. As a result, a solid understanding of the distances of the major duodenal papilla of Vater to both the pylorus and the minor duodenal papilla, or exit of the duct of Santorini, is essential when examining in this region. In six‐year study of 55 cadavers, distances between these landmarks were quantified. The expected 8 cm distance from the pylorus to the major duodenal papilla was in agreement of our multi‐year observations. However, the 2 cm distance from the major to the minor duodenal papilla was significantly shorter than what has been previously recorded in most textbooks and journals (p<0.05). This study provides insight into the accurate distances of the duodenal structures.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bilateral Multiple Renal Vessels Associated with Pyelo‐ureteral Position Anomalies. Case Report
Authors: Daescu Ecaterina , Enache Alexandra , Sztika Dorina , Zahoi Delia Elena
Published: June 2021

During the routine dissection of a 67‐year‐old male within our university's Department of Anatomy, we identified multiple renal vascular and ductal anomalies.

The left kidney was irrigated by three arteries, two with a hilar penetration and one inferior polar artery. All three of these arteries originated from the aorta – two on the anterior side, at the L1 and L3 levels respectively, and the inferior one on the posterior face of the aorta, at the same level as the median sacral artery (L5). All three arteries had accompanying veins. The upper‐most artery was accompanied by the main renal vein, the middle artery – by one of the renal vein's tributaries, and the inferior one by two veins, which joined and opened into the left common iliac vein.

The right kidney had two renal arteries, both of aortic origin – the superior one at the L1 level, 0.5cm below the superior mesenteric artery, and the inferior one on the anterior side of the aorta, at the L5 level and at the point where the aorta split into the two common iliac arteries. The superior artery entered the kidney through the sinus and was accompanied by the right renal artery. The inferior artery entered the kidney through the inferior pole, accompanied by a renal vein that drained into the left common iliac vein together with the left inferior polar renal vein.

In both kidneys, the renal pelvis was anterior of the vessels, with the ureters having a descending path on the anterior side of the kidneys.

Familiarity with such variations in renal vascular and ductal elements is critical for surgical interventions such as for total or partial nephrectomies, aortic aneurysms or renal artery reconstruction for stenosis.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Variations of the Inferior Vena Cava and Renal Veins
Authors: Klinkhachorn Penprapa , Umstot Samuel , Ritz Brianna , Zdilla Matthew
Published: June 2021

Inferior vena cava (IVC) variations signify predisposition to deep vein thrombosis and may complicate retroperitoneal surgeries including abdominal aortic surgery and nephrectomy. Failure to recognize such variations may predispose the patient to life‐threatening complications. Despite their clinical significance, few studies have detailed variations from direct observation. This study assessed 36 cadavers for variations of the IVC and left renal vein. Prevalence was determined for the following variations: double IVC, isolated left IVC, retroaortic left renal vein, and circumaortic renal vein. Two of the 36 cadavers (5.6%) were variant. The variations included a double IVC (1:36; 2.8%) and a retroaortic left renal vein (1:35; 2.9%). There were no occurrences of an isolated left IVC or a circumaortic renal vein (0:36; 0% and 0:35; 0%, respectively). Further, the double IVC and the retroaortic left renal vein were accompanied by regional pathologies—both having kidney pathology and one with an abdominal aortic aneurysm. The results of this study suggest that, IVC variations are rare and, when an IVC variation is discovered, there is typically an attending urinary system pathology.

Support or Funding Information

West Virginia University, West Liberty University, NASA West Virginia Space Grant Consortium and West Virginia Research Challenge Fund

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Implementation of a CRISPR/Cas9 course‐based undergraduate research experience (CURE) in the biochemistry curriculum at a small private college
Authors: Maki Jennifer A. , Via Zachary W. , Cavallin Jenna E. , Ankley Gerald T. , Villeneuve Daniel L.
Published: June 2021

In this course‐based undergraduate research experience (CURE), thirty students enroll in biochemistry per year and participate in a novel CRISPR/Cas9 project in collaboration with the Environmental Protection Agency (EPA) research labs in Duluth, MN. Genes of interest in fathead minnows and zebrafish are mutated to assess the validity of adverse outcome pathways based on the existing toxicological data. This work provides all biochemistry and biology majors at the College of Saint Scholastica (CSS) with an opportunity for project ownership and the consequent growth of scientific identity.

During the first semester biochemistry course, Biochemistry I, the focus is on enhancing students' understanding of the relationship between protein structure and function. Through a series of assignments, they gain experience using uniprot.org, Clustal Omega, BLAST, and PubMed. Students propose a region of their assigned fathead gene for CRISPR/Cas9 targeting and supply evidence supporting their choice. After discussing their proposals in small groups with others assigned to the same target, they prepare individual reports summarizing their findings and plan for mutation.

In Biochemistry II, students apply the data collected during the first semester to design CRISPR/Cas9 guide strands. Online bioinformatic tools are used for this purpose, and in fathead minnows, guide strands are vetted against the zebrafish genome to verify a lack of off‐target hits since the fathead genome assembly is incomplete. Students also tour the EPA research labs, meeting the scientists on the collaborative research team and learning about their work. Fish samples from previous CRISPR/Cas9 experiments are brought back to campus and students screen for mutants by extracting DNA, amplifying via PCR, and submitting the samples for sequencing. The class analyzes the sequence data using TIDE: Tracking of Indels by Decomposition (tide.nki.nl), an algorithm that delivers mutation information by position and percentage. Results from the yearlong CURE are summarized in a formal lab report and poster presentation, affording an opportunity to synthesize and reflect on the data generated throughout the yearlong experience. Implementation of this program at CSS has allowed for a significant increase in research participation by biochemistry and biology majors.

Support or Funding Information

U.S. Environmental Protection Agency, Office of Research and Development, Mid‐Continent Ecology Division, Duluth, MN The College of St. Scholastica, Faculty Development Committee Research Grant.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mutagenesis of de novo genes via CRISPR‐Cas9 in Drosophila Melanogaster
Authors: Nicosia Julia , Reinhardt Josephine
Published: June 2021

The origin of new genes helps explain how organisms have evolved to become increasingly complex over time. New genes can arise in several ways including duplication, gene fusion, horizontal gene transfer, and de novo. De novo genes arose from previously non‐coding DNA, meaning the gene began coding for a protein only recently in evolutionary history. Many de novo genes in Drosophila are expressed mainly in the testis, and surprisingly, previous work using RNA interference showed these genes may be essential to both male fertility and viability. However, we do not know whether these genes function as proteins, like most genes, or as functional RNAs. We are using CRISPR‐Cas9, a gene modification tool, to create null mutations in Drosophila melanogaster's de novo genes to answer this question. We designed CRISPR‐Cas9 targets to induce a small frame‐shifting deletion and also to delete a large section of the gene. The frame‐shifting mutation will presumably impact the protein function, but not the functional RNA. Meanwhile, the deletion will affect the function of the RNA and protein. We have created CRISPR‐Cas9 targeting constructs for multiple de novo genes, and are in the process of injecting these into Cas‐9 transgenic stocks and genotyping to detect expected mutations.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The maintenance of HM silencing is regulated by arginine residues of histone H4 tail in S. cerevisiae
Authors: Yeom Soojin , Lee Jung‐Shin
Published: June 2021

Transcriptional gene repression is one of the significant concepts for the epigenetic gene expression. Histone modifications are critical factors for the maintenance of gene silencing in eukaryotic systems. However, none of the histone modifications as epigenetic silencing markers is conserved in Saccharomyces cerevisiae. To identify global epigenetic silencing marker from yeast to human, we developed a screening method using the system for the maintenance of yeast mating type. We used the yeast histone point mutant library, which is a collection of strains containing alanine‐substituted histone residue, and yeast single‐gene knockout library. From this screening, we found histone arginine residue is required for the maintenance of HM silencing in S. cerevisiae. Among these histone residues, two basic residues of histone H4 tail including H4R17 and H4R19, are important for the HM silencing. These basic amino acids are also known to control the telomeric silencing by recruiting Dot1, which is a specific methyltransferase for histone H3 lysine 79. We found that these two amino acids can maintain HM silencing with their positive charge, but their regulation for the HM silencing is not mediated by Dot1.

Support or Funding Information

This research is supported by NRF‐2015R1D1A1A02061743 and NRF‐2015R1A4A1041105.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Changes in the Circular RNA Transcriptome During Inorganic Arsenic‐Induced Epithelial‐to‐Mesenchymal Transition
Authors: Eleazer Rebekah , Andreeva Kalina , Fondufe‐Mittendorf Yvonne
Published: June 2021

Inorganic arsenic (iAs) found in the environment is associated with diseases such as cancer. Chronic, low dose exposure to iAs induces cellular transformation and epithelial‐to‐mesenchymal transition (EMT), a hallmark of metastasis. Since iAs is not genotoxic, we hypothesize that iAs drives carcinogenesis by altering gene expression though epigenetic mechanisms. One class of epigenetic gene regulators recently discovered are circular RNAs (circRNAs), which are covalently closed and highly stable isoforms derived from precursor messenger RNAs (mRNAs). They are generated from actively transcribed genes and their biogenesis competes with that of mRNA. circRNAs are highly conserved across eukaryotes however the function of this new class of regulators is still unclear. Our lab and others have shown alterations to the epigenome as driving iAs‐mediated carcinogenesis. However the relevance of the expression of circRNAs in this process is not understood. Interestingly, our preliminary data suggest that circRNAs are differentially expressed during this process. We therefore aim to identify and profile the changes in circRNA expression that occur during iAs‐induced EMT. We have treated BEAS‐2B cells with iAs over a time‐course, measuring changes in EMT‐markers over time. We already have shown that cells move from pre‐EMT to mid EMT to late‐EMT and finally back through MET during iAs‐mediated carcinogenesis. This model was used to profile differential expression of circRNAs. To accomplish this, total RNA was isolated and enriched for circRNAs with an RNase R digest. For gene‐specific analyses, divergent primers designed to amplify across the circRNA backsplice junctions were used in reverse transcription quantitative polymerase chain reaction to identify differentially expressed circRNA transcripts. In our preliminary gene‐specific approach, we have identified a circRNA containing the 3′ untranslated region of neural cell adhesion molecule 2 (NCAM2) in BEAS‐2B cells. NCAM2 is a cell‐to‐cell adhesion molecule involved in neuronal growth and migration, and is known to be upregulated during EMT. We observed that during iAs‐mediated EMT, while the expression of the linear NCAM2 transcript increased, there was a concurrent decrease in the expression of the 3′ untranslated region. These results indicate that circRNA expression is altered during iAs‐induced EMT and suggests that iAs promotes carcinogenesis by altering steady state competition between linear and circular RNA production. We are currently using next generation sequencing to fully profile the circRNA transcriptome as cells undergo iAs‐induced EMT to gain a broader understanding of the functional significance of circRNA in iAs‐induced EMT.

Support or Funding Information

This work was supported by NIEHS grant (R01‐ES024478).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of cell‐type restricted transcriptional networks in human photoreceptor neurons
Authors: Schumacker Scott , Enke Ray A
Published: June 2021

The retina is a layered neuronal tissue lining the posterior portion of the eye that converts photons of light into visual images in the brain. Rod and cone photoreceptors are highly specialized light sensitive neurons that initiate this process of phototransduction. Though they are similar cell types, rods and cones have distinct functionalities, synaptic connections, and are affected by different blinding disease alleles. In order to characterize cell‐type specific transcriptional networks in human photoreceptors, rod and cone‐enriched samples were biopsied from the eyes of 7 post‐mortem human donors. Total RNAs were extracted from retinal samples using a Qiagen AllPrep Mini Kit. High quality RNAs were prepared into cDNA libraries representing cellular mRNAs and sequenced using an Illumina NextSeq 500 sequencer. Sequencing reads were assessed, trimmed and visualized using FastQC, Trimmomatic, and MultiQC softwares respectively. High quality reads were then aligned to the human hg38 reference transcriptome using the high speed pseudoaligner Kallisto. The sleuth statistical model and RStudio package was used to quantify read alignments and calculate differentially expressed transcripts (DETs) between rod and cone‐enriched sample groups. These analyses identified 10,875 DETs between sample groups with a FDR adjusted p‐value ≤0.05. Of these DETs, 4,365 are transcribed at higher levels in rod‐enriched retinal tissue while 6,519 demonstrate increased transcription in the cone‐enriched retina. We are currently testing the hypothesis that differential patterns of DNA methylation contribute to cell‐type restricted transcription in photoreceptor neurons. These data will contribute to a better understanding of epigenetic regulation of gene expression networks in the human retina.

Support or Funding Information

This research is supported by a JMU 4‐VA Collaborative Research Grant, Burroughs Wellcome Fund Grant #1017506, and NIH grant #1 R15 EY028725‐01A1 awarded to RAE.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High‐throughput analysis of CRISPR RNA seed sequence identity
Authors: Phan Phong T , Sashital Dipali
Published: June 2021

The type I‐E CRISPR–Cas (clustered regularly interspaced short palindromic repeat–CRISPR associated) system allows bacteria to fight off bacteriophage and other invader threats. During CRISPR interference, CRISPR‐derived RNAs (crRNAs) guide Cas effector proteins to base pair with complementary sequences in the foreign dsDNA, which leads to the cleavage of the DNA target. Target binding occurs directionally from the 5′‐end of the crRNA guide, and the first several base pairs formed between the crRNA and DNA are important to ensure binding. Mutation within this “seed” sequence often reduces the level of CRISPR interference and promotes an alternative “priming” mechanism. During priming, segments of the foreign DNA are inserted into the CRISPR array to create templates for new crRNAs. Our previous results have shown that for some crRNA sequences, mutations within the seed region can be tolerated. Here, we systematically tested the effects of the crRNA seed sequences on CRISPR interference and priming activities. Using partially randomized seed libraries, we identified the position and nucleotide‐specific effects on the mismatch tolerance for four Escherichia coli strains expressing crRNAs with different seed sequences. We show that G/C‐rich seed sequences are more tolerant of mutations than A/U‐rich seed sequences. We observe that guanine mismatches cause the strongest CRISPR immunity defects for all seed sequences tested. G/C‐rich crRNAs may promote CRISPR immunity against mutated sequences at higher rates due to the lower probability of encountering guanine mismatches. Our results indicate that crRNA sequences with higher G/C content may provide stronger immunity than A/U‐rich crRNAs.

Support or Funding Information

R01 GM115874

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Genetic Delivery of RNA Therapeutics to Alter the Expression of Oncogenic Transcripts in Glioblastoma
Authors: Hicks Martin J
Published: June 2021

Glioblastoma multiforme (GBM) is an incurable and aggressive type of brain tumor. It is the most common central nervous system malignancy with a median survival of only 14 months. It is characterized by increased activation of one or more tyrosine kinase receptors, particularly epidermal growth factor receptor (EGFR). This receptor is dysregulated in about 60% of GBM tumors. EGFR amplification, over‐expression and constitutive activation leads to uncontrolled growth and proliferation of GBM. Although a great deal is known about the aberrant biology exhibited by EGFR‐activated GBM, the application of therapies against the biologic processes is limited by the blood‐brain barrier, which restricts systemically administered therapies from reaching the brain. Although anti‐sense RNAs and small interfering RNAs can be used to target and silence gene expression, exogenously expressed RNAs are susceptible to extracellular nucleases as well as activation of cellular immunity against foreign nucleic acids. To bypass these degradatory mechanisms, we take advantage of a natural noncoding RNA gene architecture and the miRNA expression pathway along with an anti‐sense targeted approach to design and deliver DNA that encodes RNA therapeutics that alter EGFR expression. In addition, we make use of a polycistronic delivery system to express RNAs targeting splicing and alternative poly‐A signal/G‐rich elements of the EGFR transcript. DNA delivery vectors encoding RNA therapeutics were transfected into human GBM cell lines. Analysis of RNA transcripts demonstrates changes in expression of the EGFR target. Results show that our vectors were expressed at high levels with subsequent reduction in full‐length EGFR mRNA expression and concomitant activation of alternative therapeutic isoforms. Current strategies include using the polycistronic delivery mechanism to target additional oncogenic transcripts and adapting to a mouse model of GBM.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Saccharomyces cerevisiae as a viable model for the overexpression and purification of proteins involved in iron homeostasis
Authors: Hati Debolina
Published: June 2021

Iron is indispensable for the eukaryotic cell but excess of iron is toxic. Disruptions in iron metabolism leads to numerous human diseases, hence the study of iron regulation and metabolism is of high importance. Saccharomyces cerevisiae is used as a model system in our lab to study iron trafficking and homeostasis pathways to better understand iron regulation at the cellular and molecular level. In yeast, two paralogous transcriptional activators, Aft1 and Aft2, play a central role in iron regulation by activating the transcription of target genes in response to iron deprivation. Monothiol glutaredoxins Grx3 and Grx4 that utilize glutathione to bind [2Fe‐2S] clusters interact specifically with Aft1 and Aft2, transferring an Fe‐S cluster to Aft1/2 that inhibits its DNA binding activity. Aft1 is considered the primary regulator of iron homeostasis because aft1Δ mutants exhibit a stronger iron deficiency phenotype than aft2Δ mutants. Aft1 and Aft2 share 39% sequence homology in their N‐terminal DNA binding domains. The only available structure is of a truncated version of Aft2 that contains this homologous portion and includes the domains responsible for DNA binding and iron sensing. Aft1/2 have been shown to be very unstable and difficult to overexpress and purify in Escherichia coli. Hence we optimized the growth conditions to overexpress Aft1 in its native organism, Saccharomyces cerevisiae, and further try to purify Aft1 from the same. Further studies will be carried out in order to investigate the in‐vitro interaction between full length Aft1/2 and Grx3/4‐Fra2 to better understand the mechanism of Aft1/2 inhibition at the molecular level.

Support or Funding Information

Supported by Dr. Caryn E. Outten's Research Funding

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Closing the GAP Between IQGAP1 and its Yeast Homologue
Authors: Levitus Ricki Pearl , Hoeprich Gregory , Goode Bruce
Published: June 2021

IQGAP1 is actin binding protein localized to the leading edge of mammalian cells and plays an important role in cell migration. This process is common to many healthy cells including epithelial cells and neurons, but abnormal cell migration is a key contributor to metastasis of cancerous cells. Understanding the molecular basis of actin regulation during cell migration has the potential to shed light on how these cancerous abnormalities arise. Previous studies have shown increasing actin density at the leading edge when IQGAP1 is knocked down. This phenotype is consistent with knocking down Capping Protein, which blocks actin assembly. We hypothesize IQGAP1 similarly blocks actin assembly. To test this, we investigated the functions of mammalian IQGAP1 on actin assembly in vitro. Using total internal reflection fluorescence (TIRF) microscopy and bulk fluorescence assays we measured actin assembly in the presence of IQGAP1 to assess its effects. We show for the first time IQGAP1 attenuating actin elongation on single actin filaments. Our results are also consistent with the current working hypothesis that mammalian IQGAP1 attenuates actin elongation rates through a capping mechanism. Further, we provide evidence that mammalian IQGAP1 bundles actin filaments in vitro, validating previous results. Lastly, Iqg1 was identified in the yeast model organismS. Cerevisiae with sequence homology to mammalian IQGAP1. Yeast are an ideal model organism for answering fundamental questions about the function of IQGAP1 in vivo as this project expands, but in vitro functional studies are required first to show similarity between yeast Iqg1 and mammalian IQGAP1. Moving forward, we plan to repeat the in vitro experiments above with the yeast homologue to confirm that the functions of mammalian IQGAP1 are evolutionarily conserved across species.

Support or Funding Information

This research was conducted as a part of the Brandeis MRSEC SMURF Fellowship (Brandeis NSF MRSEC, DMR‐1420382).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using Site‐directed Mutagenesis alongside a Collagen binding assay to reveal the role of Polycystic Kidney Disease domain in ColH Collagenase
Authors: Caviness Perry , Mima Takehiko , Matsushita Osamu , Sakon Joshua
Published: June 2021

Hathewaya histolyticum, one of the causative agents of Gas Gangrene and previously known as Clostridium histolyticum, secretes two collagen dismantling enzymes ColG and ColH as part of its pathology. Both ColG and ColH have segmental structure consisting of a catalytic domain, polycystic kidney disease domains (single in ColG; double in ColH) and collagen binding domains (double in ColG; single in ColH). Their ability to seek out and bind to loosely wound regions of collagen make the non‐catalytic portions of ColG and ColH promising drug delivery tools. Crystal structure of dual CBDs found in ColG suggest that ColG may wedge between two collagen molecules to aid in fibril rearrangement and collagen hydrolysis while SAXS derived envelope for ColH PKD‐CBD:[GPRG(POG)12]3 suggest that ColH interacts with only collagen molecule. This may suggest that the two work together to synergistically degrade collagen. SAXS envelopes of ColH PKD‐CBD:[GPRG(POG)12]3 show that the PKD domain makes contact with the collagenous peptide. ColH PKD domains 1 and 2 contain regions that are thought to aid in collagen binding for ColH. Collagen binding assay for ColH PKD‐CBD segment bound to type 1 collagen fibrils in a biphasic manner, with one having a higher affinity (KD = 3.39 × 10−7 M) and the other having a low affinity (KD = 2.11 × 10−6 M). The PKD‐CBD segment of ColH binds to collagen much tighter than just the CBD (KD = 1.59 × 10−5). Aromatic residues (Tyr780, His782, Tyr796 and Tyr801) located on the potential collagen binding cleft of the PKD domain in Col H PKD‐CBD were mutated to Ser using site directed mutagenesis. In addition an expected tighter binding ColH PKD‐CBD mutant was developed by mutating two non‐aromatic residues (Val778 and Lys785) on the potential collagen binding cleft to Tyr. Val778 and Lys785 were chosen based on a sequence alignment study. Collagen binding assay of the ColH PKD‐CBD four Ser mutant shows a loss of one of the phases of binding and has a KD consistent with the lower affinity phase of the wild type ColH PKD‐CBD (KD = 1.87 × 10−6). Collagen binding assay of the tighter binding ColH PKD‐CBD shows a biphasic plot with the second phase binding much tighter than the wild type (KD = 5 × 10−8). Fusion proteins containing binding segments from ColG and ColH have been used to anchor bFGF and PTH antagonist at their receptors to stimulate healing of bone fractures, enhance bone allografts for spinal fusion and reverse alopecia. Our study would reveal knowledge on how ColH binds to collagen which would help the development of fusion proteins containing ColH binding segments with a higher efficacy.

Support or Funding Information

National Institutes of Health. Grant Number: 8P30GM103450

Arkansas Biosciences Institute

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.