bluuebunny/crossref_metadata_2025 huggingface dataset:
Cryo‐EM Structure of the P Element Transposase Strand Transfer Complex
Authors: Kellogg Elizabeth H. , Ghanim George , Nogales Eva , Rio Donald C.
Published: June 2021

The P element transposon encodes a cut‐and‐paste DNA transposase of the DDE class that has been broadly useful as a tool in Drosophila genetics research. Its mechanism and regulation have been extensively studied in relation to the phenomena of hybrid dysgenesis observed in Drosophila populations, as well as with biochemical in vitro studies. Among the many distinct features of the P element transposase (TNP) are the generation of unusually long (17nt) staggered cuts upon excision from its host genome and the unique requirement for the nucleotide cofactor guanosine triphosphate (GTP) to carry out transposition.

Using single‐particle cryo‐EM, we have obtained an atomic model of the P element transposase strand transfer complex (STC), consisting of a dimeric complex of the P element transposase along with two P element ends covalently linked to the target DNA. Severe preferential orientation was circumvented by collecting data on a tilted (40°) stage. The resulting cryo‐EM reconstruction refines to 3.6 Å resolution, and the details of the map are sufficient to interpret atomic details of the nucleic acid substrates and to localize GTP.

A helix‐turn‐helix domain of each monomer in the symmetric complex binds one P element end while catalyzing strand transfer on the other end, ensuring fidelity of complex formation. The RNaseH catalytic domain shares common features with other DDE transposases (so named for the catalytic triad) and the catalytic residues are identifiable. The RNaseH domain is interrupted by a non‐canonical GTP‐binding insertion domain which exhibits structural similarities to the insertion domain of other transposases, and in our structure makes contacts with target DNA. Surprisingly, we observe that single‐stranded DNA (the 17nt single‐stranded portion of the terminal inverted repeats) adopts an unusual path in our structure which appears to be stabilized by numerous protein‐DNA contacts but ultimately returns to form distorted A‐form DNA duplex leading into the active site. GTP is located close to the active site and interacts with the terminal guanosine residue of the P element DNA. We postulate that this serves to position the transposon donor in the active site for attack on the target DNA. Ultimately, the structure of the P element strand transfer complex reveals the molecular basis underlying two of the unusual properties of the P element transposition mechanism.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Genome‐wide Analysis of Enhancers in Development and Disease
Authors: Ren Bing
Published: June 2021

Transcriptional Enhancers play a crucial role in spatiotemporal expression of genes. Dysregulation of enhancers, due to mutations in transcription factors and chromatin regulators, has emerged as a major cause of human cancer. A thorough understanding of cell type specificity, function and mechanisms of action of enhancers will be valuable for development of new diagnostics and therapeutics targeting cancers. I will present our latest advances in identification and characterization of enhancers in the human and mouse genomes, across multiple stages of development, in normal and disease states, and often in single cell resolution. In addition, I will present progress in mapping distal enhancers to their target genes, via analysis of the 3‐dimensional genome architecture and chromatin loops, in order to access the role of non‐coding variants in human diseases. Finally, I will present results from large‐scale functional screenings of oncogenic enhancers in cancer cells.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of a Targeted Diversifier Allowing Mutation of All Nucleotide Types In Vivo
Authors: Dueber John , Halperin Shakked , Schaffer David
Published: June 2021

The capacity to diversify genetic codes advances our understanding and engineering of biological systems. A method to continuously diversify user‐defined regions of a genome without requiring the integration of nucleic acid libraries would enable forward genetic approaches in systems not amenable to high efficiency homology‐directed integration, rapid evolution of biotechnologically useful activity through accelerated and parallelized rounds of mutagenesis and selection, and cell lineage tracking. Here we developed EvolvR, the first system that can continuously diversify all nucleotides within a tunable window length at user‐defined loci. Our results demonstrate that EvolvR enables multiplexed and continuous diversification of user‐defined genomic loci that will be useful for a broad range of basic and biotechnological applications.

Support or Funding Information

Innovative Genomics 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.
Prevalence of Anatomical Variants of the Circle of Willis
Authors: Crofton Andrew , Beetler Danielle , Wilkerson Anthony , Rosado Christopher , Parente Ryan , Sfalanga Christina , Tailor Dharmesh
Published: June 2021

The Circle of Willis is an anastomotic structure that connects the anterior and posterior arterial networks that supply blood to the brain. It functions to maintain adequate blood supply to the brain in the event of obstruction due to injury or disease. Although it is well‐known that variants in the “textbook” structure of the Circle of Willis are common, previous studies characterizing the CoW have been limited by small sample sizes and, in some cases, reliance on cadaveric tissue in which preservation of delicate CoW structures is challenging. These shortfalls have made investigating the relationships between variants and neurological pathology difficult. Therefore, the purpose of the present study was to characterize the CoW in a large sample of living subjects to determine the prevalence of CoW variants and to identify relationships between variants and pathology. After approval for this study was obtained from the AdventHealth IRB, computed tomography angiography (CTA) scans of 1,494 cases were assessed for CoW variants and all CoW vessels measured. Inclusion criteria included patients aged 18–89 that received CTA imaging through AdventHealth Radiology between 11/15/12 and 11/14/17. One examiner assessed all CTA images via imaging software (Impax) for variants. Six other investigators measured each of the CoW vessels on the selected cases. Patient demographic information was collected via AdventHealth's medical record system. Our results indicate that CoW variants were present in 47.99% of the subjects. Of the cases with variants, 8.93% possessed symmetrical bilateral variations and 39.05% possessed multiple variants. The most commonly occurring variant was a non‐dominant vertebral artery at 17.27%. Of the non‐diminutive variants, meaning there were additions to or subtractions from the standard CoW architecture, the most commonly occurring variant was fetal origin posterior cerebral artery at 12.38%. Our results suggest that anatomical variants found in the Circle of Willis occur in just over under half of the population. Future analyses will characterize vessel diameters for the cases under study and relationships of each of the variants to neurological pathology. This study demonstrates the need for further investigation into the nature of altered CoW circulation patterns, how CoW variants may relate to brain function and pathology, and whether diminutive variants can be altered by non‐invasive interventions.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel Communications of the Infraorbital Artery: A Quantitative Analysis
Authors: Winters Jacob
Published: June 2021

Traditional descriptions of the infraorbital artery note a course inferior to the orbit that does not pass through the orbital floor before exiting at the infraorbital foramen. However, recent research suggests that the infraorbital artery may interact with structures within the orbit in some individuals. Given the various descriptions of courses and anastomoses of the infraorbital artery, a further understanding of the frequency of these variations was merited. This study aimed to quantify the prevalence of supply of the infraorbital artery to the lower orbit via the orbital floor. Cadaveric analysis (n = 36 orbits, 18 individuals) was used. Of 36 orbits, 83% exhibited infraorbital arterial supply to the orbit via the orbital floor. Of the 18 individuals, 72% demonstrated bilateral involvement. The results establish involvement of the infraorbital artery with the lower orbit in most individuals. These findings are clinically useful when considering the management of trauma in the lower orbit, as well as blepharoplasty and cosmetic fillers.

Support or Funding Information

No disclosures.

Special thanks to our donors, without whose generosity this research would not have been possible.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Displaced Descending Colon and Dolichosigmoid with Uncommon Sigmoidal Artery Branching Pattern
Authors: Kandavalli Naveen Babu , Castellanos Bedia B
Published: June 2021

Sigmoid colon is a part of large intestine and normally measures 15” in length that extends from the pelvic brim to the third piece of the sacrum where it becomes the rectum. Dolichosigmoid or redundant sigmoid colon is a term used for sigmoid colon longer than its normal size. The present case had a displaced descending colon and a redundant part of sigmoid colon. During a routine dissection class for medical students, an anatomical variation related to sigmoid colon in an 87‐year‐old male cadaver was found. The colon was carefully dissected and studied in detail the position, attachment of sigmoid mesocolon, examination of viscera in close proximity and the blood supply to each part of the large intestine. A meticulous review of the literature was conducted as well. An anomalous displacement of descending and sigmoid colons was found. The descending colon is normal in length, retroperitoneal and lying close to the mid‐line leaving out a space to its left that was occupied by loops of jejunum and ileum. The sigmoid colon is composed of redundant ascending and normal descending parts. The redundant part of the sigmoid colon is centrally placed as a loop ascending into the peritoneal cavity reaching the splenic flexure at the level of T11 approximately. The descending part of the sigmoid colon crosses the midline at L1 and runs close to the right midclavicular line continuing as rectum. The total length of sigmoid colon measures 23.2”. The blood supply to the redundant part of the sigmoid comes from a common trunk arising from inferior mesenteric that splits into left colic and two additional sigmoidal branches. The blood supply to the descending part of sigmoid is supplied by sigmoidal artery coming directly from inferior mesenteric artery. The branching pattern of sigmoidal arteries compared to previous cases in the literature makes this case very unique. Embryology of gut development is complex and often unpredictable, leading to variations in length and position. The growth and rotation of midgut is divided into four stages. An excess of growth of the caudal segment of gut tube in stage 3 can cause redundant sigmoid. This variation could increase the chances of sigmoid volvulus, constipation and may pose risk as the loops may coil around and form a knot leading to obstruction. A redundant loop of sigmoid colon may be asymptomatic or it might lead to urinary, digestive and vascular complications. The descending colon is normal in length but lies close to the midline which may compress the aorta when loaded with feces. Radiologists and surgeons must be well aware of these variations to establish a correct diagnosis and assert the appropriate management when performing colonoscopy, sigmoidoscopy and abdominopelvic procedures.

Support or Funding Information

Department of Anatomy Touro College of Osteopathic Medicine

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 in the formation of the of the trunks, Long Thoracic and Suprascapular nerves of the brachial plexus. Correlation of Those Variations with Possible Clinical and Surgical Implications
Authors: Veras Wilson R , Lopez Tarime , Diaz Jose
Published: June 2021

The Brachial Plexus The brachial plexus is a major and complicated nerve network located in the posterior triangle of the neck that supplies the upper limb and some thoracic structures. From the posterior triangle, it extends into the axilla. The brachial plexus is formed by the anterior rami of C5, C6, C7, C8, and T1 nerves. These cervical and thoracic nerves constitute the roots of the brachial plexus. The union of C5 and C6 formed the superior trunk, the continuation of C7 root form the middle trunk, and the inferior trunk is formed by C8 and T1 roots. Variations of the brachial plexus network are common, knowledge of the variant anatomy is very important to do a safe surgical procedure in this area. The brachial plexus may be damaged during trauma in the neck, bone fractures at the shoulder region, in presence of a cervical rib, a tumor of the cervical region, in obstetric procedures. Unexplained physical signs could be explained for the presence of an anatomical variation of the brachial plexus. Due to the importance of the brachial plexus, the purpose of this study was to evaluate the presence of variations in the supraclavicular part of this network and some of its branches in adult human cadavers.

Material and methods

Bilateral dissection of the upper limbs of 17 embalmed cadavers, 12 males and 5 females at the anatomy laboratory of San Juan Bautista School of Medicine.

Results

In the present study, we found five brachial plexuses prefixed in origin on the right side and one postfixed on the left side. On the right side, the long thoracic nerve was formed by C5, C6, and C7 in 3 cases, two were formed by C5, and C6. One by C5 and inferior trunk. Six by C6, and C7. One was formed by C6 and posterior division of the inferior trunk. One by C6 and Anterior division of the middle trunk. One by C5 and the inferior trunk. One arises from the upper trunk, and one was formed by C4 and C6. On the left side, the long thoracic nerve was formed by C5, C6, C7 in six specimens, one for C4, C6, C7, one by C4, C5; one was formed by C5, C6, and Lower trunk; one for C6, and inferior trunk; C6 and posterior division of upper trunk; one by C4, C6, and C7; one was formed by C5 and C7 and one was formed by branches of the Inferior trunk and the medial cord. it was not possible to determine the origin of the long thoracic nerve during the dissection in 3 cases. The Right Suprascapular arose from the upper trunk in eleven specimens. Five from posterior division of the upper trunk and one from C5. On the left side, the suprascapular nerve in seven specimens was found arising from the upper trunk, five from the posterior division of the upper trunk and two from C5.

Support or Funding Information

Support by the institution

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
This Week You Next Week Me: Rotational Peer‐to‐Peer Dissections and Presentations to Augment Undergraduate Laboratory Sessions
Authors: Parizi‐Robinson Mojgan , Moore Michael E.
Published: June 2021

Human anatomy is a challenging subject for undergraduate students due to the volume of structures and associated complex terminology. The weekly anatomy lab component adds a level of complexity due to the time required to complete each cat dissection (24 students/lab). The prosection projects started in the fall of 2016 and have since continued. The anatomy class consists of 4 labs in the fall, followed by 5 labs in the spring semester. To help students engage with the course content, improve their level of understanding and improve the quality of the dissections Baylor University introduced a rotating peer‐to‐peer teaching activity. Each week a group of 4 students completed the required dissection (i.e., brachial plexus) prior to the scheduled lab. Subsequently during the scheduled lab session this group of 4 students became temporary lab assistants by helping the other 20 students with their own dissections. Additionally, the 4 students conducted a formal presentation to their classmates about their dissection and answered functional and structural questions under the guidance of Dr. Parizi‐Robinson. End of course surveys indicated that students enjoyed both the giving and receiving portion of this activity and enjoyed the level of engagement and increased level of understanding. Therefore, this rotational peer‐to‐peer teaching activity seems to be an effective active educational tool for students' and may be beneficial for other institutions to consider.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anatomy in Medical Education: A Canadian Review
Authors: Rockarts Jasmine , Wainman Bruce , Shali Ari , Mohialdin Vian
Published: June 2021

The anatomical sciences have always been regarded as an essential element of medical education. In Canada, the methodology and time dedicated to anatomy teaching are currently unknown. In an effort to create a comprehensive view of anatomical education in Canadian medical schools, surveys were sent out to course directors and discipline leaders. Participants were asked about gross anatomy, histology, and embryology. Data was collected on total course hours, total classroom hours, large and small group hours as well as laboratory hours for the 2016/17 year. There have been two major changes in the delivery of the anatomical sciences. First, most programs deliver anatomical sciences using an integrative approach. The shift towards integration is also prevalent in the United States of America. Secondly, contact time in the anatomical sciences are at historically low levels. Compared to data published from a similar survey published in 2018, Canada has 72%, 49% and 53% of the hours of gross anatomy, histology, and embryology respectively, compared to the USA. The reasons for the difference between Canadian and USA schools is not known. Possible reasons for the disparity include the steering effects of the CanMEDS framework in Canada and the USMLE (United States Medical Licensing Examinations) Step 1 examination. The CanMEDS framework identifies six core competencies that are meant to ensure that physicians have more of a comprehensive foundation as a medical expert but an understanding of the basic sciences, including anatomy, is not a core competency rather it is assumed to be part of all competencies to some degree. Given there is no explicit role identified for the anatomical, or any other, sciences this may have led to the general decrease in curricular focus. A focus for many medical schools in the USA is student success on the USMLE Step 1 which is focused on basic sciences, including anatomy. Student performance on the USMLE drives curriculum standards. Further investigation is required to determine if the lower amount of anatomical science education received by Canadian medical students affects student performance in clerkship, residency and beyond.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PEER‐DIRECTED SMALL GROUP LEARNING IN M1 GROSS ANATOMY: ENHANCING STUDENT LEARNING WHILE REDUCING REQUIRED CONTACT HOURS
Authors: Stanley Robert Lee , Harrell Kelly
Published: June 2021

PURPOSE

Many medical schools have adopted a shortened basic science curriculum in favor of a longer clinical curriculum. In an attempt to reduce required contact hours without compromising important high‐yield content, programs have developed and implemented specific learner‐centered teaching strategies We sought to utilize peer‐directed small‐group sessions to enhance student learning in anatomy within a condensed pre‐clinical curriculum . Sessions were designed to guide learners through specific cadaveric prosections.

METHODS

Three voluntary small‐group sessions were designed to correlate with spinal cord, facial nerve/orbit, and ischioanal fossa prosections in a Medical Gross Anatomy course for first‐year medical students. There was no penalty or reward for attending these sessions, and the prosections were made available to all students, regardless of participation. A short survey was administered after each session. This survey assessed students perceived comfort level with the content area before and after the small‐group session. Student performance on specific test questions related to sessions topics was analyzed and compared between those who attended the small group sessions, and those who did not to determine patterns of performance on the specific topics.

RESULTS

There was an increase in the student perceived level of comfort with the material after each of the three small group sessions for both the 2017 and 2018 M1 classes. On a 1‐to‐5 Likert scale (1 lowest; 5 highest) of perceived comfort level with session content, averages before the three small group sessions were significantly increased for each small group session for both years. Analysis of changes in student performance regarding test performance resulted in a slight increase in performance on most questions for those who attended the small groups compared to those who did not. Word cloud analysis of the write‐in portion of the survey revealed several common themes that students thought the small group setting was a strength, and having the facilitator ask more questions of the participants, being a common area for improvement.

CONCLUSION

This work supports peer‐directed and small group learning as an effective avenue for student success in M1 Gross Anatomy. Further analysis of student performance and future studies will help determine whether this could be an effective teaching strategy utilized by other courses during the basic science years.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Written Communication Skills in Physical Therapy Students
Authors: Cline Christina , Samonds Karen , Bergan‐Roller Heather
Published: June 2021

A functional doctor‐patient relationship is predicated upon successful communication. Medical professionals (doctors, dentists, physician assistants, nurses, physical therapists) often must convey complicated scientific information to their patients who come with varying degrees of background knowledge. It is critical for students studying these professions to learn and practice best approaches to communication. Medical communication training tends to focus on verbal skills needed for patient‐physician encounters and is the focus of most medical communications training research. More work needs to be done on understanding students' written communication skills to improve both training and practice.

Here, we present a study that evaluates the written communication skills of physical therapy students enrolled in a gross human anatomy course. The study looked at the following research question: How do physical therapy students change how they communicate in writing with different audiences? Students were asked to write letters to patients, with varying levels of assumed medical knowledge. These letters summarized their analysis of the patient's symptoms as a part of case study assignments. The first patient was described as having a college education with medical background, the second patient was described as having an eighth‐grade education, and the final patient also had an eighth‐grade education. The final patient letters were compared to the previous letters through thematic analysis. Results from the qualitative coding of patient letters will present major themes present in the patient letters and if and how they change through the course of the three case studies. This work will add to our knowledge of how medical students can more effectively and efficiently learn and practice necessary communication skills that are vital for successful medical practice.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Slimed! Better understanding the role of aquaporin in the slime gland of hagfish
Authors: Lane Jim , Torenvliet Alyne , Edmunds Mary , Schimnowski Ava
Published: June 2021

Hagfish are a species of small marine vertebrates that have experienced little evolutionary change for 300 million years due to their effective defense mechanism. This mechanism has the potential to be a component of many major medical advancements as well as airbag technology and textiles. When threatened, a hagfish will rapidly deploy a slime gland exudate that expands in a fraction of a second and clogs the gills of its predator, suffocating it. This is a two‐part process involving the unraveling of thread skeins and bursting of mucin vesicles. Our research focuses on the mucin vesicles and how the membrane protein Hagfish Aquaporin 4 (hfAQP4) controls the rupturing process. When a mucin vesicle comes in contact with seawater, Ca2+ions contained in the water open Ca2+ activated protein channels within the vesicle membrane and allow for a variety of seawater ions to enter the vesicle. This changes the osmotic balance within the vesicle to favor the influx of water molecules which flood the cell through the hfAQP4 channel. The vesicle becomes overfilled with water and ruptures, releasing the slime. We continued to explore the role of hfAQP4 in this process and found research done by Cutler, et. al that compared Aquaporin 4 (AQP4) of human, rat, chicken, xenofish, zebrafish, dogfish and hagfish. This research found that hfAQP4 differs more from human aquaporin 4 (hAQP4) than any of the other aquaporins studied. In order to better understand the function of hfAQP4 in the slime release process, we will identify similarities and differences between hAQP4 and hfAQP4. Also, using the hAPQ4 pdb, 3GD8, we can use the molecular modeling software, jmol, to highlight the variations compared to hfAQP4. Our research was narrowed down to hfAQP4 due to findings that confirmed the role of hfAQP4 in the slime deployment process. When mercuric chloride (HgCl2), an aquaporin inhibitor, is added to the water that the slime is being deployed into, the vesicles membranes do not rupture which proves the involvement of hfAQP4 in this process. The (HgCl2) is very successful in inhibiting AQP4 because of the location of the amino acid: Cysteine 178. In Aquaporin 1 (AQP1), Cysteine 189 is located at the constriction of the pore, and is not easily accessible to the HgCl2. In AQP4, however, the cysteine residue is located in cytoplasmic loop D at the location of Cysteine 178, and is much more accessible to the HgCl2. This causes AQP4 to be more rapidly inhibited by HgCl2 than AQP1. The Mahtomedi MSOE Center for BioMolecular Modeling MAPS Team used 3‐D modeling and printing technology to examine structure‐function relationships of hfAQP4. The visual model will be a valuable tool in developing our story.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modelling the performance of subterranean rodent mandibles using finite element analysis
Authors: Pierzchjlo Garret , Cox Philip , McIntosh Andrew
Published: June 2021

Subterranean rodents spend most of their lives underground. As such they have developed efficient ways of excavating soil for burrow formation. Most subterranean rodents dig in one of two ways; chisel‐tooth digging, where enamel covered incisors dig through soil; or scratch digging, where keratin covered claws are used. It is thought that chisel‐tooth digging evolved in order to utilize the resilient enamel covered incisor to dig through harder soils compared to keratin filled claws, which can only dig through softer soils. Chisel‐tooth digging requires a high bite force in concert with a wide gape to achieve optimal removal of soil. A previous study compared the performance of the cranium between chisel‐tooth and scratch digging rodents at different gapes which showed the chisel‐tooth digging rodent cranium maintained higher bite forces at wider gapes. The current study compared the performance of chisel‐tooth and scratch digging mandibles using finite element analysis (FEA). To test this hypothesis, finite element models of the mandible were created and solved at varying gapes. Our results show that at small gapes, the chisel‐tooth and scratch digging mandibles show similar performance parameters. However, as gapes increase the chisel‐tooth digging mandibles outperform the scratch digging mandible. Subterranean rodents have a hard food diet and so it is no surprise that there was little difference in performance at small gapes. However, only chisel‐tooth diggers require a larger gape to excavate hard soils. The results of this study show that the mandibular morphology of chisel‐tooth digging rodents has adapted to meet its functional demands.

Support or Funding Information

Gill Dilliard Faculty Enrichment Fund ABAC Foundation

Natural Environment Research Council, Grant/Award Number: G001952

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural and functional changes in human aquaporin (AQP4) and its role in the symptoms of glaucoma
Authors: Ales Ruby , Anderson Teresa , Hueffmeier Sarah
Published: June 2021

As the human body grows older, the eyes become susceptible to an array of conditions that are detrimental to their ability to perceive the world around them, the most severe being a complete loss of vision. Glaucoma is a disease that involves the gradual build‐up of fluid in the eye, an irreversible condition that puts pressure on the optic nerve and eventually renders the victim blind. The focus of this project is to investigate possible treatments on a molecular level for primary open‐angle glaucoma in its later stages. Human aquaporin‐4 (hAQP4), a protein that serves as a water channel in the cell membrane, was identified as a potential treatment for advanced primary open‐angle glaucoma due to its involvement in the production of the aqueous humor (jelly‐like fluid which gives the eye its shape) and its presence in the optic nerve. The Mahtomedi MSOE Center for BioMolecular Modeling MAPS Team used 3D modeling and printing technology to examine structure‐function relationships of hAQP4 in the optic nerve. hAQP4 is found in the eye along with hAQP1, but hAQP4 stands out for its high water permeability, a characteristic that has been attributed to the function of the side chains of asparagines 213 and 97. In hAQP4, these side chains hydrogen bond with a single water molecule at a time as opposed to several, as occurs within other hAQPs. In this poster, we investigate possible structural changes of hAQP4 that cause a buildup of fluid in the eye and how to reverse the effects. We are also exploring the medical implications of potential inhibitors of hAQP4. Being able to activate and deactivate hAQP4 has the potential to allow doctors to drain the fluid buildup in the eye and reverse the effects of glaucoma. Currently, several potential inhibitors of hAQP4 are being investigated, but none have been confirmed.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determining Accuracy of oAb as a Mechanistic Biomarker for Alzheimer's Disease in Human Plasma
Authors: Faulk Naomi , Valencia‐Olvera Ana , Collins Nicole , LaDu Mary Jo
Published: June 2021

Alzheimer's disease (AD) is a fatal neurodegenerative disease that is the 6th leading cause of death in the United States with no cure and few palliative treatments. Currently, there is no preventative treatment, and even if such a therapeutic could be identified, without a predictive biomarker, a target population for treatment could not be determined. Although the mechanism remains unclear, AD is caused by increased levels of the amyloid‐b peptide (Ab), which aggregates to form both amyloid plaques and soluble oligomeric Ab (oAb), the latter considered a proximal neurotoxin. We believe that levels of oAb in human plasma are a prognostic biomarker, and have developed and licensed a method for their detection (LOD <5pMol). While age is the greatest risk factor for AD, APOE4 is the greatest genetic risk factor, increasing risk up to 15‐fold compared to the more common APOE3. Importantly, female (♀) APOE4 carriers have an increased risk for AD compared to male (♂) APOE4 carriers. Key to the success of this project has been the stratification of both control and AD subjects by sex within APOE genotype, with the results that plasma oAb levels are: ♀APOE4> ♂APOE4> ♀APOE3> ♂APOE3. This response was observed in two large human post‐mortem cohorts. However, stratification, the principle of personalized medicine, is simply not applied to AD populations. We now seek to threshold oAb levels using longitudinal and human AD trial data to establish prognostic potential, with the goal of partnering with the private sector for commercialization.

Support or Funding Information

LaDu lab funding includes an Anonymous Donor, the University of Illinois College of Medicine, and UIC Honors College 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.
The effects of sex and APOE genotype on the gut microbiome in EFAD transgenic mice
Authors: Maldonado Juan E. , Parikh Ishita J. , Naqib Ankur , Green Stephan J. , Estus Steven , LaDu Mary Jo
Published: June 2021

While the gut microbiome (GM), the collective genome of bacteria in the gastrointestinal tract, is primarily studied in metabolism and immune defense, the GM may also serve as a diagnostic tool and therapeutic target in neurological disorders, including Alzheimer's disease (AD). While age is the greatest risk factor for AD, the e4allele of APOE is the greatest genetic risk factor, increasing risk up to 15‐fold compared to APOE3, the common genotype. APOE4 is associated with the accelerated accumulation of the peptide amyloid‐b(Ab), which can aggregate to form both amyloid plaques and small soluble aggregates or oligomeric Ab (oAb), the latter considered a proximal neurotoxin. Importantly, among APOE4 carriers, females have an increased AD risk, rate of cognitive loss and accumulation of Ab42 compared to males. Although the GM of individuals with AD exhibit specific microbial differences compared to healthy controls, a greater understanding of how age, APOE genotype and sex contribute to the altered GM in AD may help determine the diagnostic potential of the GM composition. To study the interactions among these AD risk factors, we developed the EFAD‐transgenic mice, which express the human APOE genotypes and over‐express human Ab42. Thus, our hypothesis is that APOE genotype and sex interact to modulate the GM composition in EFAD mice. Fecal samples from 4 month (M) ♂ and ♀ E3FAD and E4FAD mice were sequenced and compared. The Shannon H index, a measure of species richness and diversity, identified a negative correlation between diversity and severity of AD pathology in the 4M EFAD cohorts of mice: ♂E4FAD > ♀E4FAD and ♀E3FAD > ♀E4FAD. Analysis of similarities (ANOSIM) demonstrated significant differences in the GM in E3FAD vs E4FAD across different taxonomic levels (operational taxonomic unit/OTU, Genus, and Family). Comparison across sex within APOE genotype revealed a significant difference between ♂E3FAD vs ♂E4FAD and ♀E3FAD vs ♀E4FAD at all taxonomic levels. Additionally, there is a significant effect for ♂E4FAD vs ♀E4FAD, though only at the OTU level. Further, heatmap analysis demonstrates clustering of ♀E4FAD samples based on 29 OTUs. Therefore, the synergistic effect of APOE4 and female sex on AD risk is exhibited by the GM, observed by the stratification of the EFAD cohorts. This study is the first step in defining the composition of the GM as a function APOE genotype and sex modulation of AD pathology.

Support or Funding Information

LaDu lab funding includes institutional funds from the University of Illinois College of Medicine, the Bridge to the Doctorate Fellowship, and the Honor's College Undergraduate Research Award at the University of Illinois at Chicago.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glycyrrhizinate Dipotassium (DPG) Effects over Skin Wound Healing
Authors: Rocha Thalita , Leite Camila Silva , Tenis Danielle Gatti , Ziegler Jussara , Priolli Denise Golçalves
Published: June 2021

Skin wound healing is a complex process involving cellular, and molecular events: inflammatory, proliferative, and remodeling phases. Infectious disease, pre‐existing diseases, medications, and aging can negatively interfere in healing. Research on leptin, collagenase, and tretinoin have been done attempting to promote healing. Glycyrrhizinate Dipotassium (DPG), derived from licorice, has anti‐allergic, antibiotic, and anti‐inflammatory effects, besides promoting the skeletal muscle regeneration by activating quiescent satellite cells, leading myoblasts and myotubes differentiation, and forming new muscle fibers. Considering these aspects, we purpose to verify the 2% DPG pharmacological effect on secondary intention wound healing in adult and aged animals. Twenty male Wistar rats were used. Two skins' excisions (2 cm diameter) of normal skin were made in the animal dorsal region (Naïve group). Topical application of 2% DPG was done on the distal wound daily for 7 days (Treated group). The proximal wound was not treated (Control group). After 14 or 21 days (n = 5 each) of wound creation, the samples were excised, fixed in 10% formol, and processed for paraffin inclusion. Slides were stained with hematoxylin and eosin (H&E), and Masson's trichrome (MT) for histological analysis. Wound healing areas, epidermal and dermal measurements were performed. Statistical analysis adopted a 5% coefficient for null hypothesis rejection. Histological analysis revealed intact skins for Naïve group. Control and Treated groups presented skin with epidermis and dermis reshaped: all epidermal layers (basale, spinosum, granulosum, lucidum, and corneum stratum), as papillary and reticular dermis. In aged rats, the wound was smaller in treated than in untreated animals, resulting in a larger scar compared with the adult rats. However, when treated wounds were compared, no differences were found between the wound areas in adult and aged rats. After 14 days the epidermis thickness of adult and aged animals treated with DPG was thicker than untreated animals. However, after 21 days, the adult epidermis thickness of animals treated with DPG was thinner than untreated animals. The dermis thickness of adult and aged animals treated or non‐treated with 2% DPG did not differ. On the 21st day, adult and aged complete reepithelialization were found in treated animals. Results indicate that 2% DPG significantly reverses wounds process to a skin normal state, even for aged animals, by reepithelization, probably acting during inflammatory and proliferative phases of healing. Such results point DPG as a promissory agent to promote skin healing.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determination of the DNA binding preference of the SIX transcription factor optix in Heliconius butterflies
Authors: Rivera Anthony Rafael , Rodriguez Jose A
Published: June 2021

Transcription factors (TFs) are sequence‐specific DNA binding proteins that determine cell state and have been shown to control cell differentiation. Evaluating comprehensive DNA specificity profiles of DNA binding proteins is a nontrivial challenge that hinders our ability to decipher gene regulatory networks. Optix is a homeodomain transcription factor that has been identified as a master regulator of red color patterning in the wings Heliconius butterflies. However, the direct gene targets of optix and its role in final wing coloration remains to be determined. We want to determine the intrinsic DNA‐binding preferences of optix. We have over‐expressed and purified full‐length Heliconius optix and its homeodomain (HD) DNA‐binding domain. We will determine the in vitro DNA‐binding specificity of optix protein of Heliconius erato using Cognate Site Identification by High‐Throughput Systematic evolution of ligands by exponential enrichment (HT‐SELEX). Our results will enable us to bioinformatically predict the genomic targets of optix and decipher its role in wing color patterning and development.

Support or Funding Information

This research is supported by: 1) Research Initiative for Scientific Enhancement (RISE) Grant#: 5R25GM061151‐172) NSF EPSCoR Research Infrastructure grant award 1736026

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Decitabine Treatment Demethylates Vast Majority of High‐Confidence Differentially Methylated Regions in HCT‐116 Colorectal Cancer Cells
Authors: Conrad William H , Anderson Jonathan , Balaram Ariane , Ganev Yoan , Gascoigne Samuel , Gimondo Christina , Palmero Brett , Quraishi Ayesha , Rodriguez Alejandro , Sadat Said Omer , Sandler Anna
Published: June 2021

Colorectal cancer (CRC) is a common, and often incurable, form of cancer. Gene silencing by CpG island hypermethylation often plays a role in CRC progression. Certain regions of the genome, called high confidence differentially‐methylated regions (DMRs), are consistently hypermethylated across numerous patient samples. In this study, we used bisulfite PCR sequencing to investigate methylation levels at DMRs in the promoter region of CDKN2A, DKK3, EN1, MiR34b, SPG20, and TLX1 in HCT‐116 CRC cells. We observed that for all DMRs except CDKN2A, the demethylating agent decitabine significantly reduced CpG methylation. Using ENCODE project data, we observed that transcriptional activator binding inversely correlates with DNA methylation at all of these sites across diverse cancers and cell types. Our data increase resolution of the methylation status at the above DMRs, show the reversibility of methylation at these sites by decitabine, and the likely role of hypermethylation at these sites in gene silencing. In the future, we plan to test if DMR any specific gene silencing protects HCT116 cells.

Support or Funding Information

This work is supported by the Lake Forest College biochemistry and molecular biology program.

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 Role of E4BP4 in Apoptosis of Human Leukemia Cells in Culture
Authors: Mitchell‐Velasquez Erick Lenin , Gomez Danny , Medh Rheem D
Published: June 2021

Apoptosis is a physiological and genetically controlled process of cell death important for development and cellular homeostasis. Several diseases can be attributed to an imbalance between cellular proliferation and attrition, brought on by inadequate or excessive apoptosis. In cancer, a lack of apoptosis causes excessive accumulation of unwanted cells. Apoptosis is primarily regulated via altered expression of key genes and modulation of signal transduction pathways. Our laboratory focuses on understanding the early apoptosis events triggered by chemotherapeutic agents in cell culture models of leukemia. Previous studies have shown that the E4BP4 (adenovirus E4 binding protein 4) gene in upregulated in correlation with apoptosis. E4BP4 upregulation has been implicated in induction of the pro‐apoptotic BIM protein, a member of the Bcl‐2 family of proteins. In studies presented here, we have utilized CRISPR‐mediated genomic editing technology to knock down E4BP4 expression. Three clones with reduced E4BP4 expression were analyzed for their response to Dexamethasone (Dex), a synthetic glucocorticoid and anti‐leukemic agent. While E4BP4 knockdown did not alter the ability of cells to undergo apoptosis in response to Dex, two of the three clones showed a reduction in the extent of BIM upregulation. Residual E4BP4 expression in our knock down clones may be sufficient to evoke an apoptotic response. Further efforts are underway to understand downstream pathways regulated by E4BP4.

Support or Funding Information

This research is funded by a NIH SCORE SC3 award to RDM (GM081099) and a MARC‐U* STAR scholarship to Erick Mitchell‐Velasquez.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Uncovering DNA binding properties of the GATA4 and TBX5 transcription factor complex
Authors: Rosado‐Rodríguez Emili Patricia , Rodríguez‐Ríos Jessica , Rodríguez‐Martínez José
Published: June 2021

Transcription factors (TFs) are essential master regulators of gene expression. These DNA binding proteins are fundamental for cellular differentiation during development. Eukaryotic TFs are notorious for not working alone and they often bind DNA as multimeric protein complexes to regulate gene expression. GATA4 and TBX5 are transcription factors that are central components of the gene regulatory network of heart development and function. Recent studies have determined the binding specificity of multiple transcription factors as monomers. However, the DNA‐binding properties of the cooperative complex between GATA4 and TBX5 remain undetermined. Based on this, we want to know the intrinsic DNA binding preferences and the emergent properties that result from the cooperative complex formed by GATA4 and TBX5. We used a cell‐free protein expression system to produce GATA4 and TBX5, and we successfully validated their DNA‐binding activity through an Electrophoretic Mobility Shift Assay (EMSA). Subsequently, we will determine the in vitro DNA‐binding specificity of the GATA4:TBX5 complex using Cognate Site Identification (CSI) by High‐Throughput Systematic Evolution of Ligands by Exponential Enrichment (HT‐SELEX). The findings of this study will help understand the DNA‐recognition rules of the GATA4:TBX5 complex and its potential roles in normal heart development.

Support or Funding Information

Grant Acknowledgement: This research is funded by the National Institutes of Health ‐ NIH SC1GM127231 and the Puerto Rico Louis Stokes Alliance for Minority Participation (PR‐LSAMP)‐Undergraduate Fellowship.

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