bluuebunny/crossref_metadata_2025 huggingface dataset:
Upregulation of cannabinoid receptor‐1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J. infection: role of NADPH oxidase (693.13)
Authors: Wang Mi , Abais Justine , Meng Nan , Zhang Yang , Ritter Joseph , Li Pin‐lan , Tang Wang‐Xian
Published: June 2021

The endocannabinoid system (CS) has been implicated in schistosomiasis‐associated liver fibrosis (SSLF). However, the molecular mechanisms mediating the action of the CS in SSLF are largely unknown. The present study hypothesized that Schistosoma J. infection upregulates cannabinoid receptor (CB) signaling via activation of NADPH oxidase leading to a fibrotic phenotype in hepatic stellate cells (HSCs). A SSLF model was developed by infecting mice with Schistosoma J. cercariae in the skin. HSCs from uninfected (control) or Schistosoma J.‐infected (SSLF) mice were isolated, cultured and identified by analyzing HSC markers α‐SMA and desmin. In HSCs isolated from control or SSLF mice, both CB1 and CB2 receptors were detected, however, only the CB1 significantly increased in HSCs from SSLF mice compared to that control mice. Such increase of CB1 in HSCs was accompanied by enhanced expression of fibrotic markers α‐SMA, collagen I, and TIMP‐1. Similarly, CB1 upregulation and enhanced fibrotic changes were observed in HSCs isolated from control mice when these cells were treated with soluble egg antigen (SEA) from Schistosoma J. Interestingly, HSCs from SSLF mice or SEA‐treated HSCs from control mice exhibited enhanced superoxide (O2.‐) production as analyzed by electron spin resonance. Importantly, inhibition of O2.‐ production by knocking down NADPH oxidase genes (Nox1 or Nox4) prevented SEA‐induced upregulation of CB1 and fibrotic markers in HSCs. Further, CB1 gene silencing blocked SEA‐induced fibrotic changes in HSCs but had no effect on SEA‐induced O2.‐ production in these cells indicating a triggering role of O2.‐ production in CB1‐mediated fibrotic signaling. Taken together, these data suggest that the fibrotic activation of HSCs upon Schistosoma J. infection or SEA stimulation is associated with NADPH oxidase‐mediated redox regulation of CB1 expression, which may be a triggering mechanism for SSLF.

Grant Funding Source: Supported by NIH grants HL057244, HL091464 and HL075316
Cardiac myocyte‐specific deletion of Heat shock protein 10 results in altered cytosolic and mitochondrial protein expression (699.6)
Authors: Suarez Jorge , Wang Hong , Ouyang Kunfu , Scott Brian
Published: June 2021

Heat shock proteins (HSPs) are molecular chaperones that prevent protein damage and proteolysis. HSP10 forms mitochondrial (Mito) chaperoning complexes with HSP60 to maintain protein quality control. Studies in vitro suggest that HSP10 plays a role in normal Mito function; however, its physiological function in the heart in vivo remains to be determined. To investigate the function of cardiac HSP10, we generated a floxed allele of the Hspe1 gene, and found that cardiac specific, tamoxifen‐inducible deletion of HSP10 in adult mice resulted in death between 12 and 18 weeks after HSP10 deletion. Increased mortality after HSP10 deletion was associated with dramatically altered protein expression patterns. For instance, cardiac hypertrophic biomarkers atrial natriuretic factor (ANF), β‐Myosin Heavy Chain (βMHC) and skeletal muscle actin were increased in the HSP10 KO mice. In Addition, there was a dramatic reduction in levels of Mito respiratory complex subunits including CI‐NDUFA9, CII‐30, COX I, COX IV, and COX Vb. Furthermore, the apoptotic pathway is activated in HSP10 KO mice as revealed by the cleavage of Caspases 9 and 3. Our results suggest that mitochondrial protein damage may be the primary pathological cause of the death. In conclusion, HSP10 plays a critical and vital role in regulating mitochondrial protein homeostasis.

Grant Funding Source: Supported by P. Robert Majumder Charitable Foundation.
Ubiquitin proteasome system controls contractile sensitivity to KCl in serum‐free organ‐cultured rat mesenteric arteries (700.9)
Authors: Morita Tomoka , Okada Muneyoshi , Yamawaki Hideyuki
Published: June 2021

Background: Organ culture of blood vessels is a useful technique to investigate the long‐term effects of drugs. Organ culture in a serum‐free condition is a better way to maintain differentiated cell function. However some functional changes may occur from freshly isolated blood vessel (fresh) such as increased contractile sensitivity to agonists and KCl. The removal of proteins (enzymes and regulatory proteins) is essential for the control of cell growth and metabolism. Failure to remove an essential protein or lack of degradation of a short‐lived regulatory protein may alter cell function in a major way. We thus hypothesized that the ubiquitin proteasome system may be a candidate which causes functional changes. The aim of the present study was to investigate the effects of inhibitors of ubiquitin proteasome on KCl‐induced contractility in serum‐free organ‐cultured rat mesenteric arteries. Methods and results: Rat isolated mesenteric arteries with endothelium were cultured for 5 days without (0% serum) or with an ubiquitin proteasome inhibitor, Bortezomib (Bort; 10 nM) or MG‐132 (125 nM). In 0% serum, sensitivity to KCl (5.4‐75.4 mM) significantly increased from fresh (EC50 for 0% serum and fresh was 18.3 and 40.3, respectively, p<0.01), while maximal contraction significantly decreased. In Bort and MG‐132, the increased sensitivity to KCl was significantly normalized (EC50 for Bort and MG‐132 was 24.9 and 23.5, respectively, p<0.01), while maximal contraction did not change from 0% serum. Conclusion: Further studies are needed to clarify how ubiquitin‐proteasome system controls the contractile sensitivity to KCl in serum‐free organ culture of isolated blood vessel. Elucidation of the mechanism might contribute to provide mechanistic insights into the role of ubiquitin proteasome system on pathogenesis of vascular disease.
Bone‐muscle crosstalk: more than mechanical (704.3)
Authors: Isaacson Janalee , Mo Chenglin , Brotto Leticia , Huang Jian , Spence Madoka , Vallejo Julian , Abreu Eduardo , Dallas Sarah , Johnson Mark , Bonewald Lynda , Brotto Marco
Published: June 2021

The intricacy of cell interactions with their microenvironment and their ability to communicate at the autocrine, paracrine, and endocrine levels continues to be revealed. Until recently, the musculoskeletal system was believed to be directed primarily by a relationship of proximity and function, dictated in large part by mechanical forces and the work of gravity. The purpose of this review is to provide an overview of the biomechanical theory of bone‐muscle interaction and to present the substantial evolution in our understanding of bones and muscles as secretory organs; including new evidence from our research labs in support of this biochemical crosstalk between bone and muscle cells. We propose that the mechanical and biochemical modes of interaction between bones and muscles are complementary and interwoven. In fact, we hypothesize that one tissue action might prime the other tissue function, thereby contributing for optimal functional unit. This perspective provides a fertile new venue for bone‐muscle crosstalk exploration, as well as the development of innovative therapies targeting musculoskeletal diseases, which currently afflict nearly 2 billion people worldwide.

Grant Funding Source: Supported by NIH P01 AG039355‐01‐A1
TNF‐α alters the release and transfer of miRNAs from endothelial cells (711.7)
Authors: Alexy Tamas , Rooney Kimberly , Weber Martina , Searles Charles
Published: June 2021

MicroRNAs (miRNAs) encapsulated within extracellular vesicles likely have a role in cell‐to‐cell signaling, but little is known as to the mechanisms by which different cell types release miRNAs. Here, we examined the release and transfer of miR‐126, miR‐21, and miR‐155 in microparticles (MPs) produced by human aortic endothelial cells (ECs) treated with TNF舉α. ECs were treated with TNF‐α (100 ng/ml), in the presence or absence of inhibitors targeting distinct MP production pathways. MPs isolated from media were assessed by flow cytometry (count) and qRT‐PCR (miRNA content). Calcein AM‐labeled MPs were used to assess uptake by recipient ECs. TNF舉α induced a 5‐fold increase in MP production after 12‐24 hours of treatment (n = 5, p < 0.05). These MPs were characterized by: 1) 70 ‐ 80% decrease in the miRNA/MP levels for miR‐126 and ‐21 but 20‐fold increase in pre‐miR‐155 (n = 10, p < 0.05); 2) 50% reduction in uptake by recipient cells (n = 3, p < 0.05); and 3) inability to transfer miR‐126 and ‐21. Co‐treatment of donor ECs with TNF舉α and caspase inhibitor (Q‐VD‐OPH; 10μM) completely abrogated TNF舉α‐induced increase in MP number, and produced MPs that had: 1) increased uptake by recipient cells; 2) enhanced miRNA/MP content; and 3) increased ability to transfer miRNA. Co‐treatment of ECs with TNF舉α and Rho‐associated kinase (ROCK) inhibitor (10 μM) produced MPs similar to those produced by TNF舉α treatment alone. Overall, our data suggest that TNF舉α induces EC release of distinct MP populations: ROCK‐dependent, miRNA‐rich MPs that can transfer their miRNA contents to recipient cells, and caspase‐dependent, miRNA‐poor MPs that do not readily transfer their contents. These data provide insight into the relationship between MP production and extracellular release of miRNA, as well as the potential of encapsulated miRNA for information transfer.

Grant Funding Source: Supported by NHLBI R01 and VA Merit Award
Maternal tobacco smoke exposure programs increased lung static compliance in female rat offspring (715.5)
Authors: Sainz Anthony , Wang Yan , Stiers Justin , Zou Chang‐Jiang , German Massiell , Albertine Kurt , Joss‐Moore Lisa
Published: June 2021

Background: Intrauterine growth restriction (IUGR) increases the risk for postnatal lung disease, in part, through programmed alterations in gene expression patterns. A consequence of altered gene expression programs in the developing lung is changes in lung compliance and resistance. In a rat model of surgically‐induced IUGR (sIUGR), male rat pups have increased compliance and decreased resistance, while female rat pups are unaffected. Maternal tobacco smoke (MTS) exposure also results in IUGR. However, the effects of MTS‐induced IUGR on lung compliance and resistance are unknown.

Objective: We hypothesized that MTS exposure alters lung compliance and resistance in rat offspring.

Design/Methods: Maternal rats received tobacco smoke exposure from gestational day 11 to term. Pups were delivered at term and cross‐fostered to a control dam until they were weaned at postnatal day 21 (d21). At d21, rats were anesthetized and paralyzed, and closed chest, lung static compliance and lung resistance mechanics were measured using the FlexiVent System.

Results: MTS significantly increased lung compliance in female (118 ± 4%, p=0.02), but not male rat offspring. Lung resistance was not affected by MTS in either male or female rat offspring.

Conclusion: MTS‐induced IUGR leads to a long‐term increase in lung compliance in female rats. We previously showed that increased compliance in male sIUGR rat pups is accompanied by decreased elastin mRNA variant expression. We therefore speculate that MTS‐induced increased compliance in female rat pups will be accompanied by decreased elastin mRNA variant expression.

Grant Funding Source: Supported by the APS STRIDE Fellowship Award
Profound lung remodeling in heart failure, a poorly understood phenomenon with a significant impact (715.6)
Authors: Arkell Alicia , Foster Andrew , Platt Mathew , Dadgar Kayla , Murrant Coral , Simpson Jeremy
Published: June 2021

Exercise intolerance, characterized by dyspnea and early onset of exertional fatigue, is a chief complaint among patients with heart failure (HF). Limb muscle dysfunction is a known contributor to exercise intolerance, however, this phenomena does not explain dyspnea. Lung remodeling has been reported in end‐stage HF patients which could contribute to dyspnea and exercise intolerance, underscoring the importance and urgent need to better understand its role in HF. Our aim is to characterize the development of lung remodeling and its impact on exercise intolerance in HF. We hypothesize that lung remodeling occurs early in the development of HF, causing exercise‐induced hemoglobin (Hb) desaturation, affecting exercise performance. Methods: Male CD1 mice were subject to either transverse aortic constriction (TAC) or sham surgery. Exercise tolerance and Hb saturation were measured during a graded exercise test. The animals were sacrificed and lungs excised and fixed for histological evaluation. Results: Time to exhaustion was decreased by 40% in TAC. Hb saturation in TAC decreased during exercise while sham remained unchanged (p<0.05). Hb levels were similar between sham and TAC animals. Lung compliance was significantly reduced in 9 week TAC compared to sham (p<0.05), which further declined by 18 weeks (p<0.01). Histological assessment revealed significant remodeling including a 43% increase in fibrosis (p<0.01), marked inflammation and distended alveoli. Conclusions: Lung remodeling plays a significant yet under acknowledged role in exercise intolerance in HF patients.

Grant Funding Source: Ontario Thoracic Society
Three conserved domains in the 3’UTR region of αENaC mRNA modulates the stability of the transcript in alveolar epithelial cells (716.1)
Authors: Migneault Francis , Pascariu Mihai , Laperle Jonathan , Dagenais André , Berthiaume Yves
Published: June 2021

The epithelial sodium channel (ENaC) plays an important role in the alveolar epithelium by mediating Na+ reabsorption, an essential process for resolution of pulmonary edema in acute respiratory distress syndrome (ARDS). Several proinflammatory conditions such as TNF‐α leads to a reduction of ENaC expression and activity that contributes to development of ARDS. To study how pro‐inflammatory conditions could modulate αENaC mRNA stability, we developped a Tet‐Off model that allows the specific inhibition of αENaC mRNA expression in presence of doxycycline. Using this model, we found that the half‐life (T1/2) for the transcript was about 1 hr. TNF‐α, a proinflammatory cytokine found in broncho‐alveolar lavage of ARDS patients, decreased T1/2 to 15 min, suggesting that destabilisation of αENaC transcript plays an important role in downregulation of ENaC expression in pro‐inflammatory conditions. The 3’ untranslated region (3’UTR) of a mRNA plays an important role in modulating mRNA stability. αENaC mRNA 3’UTR is very long (~30% of the transcript). Using a bioinformatic approach, we could define three domains (D1, D2, D3) able to form secondary structures that were highly conserved in different species. Alveolar epithelial cells were co‐transfected with 3’UTR deletion mutants inserted in pTRE‐tight vector along with the Tet‐Off vector. Deletion of the distal part of 3’UTR (D3) leaded to stabilization of the transcript with a T1/2 of 2.6 hrs. A longer deletion to remove also D2 showed a T1/2 of 4.6 hrs, while deletion of D1 decreased the half‐life to 0.5 hr. These results showed that the conserved domains have stabilizing (D1) and destabilizing (D2, D3) properties on αENaC mRNA expression. The hairpin structure of these domains could recruit RNA‐binding proteins responsible for modulation of αENaC mRNA stability. Altogether, our results suggest that modulation of αENaC mRNA stability via its 3’UTR could play an important role in regulation of ENaC expression during pro‐inflammatory conditions.

Grant Funding Source: Supported by FRSQ, CFC and CIHR.
ENaC activity is decreased in alveolar epithelial cells from protein kinase C‐α knockout mice (716.2)
Authors: Eaton Amity , Bao Hui‐Fang , Yue Qiang , Eaton Douglas
Published: June 2021

ENaC open probability (Po) requires the association of ENaC with anionic phospholipids like PIP2. Protein kinase C is also alters ENaC activity, but the mechanism is unclear. Recently we showed that PKC can affect ENaC through phosphorylation of myristolyated‐alanine‐rich‐C‐kinase‐substrate protein (MARCKS). MARCKS acts to sequester PIP2 in close proximity to ENaC in the membrane and phosphorylation of MARCKS by protein kinase C (PKC) causes MARCKS to dissociate from the membrane. Without MARCKS in the membrane, PIP2 no longer associates with ENaC and ENaC Po decreases. To test whether PKC‐α influences ENaC activity in the lung, we examined ENaC regulation in a PKCα‐/‐ murine model. Primary cultures of alveolar type II cells were prepared from wild‐type and knockout animals, and were subjected to cell‐attached patch clamp. In PKC‐α KO, ENaC Po was significantly less (0.100 ± 0.0241, n = 18) compared to wildtype SV129 mice (0.200 ± 0.0310, n = 28, p = 0.025). The number of channels per patch (N) also decreased. Evan’s blue assays showed a two‐fold decrease in ENaC‐mediated fluid uptake in the KO mice, while wet‐dry assays also showed a decrease in ENaC activity in the KO animals. To confirm this decrease, a lung from each animal was perfused with biotin and membrane protein pulled down with streptavidin. While total ENaC protein did not change in PKCα‐/‐ mice compared with wildtype, membrane localization of all three ENaC subunits decreased. Using confocal microscopy, we observed an increase in reactive oxygen species (ROS) in the alveolar type II cells of PKCα‐/‐ mice. The increased ROS stimulated PKCδ which phosphorylatyed MARCKS in lieu of PKCα and decreased ENaC in Po in the knockout animals.

Grant Funding Source: Supported by NIH
Network of medical school educators for best practices in case‐study Problem Based Learning (719.7)
Authors: Packer C
Published: June 2021

Medical schools are reforming curriculum to integrate all competencies throughout all years. Interdisciplinary student‐centered case‐study PBL curriculum should result in students: 1. Gaining proficiency and understanding the process in clinical problem solving. 2. Recognizing knowledge gaps, learning to use reliable information, applying new knowledge. 3. Interacting effectively with peers, demonstrating professionalism appropriate to physician/patient interactions. Many schools introduced variations into case‐study content and PBL format and produced numerous assessment tools. Accordingly, the project objective was to form a collaborative network of medical school PBL case‐study teachers to share best strategies in facilitating PBL and develop/revise assessment tools to better address student engagement, learning outcomes, sense of value of PBL. Visits to Indiana University School of Medicine Centers of Medical Education in Terre Haute, Lafayette, Gary, and to University of Louisville and Northern Ontario School of Medicine were conducted. Materials and ideas were also shared electronically. Results include establishment of a collaborative network, sharing best practices and curricular materials, initiation of collaborative projects. An outcome of this work will be a model for other schools who wish to adopt/reform a PBL case study course or curriculum.

Grant Funding Source: Supported by an APS Teaching Career Enhancement Award (TCEA).
Brainstem 101: an interactive neuroanatomy application for iOS Platform (720.7)
Authors: Zebarjadi omid , Amirnovin Ramin , Ehrlich Cameron , Puder Barbara
Published: June 2021

Brainstem101, is an innovative computer application that combines art and neuroanatomy ‐‐ brainstem structures, functionalities and clinical highlights‐‐ together to create an interactive learning module for medical students and professionals. The Application features an atlas of human brainstem that presents 9 horizontal Cross‐Sections (CSx) along with an index of structures. Selection of a CSx will provide a list of its structures, while selection from the index displays the structure in CSx(s) where present. Each CSx can be viewed individually in the “Profile Page”, where one can select a structure and read about functions and clinical relevance. The application allows user interaction not only by tapping to select the structures of a CSx, but also by allowing the user to put any CSx onto a “Draw Pad” where he can take notes/collaborate by writing on top of the diagram.

Another major feature is the “Clinical Page”; this component provides a list of syndromes to choose from, with a detailed description of the syndromes' features (and structural origins) and artfully displays the physical manifestations of that syndrome on a human model. The associated vascular compromise is outlined on a 3D brainstem model.

Other features include “Quiz Mode”, radiology imaging, search function, and supplemental schematics.

Grant Funding Source: none
Advanced dissection techniques: an elective for 4th‐year students to revisit anatomy, prepare for residency, and aid 1st‐year medical students beginning anatomy (721.1)
Authors: Viscomi Natalie , Saulog Alison , Bradley Melissa , Volkman Emma , Campbell Meadow , Clough Rich
Published: June 2021

Our advanced dissection techniques course is designed for 4th‐year students to review and gain more advanced anatomy training especially for students embarking on “anatomy‐intense” residency programs such as surgery, physiatry, obstetrics & gynecology, urology and radiology. The duration of the electives are typically two weeks and they are specifically tailored to the students interests. The dissections conducted by the 4th‐year students are structured and targeted and not all inclusive as were the dissections performed during their first year of medical school. The elective dissections are designed to introduce new approaches, angles, and ways to see relationships between anatomical structures. These dissections, and expositions by the senior students, are also used to supplement the learning of anatomy for our first year students. The opportunity for first‐year students to see specialized anatomy, as presented in the electives, provides a unique exposure otherwise unavailable during the first year. This elective has a two‐fold benefit: it improves confidence in anatomy for 4th‐year students embarking on residency and these dissections are used as valuable teaching aids for the first year curriculum.
Peer teaching in gross anatomy: reducing contact hours while maintaining student learning (721.12)
Authors: Dunham Stacey
Published: June 2021

In an effort to reduce course contact hours in Gross Anatomy Lab at Indiana University Bloomington, students now dissect in every other lab and peer teaching is used to ensure all students are familiar the entire dissection. In traditional dissections, students were present for every dissection. This study examines the peer teaching process to determine if it is as effective or more effective for student learning as traditional lab dissection. Interviews were conducted with individuals (i.e. instructors and select students) who have experienced both peer teaching and traditional dissection in lab. Interviews revealed both advantages and disadvantages to the use of peer teaching in lab; however, instructors and students indicated an overall preference for peer teaching. Additionally, a survey was completed by current medical students examining their perceptions of peer teaching. Inclusion of peer teaching has had positive benefits in the gross anatomy lab, but additional research concerning best peer teaching practice is necessary.
Gender differences in opposite dissection group peer feedback among interprofessional allied health students in the gross anatomy lab (721.20)
Authors: Chen Vince , Martin Charys , Nichols Carol , Edmondson Anna
Published: June 2021

Giving professional feedback is uncomfortable, but the small group setting in the anatomy lab provides an ideal environment to develop these skills. Lab groups alternate dissections, and allied health (physician assistant, occupational & physical therapy) students provide feedback to the opposite lab groups based on interactions & dissection reviews. To assess the narrative feedback given by allied health students to peers in opposite lab groups, feedback comments were quantified and sorted into themes and divided by gender. Most comments were positive; the majority associated with themes involving professionalism & leadership skills (48%), followed by knowledge (35%) & personal behaviors (5%). Similar results were seen with constructive criticisms. Females left more comments than males (4.03 vs. 2.89 per student) and emphasized knowledge more in both positive (38% vs. 27%) & constructive (40% vs. 29%) comments than males. Males focused more on professionalism/leadership skills in their constructive criticisms compared to females (71% vs. 59%). Some of these differences may be attributed to the fact that males gave more generic & neutral comments than females (18% vs. 8%) and that there were more females enrolled in the course (93 vs. 35). However, these differences may also be attributed to intrinsic traits amongst genders, including detail‐oriented skills, thoroughness, and insightfulness.
Use of heart rate monitors to assess student engagement in lecture (721.25)
Authors: Darnell Diana , Krieg Paul
Published: June 2021

Some years ago, Bligh carried out a series of classroom lecture studies showing that student heart rates decreased over the course of a 50‐minute lecture (Bligh 1972, 1998, 2000). The decline in heart rate was interpreted as a measure of decreasing arousal, which is one component of engagement. In addition, Bligh reported a single event where a question from a student resulted in elevation of heart rate in observers. It appears that these studies are unique in using a physiological readout as a measure of student engagement or attention in the classroom. We plan to repeat and extend this study using current technology in interactive lecture, allowing more students to participate, more data to be collected, and additional analysis of the impact of interruptions and activities.

We propose that lightweight heart rate monitors with wireless connectivity (Mio Alpha; resembling fat wrist watches), may be used to measure student engagement in the classroom setting. In preliminary studies using seven monitors worn by medical student volunteers, we have replicated Bligh's observations and observed, 1) a decrease in average heart rate between across a 50‐minute lecture class, and 2) a temporary increase in heart rate in response to student questions. In addition, we have observed that brief pair‐share sessions (an interactive learning method) resulted in elevated average heart rate. Results will be presented from analysis of heart rate data from more than 20 lecture classes. The long‐term objective is to use heart rate to assess the relative effectiveness of different interactive and non‐interactive teaching methods to increase student engagement.

Bligh DA. What’s the Use of Lectures? Jossey‐Bass Publishers, SF (2000). Or Intellect Books (1998). Originally published in 1972.

Grant Funding Source: Supported by UA AMES Education grant
Understanding the temporal bone and its internal pathways through an animated diagrammatic approach (721.3)
Authors: Chou Benjamin , Nielsen Mark , Morton David
Published: June 2021

Understanding the topography of the cranial nerves within the temporal bone is a challenge for both medical students and residents. Unlike most other areas of anatomy, this small, confined boney space is not easy to dissect. This makes it difficult to study and to visualize its internal anatomy. Students are then left to wordy textual descriptions and accompanying illustrations, which can be very excellent but also very confusing for the beginning learners. To address this problem, we created a temporal bone tutorial that demonstrates all the cranial nerve pathways and temporal bone structures on a simplified three‐dimensional illustration. The entire presentation is focused on the three‐dimensional spatial orientations of the tympanic cavity within the temporal bone and the topography of the cranial nerve pathways through the temporal bone. Student feedback has been very positive and most students respond that this helps them better understand this difficult region of anatomy.
Tetris as homework: does videogame training improve spatial anatomy comprehension? (725.4)
Authors: Labranche Leah , Johnson Marjorie , Allman Brian , Nguyen Ngan
Published: June 2021

Spatial ability, particularly spatial visualization (Vz), is a significant predictor of success in human anatomy. There is evidence that Vz can be improved through training with videogames, such as Tetris. The present study investigates the relationship between videogame training, Vz, and visuospatial anatomy comprehension. Participants (n=27) completed the Mental Rotations Test (MRT) and the Spatial Anatomy Task (SAT) in order to assess baseline levels of Vz and visuospatial anatomy comprehension, respectively. According to MRT scores, the participants were semi‐randomized into a Control (n=12) or Training (n=15) group, with both low‐ and high‐Vz individuals in each group. Participants in the Training group played five, one‐hour sessions of Tetris over five consecutive days. At least one week after baseline testing, all participants again performed the MRT and SAT. Participants in both the Control and Training groups showed significant improvements on their post‐MRT and SAT; however, contrary to our hypothesis, videogame training did not improve Vz and visuospatial anatomy comprehension beyond that observed in the Control group. Moreover, this improvement was independent of participant sex differences. Additional subjects are being recruited to help us further explore the potential utility of videogame training for anatomically‐demanding fields.
Identification of the efferent neurons to the lagena of the chick (726.5)
Authors: Mahmoud Amany , Maklad Adel
Published: June 2021

The efferent neurons of the inner ear are part of a descending pathway from the hindbrain. Although the efferent axons innervate sensory epithelium, the inner ear efferent neurons are derived from the motor column. To date, some problems about the anatomy and function of the efferent vestibular system are still unsolved. In birds, efferent cochlear neurons lie in two areas in the brainstem a dorsal group medial to the facial motor nucleus and a ventral group medial to the lateral superior olive. However, the location of the vestibular efferent neurons in birds is still unclear. The body of work on this point found that the vestibular efferent neurons are one group located medial to facial motor nucleus. However, in one study the vestibular efferent neurons lie in two main groups as the auditory efferent neurons. To solve this problem we implanted Neuro Vue Red (lipophilic tracer) in the macula lagena of post fixed E 19 chick. We found that the efferent vestibular neurons in the chick lie in two nuclei, the dorsomedial nucleus located medial to the facial motor nucleus and the ventrolateral nucleus located medial lateral superior olive. Our results suggest that the vestibular and auditory efferent neurons are developed from a single nucleus in the hindbrain
Turning selfish proteins into programmable genome editors (733.2)
Authors: Richter Jazmine , Shen Betty , Lambert Abbie , Stoddard Barry , Kaiser Brett
Published: June 2021

Though no suitable methods for genome editing yet exist, the most promising approaches utilize targeted nucleases and take advantage of a cell’s natural repair processes that either disrupts the cleaved sequence or repairs it using a donor DNA template. LAGLIDADG Homing Endonucleases (LHE’s) are naturally occurring selfish proteins with two key features ideal for genome editing: recognition of a long DNA target site (~22 bp) and double‐stranded cleaving activity. Originally thought to be impossible to engineer due to their size and complexity, recent approaches based on experimentally determined structural models to direct the bioengineering process have proven successful. The aim of my research project was to solve the three dimensional structures of various LHE’s using x‐ray crystallography. Using E. coli‐expressed proteins, I solved the structures of two LHE’s (I‐GpeMI and I‐CpaMI) bound to their target DNA. With these structures we are able to identify DNA‐interacting amino acids and focus our engineering efforts of the enzyme scaffolds towards new DNA target sites.

Grant Funding Source: Murdock Trust
Tension characteristics of the stress‐relaxed collagen matrix (733.3)
Authors: Vaughan Melville , Spencer Chelsea
Published: June 2021

The stress‐relaxed collagen matrix is a simple yet appropriate experimental model to study the myofibroblast of wound healing and pathological contractures, because cells are able to generate forces necessary to stimulate myofibroblast differentiation. Although the model has been used for a number of years its tensile properties have not been fully characterized. Our goal was to demonstrate tension maturation on a daily basis, determine the maximum tension generated by an individual cell culture, and determine what problems are associated with long‐term culture. Human and rat fibroblasts were cultured within type‐1 collagen matrices; we indirectly quantified tension by measuring the contraction of the matrix after release from the dish at various times after setup. We plotted a line graph of time versus area. We observed a characteristic curve composed of two slopes, one due to stress fiber‐related contraction and a subsequent slope related to tractional‐force diameter reduction. Over time the ratio of contraction versus tractional force increased to a maximum; at a designated time beyond the maximum, the ratio decreased, suggesting a reduction of tension related to failure of the matrix. The failure may have been caused by attachment slippage or migration of cells out of the matrix. The contraction/tractional force ratio can be used to determine the tension maturity within a given experiment. Investigators using this model will likely need to determine whether this matrix can support the cell’s maximum tension generation before mounting a large‐scale study.
Encapsulation of biological stains for drug delivery imaging and microscopy in dentin tubules (733.6)
Authors: Lau Michael , Haseeb Ridwan , Montagner Francisco , Rodrigues Danieli
Published: June 2021

Encapsulation of Biological Stains for Drug Delivery Imaging and Microscopy in Dentin Tubules
Michael Lau1, Ridwan Haseeb1, Francisco Montagner2, Danieli Rodrigues1: 1Department of Bioengineering, University of Texas at Dallas, Richardson, TX; 2Department of Conservative Dentistry, Federal University of Rio Grande do Sul, Brazil; Introduction: Bacteria are able to remain into dentin after the root canal disinfection. Drug penetration into the tubules can be a key component for infection control. This study uses the synthesis of poly(ethylene glycol)‐block‐poly(L‐lactide) PEG/PLA encapsulated biological stain particles to characterize the penetration depth of drug delivery systems with Confocal Laser Scanning Microscopy (CLSM).

Methodology: Encapsulation of the biological stain was done with an oil‐water emulsion‐evaporation technique. The PEG/PLA copolymer and the stain were dissolved in the oil phase while the surfactant was dissolved in water. The oil phase and water phase was combined and emulsified then stirred. The particles were centrifuged, washed, and lyophilized. Characterizing the particle size and fluorescence was done with CLSM (VK‐X200 Keyence Laser Scanning Microscope).

Results: The microscopy has shown particle formation in the 0.3‐1.0 m range, which depended on the method of emulsification: homogenizing or sonication. CLSM revealed that the particles fluoresce under light with 525‐605 nm wavelengths.

Conclusion: The encapsulation technique effectively encapsulated the biological stain to penetrate the dentin tubules. With particle size similar to dentin morphology, the encapsulated stain proliferation into the dentin tubules will model dentin penetration of drug delivery systems of similar size.
Three distinct pathways for double‐strand break formation at palindromic repeats in yeast (735.10)
Authors: Sheng Ziwei , Saini Natalie , Haber James , Lobachev Kirill
Published: June 2021

Palindromic DNA sequences capable of forming cruciform and hairpin structures are strong hot‐spots for chromosomal rearrangements in a variety of organisms including humans. Previously, we demonstrated that palindromes induce chromosomal instability by triggering double‐strand breaks (DSBs) in yeast. In this study, we uncovered three mechanisms for DSB formation at palindromic loci.

In E. coli and S. pombe, the SbcCD and Rad23‐Rad50‐Nbs1 complex were implicated in metabolism of palindrome during replication. On the other hand, the S. cerevisiae homolog Mre11/Rad50/Xrs2 (MRX) complex does not induce DSBs at the Alu quasi‐palindrome. We found that the perfect palindromes, compared to quasi‐palindrome, create a more prominent replication barrier and at the same time, are subject to MRX attack. Mus81‐Mms4 generates breakage in mrx mutants at palindromes formed by two URA3 genes but not at other perfect palindromes. The fact that hairpin‐capped DSB formation is not eliminated in mrx or Δmus81 mutant implies the existence of an alternative pathway for chromosomal breakage at palindromic loci. We developed a system that allows creating a quasi‐palindrome in a controllable manner throughout the cell cycle. The advantage of this system is that the pre‐existing population of DSBs is absent therefore allowing the visualization of DSBs in each cell cycle stage. Surprisingly, we found that, break formation is tightly regulated and occurs mainly in the G2 stage. We are using this system to examine the roles of cell cycle kinases and the other known structure‐specific nucleases in the generation of hairpin‐capped breaks.
A possible role for adenosine monophosphate‐activated protein kinase in regulating tubulin acetylation (739.10)
Authors: Rand Sarah , Lawrence Kayla , Danowski Barbara
Published: June 2021

Microtubules (MTs) play important roles in cell motility, mitosis, and intracellular vesicle transport. These functions are regulated by microtubule associated proteins and/or tubulin posttranslational modifications, which can alter the structure and dynamics of the microtubules. Prior research showed that ATP depletion results in microtubules that are stable to the effects of MT‐depolymerizing drugs. The stable microtubules were also shown to be enriched in detyrosinated tubulin, but no information existed on other tubulin posttranslational modifications. We investigated whether microtubules in ATP‐depleted C3H10T1/2 mouse fibroblasts are more heavily acetylated, and if so, whether the acetylation is dependent on the activity of Adenosine Monophosphate‐Activated Protein Kinase (AMPK). We found that depleting cells of ATP by treatment with sodium azide and 2‐deoxyglucose resulted in a substantial increase in acetylated tubulin, as seen by western blot analysis and immunofluorescence microscopy. Most but not all stable microtubules showed tubulin acetylation. Since various energy stressors are known to activate AMPK, we next ascertained by western blotting that ATP‐depletion led to an increase in phosph‐AMPK, the active form of the enzyme. Cells treated with the AMPK inhibitor, Compound C, prior to ATP depletion showed less tubulin acetylation than cells treated with ATP‐depleting drugs alone. These results suggest a link between the level of tubulin acetylation and the metabolic state of the cells.
Sumoylation of the transcription factor Nrf2 (739.3)
Authors: Walters Treniqka , Arinze Ifeanyi
Published: June 2021

The transcription factor Nrf2 induces transcription by binding to the antioxidant response element(s) in target gene promoters, enabling oxidatively stressed cells to mount a response to oxidative stress in order to restore redox homeostasis. To act as a transcription factor, Nrf2 must be separated from its inhibitory protein Keap1. Post‐translational modifications (PTMs) that mediate separation of Nrf2 from Keap1 have been extensively studied but PTMs that impact its biology after this separation are just beginning to emerge. Using in vitro sumoylation assay and other methods, we demonstrated that Nrf2 is a SUMO‐targeted protein [Malloy et al. (2013) JBC 288, 14569‐14583]. Here, we have used three SUMO site‐predicting algorithms (SeeSUMO, SUMOsp and SUMOplot) to locate putative SUMO‐binding sites in mouse Nrf2. Three of the fifteen putative sumoylation sites identified by these algorithms have high probability scores on all three prediction algorithms. Using site‐directed mutagenesis, Co‐IP, and in vitro sumoylation assays, we investigated which of these sites is/are authentic for SUMO conjugation. We also assessed whether sumoylating Nrf2 impacts its transcriptional activity.

Grant Funding Source: Supported by NIH grants SC1CA143985, 5T32 GM07628‐34 and CTSA award No.UL1TR000445 from the NCATS.
Profiling the O‐GlcNAcylation of the kinome using kinase microarrary (739.8)
Authors: Han Guanghui , Neiswinger Johnathan , Hardiville Stephan , Sakabe Kaoru , Zhu Heng , Hart Gerald
Published: June 2021

Mutations and dysregulation of kinases play causal roles in human disease development, signaling, and metabolism. Understanding the function of kinases is of an outstanding interest for biomarker discovery and also necessary for the development of agonists and antagonists for the use in disease therapy. O‐linked beta‐N‐acetylglucosamine (O‐GlcNAcylation) is a post‐translational modification known to regulate different aspects of a proteins function including localization, activity, and stability. Like phosphorylation, O‐GlcNAcylation, which modifies serine and threonine residues on nuclear and cytoplasmic proteins, is a ubiquitous, reversible process that regulates numerous cellular processes. Recent evidence indicates that site‐specific crosstalk between O‐GlcNAcylation and phosphorylation and the O‐GlcNAcylation of kinases play an important role in regulating cell signaling. Hence, it is very important to investigate the O‐GlcNAcylation of the kinome. Previously, we identified 42 kinases that are substrates for O‐GlcNAcylation using an in vitro OGT assay with [H3] radiolabel on a functional kinase array. However, using [H3] has serve senstitive limiations suggesting a more sensitive approach is needed to probe large libraries of proteins. Herein, using a novel kinase microarray coupled with an in vitro OGT labeling assay with immuno fluorophore detection technique, we report a simple, yet sensitive, strategy by which this method can be used to profile the entire kinome for O‐GlcNAcylated proteins.

Grant Funding Source: NIH R01CA42486, R01DK61671; N01‐HV‐00240; P01HL107153, R24DK084949
ARE‐mediated posttranscriptional regulation of human IL‐3 in T cells (749.1)
Authors: Gonzalez Pablo , Hernandez Marimar , Gonzalez‐Feliciano Jose , Gonzalez Carlos
Published: June 2021

Interleukin‐3 (IL‐3) is a lymphokine that supports a broad range of hematopoietic cells and is involved in the body’s inflammatory response. Aberrant expression of this lymphokine is implicated in chronic inflammation and several types of cancer. IL‐3 is a member of a class of transiently expressed mRNAs that harbor Adenosine/Uridine‐Rich Elements (ARE) in their 3’‐Untranslated Regions (3’‐UTR). Previous results from our laboratory suggest a regulatory role for the IL‐3 AREs. We identified four different ARE clusters within the IL‐3 3’‐UTR. To understand the role of these sequences in translational regulation, we conducted site‐directed mutagenesis to interrupt each ARE cluster. Firefly luciferase reporters harboring different mutations in the ARE clusters were transfected into HeLa and Jurkat cell lines. The luciferase chimeras lacking the IL‐3 ARE region showed an increase in luciferase activity, suggesting a loss of translational repression. The chimeric reporter with a mutated ARE3 cluster, which is a nonamer class ARE, appears to be mainly responsible for the 3’‐UTR exerted repression. We also investigated the possible role of the 3’‐UTR in the interaction between the IL‐3 mRNA and ribosomes via polysome profile analysis. These results showed a reduced ribosomal interaction in chimeras harboring the IL‐3 3’‐UTR, suggesting that the ARE can inhibit the translational capability of the IL‐3 transcript.

Grant Funding Source: Supported by U54 CA96297, PR‐LSAMP, UPR‐MSC and RISE 2R25GM61151
Novel adenosine/uridine‐rich element‐binding proteins recognize interleukin‐3 ARE in leukemia T cells (749.4)
Authors: Martinez Marina , Gonzalez Jose , Lopez Armando , Gonzalez Carlos
Published: June 2021

Human Interleukin‐3 (hIL‐3) is a cytokine that promotes myelopoiesis, differentiation of macrophages and granulocytes. Aberrant expression of this lymphokine has been associated with several hematological cancers. IL‐3 3’‐UTR harbors Adenosine/Uridine‐Rich Elements (AREs) involved in its post‐transcriptional control. These regulatory sequences are recognized by specific ARE‐Binding Protein (ARE‐BP) complexes. Previous results from our laboratory estimated five ARE‐BP complexes from 34 to 88 kDa binding to the hIL‐3 ARE using UV‐crosslinking assay. Our goal is to identify novel ARE‐BPs that mediate the post‐transcriptional regulation of hIL‐3. To achieve this goal, RNA affinity purification coupled with MS/MS analysis was performed. Also, an immunoblot analysis to confirm the presence of specific proteins was carried out. These results identified ~40 proteins between 12 to 82kD interacting with the hIL‐3 ARE in acute lymphoblastic leukemia cells. MS/MS results in accordance with previous results from our laboratory showed that HuR and p42hnRNP C1/C2 are components of the ARE‐BPs that recognize the hIL‐3 ARE. In addition, our results identified KSRP and SAM68 as novel hIL‐3 ARE‐BPs. Besides, we found that p45AUF‐1 protein interacts with hIL‐3 ARE. Taken together, these data imply that novel ARE‐BP complexes can play an important role in the IL‐3 post‐transcriptional regulation. Ultimately, elucidating the role of these ARE‐BPs in IL‐3 expression can provide new insights about the ARE‐mediated post‐transcriptional regulatory pathway and blood cancer.

Grant Funding Source: MBRS‐RISE Program R25GM061838, UPR‐PES Intitutional funding
Small RNA regulation of a multidrug efflux pump (750.1)
Authors: Parker Ashley , Gottesman Susan
Published: June 2021

Multidrug efflux pumps are a major mechanism used by bacteria to export drugs and toxic compounds from the cell. One of the most important efflux pumps in E. coli is the AcrAB‐TolC system. In E. coli and among other enteric organisms, acrAB and tolC deletion mutants are known to be hypersensitive to antibiotics, and very little has been done to investigate regulation at the post‐transcriptional level of these efflux genes. Small ribonucleic acids (sRNAs) are known to be major post‐transcriptional regulators in bacteria, they usually enhance or repress translation by binding to the 5’ untranslated region (UTR) of mRNA targets with the help of a small chaperone protein, Hfq. In this study we hypothesized that Hfq‐dependent sRNAs might regulate acrA, acrB, and/or tolC. Translational fusions of the 5’ UTR and start of the open reading frame for each gene with lacZ were constructed and transformed with a sRNA plasmid library to test for sRNA regulation, measured by beta‐galactosidase assays. RyeB, a sRNA expressed during stationary phase, was found to repress the expression of tolC, which encodes the outer membrane protein of many multidrug resistant efflux pumps. Compensatory mutants were used to validate that the repression was by direct base pairing, upstream from the ribosomal binding site. We have confirmed by Western blot analysis that RyeB regulation of tolC at the mRNA level leads to a decrease in TolC protein levels. Our results suggest that the decreased amounts of TolC in stationary phase is due to the increase in expression of RyeB, and this may limit efflux of some compounds under these conditions.

Grant Funding Source: National Institutes of Health, Intramural Research