bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of Gaze Tracking and EEG to Measure Short‐ and Long‐term Learning by Medical Gross Anatomy Students
Authors: El‐Shaar Ala'a , Nagle Corinne , Tat Michael , Zumwalt Ann , Budson Andrew
Published: June 2021

The goal of medical educators is to teach their students in a manner that is effective for long‐term, accurate knowledge retention, but measurement of long‐term retention is difficult. Recent work by our lab has explored the use of gaze tracking to document and measure learning in medical gross anatomy students. In this study, we combine gaze tracking and EEG to examine knowledge retention by these students. Medical gross anatomy students (n=22) were asked to identify anatomical structures displayed on a computer screen immediately following the gross anatomy course and again 6 months after the course ended. In this experiment, the participants were instructed to visually fixate on the named structure of interest for more than 1 second, or to indicate uncertainty by fixating on the upper left corner of the screen. Immediately after the course ended, the students correctly fixated on the structures 70% of the time, incorrectly fixated 26.5% of the time, and indicated uncertainty 3.5% of the time. Preliminary results indicate that six months after the end of the course, the students' performance at this task had not diminished (67% correct, 26% incorrect, 7% uncertain). However, the speed with which the students made their final decision was significantly longer 6 months after the course ended. The average time it took to identify the structure was 2.22s immediately after the course and 3.0s at the 6 month follow up (p<0.001). These results indicate that 6 months after the end of the course, the subjects have solid knowledge retention but require more time to think before answering correctly. EEG data indicating subject recollection and familiarity with this material will also be reported.
The Influence of Spatial Ability on High and Low Order Anatomy Examination Questions
Authors: Phelps Taylor , Nguyen Ngan , Edmondson Anna , Martin Charys
Published: June 2021

Students with high spatial visualization ability (Vz) outperform students with low Vz in anatomy; yet, the influence of Vz on different Bloom's Taxonomy levels of multiple‐choice questions (MCQs) has not been established. This study aimed to assess the influence of Vz on different Bloom's Taxonomy levels of MCQs & whether studying anatomy improves Vz. It is hypothesized that students with high Vz will outperform students with low Vz on higher order MCQs. Allied health students (n=47) completed the Mental Rotations Test (MRT) to establish Vz. The mean Vz was 9.1±3.6, which divided students into high Vz (n=23; avg MRT=11.8±2.9) & low Vz (n=24; avg MRT=6.5±1.6) groups. The MCQs were classified into four Bloom's levels: knowledge, comprehension, application & analysis. Data indicate that students with high Vz (avg=87.6±6.1) outperform students with low Vz on MCQs (avg=82.1±7.6; p<0.05). Students with high Vz performed better on knowledge, comprehension, & analysis MCQs (p<0.05), while there was no difference on application MCQs. Upon completion of the course, students repeated the MRT; the overall mean Vz increased from 9.1±3.6 to 12.5±4.6 (p<0.05). Both the high & low Vz groups demonstrated improvement (high Vz: 11.9±3.0 ‐ 15.3±4.4; low Vz: 6.7±1.6 ‐ 9.9±3.1, p<0.05). Results suggest that Vz plays a role in MCQ performance in both high & low order Bloom's levels & that participation in anatomy can assist in training Vz.
Evaluation of the Impact of Peer Teaching within the Gross Anatomy Laboratory on Student Academic Performance
Authors: Wheeler Kelli , Martin Charys , Edmondson Anna
Published: June 2021

The gross anatomy lab provides an opportunity for small group peer teaching, enabling medical students to develop communication skills & enhance content understanding. Students are divided into teams that alternate dissections. Team members rotate leadership responsibilities for guiding peers through dissection & providing a review to the opposite group. This study assessed the impact peer teaching through team leading has on academic performance in anatomy. We hypothesized team leaders would outperform peers on written & practical exam questions related to sessions they led, & lower performing students would benefit more from peer teaching than higher performing students. Results showed team leaders significantly outperform non‐leaders on lab practical (χ2=60.2, p<0.01, ϕ=.03) and written exam (χ2=14.7, p<0.01, ϕ=.02) questions related to the lab they led. The class was divided into high & low performers based on final practical & written grades. On lab practicals, team leaders significantly outperformed non‐leaders in both subsets (High: 90% vs 87%; Low: 83% vs 77%), with a greater difference seen in the lower performing group. Lower performing leaders also significantly outscored non‐leaders on written exams(Low: 80% vs 76%). Results suggest peer teaching through team leading significantly impacts leader performance on lab & written exams and may be more beneficial to lower performing students.
Transition Journey: Conventional Teaching to Integrated Teaching in Third World Countries
Authors: Potaliya Pushpa , Ghatak Surjit
Published: June 2021

Introduction

Most of third world countries have started moving on from non‐integrated discipline based system where major focus is on didactic lectures, tutorials and subjective assessment with very less emphasis on interactions. To have better diagnostic abilities among medical students the ideal approach is the integration of basic preclinical subjects along with developing an understanding of correlating them with clinical cases.

Objective

1. To compare and evaluate integrated teaching with traditional education system.

2. To observe its effects on performance of students by an experiment model.

3. To evaluate how it can improve quality of medical education in developing countries.

Material and Method

Target learners were divided in two Groups A and B to undergo traditional Conventional and Integrated teaching respectively for a particular topic. Earlier whole lot was undergoing same conventional method for various topics. Pre and post session questionnaires were given to students. Faculty feedback was also considered and noted. Statistical analysis was done for obtained data.

Result

Both short and long term outcome of implementing such module will be discussed at time of conference. Along with that discussion would emphasize on its role in the medical education system of developing countries and difficulties encountered to implement it there.

Conclusion

By executing integrated system, a tremendous responsibility of the institutions to provide quality care to people can be achieved with ease and for long term in present scenario. This will definitely be a boon to the healthcare sector of third world countries.
Changing the course: Evaluation of a Novel Pedagogical Approach to Clinical Musculoskeletal Anatomy Education
Authors: Rascoe Alexander , Anderson Daniel , Black, MD Kevin , Lazarus, PhD Michelle
Published: June 2021

Musculoskeletal (MSK) complaints, including back pain, represent a large percentage of physician office visits. Despite the disease burden, current literature suggests physicians are not adequately trained to care for these conditions. Enabling future providers to address MSK complaints requires revising the current medical school curriculum.

Here we report a novel curriculum developed through the application of contextual learning theory and a targeted needs assessment. The course emphasizes clinical reasoning in the primary care setting and intra‐specialist communication through application of a “multi‐disciplinary” teaching approach. Throughout the course, students dissect cadavers, practice clinical reasoning skills and discuss surgical treatment options. Course participants improved by an average of 24% (p<0.05) on a validated MSK clinical anatomy free‐response examination; while a control group showed no change. Pilot data suggests this pedagogical approach results in a significant 6 month retention of the clinically applicable anatomy above that obtained by historical peers. This study demonstrates that vertical integration of basic science into clinical year medicine is an effective method for quantifiable knowledge improvement and retention. Moreover, the course design and implementation may serve as a blueprint for further vertical integration of basic sciences into clinical year medical education.
Use of a Rotating Gross Dissection‐Clinical Training Schedule to Incorporate Ultrasound Training in a Medical Anatomy Course
Authors: Ressetar Holly , Minardi Joseph , Palmer Bruce
Published: June 2021

Increased use of clinical ultrasound (US) has fostered its integration early in the medical curriculum. Our objective was to smoothly integrate fundamental hands‐on US training to over 100 students in a first year medical human structure course without overwhelming students or compromising anatomy study. For the past 2 years, we taught US as a pass/fail component to our combined gross anatomy/histology course. Students attended 6 US lectures and 6 lab sessions covering limbs, thorax/abdomen, pelvis/kidney and US procedures. US and gross anatomy labs coincided and a database system was used to make assignments, assuring that each dissection group of 5 students never had more than 2 team members simultaneously attending US lab. Dissection groups rotated 3 times a semester so it was essential to proctor students to ensure they adhered to assignment. US labs consisted of facilitator‐mediated hands‐on practice using standard patients. Appropriate assessment of US skills in a basic science course was challenging and raised the most student debate. Anatomy‐based case study exam questions that required US interpretation counted towards their final anatomy grade while a separate pass/fail grade was based on quizzes and final practical covering US theory and technique. Some students felt US material should have been assessed separately while others felt the time commitment warranted recognition as part of the anatomy grade. Year‐end surveys indicated that students enthusiastically accepted the extra time commitment and highly rated the US experience. It reinforced their gross anatomy learning and provided their first patient encounter.
Anatomy Outreach: Long‐term Investment in the Future of Medicine
Authors: Schindel Benjamin , Narcisse Patrick , Minko Elizaveta , Pagano Anthony , Marquez Samuel
Published: June 2021

SUNY Downstate Medical Center has one of the most diverse student bodies in the country, yet medicine today does not reflect this ethnic diversity. To ensure that underrepresented students receive exposure to the allied health fields, the Downstate Health Professions Recruitment and Exposure Program (HPREP) recruits 30 minority high school students from Brooklyn to attend lectures on physiology, perform anatomical dissections, and learn clinical skills, all facilitated by medical students. A five question pre‐ and posttest evaluating the different activities is given at every session to quantify student learning. The average pretest score was 27.33% (STD=1.711), rising to 80.74% in the posttest (STD=1.369), with an average improvement of 58.5%. The perceived value of the program as a critical confidence builder for students' long‐term career goals is demonstrated by students' active engagement and pertinent questions. The strength of the education outreach program lies with medical students leading the program using anatomy as the driver. The success of this anatomy outreach program lies not only in the short‐term gains for individual students but, more importantly, in its benefit to society in approximately a decade when the young students complete their educations and bring greater diversity to the allied health professions.
Impact of a Pre‐Course and Real‐Time Dissection Demonstrations on Student Learning in Veterinary Gross Anatomy
Authors: Sparks Cathryn , Taboada Joseph , Daniel Annie , McNulty Margaret
Published: June 2021

Currently, no veterinary school in the US requires anatomy as a pre‐requisite for admission into the professional program. As curricular changes devote less time to anatomy, instructors have begun supplementing or replacing dissections with multi‐media demonstrations. The purpose of this study was to compare the effectiveness of an anatomy pre‐course and dissection demonstrations on learning. A one‐week anatomy pre‐course was offered before start of classes to the incoming first year veterinary class at LSU. Students were placed in one of 3 groups that learned canine anatomy through 1) dissection only, 2) instructor demonstration via a live camera, and 3) a combination of short demos followed by dissection. At the end, students were given a written & practical exam. In addition, exam grades in the subsequent fall professional veterinary anatomy course were compared among the 3 groups and those who took the pre‐course vs. those who didn't. Material covered in the pre‐course was included in the 1st exam of the professional course, but only new material (not included in pre‐course) was in the 2nd exam. No significant differences were identified among pre‐course exam scores, however the average on the practical exam for dissection‐only group was 15% lower than the other groups, indicating that demonstrations enhance understanding of structures. However, students who took the pre‐course did significantly better not only on the first exam in the professional anatomy course, but on the second exam as well, indicating prior exposure to anatomy is very beneficial to student learning.
Impact of Clinical Anatomy League of University of São Paulo Medical School (FMUSP) on motivation and performance of anatomy learning
Authors: Iuamoto Leandro , Nogueira Thiago , Souza Braian , Ribeiro João , Hojaij Flávio , Akamatsu Flávia , Andrade Mauro , Jacomo Alfredo
Published: June 2021

Undergraduate medical students learn anatomy basically by textbooks, lectures and cadavers dissection. The Clinical Anatomy League of University of São Paulo Medical School (CAL‐FMUSP) is an initiative to strengthen the anatomy learning and to complement the knowledge acquired during graduation.

To describe the activities of the CAL‐FMUSP and analyze its impact on student's interest, motivation and learning.

Questionnaires were answered by the League's introductory course attending students (n=53). In a second moment (2014), a 20‐question exam was applied to assess students' ability to identify and describe important anatomical structures. 33 students agreed to take the exam. Fourteen (42.4%) were League members – intervention group – and 19 (57.6%) composed the control group. Also we compared student's baseline grades Anatomy Course.

Students' motivations in becoming a League member were: improve their anatomical knowledge (34.6%) and be able to understand the anatomy involved on surgical procedures (21.0%). The expectations on League's activities were: improve surgical technique and dissection on fresh cadavers such as fixate their knowledge (26.7%). Students' moderate interests on the League went from 29.4% to 14.0%, intense interest raised from 49.0% to 54% and very intense interest increased from 21.6% to 32.0%. In the exam‐based experiment, League members' performance on the exam was better in comparison to the control group performance (p = 0.044).

The CAL‐FMUSP with its course and activities stimulates medicine students' interest in anatomy and improves the anatomical knowledge fixation.
Dentition Influences Shape of Oral Jaws in Fish
Authors: Hammer Christine , Atukorala Atukorallaya Devi Sewvandini , Franz‐Odendaal Tamara
Published: June 2021

The interactions between bone and tooth development are poorly understood and largely understudied. Although the teleost lower jaw (mandible) has been described morphologically, this information has not been quantified and the upper jaw (premaxilla) has largely been ignored in the literature. The purpose of this study is to understand how jaw shape and tooth presence/absence correlate with one another. We describe the development of the jaw bones of two related teleosts, one with extensive dentition (Mexican tetra; Astyanax mexicanus) and the other without oral teeth (Zebrafish; Danio rerio). We collected a growth series for each species, used an acid‐free double stain to visualise the skeleton, and then analyzed samples using outline shape analyses. Differences between species were observed throughout growth. Variation in bone shape was detected along the occluding edge of the premaxilla, as well as the rostral and caudal most regions of the mandible. In the premaxillae, this variation was statistically significant between adult Mexican tetra and zebrafish, while for the mandible only the caudal region had significant shape differences. This study provides insights into the potential cross talk between bone and tooth development, providing essential information in the field of vertebrate anatomy and evolution. This research was funded by the Natural Sciences and Engineering Research Council of Canada.
Three‐dimensional Functional Morphology of the Australopithecus afarensis Fourth Metatarsal
Authors: Kuo Sharon , Kimbel William , Johanson Donald , Congdon Kimberly , Ward Carol
Published: June 2021

The presence of an inflexible foot with human‐like arches has been debated for Australopithecus afarensis based on fossilized footprints, and has been recently inferred from the first complete fourth metatarsal discovered for this species (A.L. 333‐160, Ward et al. 2011. Science 331,750‐753). Initial interpretation of this fossil was made on a limited comparative sample using two‐dimensional metrics that did not capture key functionally relevant aspects of metatarsal form. To further explore the functional morphology of this fossil, we compared articular surface orientation and surface topography of A.L. 333‐160 with that of a larger sample Pan troglodytes (n=26), Gorilla gorilla (n=17), and Homo sapiens (n=31) using novel three‐dimensional measurements of in silico polygonal models of fourth metatarsals. Humans differ from both chimpanzees and gorillas in torsion, convexity and orientation of the base, as well as convexity of dorsal and plantar portions of the head. A.L. 333‐160 falls within the range of humans in all measures. Discriminant function analysis cleanly separates humans from apes, and demonstrates that Australopithecus afarensis groups only with humans. These data support the hypothesis that the Australopithecus afarensis foot shared the stiff midfoot and lateral arch characteristic of later hominins.
The Post‐surgical Stability of the Nitinol Shape Memory Staple in Orthopaedics
Authors: Shenoy Abhishek , Gordon Stanley , Hayes Westley , Saha Subrata , Marquez Samuel
Published: June 2021

The Nitinol shape memory compression staple has been used in surgeries & its unique mechanical properties may strongly influence a fracture's post‐operative stability. This study examines the biomechanical behavior of Nitinol compression staples (Nts) assessing its efficacy over standard orthopaedic staples in bone fixation techniques in transverse scaphoid & humeral fractures. Bones were dissected, disarticulated & cleaned from 8 embalmed donor bodies. Sectioning of scaphoid & humeral samples (to mimic transverse fractures) was undertaken with subsequent realignment with embedded stapling. A set of four humeral mid‐shaft sectioning was performed yielding two pairs of bony segments, which were fixated with standard staple and the Nts with current applied to the staple before strength of staple tests were assessed. Results showed carpal bones too fragile fracturing before accurately measuring the staples' effectiveness under the Instron 8874 Servohydraulic machine loads of compression failure test. Nts used on the humeral sample, however, showed greater resistance to load of failure than standard staples lending support that compression staples in Orthopaedic surgeries may yield more favorable surgical outcomes. Our findings show promising potential but further studies on Nts are required to strengthen their favorability among methods of fixating small bone fractures.
Identifying Mechanisms and Evolutionary Implications of Mechanosensitive Growth in the Embryonic Limb Skeleton
Authors: Pollard Andrea , Charlton Bethany , McGonnell Imelda , Pitsillides Andrew
Published: June 2021

The developing skeleton experiences loading as a result of embryo movement. This is necessary for normal longitudinal growth of limb skeletal elements. Herein, we address the hypothesis that mechanical input can alter limb proportions by modifying growth.

We aimed to identify mechanisms which underpin altered growth in response to mechanical input and demonstrate that environmental factors such as incubation temperature can alter motility in oviparous species, altering limb proportions.

Embryonic chicks were immobilised with a neuromuscular blocker between day 10‐18 of development and growth monitored by repeat MRI scans. Control embryos were treated with vehicle only. Crocodile eggs were incubated at 28°C and 32°C and movement monitored throughout development. Embryos were euthanized at near‐hatching age and body size and limb proportions measured. Proliferation, hypertrophy and apoptosis, components of endochondral ossification, were evaluated in limb growth cartilage of all groups.

Growth was reduced by immobilisation predominantly in the metatarsals and femur in chicks. The distribution of proliferation was altered, volume of hypertrophic chondrocytes decreased (29% P<0.05) and apoptosis increased in these elements, suggesting that mechanical input is necessary to direct and regulate these processes. Temperature influences motility of embryonic crocodiles and alters limb proportion, with greater temperatures producing longer tibias and shorter digits. This may explain the variation in limb proportionality observed within some reptile species and our data suggest that plasticity in limb form during development may be evolutionarily advantageous.

This work is funded by the Anatomical Society.
Improving Spider Phylogeny Using 18S Ribosomal DNA Secondary Structure Modeling
Authors: Faso Vincent , Spagna Joseph
Published: June 2021

Spiders are key predators in most terrestrial ecosystems and produce silks and venoms, making them important in the fields of biomaterials and neuropharmacology. Despite their importance, the basal evolutionary relationships of spiders remain poorly resolved. The objective of this study is to gain better resolution and understanding of these relationships by examining individual genes more rigorously, using secondary structure models for alignment of individual ribosomal genes and assessing the self‐complementary pairs in a doublet model. This model was used to build a matrix of 18S ribosomal sequence from 37 spider taxa. The matrix contained 2,002 characters, 962 loop characters and 1,040 stem characters, which we analyzed utilizing a partitioned Bayesian analyses. We obtained three trees: a tree derived from the comparison of loop structures alone (under the GTR model), a tree derived from the comparison of stem structures alone (doublet model), and a combined‐data tree with both model partitions included. Recent genomic studies utilizing large numbers of protein‐coding genes have concluded that contrary to previous gene and morphological studies, orb weaving spiders are not monophyletic; these studies conclude that cribellate deinopoid orb weaving spiders are more closely related to non‐orbweavers than they are to araneoid orb‐weaving spiders. Our analyses using the doublet model, both combined and restricted to the single stem partition, support a more traditional grouping, with a single origin for all orb‐weaving spiders. This work was supported by the William Paterson University Assigned Release Time program.
The mRNA Binding Protein AUF1 Controls the Regenerative Potential of Activated Skeletal Muscle Stem Cells
Authors: Chenette Devon , Cadwallader Adam , Olwin Bradley , Schneider Robert
Published: June 2021

AU‐rich mRNA binding factor 1 (AUF1) is a promoter of mRNA decay. AUF1‐/‐ mice show loss of skeletal muscle mass and muscle weakness similar to limb girdle muscular dystrophy (LGMD). A mutation in a family cohort affected with LGMD was association‐mapped to the chromosomal location of human AUF1. We found that AUF1 levels increase in satellite cells following injury. We compared the rate of skeletal muscle regeneration between wild type (WT) and AUF1‐/‐ mice. 15 days following injury WT mice show repair while the AUF1‐/‐ mice show no regeneration. Mouse fiber culture studies show that AUF1‐/‐ satellite cells are unable to express the late stage myogenic regulatory factor, myogenin. We also observed a loss of Pax7, an early satellite cell marker, in AUF1‐/‐ satellite cells. This suggests that in the absence of AUF1 satellite cells cannot differentiate or return to quiescence. To understand the molecular role of AUF1, we completed studies using C2C12 cells. When AUF1 is partially silenced we observe delayed myogenesis due to reduced expression of Myogenin. The expression of nascent Myogenin was also reduced leading us to examine Myogenin regulating transcription factors. When AUF1 is partially silenced there is a 2.5 fold increase in Twist1, a Myogenin transcriptional repressor. Using RNA immuno‐precipitation we identified a direct interaction between AUF1 and Twist1 mRNA. This suggests that AUF1 mediated decay of Twist1 mRNA is crucial for the ability of activated satellite cells to express Myogenin and complete regeneration. Without AUF1 the satellite cell population maintains a “stem‐like” phenotype and depletes the quiescent population.
Characterization of the Oligomeric Transition of E. coli γ‐Glutamylcysteine Ligase
Authors: Wiese Elizabeth , Diederichs Kathryn , Kelly Brenda
Published: June 2021

Gamma‐glutamylcysteine ligase (γ‐GCL) is the enzyme that catalyzes the rate‐limiting step in the synthesis of glutathione. Glutathione is necessary for the survival of most organisms, as it functions to detoxify cells. In vitro, purified E. coli γ‐GCL transitions from a monomeric (47kDa) to a dimeric (88kDa) form over time. In order to study whether oxidation and reduction chemistry is important in the transition from monomer to dimer, native polyacrylamide gel electrophoresis was used to monitor transitions between each form. Transitions were induced through the addition of the reducing agents Tris(2‐carboxyethyl)phosphine (TCEP) and dithiothreitol (DTT), and the oxidizing agent ascorbic acid. The addition of DTT and TCEP results in the conversion from dimer to monomer in a concentration dependent manner; however, differences in migration of these induced oligomers suggests there are shape differences between the native and induced forms. A transition to a higher order oligomer is observed upon the addition of the oxidizing agent ascorbic acid. The characterization of each oligomer and the oligomerization process will be important to advance our understanding of this enzyme system and will inform in vivo and in vitro studies with E. coli γ‐GCL.
An Allosteric Network in LRH‐1 Mediates Phospholipid‐Driven Gene Regulation
Authors: Kohn Jeffrey , Musille Paul , Kossmann Bradley , Ivanov Ivaylo , Ortlund Eric
Published: June 2021

The orphan nuclear receptor (NR) liver receptor homolog‐1 (LRH‐1) has been adopted after the discovery that phospholipids (PLs) could bind to its ligand binding pocket (LBP) and drive the regulation of many genes involved in metabolism, steroidogenesis, cell cycle, and the unfolded protein response. The transcription or repression of these genes is contingent upon the properties of the bound PL: mid‐chain phosphatidylcholines such as DLPC are LRH‐1 agonists and have antidiabetic effects, while long‐chain saturated PCs and PLs with other head groups do not, despite binding to LRH‐1. Like other NRs, LRH‐1 regulates genes via the recruitment of transcriptional coregulators. The PL bound to LRH‐1 dictates its preference for coactivators or corepressors. Using the combination of X‐ray crystallography and molecular dynamics, we have identified an allosteric network that connects the mouth of the LRH‐1 LBP to the AF‐2 coregulator binding surface via helices 3‐6, which traverse the LBP. Clustering analyses reveal that the molecular motions between these two areas become coordinated upon the binding of an activating PL, stabilizing the AF‐2 to favor coactivator binding. In the apo state, or when bound to nonactivating PLs, corepressor binding is favored. Ligand‐coregulator disagreement weakens this network, disfavoring the binding of a coregulator whose activation or repression state does not match that of the ligand. This network allows PLs to finely tune the expression of entire gene programs that control many aspects of cellular and metabolic activity, and may be exploitable by small molecule therapeutics for the treatment of human diseases, including metabolic disorders and cancer.
Protective role of phosphatidic acid in the cellular response to lysophosphatidylcholine analogues
Authors: Ganesan Suriakarthiga , Fernandez Murray Pedro , McMaster Christopher , Zaremberg Vanina
Published: June 2021

The synthetic lysophosphatidylcholine (LPC) analogue edelfosine and the naturally occurring platelet activating factor (PAF) are structurally related lipids. Edelfosine is a potent antitumor drug, while PAF is a neurotoxic lipid that has been implicated in Alzheimer's disease. These LPC analogues are also toxic to budding yeast, which can be used as an excellent model system to investigate their mode of action.

LPC analogues induce alterations in membrane organization, lipid metabolism and signaling in yeast. Phosphatidic acid (PA) produced by the activity of PLD alleviates sensitivity of yeast cells to PAF and edelfosine. PA is a key lipid signalling and precursor of all glycerolipids. Recent lipid analysis indicates that diacylglycerol (DAG) accumulates in cells treated with LPC analogues and this could contribute to their toxicity. In fact, sensitivity assays in yeast genetically manipulated to alter PA and DAG levels clearly support a protective role of PA in the response to these lipid drugs. Altogether, the evidence suggests that cells are able to sense altered PA: DAG ratios induced by these metabolically stable lipids. While DAG binding proteins have not been clearly identified in yeast, several yeast proteins recognize PA with high specificity. One such protein is the transcriptional repressor Opi1, which regulates expression of phospholipid biosynthetic genes through its interaction with PA. We provide evidence supporting a role of Opi1 in sensing the membrane changes induced by edelfosine, including i) altered sensitivity of cells lacking Opi1, ii) prevalent nuclear localization and iii) Opi1‐dependent UASINO gene repression induced by edelfosine.
Phosphatidylinositol Phosphates Modulate STARD4 Sterol Transfer between Membranes
Authors: Iaea David , Maxfield Frederick
Published: June 2021

The steroidogenic acute regulator‐related lipid‐transfer (START) domain containing proteins are involved in several pathways of non‐vesicular trafficking of sterols. Among the soluble START proteins, STARD4 is expressed in most tissues and has previously been shown to transfer sterol. However, the mechanism of membrane interaction and sterol binding of STARD4 are unclear. The activity of two sterol transport proteins were found to be modulated by PI(4)P. This suggested a potential role of phosphatidylinositol phosphates (PIPs) to act as organelle‐specific signals that could target and modulate soluble sterol transporters such as STARD4. To investigate this, the anionic lipids of donor and acceptor liposomes were replaced with physiologic levels of individual PIPs, and STARD4 sterol transport activity was measured. Utilizing bulk phosphatidylserine (PS), STARD4 transfers 7 molecules of sterol/STARD4/minute. Interestingly, we found that PI(4,5)P2, only in donor membranes, and PI(5)P or PI(3,5)P2, only in acceptor membranes, increase STARD4 activity 10‐fold to 1 molecule of sterol/STARD4/second. The sterol transfer rate for STARD4 is relatively slow from 0‐1.5 mol% PIP, with an increase in sterol transfer rate that is maintained from 2‐10 mol%. These studies identify three membrane specific PIPs that modulate STARD4 sterol transfer activity. The increase in activity is dose‐dependent, insensitive to inhibition using soluble inositol phosphates, and maintained in membranes containing bulk anionic lipids. Physiologically, these studies suggest a mechanism of rapid vectorial transport as STARD4 extracts sterol from PI(4,5)P2 donor membranes and delivers to PI(5)P and PI(3,5)P2 acceptor membranes to maintain sterol homeostasis.
Cysteinylation of the ALS‐Associated Protein SOD1 Confers Resistance to Oxidation
Authors: Auclair Jared , Ringe Dagmar , Petsko Gregory , Agar Jeffrey
Published: June 2021

Natural by‐products of aerobic metabolism are reactive oxygen species (ROS). ROS arise from the incomplete reduction of molecular oxygen, resulting in the production of hydrogen peroxide (H2O2) and the superoxide anion (O2‐.). Cells combat harmful ROS species with a multi‐faceted anti‐oxidant defense mechanism that includes catalytic removal of ROS by enzymes such as copper‐zinc superoxide dismutase (SOD1). Loss of SOD1 function ‐ by oxidation, for example ‐ can cause an increase in superoxide anions, leading to such negative‐effects as neurotoxicity. Preventing oxidation may aid in preventing these negative effects. Recombinant human SOD1 was expressed and purified from yeast and brain or spinal cord either aerobically or anaerobically. Profile spectra of unmodified versus modified SOD1 samples were obtained using a 9.4T FTMS. The 2.5 Å resolution crystal structure of cysteinylated SOD1 (SOD1cys) was determined using molecular replacement. Profile spectra showed post‐translational modifications (PTMs) of SOD1 were present, and that cysteinylation and peroxide‐mediated oxidations appear to account for the majority of observed in vivo PTMs. In addition, we observed that SOD1cys is protected from oxidation in vitro and localized the site of cysteinylation to Cysteine 111. This was further confirmed by the crystal structure, which showed only one cysteine modification per dimer. In addition, we demonstrated SOD1 purified anaerobically had a 50‐fold reduction in cysteinylation compared to an aerobically purified sample, thus suggesting some of the S‐thiolation modifications may be artifacts. We conclude SOD1 cysteinylation is one possible way to combat the negative effects associated with life in an oxidizing environment.
Regulation of the Hsp70 BiP Molecular Chaperone Activities by Glutathiolation
Authors: Wang Jie , Sevier Carolyn
Published: June 2021

Elevated levels of reactive oxygen species (ROS) within cells can be detrimental to cell function. To gain insight into how cells normally prevent damage associated with ROS build‐up, our lab has focused on understanding the signaling pathways that serve to sense and respond to ROS accumulation (oxidative stress). Previously, we have shown that a cysteine in the molecular chaperone BiP, a Hsp70 within the endoplasmic reticulum (ER), is susceptible to oxidation by peroxide during ER‐derived oxidative stress, forming a sulfenic acid (‐SOH) moiety. Sulfenylation of BiP decouples the ATPase and peptide‐binding activities of BiP, enhancing BiP's capacity to bind polypeptides while also limiting BiP's ability to hydrolyze ATP. Oxidation of the BiP cysteine is necessary to promote cell survival during stress. Here we demonstrate that BiP's cysteine is also susceptible to glutathione modification (‐SSG). Increased levels of glutathiolated BiP correlates with changes in the cellular glutathione pool. Specifically, higher levels of oxidized glutathione in cells are associated with an increased pool of glutathiolated BiP. Similar to sulfenylation, we observe glutathiolation decouples BiP ATPase and peptide‐binding activities, turning BiP from an ATPase dependent “foldase” into an ATP independent “holdase”. We propose the susceptibility of BiP to modification with glutathione may be a means to prevent irreversible oxidation of BiP by peroxide. Under high levels of peroxide, sulfenic acid, which is reversible, can be further oxidized to irreversible sulfinic (‐SO2H) and sulfonic (‐SO3H) acid adducts. We are exploring whether the capacity for BiP glutathiolation is necessary to limit irreversible modification of BiP by peroxide during stress.
Sweet Tooth of a Glucose Transporter: GLUT1 C‐terminal Phosphorylation and O‐GlcNacylation effect in Breast Cancer Cell Lines
Authors: Padilla‐Meier Gay Pauline , Mishra Suresh
Published: June 2021

Cancer cells have altered metabolism that is largely dependent on aerobic glycolysis. In cancer cells elevated levels of glucose transporters (GLUT) facilitate the high glucose requirements of cancer cells. GLUT proteins allow for the passive transport across the plasma membrane down its concentration gradient. In various cancer tissues as well as human breast cancer cell lines elevated expression of GLUT1 has been reported. Another hallmark of cancer cells is hyper O‐GlcNacylation, which is a post translational modification (PTM) which adds a single N‐acetylglucosamine to either a serine or threonine residue. Only one O‐GlcNacylation site has been identified for GLUT1 (S465). The O‐GlcNacylation site of GLUT1 is in proximity of identified phosphorylation sites suggesting a possibility of interplay among the PTM sites of the C‐terminus. The aim our study is to discern if O‐GlcNacylation of GLUT1(S465) is affected by phosphorylation of neighboring residues and to determine if PTM of these sites regulate the function of GLUT1 and cancer cell metabolism. The single O‐GlcNacylation site in GLUT1 (S465) and neighboring serine/threonine residues were mutated and overexpressed in breast cancer cell lines. We investigated the molecular pathways involve in breast cancer cell metabolism and the effect of overexpression of GLUT1 variants. The GLUT1 variants showed altered levels of O‐GlcNac transferase, O‐GlcNacase and total O‐GlcNacylation of proteins in the cell. Breast cancer cell lines transfected with GLUT1 or it variants showed variable levels of glucose uptake. The results show that PTM of the c‐terminus of GLUT1 generally affects O‐GlcNacylation levels and metabolism of breast cancer cell lines.
Targeting the SUMO E1‐E2 Enzyme Interaction in Plasmodium falciparum
Authors: Reiter Katherine , Bosch Jurgen , Matunis Michael
Published: June 2021

Resistance to first‐line anti‐malarial drugs has accelerated the need for new drugs with novel targets for effective malaria treatment. Many of the current anti‐malarial drugs work in part by overwhelming the parasite stress response. In eukaryotes, the oxidative stress response machinery includes antioxidants as well as SUMOylation, a post‐translational modification that involves the covalent attachment of small ubiquitin‐related modifiers (SUMOs) to proteins. We hypothesize that SUMOylation plays an essential role in parasite cell stress survival, and that parasite‐specific SUMOylation inhibitors could prove effective in combination with current anti‐malarial drugs. We have previously demonstrated that human and Plasmodium falciparum (Pf) SUMO conjugation systems have biochemically distinct E1‐E2 enzyme interactions, suggesting a target for drug development. Here, we use x‐ray crystallographic studies to further define and compare the human and Pf E1‐E2 interaction. We have identified non‐conserved and structurally distinct surface residues that will help guide the identification of parasite‐specific inhibitors of SUMOylation.

This research was funded in part by the National Institutes of Health (GM060980), the Johns Hopkins School of Public Health Sommer Scholar's Program, and the Johns Hopkins Malaria Research Institute.
Structural and Functional Features of Recognition by Toll‐Like Receptors
Authors: Jerala Roman
Published: June 2021

Toll‐like receptors are a family of membrane receptors that recognize structural motifs of pathogens as well as endogenous danger signals that comprise structural features that are not present or are underrepresented under the normal physiological conditions. TLR agonists span from small nucleotide analogues to polynucleotides and proteins. Crystal structures of several ectodomains of TLRs have been determined that exhibit some similarities but also a significant degree of difference in the structural motif of the recognition of their respective agonists. With the exception of heterodimers of TLR2 all other known TLRs recognize the ligands in symmetric 2:2 stoichiometry, employing the composite recognition pockets contributed by the two binding sites of the receptor or coreceptor molecule. Those motifs allow us identify endogenous agonists, signaling danger for TLR4 and propose the CpG ssDNA recognition motif of TLR9. Based on the crystal structure of fragments of TLR5 with a fragment of inactive flagellin we used molecular modeling guided mutational studies of the receptor and flagellin to demonstrate that the recognition of the protein agonist involves multipartite recognition of several segments of the receptor. This type of recognition targets the structural elements of microbial molecules that are essential for their function and are therefore difficulty to modify to evade recognition.
Effects of Bacterial Endotoxins on the Toxicity of Extracellular Histones to Mammalian Cells
Authors: Burris Deon , Sharma Lokesh , Mantell Lin
Published: June 2021

During Bacterial infections, endotoxins are released from the bacterial cell wall and histones from damaged host cells into the extracellular milieu. Extracellular histones are known to act as potent bactericides against a wide range of pathogens. Additionally, deleterious effects of histones on mammalian cells were recently reported. Unpublished data from our lab show that the bactericidal effects of histones can be blocked by the presence of bacterial lipopolysaccharide (LPS). However, the effects of LPS on histone toxicity to the viability of mammalian cells are unknown. Using macrophage‐like RAW 264.7 cells, we studied the effects of extracellular histone in the presence of LPS on host macrophages. We used Trypan blue exclusion and MTT assays as indicators of cell viability. In accordance to previous studies, a substantial decrease in cell viability was found in cells treated for 24 h with histone alone. However, simultaneous treatment with LPS significantly blocked histone‐induced cell death. These results demonstrate that co‐treatment of extracellular histone with bacterial endotoxin decreases its detrimental effects on cell viability; suggesting, bacterial products may interfere with histone‐mediated cell death. We are currently investigating potential mechanisms underlying these observations. These studies give new insights on host‐pathogen interactions during infections.
Three‐dimensional Collagen IV and Hormone Prolactin Regulate Breast Cancer Cell Invasion: Role of PAK1 and Matrix Metalloproteinases
Authors: Diakonova Maria , Oladimeji Peter , Rider Leah
Published: June 2021

Three‐dimensional (3D) matrix is a critical component of mammary tissue development not only under physiological but also in pathophysiological conditions. The hormone/cytokine prolactin (PRL) also contributes to the pathogenesis of breast cancer via activation of JAK2/ STAT‐ and MAPK‐dependent pathways. Serine‐threonine kinase PAK1 is one of the targets of PRL‐activated JAK2. We showed previously that tyrosine kinase JAK2 phosphorylates PAK1 in response to PRL on three tyrosines (Tyr 153, 201 and 285). We show herein that the PAK1 phosphorylation is required for maximal PRL‐dependent invasion of human breast cancer cells through Matrigel and that 3D collagen IV acts synergistically with PRL‐activated PAK1 to induce the expression and secretion of MMP‐1 and MMP‐3. In contrast, PRL‐activated PAK1 decreases the 3D collagen IV induction of MMP‐2. MMP‐9 expression and secretion are stimulated by 3D collagen I, not collagen IV, and are not affected by PRL but are down‐regulated by PAK1. Using shRNA or pharmacological inhibitors, we show that MMP‐1 and ‐3 are required and MMP‐2 contributes to PRL‐ dependent invasion. Erk 1/2 signaling appears to be required for the enhanced expression and secretion of MMP‐1, and ‐3 and enhanced PRL‐dependent invasion. p38 MAPK and JNK 1/2 pathways participate in production of MMP‐1 and ‐3 as well as in PRL/PAK1‐dpendent cell invasion. The NFkB pathway is required for MMP‐3 and ‐9 secretion. Together, these data illustrate complex interaction between cellular substrate and PRL regulation of breast cancer cell progression, implicating a pivotal role for PAK1 tyrosyl phosphorylation in MMP regulation.

This work was supported by National Institutes of Health Grant R01 DK88127 (to MD).
The Role of Complement and the Extracellular Matrix in Early Stage Macular Degeneration
Authors: Fernandez‐Godino Rosario , Pierce Eric , Garland Donita
Published: June 2021

Gene targeted Efemp1R345W/R345W knockin mice are a documented model of the Efemp1‐associated macular degeneration. These mice develop features of macular degenerations including extensive basal deposits when the complement system is active. The aim of this project was to use cultured primary RPE cells to study the mechanisms involved in the pathogenesis of basal deposit formation and the role of the complement system.

Mice were used in conformance with the FASEB Statement of Principles for the use of animals in research and education. Primary RPE cells from wild type, Efemp1R345W/R345W, and Efemp1R345W/R345W:C3‐/‐mice were grown on transwells. Cell phenotypes were characterized by electron microscopy. The presence of complement components and factors, extracellular matrix proteins and pro‐inflammatory molecules were quantified by qRT‐PCR, immunofluorescence and ELISA. The activities of specific proteins were modulated with antibodies and recombinant proteins.

We conclude that cultured primary RPE cells from Efemp1R345W/R345W knockin but not from wild type or double mutant Efemp1R345W/R345W:C3‐/‐mice form extracellular deposits. The deposits contain extracellular matrix and complement proteins. The formation of deposits can be inhibited by neutralizing C3a, IL‐1B, IL‐6 or EFEMP1 in Efemp1R345W/R345W cultures. Wild type cells make deposits in the presence of C3a or recombinant mutant EFEMP1.

We conclude that RPE cells from Efemp1R345W/R345W knockin mice generate the extracellular deposits through a C3a dependent mechanism that is activated by EFEMP1R345W. These cells thus provide a model system to investigate the early mechanisms responsible for RPE dysfunction in macular degeneration.