bluuebunny/crossref_metadata_2025 huggingface dataset:
Objective structured skill examination in physiology education (531.6)
Authors: Matsuo Osamu , Kawao Naoyuki , Tamura Yukinori , Okada Kiyotaka , Nagai Nobuo , Ueshima Shigeru
Published: June 2021

We tested the efficacy of objective structured skill examination (OSSE) as a new and useful examination. The second grade students at Kinki University Faculty of Medicine were prospectively eligible. Students took problem‐based learning (PBL) tutorials in addition to classroom lectures and laboratory practice in each session. The sessions were on: cardiac, circulation, hematological, and renal systems. After all sessions, OSSE was performed to evaluate the students’ performance. The technical skills evaluated in OSSE were as follows: electrocardiogram recording, blood pressure measuring, use of centrifuge, pipette technique, hematocrit measurement, urinary specific gravity determination, and measurement of urine volume. One order sheet and the experimental apparatus for OSSE were positioned at each station and students terminate the maneuver in 2 min. The score from OSSE was compared to that from the conventional evaluation. Significant positive correlation was not observed. More than half of the students considered OSSE to be meaningful. In conclusion, we developed a new structured examination to objectively assess the students' technical skills in laboratory practice.

Grant Funding Source: None
Transitions in teaching: he experiences of basic scientists changing to an integrated anatomy curriculum (532.5)
Authors: Hopkins Robin
Published: June 2021

Objective: The purpose of this study was to understand what the experience of transitioning to an integrated curriculum is like for anatomy educators with a basic science background, and to interpret what it means for their teaching.

Methods: Lillian Douglas School of Medicine (pseudonym) was used as a case study of an anatomy program that has successfully transitioned to teaching anatomy in a clinically relevant way. A phenomenological approach was used to explore what it was like for basic scientists to change to teaching in an integrated curriculum. The primary investigator spent one month with the program during which she interviewed participants and observed lectures and labs. Data was analyzed using the reiterative cycle of hermeneutics.

Findings: Using Daniel Pratt’s General Model of Teaching as a conceptual framework, transitioning to an integrated curriculum entailed a shift in the elements of teaching from emphasis on the teacher and content to students and the context of clinical practice. The foregrounding of students and context included both a change in content in terms of clinical cases, and a change in teaching towards a more student centered “Socratic” approach. These changes involved collaborating with physicians and pedagogical experts respectively, as well as ceding control in terms of deciding what to include in the course and how they would go about teaching it.

Grant Funding Source: Supported by the Social Sciences and Humanities Research Council of Canada
The effect of a 4th year anatomy teaching course on anatomical knowledge and teaching confidence (533.2)
Authors: Lufler Rebecca , Stefanik Joshua
Published: June 2021

There has been a push by the Liaison Committee on Medical Education (LCME) and Accreditation Council for Graduate Medical Education (ACGME) to integrate the basic sciences into the 4th year medical curriculum and inculcate medical students with teaching skills. The objective of this study is to determine the effectiveness of a 4th year medical school elective course focused on teaching gross anatomy to address these concerns.

4th year medical students enrolled in the course were administered a pre‐test to assess their gross anatomy knowledge and a survey to assess their confidence in gross anatomy knowledge and teaching skills. After a 4‐week course involving dissection and teaching 1st year medical students, they were given the same confidence survey and a post test assessing the same anatomical concepts using different questions.

11 students completed the course, 81.8% (9) were male and 18.2% (2) were female. The mean age (standard deviation) of the students, anatomy knowledge pre‐test score and post‐test score was 27 (2.2), 53.9 (15.7) and 88.2 (7.2), respectively. The post‐test score was significantly higher than the pre‐test score (p <0.001). The median anatomy confidence before and after the course was 8.0 and 8.0, respectively. The median teaching confidence before and after the course was 8.0 and 9.0, respectively.

Based on these preliminary results, a 4th year medical school elective course focused on teaching gross anatomy appears to increase anatomy knowledge but not measures of self‐reported confidence in teaching and anatomy knowledge.

Grant Funding Source: Supported by Tufts University School of Medicine Innovations in Education Intramural Grant
Measure concepts, not questions: quantitative analyses of students’ performance on anatomy exams (533.5)
Authors: Orsbon Courtney , Greenwald Martin , Ross Callum
Published: June 2021

A competency‐based, pre‐clinical anatomy curriculum must provide medical students with the knowledge needed in a variety of medical and surgical specialties, and such knowledge is typically assessed through testing. While conventional testing methods give a general sense of whether a student understood the material, they are often unable to determine the specific strengths or weaknesses of both the students and the curriculum. For example, one would not know whether students pass exams by satisfactorily understanding all of the material, as opposed to ignoring some portion of it but studying another sufficiently to pass. To address this uncertainty, we developed a method to quantitatively analyze student performance on anatomy exams using resources readily available to many medical educators. Every exam question was assigned one or more anatomical concepts presented during lectures. Data were downloaded from the Blackboard Learning Suite, and student performance on these concepts were aggregated and analyzed independently of their question using code developed for R. This code generates statistics for each exam question and concept, quickly identifying those individual questions which are potentially too easy (100% correct) and too hard (0% correct) as well as the overall concepts that the class both succeed and struggled in demonstrating competence. We also compare students’ conceptual performance to physicians’ Likert ratings of the clinical importance of those concepts reported in a prior study. Our method of categorizing questions by concepts gives medical educators the ability to discover and correct potential weak points in the both individual students’ knowledge base and the competency‐based curriculum.
Using in‐class drawing exercises to teach anatomy and visual skills (534.10)
Authors: Barger J. Bradley
Published: June 2021

Students need to be able to understand and mentally manipulate visual images in order to learn anatomy. It is often assumed that students will come to class already well equipped with the visual skills needed for success, if this set of skills is considered at all. This research explores the pre‐existing visual skills undergraduate students have when entering an anatomy course, and if those visual skills change over time. Basic human anatomy is a 200 level course with lecture and lab components, and is taught in two nearly identical sections of ca. 200 students each. One lecture section was given additional drawing lessons as a way of improving visual skills while also learning anatomy course content. The other lecture section (control) learned the same anatomy course content via traditional pedagogical methods without drawing lessons. Both sections were surveyed at the beginning and end of the semester about their study habits, visual approaches to learning, and mental rotation abilities. Participation in the drawing lessons was then correlated with changes in mental rotation scores, study habits, and final grades. Students in the drawing section showed an increase in mental rotation scores, and increased frequency of visual approaches to studying, but no significant differences in final course grades were found.
The impact of pre‐matriculation summer educational enrichment program on student performance in medical gross anatomy course (534.6)
Authors: Lu Yuefeng , Baldwin Stanley , Sinning Allan , Case Steven , Conway Marianne , Lynch James , Lehman Michael
Published: June 2021

The University of Mississippi Medical Center offers a seven‐week summer program to students who have been accepted into medical or dental schools but may encounter difficulty because of low scores on MCAT or other adverse factors. Besides concrete academic contents from first year basic science courses (Gross anatomy, Biochemistry and Histology), the program also emphasizes study skills and test‐taking strategies. In the current study, grades in the Medical Gross Anatomy course from the past three years were analyzed. Since contents covered in the summer program match well with the actual Block 1 (back & upper limb) and Block 4 (head & neck) sections, we compared the differences between these two blocks with the other two un‐targeted blocks (2‐thorax & abdomen; 3‐pelvis & lower limb). Result 1: Compared with the rest of the class, summer participants performed either at or above the class average for Block 1 and 4 exams; in contrast, their grades were lower or not different for Block 2 and 3 exams. Result 2: Compared with themselves after being normalized with the rest of the class, summer participants did significantly better in covered blocks. In conclusion, the summer program is a valuable opportunity for educationally disadvantaged students, providing a useful simulation of the rigors of medical school and preparing them to achieve better results.
Accessing gain and knowledge retained in cross‐sectional anatomy (535.11)
Authors: Ribeiro João , Akamatsu Flávia , Hojaij Flávio , Andrade Mauro , Jácomo Alfredo
Published: June 2021

Broad knowledge of cross‐sectional anatomy is essential for medicine students. This study aims to measure the gain of knowledge concerning sectional anatomy in a group of students who had no prior contact with such a concept. We asked 4th semester medicine school students to voluntarily identify 20 anatomical structures in plastinated slices, projected on a screen, and given 30s for each structure. After this first trial, students attended weekly classes for 2 months in the Discipline of Structural Human Topography in University of São Paulo, alternating lectures and laboratory activities focused on the upper limb, inferior limb and back. Following this, they underwent a formal evaluation and started learning new contents for further 4 weeks, when a new trial, identical to the first one, was held. 70 students answered questions in the first trial and 52 in the second one. First trial´s average score and standard deviation were 5.67 (±2.38), while second trial´s were 13.02(±3.24). Concerning structures of the forearm, in the first trial, students reached only 4.8% right answers, against 60.9% afterwards. Corcerning foot structures, results were 36.1% and 52.4%. A relevant enhance in the hability of correctly identifying anatomical structures in plastinated can be obtained in short educational interventions, combining lectures and laboratory activities.
Understanding anatomical competence: perceptions of residents and program directors using a mixed methods survey (535.6)
Authors: Fillmore Erin , Brokaw James , Kochhar Komal , Nalin Peter
Published: June 2021

Over the past two decades, the teaching of anatomy to medical students has undergone major transformative changes. The purpose of this study was to better understand current perceptions of anatomical competence of residents. We examined residents’ perceptions of their own anatomical competence in clinical practice as well as program directors’ perceptions of their residents’ anatomical competence in clinical practice. A mixed methods survey was sent to both residents and program directors of 15 residency programs at Indiana University School of Medicine. The survey response rates for residents and program directors were 46% (n = 385) and 71% (n = 61), respectively. Results indicate that residents believed their gross anatomy education better prepared them to be anatomically competent, in contrast to their program directors perception (P = <0.0001). However, program directors believed that their residents were developing anatomical competence at an appropriate rate, in contrast to the residents who were less satisfied with their rate of progress (P = <0.0001). Finally, both residents and program directors agreed that there is a need for more formal anatomy educational opportunities during residency. Exploring these results within the context of a changing anatomy curriculum brings to the forefront important perspectives on anatomical competence.
Trends in gaze tracking patterns with increasing exposure to anatomy (535.8)
Authors: Zumwalt Ann , Iyer Arjun , Ghebremichael Abenet
Published: June 2021

The process of learning is complex and multifaceted. It is therefore worthwhile to define objective markers to indicate a learner’s progression. The goal of the present study is to determine whether gaze tracking patterns differ as a function of knowledge acquisition. The eye movements of 31 Medical Gross Anatomy students were recorded while they identified anatomical structures on computer slides. All participants completed the experiment two times: once before they started the class (baseline) and once after one of three course sections (revisit; 10‐11 subjects per section). Participants viewed tagged structures from all three sections of the course and obscure images (animal anatomy). Therefore all subjects viewed some structures about which they had learned and some structures they have never encountered. We predict that with experience the students will attend faster and fixate longer on cognitively salient areas of interest (cAOIs) on images from the section(s) of the course they have completed at the time of revisit. Preliminary analyses of this ongoing experiment indicate that successful students may develop intelligent search strategies with increasing exposure to the course. Subjects who correctly identify the structure fixate for 60‐150% longer on cAOIs, even on obscure images, and take 20‐90% more time to answer than they did at baseline. These trends appear to get stronger later in the course. There are no apparent trends in the speed with which subjects attend to cAOIs. These preliminary results indicate intriguing trends in successful image search strategy with increasing anatomy knowledge. Final analysis will include students’ exam scores, course grades, and visuospatial abilities in the context of gaze variables to compare and contrast the visual search strategies of successful and less successful anatomy students.
Can stereoscopic technologies build better surgeons? The effects of stereoscopy and spatial visualization ability on laparoscopic performance in surgical residents (536.12)
Authors: Maini Arjun , Roach Victoria , Pautler Stephen , Wilson Timothy
Published: June 2021

Acquisition of surgical skill is a multifaceted process, in which practice, and innate human factors such as spatial visualization ability (Vz) play key roles. Elevated Vz (HVz) is thought to facilitate surgical skill acquisition. The benefit of viewing laparoscopic skills in stereo (3D) is undetermined in experienced populations, and may show differing effects between those of variable Vz. The Vz of surgical residents will be evaluated using a Mental Rotations Test, and then surgical skill will be measured via the McGill Inanimate System for Training and Evaluation of Laparoscopic Skills (MISTELS). Participants will complete the MISTELS tasks in both 3D and monoscopic format (2D) while being randomly assigned to complete the task in either 2D first or 3D first. Scoring of the MISTELS is based on time, completion and efficiency. It is hypothesized that HVz individuals will score higher than LVz counterparts. Prior research in novice subjects suggests that the effects of 3D and 2D viewing yields variable results based on an individual’s Vz, such that LVz trainees may benefit more than HVz from 3D viewing after a period of experience. If 3D viewing enhances surgical skill in our subjects, it may suggest future use in laparoscopic training curricula. Comparison of current results with those of novice subjects may suggest the intervening effects of experience on the effect of Vz and/or viewing modality.
Face and content validity of three assessment tools developed to evaluate cerebral angiography performance (536.4)
Authors: Nguyen Ngan , Eagleson Roy , Boulton Mel , de Ribaupierre Sandrine
Published: June 2021

Cerebral angiography (CA) is considered the gold standard for diagnosing primary neurovascular diseases. Although guidelines have been established to ensure adequate CA experience before independent practice, current tools used to assess CA performance during training have questionable validity and reliability. The initial phases of validation often involve determining whether the tool appears to measure the construct of interest (face validity; FV) and whether the items in the tool cover a representative sample of the construct (content validity; CV). This study proposes to establish the FV and CV of 3 assessment tools (task‐specific checklist, error‐based scale, and global rating scale) developed to evaluate CA performance. FV and CV were established by asking expert catheter‐based physicians to judge whether each tool appears to be a practical and pertinent measure of CA performance and to evaluate the appropriateness of each tool item. Based on experts’ recommendations, each tool was modified to improve its ease‐of‐use, organization, and clarity. Confirmation of the FV and CV of the 3 tools will contribute to ensuring that future assessment of CA performance is both accurate and consistent.

Grant Funding Source: Supported by MITACS
Increased X‐linked inhibitor of apoptosis‐associated factor 1 and decreased activation of the X‐linked inhibitor of apoptosis protein are associated with increased apoptosis in the preeclamptic placenta at term (541.11)
Authors: Mejia Camilo , Arroyo Juan , Price Matthew
Published: June 2021

Preeclampsia (PE) is associated with placental insufficiencies and is characterized by an increase in placental apoptosis. Several mechanisms had been suggested but not much information is available on the role of the inhibitor of apoptosis family (IAP) of proteins. The X‐linked inhibitor of apoptosis protein (XIAP) is one of the most potent members of the IAP family of proteins. Its activation is controlled by the X‐linked of apoptosis associated factor 1 (XAF1). Our objective was to determine the level of XAF1 and its correlation with active XIAP protein in PE placenta compared to controls. Control and PE placental samples were collected shortly after delivery. Tissues were frozen in liquid Nitrogen for western blot studies. We observed: 1) No significant differences in XIAP protein expression; 2) a significant decrease in active (phospho) XIAP protein (30%; p<0.05) in the placentas of PE women; and 4) a significant increase (38%; p<0.008) for XAF1 during preeclampsia. We conclude that the decrease in XAIP activation during PE could potentially account for the increased apoptosis observed in this tissue at term. Also, that a mechanism for this decreased XIAP activation involves increasing placental XAF1 during preeclampsia. This result gives insights into pathways associated with the increased placental apoptosis and could be useful in the understanding of potential mechanisms that lead to placental insufficiency associated with this disease.
CCN2 induction by TGF‐β1 requires osteoblast specific promoter motifs (541.6)
Authors: Martinkovich Stephen , Silverstein Rachel , Geisinger Max , Planey Sonia , Arnott John
Published: June 2021

Connective tissue growth factor (CCN2; formerly CTGF) is a 38kDa, cysteine rich, extracellular matrix protein that belongs to the CCN family of proteins. CCN2 acts as an anabolic growth factor to regulate osteoblast differentiation and function. CCN2 is induced by TGF‐B1 and acts as a mediator of TGF‐B1 induced matrix production in osteoblasts; however, the molecular mechanisms that control CCN2 induction are poorly understood. In this study, we investigated the role of CCN2 promoter motifs involved in CCN2 induction by TGF‐B1 in primary osteoblasts. We used a combination of luciferase promoter reporter assays and gel shift studies to demonstrate the novel requirement of both the TGF‐B1 response element (TRE) and Smad binding element (SBE) for CCN2 promoter trans‐activation in osteoblasts. We propose that the TRE and SBE function as cognate motifs for CCN2 induction by TGF‐B1 in an osteoblast specific manner.

Grant Funding Source: Supported by NIAMS
The type III TGFβ receptor is required for hyaluronan‐mediated epicardial invasive cell motility (542.5)
Authors: Allison Patrick , Espiritu Daniella , Barnett Joey , Camenisch Todd
Published: June 2021

The epicardium originates from the proepicardial organ (PEO). Both the PEO and epicardium require directed cell‐motility to cover the heart and instruct epicardially derived cells for coronary vasculature formation. The type III transforming growth factor‐beta receptor (Tgfbr3) is required for epicardial cell invasion and development of coronary vasculature in vivo. Utilizing a primary epicardial cell line derived from Tgfbr3(+/+) and Tgfbr3(‐/‐) e13.5 mouse embryos , we show that Tgfbr3(‐/‐) epicardial cells are 34.4% deficient in 2D migration relative to wild type. Furthermore, we show that high‐molecular weight Hyaluronan (HMWHA) drives 3D cellular invasion in wild type epicardial cells, but not Tgfbr3(‐/‐) epicardial cells. We show Src kinase is required for HMWHA stimulated filamentous actin (f‐actin) polymerization in wild‐epicardial cells. Furthermore, HMWHA stimulates Y416Src phosphorylation in wild type, but Src is not activated in Tgfbr3(‐/‐) epicardial cells. RhoA and Rac1 GTPase are downstream of Src kinase, and directly upstream of f‐actin polymerization; HMWHA robustly induces RhoA and Rac1 GTP binding and activity in wild type, but not Tgfbr3(‐/‐) epicardial cells. This deficit in RhoA and Rac1 activation results in reduced formation of f‐actin stress fibers in HMWHA stimulated Tgfbr3(‐/‐) epicardial cells. Taken together, we demonstrate that the type III TGFβ receptor is required for activation of basic cell motility pathways stimulated by Hyaluronan that are required for proper coronary vasculature formation.

Grant Funding Source: NIH ES 04940; ES06694; SWEHSC P30ES006694
Transcriptional regulation and proteolytic processing of perlecan in mast cells lead to the production of forms that contain the alpha2/beta1 integrin binding site: roles in wound healing and tissue regeneration (544.3)
Authors: Whitelock John , Jung MoonSun , Cheng Bill , Lord Megan
Published: June 2021

This study demonstrated that primary human mast cells as well as the rat and human mast cell lines, RBL‐2H3 and HMC‐1, produce the heparan sulfate proteoglycan (HSPG), perlecan, with a Mr of 640kDa as well as smaller molecular weight species of 300 and 130kDa. Utilizing domain‐specific antibodies and N‐terminal sequencing we confirmed that both forms contained the C‐terminal module of the protein core, which were generated by mast cell‐derived proteases. Fractions from mast cell cultures that were enriched for these fragments were shown to bind endothelial cells via the alpha2/beta1 integrin and stimulate the migration of cells in “scratch assays”. Both of these activities were inhibited by incubation with antibodies reactive to the C‐terminus of the perlecan protein core. This study showed that the transcriptional regulation of the HSPG2 gene is more complex than first thought, particularly in mast cells. Domain specific RT‐PCR experiments demonstrated that these cells were capable of splicing the perlecan gene (HSPG2) and when we performed quantitative PCR experiments, we demonstrated that the C‐terminal region was over‐represented compared to the N‐terminal region. This together with the data demonstrating proteolytic processing suggest that the C‐terminal region of perlecan, which contains the alpha2/beta1 integrin binding site may have downstream biological functions that regulate angiogenesis and matrix turnover and thereby promote wound healing and tissue regenerative processes.
Response analysis of hemodynamics parameters to tyramine infusion post‐physical training in spountaneously hypertensive rats (546.5)
Authors: Miranda Munique , Melo Poliana , Lemos Marina , Almeida Thalles , Motta Gustavo , Júnior Moacir , Dias Valdo , Neto Octávio
Published: June 2021

The aim of the present study was to assess the effects of exercise training in the vascular control after sympathetic stimulation by tyramine infusion in spontaneously hypertensive rats (SHR). 33 male SHRs and WKY were submitted or not to a swimming protocol during 8 weeks, resulting in four groups: sedentary normotensive (SN, n=7), trained normotensive (TN, n=8), sedentary hypertensive (SH, n=8) and trained hypertensive (TH, n=10) rats. Arterial pressure (AP), heart rate (HR), means of power spectral analysis of HR (HRV), systolic AP variability (SAPV) and diastolic (DAPV) were recorded in baseline conditions. Following, the infusion of tyramine in three doses was administrated. Exercise training attenuated AP in TH vs. SH (p<0.001). The variance of HRV was lower in SH vs. TH (p=0.018). The other hand, the variance of SAPV and DAPV were higher in SH vs. TH (p=0.05). The LF component of HRV, SAPV and DAPV were lower in TH than SH (p<0.05). LF/HF was lower in TH than SH (p<0.05). The TN and TH presented an increased parasympathetic modulation compared with SN and SH (p<0.05). Pressor response to tyramine was enhanced in TH, but not in WKY. In conclusion, the results points to a sympatolitic effect of exercise training. This conclusion is supported by the impaired tyramine pressor response, suggesting an up regulation of α1‐receptors in vascular smooth muscle, once the administration of an agonist showed enhanced and the norepinephrine releaser showed impairment or the pressor response. These findings suggest that exercise training may be a therapeutic tool for improving function in hypertensive individuals.
To activate or destroy: regulation of the RNA Pol II elongation factor and ubiquitin ligase Elongin (550.3)
Authors: Weems Juston , Hall Shawn , Slaughter Brian , Unruh Jay , Gilmore Joshua , Washburn Michael , Florens Laurence , Conkright Julianna , Weems Lauren , Conaway Joan , Conaway Ronald
Published: June 2021

Lesions induced by a variety of DNA damaging agents can block RNA polymerase II (Pol II) elongation. Mechanisms that reactivate or remove Pol II that has become stalled or arrested at such lesions are critical for resumption of gene expression after DNA damage repair and, in addition, may contribute to efficiency of the DNA repair process by enhancing access of the damage repair machinery to the lesion. Following exposure of cells to UV irradiation or other DNA damaging agents, arrested Pol II is ubiquitinated and degraded by the proteasome.

Elongin was first identified and purified showing an activity that stimulates the rate at which Pol II elongates nascent transcripts in vitro and is composed of 3 subunits, Elongin A (EloA), B (EloB), and C (EloC). Although the majority of Elongin A isolated from cells contains the three subunit Elongin complex, it can also be linked to a heterodimeric module composed of the Cullin family protein Cul5 and the RING finger proteins Rbx1 or Rbx2 to form a larger complex that targets stalled Pol II for ubiquitination. A previous study used indirect immunofluorescence to provide evidence for increased nuclear colocalization of EloA and Cul5 after UV‐induced DNA damage, raising the possibility that DNA damage may promote assembly of the EloA ubiquitin ligase complex. Using a combination of MudPIT‐based mass spectrometry and FRET‐based approaches, we have obtained evidence that supports this model. In ongoing studies, we seek to define mechanisms by which stalled Pol II regulates assembly and activity of the Elo A ubiquitin ligase complex.
Comparative mutagenicity amongst c‐family bacterial DNA pol III’s (551.2)
Authors: Morris Justin , Mukherjee Purba , Pata Janice
Published: June 2021

As evidence points to the use of multiple DNA pol III’s by gram‐positive bacteria, a question remains: which biochemical factors (domain composition, DNA oligonucleotide (oligo) length, etc.) serve as the strongest determinants of fidelity? We have characterized various patterns of relative mutagenicity amongst the essential C‐family bacterial DNA polymerases found in Staphylococcus aureus. These include two PolC constructs: PolC‐Exomut and PolC‐ΔNΔExo, as well as DnaE. In vitro assays were performed using various primer‐template (p/t) DNA substrates and nucleotides. Additional assays paired the polymerase with the β clamp. DnaE showed the highest frequency of nucleotide misincorporation, especially on longer p/t DNA substrates, as well as in bypassing 8‐oxoguanine lesions. PolC‐Exomut, the construct with both N‐terminal and Exonuclease (Exo) domains, consistently showed the most fidelity. A direct relationship between fidelity and DNA substrate length was also observed. The presence of secondary proteins consistently increased overall addition, but not fidelity. Overall, these assays indicate DnaE as a more error‐prone polymerase compared to its PolC counterparts. Additionally, oligo length was shown to influence the accuracy of nucleotide addition. Future assays will further examine the effects of oligo length as well as fidelity patterns specifically attributable to the N‐terminal domain.

Grant Funding Source: Supported by NIH Grant R01‐GM080573
Mammalian protein arginine methyltransferase 7 (PRMT7) specifically targets R‐X‐R sites in lysine and arginine‐rich regions (555.1)
Authors: Feng You , Clarke Steven
Published: June 2021

The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remains controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω‐monomethylarginine residues and confirm its activity as the prototype type III PRMT. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post‐translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine‐ and arginine‐rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (R‐X‐R motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7.

Grant Funding Source: Supported by NIH R01GM026020
A new family of lysine methyltransferases preferentially targets molecular chaperones to orchestrate the functional organization of the human proteome (555.12)
Authors: Cloutier Philippe , Coulombe Benoit
Published: June 2021

In efforts aimed at identifying and characterizing protein substrats and regulators of all known and putative human methyltransferases using cell compartment affinity purification coupled with mass spectrometry (MCC‐AP‐MS), our laboratory has discovered a novel family of lysine methyltransferases that preferentially target and regulate molecular chaperones. Our results indicate that methylation of molecular chaperones VCP by METTL21D and Hsp70 by METTL21A regulates the activity of both chaperones. In the case of VCP, trimethylation of lysine 315 by METTL21D is stimulated by the addition of the UBX cofactor ASPSCR1, which we show directly interacts with the methyltransferase. This stimulatory effect was lost when we used VCP mutants (R155H, R159G and R191Q) known to cause Inclusion Body Myopathy with Paget's disease of bone and Fronto‐temporal Dementia (IBMPFD) and/or some familial forms of Amyotrophic Lateral Sclerosis (ALS). Lysine 315 falls in proximity to the Walker B motif of VCP's first ATPase/D1 domain. Our results indicate that methylation of this site negatively impacts the ATPase activity of this molecular chaperone. In the case of Hsp70, methylation of lysine 561 by METTL21A was shown to decrease formation of stress granules in response to arsenite treatment, a model that is widely used to study improper protein folding in degenerative disorders. In addition to having implications for the development of therapeutics for degenerative neuromuscular disorders such as ALS and IBMPFD, the discovery of a family of chaperone‐targeting methyltransferases led us to propose the existence of a chaperone posttranslational modification code, that we termed the “chaperone code”, which is at play to orchestrate the proper folding and assembly of protein complexes that make up the human proteome. We will report on our most recent results relating to decryption of the chaperone code and its role in disease.

Grant Funding Source: Muscular Dystrophy Association
Arkadia mediates sumoylation‐dependent degradation of Nrf2 in promyelocytic leukemia‐nuclear bodies (555.5)
Authors: McIntosh Deneshia , Arinze Ifeanyi
Published: June 2021

The cellular abundance of the transcription factor Nrf2 is largely controlled by Keap1, an inhibitory protein that functions as a substrate adaptor for ubiquitylation of Nrf2 by the Cullin3/RING box1 (Cul3‐Rbx1) E3 ubiquitin ligase complex, thereby targeting Nrf2 for proteasome‐dependent degradation in the cytoplasm. It is becoming increasingly apparent that degradation of Nrf2 can also occur by Keap1‐independent mechanisms. Recently, we showed that Nrf2 is a SUMO‐modifiable protein and that polysumoylated Nrf2 can be polyubiquitylated by the poly‐SUMO‐specific E3 ubiquitin ligase known as RING finger protein 4 (RNF4) and subsequently degraded in PML‐NBs. The role of sumoylation in the turnover of Nrf2, independent of Keap1, is a novel aspect of the biology of Nrf2. Here, we explored whether sumoylated Nrf2 is a substrate for Arkadia, another polySUMO‐specific E3 ubiquitin ligase (also known as RING finger protein 111 [RNF111]). Our results indicate that Arkadia can ubiquitylate polysumoylated Nrf2 in PML‐NBs, suggesting that Arkadia can target polysumoylated Nrf2 for degradation in these nuclear domains. This work provides support to the idea that post‐translational modification of Nrf2 by sumoylation impacts the turnover of Nrf2 in the nucleus.

Grant Funding Source: Supported by NIH grants # SC1CA143985, 5T32HL007737, and 2 SD1MD000104
Sgtα: a novel independent regulator of steroid hormone receptors (567.8)
Authors: Gutierrez Omar , Garcia Yenni , Cox Marc
Published: June 2021

Steroid hormone receptors (SHRs) are essential for the regulation of developmental, reproductive, and physiological homeostasis in eukaryotes. SHRs belong to the nuclear receptor superfamily, and comprise the androgen (AR), glucocorticoid (GR), progesterone (PR), mineralocorticoid (MR), and estrogen receptors (ER). Understanding the regulation of SHRs is critical for the development of novel therapeutic strategies for SHR‐mediated diseases. Heat shock protein (Hsp) 90/70 kDa assembly is essential for the dynamic chaperone complex that directs SHRs folding. A recently identified cochaperone, human small glutamine‐rich TPR‐containing protein alpha (SGTα), has been shown to negatively regulate AR by retaining AR in the cytoplasm. Using functional yeast and mammalian reporter assays, we have demonstrated that SGTα can also regulate GR and PR. Interestingly, when co‐expressed with immunophilin FKBP52, a positive regulator of the same receptors, SGTα continues to abrogate receptor activity. Another TPR‐containing protein present in yeast, CPR7, can positively regulate AR. Thus, we assessed the effects SGTα has in a yeast model system using a CPR7 deletion strain. We observed the same pattern seen with FKBP52, suggesting that the regulation by SGTα is not solely based on its TPR domain. Future studies aim to determine the role for SGTα at the transcriptional level.

Grant Funding Source: Supported by the NIH/SCORE grant from the NIGMS (1SC1GM084863) and COURI
Characterizing an engineered release factor capable of reading all three stop codons (569.2)
Authors: Korkmaz Gürkan , Lind Christoffer , Åqvist Johan , Sanyal Suparna
Published: June 2021

Translation termination is the last step of protein synthesis where the newly synthesized protein is released from the ribosome. For this, a class I release factor (RF1 and RF2) in bacteria binds to one of the three stop codons (UAA, UAG and UGA) and hydrolyze the ester bond between nascent protein and the peptidyl‐tRNA. RF1 and RF2, although similar to each other, read the stop codons in a semi specific manner i.e. RF1 reads UAA and UAG, and RF2 reads UAA and UGA codons. According to current literature [1] these factors discriminate highly against sense codons. Although molecular dynamics simulation experiments [2] have proposed a network of interactions through which RF1 and RF2 recognizes their respective stop codons, there exist very little biochemical experiments to explain how this high specificity is achieved.

Using site directed mutagenesis we generated several variants of RF1 and RF2 and characterized those in competition and kinetic assays. One of our engineered release factors showed omnipotent property in stop codon recognition. We have characterized this engineered release factor with respect to other related codons especially in comparison with other release factors. Further we were able to validate our biochemical experiments using computer simulations. Our experiments pinpointed the crucial residues on the class‐I release factors for stop codon specificity and enabled us to engineer a class I release factor capable of reading all three stop codons.

Grant Funding Source: Supported by; Swedish Research Council, Sven and Lilly Lawski Foundation
Regulation of glucose transport by insulin/IGF‐like signaling in Caenorhabditis elegans (576.6)
Authors: Kitaoka Shun , Morielli Anthony , Zhao Feng‐Qi
Published: June 2021

Caenorhabditis elegans (C. elegans) has well conserved insulin/IGF‐like signaling (IIS). In our previous study, we have identified FGT‐1 as the only mammalian facilitative glucose transporter (GLUT) homologue in C. elegans which shows the transport activity to glucose in Xenopus oocytes (Kitaoka et al. PLoS ONE 2013, 8: e68475). To explore C. elegans as a model organism to study the regulation mechanism of glucose transport by IIS, we have studied the regulation of FGT‐1 by IIS in C. elegans. The growth of fgt‐1 mutants or fgt‐1;daf‐2 (mammalian insulin receptor homologue) double mutants showed no difference from the wild‐type (wt) animals or daf‐2 mutants, respectively, in normal medium or in the medium supplemented with 5 mM glucose. Similarly, the lifespan of fgt‐1 mutants had no difference from the wt animals in normal medium or in the glucose‐supplemented medium. However, the rate of development and the lifespan were significantly lower in wt animals than in fgt‐1 mutants when cultured in the medium supplemented with 5 mM 2‐deoxy‐D‐glucose (2‐DG), and the protective effect of fgt‐1 mutants on 2‐DG was lost in daf‐2 background. These data indicate that FGT‐1 is involved in glucose uptake in C. elegans, and although the FGT‐1 function did not appear to beregulated by IIS, glucose metabolism may be regulated by IIS in C. elegans.
In vivo receptor mediated near‐infrared transferrin uptake using fluorescence lifetime fret: implications in the targeted drug delivery (58.9)
Authors: Rajoria Shilpi , Zhao Lingling , Intes Xavier , Barroso Margarida
Published: June 2021

Traditional cancer therapy generally leads to harmful side effects, thus warranting development of targeted therapies which are better tolerated by cancer patients. The main objective of current study is to develop unique in vivo non‐invasive optical imaging assays for the optimization of targeted therapy and diagnostic tools. We propose to quantify the internalization of therapeutic drugs conjugated to transferrin (Tfn), the native ligand of transferrin receptor (TfnR) using a Förster Resonance Energy Transfer (FRET) based imaging technology in vivo. Quantification of in vivo FRET donor % (FD%) in tumor xenografts, indicates a higher proportion of FD% with increasing acceptor:donor ratio demonstrating tumor uptake of NIR‐Tfn. Immunohistochemistry analysis of tumors suggests that the injected NIR‐Tfn is translocated to the tumor site. In conclusion, our novel NIR FRET assay successfully demonstrates a quantitative receptor‐mediated uptake of Tfn in human breast tumors in vivo, allowing for the development of Tfn‐based targeted therapy by means of not only delivering the drug at target site but also potentially block the iron‐internalization function of TfnR, thus directly killing the cancer cell. More importantly, our data supports the quantitative accuracy and sensitivity of a novel NIR FLIM FRET imaging assay to be used non‐invasively through living tissues in a small animal model.

Grant Funding Source: National Institutes of Health grant R21 CA161782‐01
Biochemical characterization of 2, 3‐butanediol dehydrogenase from Taiwanofungus camphorata (583.2)
Authors: Wen Lisa , Tsai Wei‐Wei , Huang Jenq‐Kuen , Lin Chi‐Tsai
Published: June 2021

The enzyme 2,3‐Butanediol dehydrogenase (BDH) plays important roles in reduction of acetoin to 2,3‐butanediol which is an important platform chemical with many industrial applications. Here, a TcBDH cDNA (1348 bp) encoding a putative BDH was cloned from Taiwanofungus camphorata. The deduced amino acid sequence is similar to the BDHs from other species. A 3‐D structural model of TcBDH has been constructed based on the known structure of Pseudomonas putida formaldehyde dehydrogenase (PpFdh, PDB code 1KOL). To characterize the TcBDH protein, the coding region was subcloned into an expression vector pYEX‐S1 and transformed into Saccharomyces cerevisiae. The recombinant His6‐tagged TcBDH was expressed and purified by Ni2+‐nitrilotriacetic acid Sepharose. The purified enzyme showed a single band of ~50 kD on 12% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The Michaelis constant (KM) value of the recombinant enzyme for acetoin was 8.46 mM. The enzyme’s optimal pH was 6.

Grant Funding Source: National Science Council of the Republic of China, Taiwan under grant NSC 100‐2313‐B‐019‐003‐MY3
Biochemical characterization of a functional recombinant aryl‐alcohol dehydrogenase from Taiwanofungus camphorata (583.4)
Authors: Huang Jenq‐Kuen , Chang Che‐Chi , Ken Chuian‐Fu , Wen Lisa , Lin Chi‐Tsai
Published: June 2021

A TcAAD cDNA encoding a putative aryl‐alcohol dehydrogenase (AAD) was cloned from Taiwanofungus camphorata (Tc). The deduced amino acid sequence is conserved among the reported AADs. A 3‐D structural model of the TcAAD has been created based on the known structure of voltage‐dependent potassium channels subunit beta‐2 (PDB code: 3EAU). To characterize the TcAAD, the coding region was subcloned into an expression vector and transformed into Saccharomyces cerevisiae. The recombinant His6‐tagged TcAAD was overexpressed and purified by Ni affinity chromatography. The purified enzyme showed a band of approximately 39 kDa on a 12 % SDS‐PAGE. The molecular mass determined by MALDI‐TOF is 40.58 kDa which suggests that the purified enzyme is a monomeric enzyme. Using veratraldehyde as a substrate, the KM, Vmax of TcADD was determined at pH 6.0. Using benzyl alcohol derivatives as substrates, the oxidizing power of TcADD via NAD+ at pH 9.6 was studied.

Grant Funding Source: National Science Council of the Republic of China, Taiwan under grant 100‐2313‐B‐019‐003‐MY3
Enzyme‐catalyzed transesterification of lipids in methyl acetate using a methanol tolerant lipase from Proteus mirabilis (583.5)
Authors: Carey Demetrius , Witherow D. Scott
Published: June 2021

Recombinant lipase catalyzed biodiesel production has the potential to be more economically efficient than traditional acid and/or base catalyzed methods. Enzyme catalysis may require smaller loads, lower temperatures, less time and can be repeated if immobilized enzymes are utilized. However, many lipases expressed in E.coli are found in inclusion bodies, making recombinant production difficult. Further, often they are denatured in the methanol, the most common acyl acceptor. Lipase from Proteus mirabilis is naturally soluble in E.coli and has previously been mutated for methanol tolerance. These “dieselzymes” have not been previously characterized or optimized in reactions using alternate acyl acceptors. Mutated “dieselzymes” have been expressed in E.coli using the pET28 bacterial expression vector, purified using immobilized metal affinity chromatography and characterized for transesterification activity in reactions using methyl acetate, p‐nitrophenyl palmitate, and synthetic triacylglycerides at varying pHs and temperatures
High throughput screening of natural products for LDH inhibition as an anti‐mitotic therapeutic target (585.3)
Authors: Deiab Shihab , Mazzio Elizabeth , Zarmouh Najla , Mack Nzinga , Soliman Karam
Published: June 2021

A pathological feature hallmark of solid tumors is an over‐functional energy metabolism which results in high level production of lactic acid from glucose. The key enzyme in the production of lactate is LDH, which is highly expressed in tumor tissue and regulates substrate utilization of pyruvate to form lactate. In this study, LDH isolated from human muscle was screened using over 905 plant derived extracts to discover natural products [0.0005‐.77mg/ml] with capability to inhibit LDH. Extracts were tested using an enzyme micro‐array format and extracts containing inhibitory properties were then ranked according to their IC50's The data clearly showed that the most potent plant based LDH inhibitor was Rhus chinenesis. Through bioactivity guided chemical separations and identifications as well as kinetic and docking studies, we showed that pentagallolyl glucose, PGG, is the entity responsible for the non‐competitive inhibition of LDH‐A. We further examined several cancer cell lines viability and lactic acid production in the presence of PGG and found a decrease in viability consistent with the decrease in lactic acid production. However further studies will be required to assess if and what role inhibiting the LDH enzyme can provide in terms of adaptability and survival mechanisms involved with energy metabolism of tumor cells.

Grant Funding Source: Supported by NIH grants from NIMHD G12 MD007582 and P20 MD006738
AraL from B. subtilis is a sugar phosphatase and member of the nitrophenyl phosphatase family of the HAD superfamily (585.6)
Authors: Armeli Jordan , Madaio Michael , Hill Jacqueline , O'Handley Suzanne
Published: June 2021

The HAD (Haloacid Dehalogenase) superfamily is a diverse superfamily with a majority of the enzymes being phosphatases. One family of the HAD superfamily is the nitrophenyl phosphatase family, so named because the first family members were yeast enzymes found only to hydrolyze p‐nitrophenyl phosphate. Since that time, a number of enzymes with a variety of activities have been identified and characterized, most recently, a sugar phosphatase from B. subtilis. We have cloned the araL gene, expressed and purified the AraL protein, and partially determined its activity. Like the other family members, AraL is a phosphatase, but unlike any other member, it is the first sugar phosphatase to be reported for this family. Currently, we have found activity on ribose 5‐phosphate, but this may not be the best substrate, as araL is within the arabinose operon. If AraL degrades sugar phosphates found in the arabinose pathway, its cellular role may be to help regulate this important sugar metabolism pathway.

Grant Funding Source: Supported by an NIH AREA grant.