bluuebunny/crossref_metadata_2025 huggingface dataset:
A faculty learning community as a vehicle for change: the effect of iterative course change on student learning and attitudes in introductory biology (530.4)
Authors: Elliott Emily , Coffman C. , Reason R. , Raker J. , Becraft P. , Essner J. , Gangloff E. , Howell S. , Kukday S. , Peterson T. , Sakaguchi D. , Vleck D. , Yin Y. , Powell‐Coffman J. , Ogilvie C.
Published: June 2021

Students learn concepts and skills more efficiently when they actively engage with content, and introductory biology courses nationwide are shifting towards student‐centered classes. However, incorporating student‐centered learning into a large‐enrollment course presents unique challenges. Here we describe a Faculty Learning Community (FLC) where faculty instructors collaborate to transform a large enrollment introductory biology course from a traditional lecture format into a student‐centered course. Activities and strategies are incorporated and improved over time as faculty discuss successes and new approaches, allowing the FLC to optimize activities and build up a resource pool. We evaluate the effect of this iterative change on the classroom environment, student learning, and student attitudes utilizing a pre‐ and ‐post survey design. Preliminary results suggest that student content learning is increasing over time. Further, certain types of activities may be particularly effective at improving learning. Longitudinal data from three semesters of the course will be used to explore 1) how iterative change affects student learning, 2) which approaches are most effective, 3) how to target difficult concepts and 4) how different student populations are impacted by the changes. Results will provide a template for other institutions seeking to incorporate similar course improvements.

Grant Funding Source: Supported by HHMI “Engage to Excel” grant at Iowa State University
Reproducible gains in student attitudes toward biology from a single in‐class case study (531.1)
Authors: Hines Justin , Serrano Antonio , Liebner Jeffrey
Published: June 2021

The study objective was to test a case study which simulates scientific inquiry within the limits of a single course period and which can be used in large lecture‐format courses. Students unknowingly take the roles of the initial investigators of the Kuru epidemic of Papua New Guinea in 1957 and work in groups to iteratively conduct simulated investigations, evaluate the results, and form hypotheses regarding the nature of the disease. The activity culminates in a mock‐scientific conference, in which student groups collaborate by sharing findings, mirroring real events which led to the discovery of prion diseases. Learning goals focus on student opinions, attitudes and skills related to the nature of science, causes of disease epidemics, and prions as atypical infectious agents. The activity has been implemented successfully at a variety of schools and courses which span five fields: biology, microbiology, genetics, zoology, and biochemistry. Aggregated data from five courses shows overwhelmingly positive affective responses from students. Most importantly, in two sequential offerings of Introductory Biology in 2012 and 2013 we were successful in using a modified CLASS‐BIO survey to reproducibly measure statistically significant objective changes in attitudes toward biology as a result of the activity alone. We hope that both the activity and approach can be used broadly by life sciences educators.
Newer teaching and learning techniques in the advancement of physiology education (531.15)
Authors: Gopalan Chaya , Glasheen Megan
Published: June 2021

Instant access to content information is revolutionizing both the learning and teaching in higher education. The teacher‐centered instruction is being replaced by active learning and other student‐centered teaching methods. At St. Louis College of Pharmacy, a Flipped Classroom model is tested in the Advanced Physiology course where students are assigned homework in the form of lecture videos, PowerPoint presentations and reading assignments prior to their class meetings. The class time is dedicated to mini lectures, Team‐Based Learning (TBL) activities and individual assessments. Clinical scenarios in the case discussion and the hands on lab activities are designed to provide ample opportunities to apply and review the lecture content. Additional support to help students’ cognitive skills is provided by weekly review sessions, walk‐in tutoring opportunities and the availability of tutors for one‐on‐one instruction. The assessment includes a combination of factual details, problem solving, and comprehension skills. Student survey indicated that the homework assignments helped students come prepared (75 %), and changed the way they studied (65 %). Students also believed that the TBL session helped them prepare and understand the concepts better (83 %). Students’ preparedness and the level of engagement during class were improved compared to didactic mode of teaching.
A discrepancy in the improvement of critical thinking skills exists between ESL and non‐ESL students after content‐based instruction and peer discussion (531.19)
Authors: Hryciw Deanne , Burder Ronan , Tangalakis Kathy
Published: June 2021

In students with English as a second language (ESL), language instruction based on relevant discipline specific content can be used to assist students to achieve better learning outcomes. However, a limitation is that it may not develop higher order critical thinking skills. Here we describe a pilot study that uses a peer discussion activity in addition to content‐based instruction, to enhance critical thinking skills in both ESL and non‐ESL Biomedical Science undergraduate students.

Students were provided with a protocol for the analysis of a scientific manuscript. Then, they participated in a peer discussion following a Socratic format. Students wrote evaluative pieces to summarize the manuscript’s topics before and after the discussion.

Using a Bloom’s taxonomy score to rank the students evaluative pieces, only 20% of ESL students improved their critical thinking score, compared to 42% of non‐ESL students. Despite this, survey data demonstrated that all students agreed that the peer discussion improved their understanding of the content in the manuscript and enhanced their ability to review scientific literature. Importantly, ESL students believed that the discussion made them feel positive about their ability to read scientific literature. Thus, further development of specific tools for enhancing critical thinking in the ESL student group is warranted.
The FUTURE Program: ensuring that underserved populations become the scientific and civic leaders of tomorrow (531.24)
Authors: Roberts Rebecca
Published: June 2021

The Center for Science and the Common Good (CSCG) at Ursinus College provides a framework for all Ursinus science majors to think, speak, and write about the impact of science on the common good. One target group of the CSCG is the FUTURE Students (Fellowships in the Ursinus Transition to the Undergraduate Research Experience), members of underserved populations in science who are given research opportunities early and often to ensure that they can become scientific and civic leaders. We include underrepresented minorities (URMs), women, first‐generation college students, and disabled students in this program. Students in the program enter a research experience at an early point, participate in a team‐taught course titled “Science and Mathematics in Society”, continue research in subsequent years, and are closely mentored by a faculty member and an upper class FUTURE Mentor. FUTURE students are encouraged to become FUTURE Mentors and to participate fully in the CSCG. The CSCG was founded in 2012 through an HHMI grant following a successful pilot FUTURE cohort in 2010. The program has had a positive impact on students, making them more resolute in their career choice, giving them confidence in their ability to communicate and make connections with faculty members, and increasing their awareness of the role of science in the common good.

Grant Funding Source: Supported by the Center for Science and the Common Good with support from HHMI
A simple approach for teaching a complex topic by asking students to participate in an engaging narrative to improve attention and retention (531.4)
Authors: Nguyen Jessica , Carvalho Helena
Published: June 2021

Didactic teaching, while it is the most widespread form of instruction, is not the most effective form for many learning styles. Topics in physiology are complex and in some cases counterintuitive. To teach ‘changes in fetal circulation post partum’ an innovative approach was developed and applied in small group setting. A narrative story line lead the group to imagine circulation from the perspective of the fetal red blood cell (RBC). Students were asked to pretend to be RBCs and ‘travel’ the intrauterine fetal circulation while following verbal instructions. Starting at the placenta they were walked through the transfer of oxygen from mother to infant what reinforced the comprehension about oxygen affinity to fetal hemoglobin (Hb) versus adult Hb. As oxygenated blood the students traveled through the liver via a prenatal bypass, the ductous venosus. They then traveled to the heart and were presented with two other prenatal passages; the foramen ovale and the ductous arteriosus. Rationale for these bypasses were discussed. The students were then presented a conflict they must resolve; these prenatal pathways are subsequently shut down after birth. The students had to problem solve as a team to find their new path of circulation.
Medical gross anatomy: conversion of a traditional discipline‐based course to a briefer and more clinically focused foundational module in a revamped preclinical curriculum (532.1)
Authors: Davies David , Malak Sharp , Jensen Joseph , Akhter Noor , Beneš Helen , Childs Gwen , Daniel Joseph , Hayes Helen , Kandavalli Naveen , Phelan Kevin , Stanley Laura , Syed Mohsin
Published: June 2021

Here we report ongoing work to replace discipline‐based courses with foundational and system‐based modules in a new curriculum. As part of this effort, a new Human Structure module was designed to teach clinically relevant anatomy as a basis for subsequent courses. Multiple steps were employed to achieve these goals. Specifically, course duration was decreased from 19 to 8 weeks, instructional time was trimmed 25% (168 hrs to 125 hrs), scheduled lab sessions were cut 40% (45 labs to 27 labs), and embryological topics were moved to other modules. Clinical lectures were increased 57% (7 hrs to 11 hrs): 11 radiologists and surgeons contributed lectures, led case discussions, and/or worked in the dissection laboratory. Five clinical case studies and four histology lectures were also added. Three outcomes gauge the effects of these changes. On a customized NBME final exam, students achieved a mean score of 87.6% ± 7.4 (STD, n = 172). Students evaluated the course with an online survey as 4.44 ± 0.85 (Likert Scale, 5 = outstanding). Similarly, student evaluations of 19 instructors yielded a mean score of 4.38 ± 0.78. Informal faculty assessment indicated that student participation, performance, and morale were equivalent to that in the traditional gross anatomy course.

Grant Funding Source: Supported by: UAMS Dept. of Neurobiology and Developmental Sciences.
Creation and implementation of a community service honor society (534.2)
Authors: Puder Barb , Elliott Craig
Published: June 2021

An honor society acknowledging students who demonstrate excellence in community service/ outreach/volunteerism has been established at Samuel Merritt University. This honor society was developed from the observance that although most institutions recognize outstanding academic performance, students who have the ability to volunteer their time and maintain good academic standing should be recognized as well. Students are required to self‐enroll onto the online community service webpage. The webpage is an online course management system called Canvas that has been designed to serve 2 major purposes: 1. To announce upcoming community service opportunities, and 2. Create a method to track and record community service hours. Upon enrollment, students view and sign up for the upcoming outreach activities. After completion of the service activity, students submit a self‐reflection online to receive credit. Prior to graduation, students apply to be accepted into the honor society and several criteria are used to evaluate the student’s performance. Students can use the information saved on the community service website to aid in applying for scholarships, grants, residencies, and employment.
Premedical anatomy experience impact on study time for medical students (534.8)
Authors: Bohn Jacqueline , Harper Noah , Jensen Jeff , Folson Susan , Morton David
Published: June 2021

Most medical schools do not require gross anatomy as a prerequisite for admission. Our previous study questioned whether students who had taken an anatomy course prior to medical school performed better in anatomy and in overall academics than students who had not. Data demonstrated that 1st year students at the University of Utah School of Medicine (UofUSOM) showed no statistically significant difference in anatomical or overall academic performance between students who had anatomy and students who did not have anatomy prior to entering the UofUSOM (with the exception of the final practical cadaver exam). A limitation of this previous study was that the survey did not address the relative number of hours spent studying each week by students who had exposure to anatomy prior to entering the UofUSOM to those that did not. The current survey assessed the average number of anatomy and overall weekly study hours self reported by freshman medical students in order to better understand academic performance by students with and without premedical anatomy exposure.
Discovery and characterization of an essential Francisella tularensis protein required for tularemia disease development (538.5)
Authors: Lo Karen , Visram Shyanne , Guttman Julian
Published: June 2021

Potential bioterrorism agent Francisella tularensis subspecies tularensis (F. tularensis) causes tularemia; a disease that can cause up to 60% mortality. An essential step to developing tularemia is the ability of F. tularensis to escape the vacuole that it initially occupies to replicate in the cytosol of epithelial cells. We hypothesize F. tularensis possesses virulence factors (VFs) that facilitate this process. In this study, we identified a crucial VF for tularemia development. To identify this VF, we screened a 3,050 Francisella mutant library for microbes deficient in bacterial replication once inside host cells. By using subsequent bioinformatics analysis we identified proteins in a subset of mutated bacteria that fit traits of VFs and we tested their ability to cause tularemia‐induced death in mice. Mice infected with bacteria inactivated in 1 novel gene appeared uninfected whereas wild‐type infected mice died within 2 days. Electron and immunofluorescence micrographs of infected hepatocytes showed that this gene aids the bacteria in exiting lysosome‐associated membrane protein‐1 (LAMP‐1) positive membrane vacuoles leading to bacterial replication in the cytosol. Our findings demonstrate this novel VF enables F. tularensis to escape into the cytosol and ultimately cause disease.
Auto‐regulatory interactions between NFI occupancy and ETV1 direct the timing of gene expression in late maturing neurons (539.10)
Authors: Ding Baojin , Cave John , Kilpatrick Daniel
Published: June 2021

Developmental timing mechanisms play an essential role in nervous system development, and their disruption during synapse maturation is implicated in neurodevelopmental disorders. To examine how the timing of gene expression related to synapse formation is regulated during neuronal maturation, we used cerebellar tissues from gene knockout mouse and lentiviral transduction of mouse cerebellar granule neuron (CGN) cultures together with microarray, qRT‐PCR, and chromatin immunoprecipitation assays. We found that Nuclear Factor One (NFI) regulates the temporal expression of genes required for dendrite and synapse formation via its delayed binding to their promoters in developing CGNs. Further, a second transcription factor, ETV1, directly binds to and is required for the NFI occupancy and expression of numerous NFI‐dependent genes in vitro and in vivo. ETV1 binds to and activates the Etv1 gene itself, and this auto‐regulatory loop is induced prior to the activation of downstream target genes. We propose that ETV1 activates NFI temporal occupancy of late‐expressed genes in a stepwise manner by initial positive feedback up‐regulation of the Etv1 gene followed by activation of downstream late genes as ETV1 expression increases. Sequential transcription factor auto‐regulation and subsequent binding to downstream promoters provides a developmental timing mechanism for dendrite/synapse gene expression.

Grant Funding Source: Supported by PHS grant NS063047 to DLK
A novel cytoplasmic tail fqqi motif mediates internalization and intracellular trafficking of guanylyl‐cyclase/natriuretic peptide receptor‐A (539.4)
Authors: Mani Indra , Tripathi Satyabha , Pandey Kailash
Published: June 2021

Cardiac hormone, atrial natriuretic peptide (ANP) modulates blood pressure and blood volume by activation of guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA), which produces the second messenger cGMP. The aim of the present study was to examine the role of small sequence motifs in the internalization and intracellular trafficking pathway of NPRA by immunofluorescence confocal microscopy. A chimeric protein of enhanced green fluorescence protein (eGFP) and NPRA (eGFP‐NPRA) was used to study the NPRA trafficking in the intact mouse mesangial cells (MMCs). We have identified a FQQI (Phe790, Gln791, Gln792 and Ile793) motif in the cytoplasmic tail of NPRA sequence and mutated with alanine residues (FQQI/AQQA) and cells were transiently transfected with both constructs. In the ANP‐treated cells, colocalization of wild‐type eGFP‐NPRA with pan‐Cadherin, a plasma membrane marker, indicated that internalization of receptor accounted for 59.2%, 70.0%, 71.8%, and 77.4% at 5, 10, 15, and 30 min, respectively of total fluorescence intensity compared with untreated cells. Colocalization of eGFP‐NPRA with early endosome antigen‐1 (EEA‐1) marker was maximum at 5 min (64.5%), and gradually decreased at 30 min (24.4%). However, the mutation of FQQI/AQQA decreased the receptor internalization by 45% (P< 0.001) and the receptor colocalization into endosome by 58% (P<0.001), respectively, compared with wild type receptor. Our, study suggest that a novel FQQI motif in the cytoplasmic tail mediates the internalization and intracellular trafficking of NPRA, which plays critical roles in hypertension and cardiovascular homeostasis.

Grant Funding Source: Supported by the NIH grant (HL57531)
Subcellular gravin localization regulates PKA compartmentalization and PKA‐dependent signaling (539.7)
Authors: Schott Micah , Maliske Ben , Thompson Gonowolo Faith , Grove Bryon
Published: June 2021

Scaffold proteins play a critical role in cellular homeostasis by positioning signaling molecules in proximity to downstream effectors. However, some scaffold proteins do not assemble static enzyme complexes, but form dynamic signalosomes that traffic to different subcellular compartments in response to stimuli. Gravin (AKAP12) ‐ a multivalent scaffold linked to cell cycle progression, cellular migration, β2‐adrenergic receptor regulation, and hippocampal learning and memory ‐ anchors PKA and other enzymes to the plasma membrane but is redistributed to the cytosol after [Ca2+]i elevation. We postulate that gravin redistribution represents a novel mechanism for cross‐talk between Ca2+‐dependent and cAMP‐dependent signaling pathways. To assess this, we measured the impact of gravin localization on compartmentalized PKA activity using the PKA FRET biosensor AKAR3. Expression of gravin in cells lacking endogenous gravin caused an increase in forskolin‐dependent PKA activity in AKAR3 constructs targeted to the plasma membrane when compared to control cells lacking gravin or expressing a gravin construct lacking the PKA‐binding domain. Expression of a gravin mutant with reduced membrane localization showed no increase in membrane PKA activity compared to control cells lacking gravin. Interestingly, gravin expression caused a decrease in forskolin‐dependent PKA activity in cytosolic AKAR3 constructs compared to control cells lacking gravin. These results confirm that gravin localization regulates PKA signaling and support the hypothesis that gravin mediates cross‐talk between Ca2+ and cAMP‐dependent signaling in functions such as in cell cycle progression, cellular migration, β2‐adrenergic receptor regulation, and hippocampal learning and memory. Based on these results, dynamics in scaffold protein localization likely represents an important paradigm for the regulation of cellular signaling networks.

Grant Funding Source: NIH P30GM103329
Cis‐regulatory control of Hedgehog target genes (541.1)
Authors: Rivera‐Lugo Rafael , Ramos Andrea , Blake Victoria , Barolo Scott
Published: June 2021

The Hedgehog (Hh) signaling pathway is an important regulator of embryogenesis and tissue homeostasis during adulthood. This pathway is conserved from flies to humans, where it controls the transcription of key target genes. Cubitus interruptus (Ci), a member of the Gli family of transcription factors (TFs), regulates the transcription of Hh target genes by binding to regulatory sequences known as enhancers. In the Drosophila wing, preliminary data show that enhancers that contain consensus Ci binding sites, GACCACCCA, are repressed in cells that lack Ci. We hypothesized that an unidentified transcriptional repressor binds preferentially to consensus Ci binding sites to refine the expression patterns of some Hh target genes. In this project, we identified in silico nine related TFs that have similar Ci binding motifs, and tested whether they selectively recognize Ci binding sites in vitro. We transcribed and translated the DNA binding domains of Button head, Drosophila Specificity Protein 1, Lameduck (Lmd), Sugarbabe, Odd paired (Opa), Klumpfuss (Klu), Stripe, Scratch, and Longitudinals Lacking. Using Electrophoretic Mobility Shift Assays, we tested if these proteins differentially recognized consensus Ci binding sites versus non‐consensus binding sites, which are sequences that deviate from the consensus by one or more base pairs. We found that Opa, Klu, and Lmd recognized consensus Ci sites, but not non‐consensus sites. In addition, we performed in vivo genetic experiments to knockdown these proteins in the wing and the embryo. Knocking down Klu in the wing, resulted in a posterior cross vein phenotype, and knocking down Opa resulted in lethality. Knocking down Klu and Opa in embryos caused derepression of the Hedgehog target gene patched. These results posit a potential role for these TFs in refining the expression of Hh target genes.
Exercise training attenuates alterations in blood pressure and heart rate variability in old ovariectomized rats under fructose consumption (546.2)
Authors: Machi Jacqueline , Bernardes Nathalia , Dias Danielle , Moraes‐Silva Ivana , Santos Fernando , Wichi Rogerio , De Angelis Katia , Irigoyen Maria Claudia
Published: June 2021

The aim of the present study was to investigate the effects of exercise trainig (ET) in metabolic and cardiovascular parameters of old Wistar female under 24 months old. Female rats were ovariectomized and trained on a treadmill (1 h/day; 5 days/wk for 8 wk). The animal had free access to standard chow and fructose solution (100g/L) in drinking water and divided into 3 groups: ovariectomized (O, n= 8), ovariectomized fructose (OF, n= 8) and trained ovariectomized fructose (TOF, n= 8). Adipose tissue, insulin resistance, physical capacity, blood pressure and heart rate variability were evaluated at the end of the protocol. The metabolic results showed that ET decreased the adipose tissue in OFT (TOF: 5.28±0.6; O: 6.5±0.7 vs. OF: 10.07±0.6 g) and insulin resistance (TOF: 5.0±0.2 vs. OF: 3.2±0.6 mg/dl/min). The physical capacity was increased in (TOF: 1.8±0.1) compared with sedentary (O:1.1±0.05; OF: 1.1±0.05 Km/h). ET attenuated the increase in MBP (TOF: 107±1: O:119±2; OF:119±2 mmHg). The same behavior was observed in SBP (TOF: 129±1; O:142±2; OF:143±2 mmHg) and DBP (TOF: 89±1; O:100±2; OF:98±2; mmHg). TOF group showed increased pulse interval variance (133.3±36 ms2) when compared with O (88.3±22 ms2) and OF (114±22 ms2). The cardiac sympathetic modulation was decreased in TOF (1.95±0.4 ms2) when compared with OF (4.18±0.8ms2.) while cardiac vagal modulation was increased (TOF: 76±2.7 vs. OF: 69±3.6 n.u.). Consequently, sympatho‐vagal balance was decreased in OFT (0.33±0.04) vs. OF group (0.52±0.09). A similar behavior was observed for sympathetic modulation of blood pressure (TOF: 30±2.5 vs. O 37±3.4; OF: 40±4.1). In conclusion, exercise training effectively improves metabolic and cardiovascular parameters (blood pressure increase and heart rate variability) altered by excessive fructose consumption in aged rats submitted to ovarian hormone deprivation.
Oxidative control of oligomeric structure formation in Vibrio cholerae cryptochrome 1 (549.2)
Authors: Velez Julian , Gindt Yvonne
Published: June 2021

Cryptochrome is a member of the blue‐light photoreceptor family, and it appears to play a role in the regulation of the circadian rhythm in organisms. The oligomeric structure of the protein appears to play a key role in the signaling mechanism. Vibrio cholerae Cryptochrome 1 (VcCry1) will be used as model protein for this study; it may be involved in both DNA repair and blue‐light signaling in vivo. From our preliminary data, the oligomeric structure in VcCry1 appears to be related to the oxidation state of the active site FAD cofactor. Chemical cross linking will be used to examine the link between FAD oxidation state and oligomeric structure.
The role of water dynamics in mediating protein‐DNA interactions (549.4)
Authors: Naughton Brigitte , Pollak Adam , Han Songi , Reich Norbert
Published: June 2021

The role of hydration water on protein and DNA surfaces is fundamental to their interaction and processes, yet experimentally challenging to study. We recently applied a novel Overhauser dynamic nuclear polarization‐enhanced NMR method to the study of biological hydration water which provides unprecedented insights into ps to sub‐ns water dynamics within 5‐10 Angstrom of molecular surfaces: waters loosely associated with DNA have surprisingly high degrees of mobility, nearly unperturbed, while waters surrounding protein surfaces are significantly fortified, compared to the dynamics of bulk water. Here we investigated how water mobility contributes to the site location mechanisms of several DNA binding proteins. Dimethyl sulfoxide increases this water mobility on molecular surfaces (DNA or protein) and is predicted to increase the site location efficiency of proteins that rely extensively on a sliding mechanism; we confirmed this to be the case with EcoRI endonuclease when adding 1% DMSO. In contrast, glycerol is predicted to have a retarding (or sometimes neutral) effect on surface water mobility and thus sliding translocation kinetics, which we again confirm to be the case with EcoRI endonuclease when adding 1% glycerol. In contrast, the translocation efficiency of DNA adenine methyltransferase (Dam) that does not rely extensively on sliding is not altered with either osmolyte addition. The observed effects on processivity have been corrected for minor changes in catalytic efficiency in the presence of osmolytes. Our results reveal that water’s fast moving properties around DNA are important for protein‐DNA interactions and in particular, the translocation along the double helix, whose efficiency can be slowed or enhanced when the water barrier on the DNA or protein surfaces is strengthened or weakened by the addition of glycerol or DMSO.
Structure/function studies on the allosteric mechanism of Rad51 recombinase (549.8)
Authors: Morrical Scott , Mandigo Torrey , Silva Michelle , Maher Robyn , Villanueva Nicolas , Eckenroth Brian , Zahn Karl , Nguyen Xinh‐Xinh , Averill April
Published: June 2021

Eukaryotic Rad51 recombinase plays a central role in maintaining genome stability by promoting homology‐directed repair of DNA double‐strand breaks and stalled replication forks. Rad51 forms presynaptic filaments on single‐stranded DNA, which activates the ATPase, homologous pairing, and DNA strand exchange activities of the enzyme. Structure/function studies of yeast Rad51 reveal that coordination between the ATP and DNA binding sites is mediated by residues at the protomer‐protomer interface and in DNA binding loops L1 and L2. Conserved interface residues Phe‐187 and His‐352 play central roles in sensing the ATP‐bound state of Rad51‐ssDNA filaments and inducing conformational changes in the filament that are necessary for DNA strand exchange. An F187A mutant is proficient in ssDNA‐dependent ATPase activity but deficient in DNA strand exchange, suggesting an inability to couple ATP binding to the homology search function of Rad51. Mutations at the His‐352 locus affect ssDNA‐binding affinity and catalytic turnover. An H352Y mutant catalyzes a single round of DNA strand exchange and subsequently fails due to a nucleotide exchange defect. Meanwhile, new X‐ray crystallographic structures reveal previously unseen details of the DNA and ATP binding surfaces of Rad51. The structures indicate that conserved aromatic residue Phe‐290 is important for stabilizing a conformation of L1 that allows optimal contacts between conserved Arg‐293 and DNA. Results of this work will facilitate modeling of the structural transitions that occur in Rad51 filaments during recombination.

Grant Funding Source: NIH grant no. P02 CA098993
Investigating the role of 53BP1 in DNA repair (554.1)
Authors: Flores Desirey , Grosesser Torsten , Hatherill J. Robert
Published: June 2021

Hypothesis: 53BP1 foci will return to baseline 24 hrs after radiation exposure, but it will take the BALB/c longer due to their increased radiosensitivity. P53 binding protein No.1 (53BP1) has been shown to accumulate in nuclear areas of a cell that have extensive DNA damage such as double strand breaks (DSB). DSBs are the worse type of DNA damage and cells that have been exposed to damaging agents such as ionizing radiation are susceptible to developing cancer. Micronuclei (MN) were scored as markers of induced DNA damage, which appear as extra nuclear bodies that are formed from un‐ or mis‐rejoined DSBs. In the present studies cells from BALB/c and SPRET/EiJ mice were used to investigate the effects of acute X‐rays and prolonged 32P exposures. 53BP1 foci were imaged after acute exposures and MN were scored after prolonged exposures. BALB/c and SPRET/EiJ cells were challenged with an X‐ray dose of 1 Gy and immunostained to evaluate the number of foci. Both cell lines were also exposed to 32P over a prolonged amount of time to receive a total dose of 0.4 Gy and were then scored for MN. After the acute exposures the number of foci was higher in the cells derived from BALB/c mice compared to SPRET/EiJ cells and after 24 hrs the amount of foci returned to baseline in both cells lines. We conclude that cells from BALB/c mice exposed to radiation sustained more DNA damage than the SPRET/EiJ cell line and 53BP1 foci are localized to areas of DNA damage.

Grant Funding Source: NSF ATE DUE 1205059
Posttranslational arginylation is essential for stress response (555.15)
Authors: Kumar Akhilesh , Zhang Fangliang
Published: June 2021

Arginylation consists of addition of an extra arginine to an existing peptide chain, thus changing the de facto primary sequence as well as the surface change. This process is evolutionarily conserved and mainly mediated by arginyltransferase1 (Ate1). While earlier studies found that the overall arginylation activity often increases during stressing conditions including heat shock, oxidative stress, and injury, little is known about the role of arginylation or Ate1 in stress response. By using multiple types of eukaryotic cells as test models, we found that Ate1 is a pro‐apoptotic factor. Knockout of Ate1 in cells leads to a retardation of stress response and the diminish of apoptosis and cell death during exposure of high doses of various stressing factors including heat shock, oxidative and osmotic stress, and heavy metal. Oppositely, over‐expression of Ate1 leads to cell death. Intriguingly, we also found that the activity of arginylation is essential for the wellbeing of the cells during mitochondria‐dependent metabolism as well as nutrient‐deprivation. Our data suggests that arginylation is a global regulator for metabolism and stress response, and provides an explanation for the observed role of Ate1 in carcinogenesis and injury.

Grant Funding Source: NIGMS
Alterations to O‐GlcNAc cycling disrupt mitotic phosphorylation (555.16)
Authors: Whelan Stephen , Tan Ee Phie , Costello Catherine , McComb Mark , Slawson Chad
Published: June 2021

O‐GlcNAc is a post‐translational modification (PTM) consisting of a single N‐acetylglucosamine moiety attached to serine and threonine residues in nuclear and cytoplasmic proteins. O‐GlcNAc levels can cycle dynamically in response to hormones, nutrients, or stress in order to regulate a multitude of cellular processes. For instance O‐GlcNAc cycling is an important regulator of mitosis. Recently, we demonstrated that cells with a gain of function in either O‐GlcNAc transferase (OGT) or O‐GlcNAcase (OGA) have disrupted mitotic spindles and mitotic exit defects. OGT/OGA gain of function caused the formation of large poorly condensed spindle chromosomes, misaligned kinetochores, and an increase in multipolar spindles. This phenotype was caused partially by a disruption in phosphorylation. In this current study, our objective is to determine how alterations in O‐GlcNAc cycling disrupt mitotic phosphorylation cascades. We employed quantitative proteomics in order to identify changes to mitotic phosphorylation. We were able to quantitate both protein expression changes as well as alterations in PTM’s in OGT or OGA gain of function cells. We identified over 1000 proteins whose expression as well as PTM status was altered by aberrant O‐GlcNAcylation. These results demonstrate the cross‐talk between protein modifications involved in regulating mitotic progression and suggest that diseases that disrupt one PTM will also alter the actions of other PTM’s.

Grant Funding Source: NIH‐NHLBI Contracts: HHSN268201000031C ‐ CPC; NIH Grants: P41 RR010888/GM104603 – MSR R21 HL107993 ;
Heat‐induced degrons and the co‐chaperone Ydj1‐adaptor mediate the ubiquitination of misfolded proteins by the Rsp5 ubiquitin ligase upon stress (558.1)
Authors: Fang Nancy , Mayor Thibault
Published: June 2021

The ubiquitin proteasome system is critical for the prompt elimination of misfolded proteins, which pose a deleterious risk to the cell. Indeed numerous diseases are associated with protein misfolding and aggregation. The heat‐shock response mediates several cellular events to prevent accumulation of misfolded proteins including upregulation of heat‐shock proteins and increased proteolysis. We found that upon heat‐stress Rsp5 is the main E3 ligase that ubiquitinates cytosolic misfolded proteins to target them for proteasome degradation. Notably, degradation of misfolded proteins is dependent on both Ubp2 and Ubp3 deubiquitinases. The absence of either deubiquitinase leads to reduced cell fitness upon misfolding stresses. We also found that the association of Rsp5 with the Hsp40 co‐chaperone Ydj1 increases upon stress and that association is required for the ubiquitination of heat‐induced Rsp5 substrates. In addition, PY Rsp5‐binding motifs are also enriched among heat‐induced Rsp5 substrates and found primarily in structured regions, often less accessible. Our results support a bipartite recognition mechanism combining chaperone‐dependent and heat‐induced degrons that mediate ubiquitination of cytosolic misfolded proteins by Rsp5.
Novel protein engineering of lipase by protein folding memory (567.1)
Authors: Satomura Atsushi , Kuroda Kouichi , Ueda Mitsuyoshi
Published: June 2021

Some hydrolytic enzymes including lipases and proteases have propeptides in their N‐ or C‐ terminals. Such propeptides seem to function as an intramolecular chaperone to facilitate the folding of their active domain and are cleaved off after completion of the function as chaperone. By introducing mutations into propeptides of lipases and altering the chaperone functions, we have successfully created altered lipases which showed improved activities and stabilities, in spite of the same primary sequences as the original lipase. We named this phenomenon “protein folding memory”, because mutations in the propeptide imprinted memories into the mature enzyme structures [1]. Our study aimed to alter a substrate recognition of Rhizopus oryzae lipase (ROL) by protein folding memory. Eight hydrophilic amino‐acids were introduced into a part of the propeptide essential for the folding of ROL. This propeptide‐mutated ROL showed a 1.3‐fold higher lipase activity than that of WT ROL. In addition, the propeptide‐mutated ROL remarkably exhibited a peptidase activity, which was absent in the original form [2]. Here, we report the structural and functional differences between WT ROL and propeptide‐altered ROL with the same primary sequence.

[1] M. Nagayama, et al., Biochemistry, 51, 3547‐3553 (2012) [2] A. Satomura, et al., submitted
Analysis of co‐chaperone interactions with Hsp90 (567.7)
Authors: Wolmarans Annemarie , LaPointe Paul
Published: June 2021

Heat shock protein 90 (Hsp90) is a highly abundant, ATP‐dependent molecular chaperone that is present in all eukaryotic cells. It is essential for the maturation and regulation of a specific set of substrate (client) proteins, many of which are key players in oncogenesis. Hsp90, as a homodimer, acts in the context of a complicated ATPase cycle. Co‐chaperone proteins guide and modulate Hsp90 through its functional cycle in order to mature clients, by binding to one or more of Hsp90’s domains in a sequential manner. To address Hsp90’s conformational requirements for cycle progression, we analyze two specific co‐chaperones, Aha1 and Sba1, which bind to Hsp90 in its closed, ATP‐bound state. We have constructed 14 Hsp90 variants harboring mutations in the N terminus of Hsp90 which have the potential to block the binding of Aha1 and Sba1. Several N‐terminal mutants show promising results as they are weakly stimulated by Aha1 or cannot be inhibited by Sba1 from the Aha1‐stimulated rate. Interestingly, one specific mutant harboring the D132K mutation does not interact with Sba1. Analysis of these five mutants in combination with previously characterized Hsp90 mutants will allow for the restriction of either Sba1 or Aha1 to one monomer of Hsp90 or the other. This subunit complementation strategy will bring insight into how Aha1 and Sba1 act asymmetrically on Hsp90.

Grant Funding Source: Supported by CIHR
Structure, function and evolution of the mitochondrial replicative DNA helicase (570.1)
Authors: Kaguni Laurie , Stiban Johnny , Farnum Gregory , Hovde Stacy , So Minyoung
Published: June 2021

Mitochondrial DNA (mtDNA) helicase is an essential part of the mtDNA replisome. The mtDNA helicase shows high amino acid sequence homology with bacteriophage T7 gene 4 protein (T7gp4), which has both DNA primase and DNA helicase activities in its N‐terminal domain (NTD) and C‐terminal domain (CTD), respectively. Although the NTD of mtDNA helicase in metazoa does not have detectable primase activity, retention of extensive amino acid sequence homology throughout most eukaryotic lineages suggests other important roles, such as in protein stability and/ or protein‐protein interactions. Here, we investigate the structural and biochemical properties of the NTD of Drosophila mtDNA helicase by constructing two N‐terminal truncated variants: a full‐length NTD (including both the zinc binding domain (ZBD) and the RNA polymerase domain (RPD)), and a ZBD only construct. We show that NTD and ZBD constructs of Drosophila mtDNA helicase contain an iron‐sulfur cluster, most likely a 2Fe‐2S, as determined by mass spectrometry, and UV‐visible and atomic absorption spectroscopy. Mutagenesis studies show that the four conserved cysteine residues in the ZBD responsible for binding zinc in T7gp4, bind the iron‐sulfur cluster. We extend our biochemical studies in vivo through several approaches to identify partner proteins of the Drosophila mtDNA helicase that may elucidate the functional role of its iron‐sulfur cluster.

Grant Funding Source: NIH GM45295
Structure‐function relationships in the mitochondrial single‐stranded DNA‐binding protein (573.2)
Authors: Neitzke Orrin , Kaguni Laurie
Published: June 2021

Mitochondrial single‐stranded DNA‐binding protein (mtSSB) plays a key role at the mitochondrial replication fork. Previous studies examining variant human mtSSBs showed a significant decrease in their ability to stimulate the human mitochondrial replicase, DNA polymerase γ (pol γ) in vitro, suggesting that specific structural features are involved in direct interactions with pol γ (Oliveira, M. O., Kaguni, L. S. (2011) J. Biol. Chem. 286, 40649‐40658). We are currently studying the effects of the corresponding variants of the Drosophila melanogaster mtSSB (Dm mtSSB) in coordinating DNA polymerase γ function and by doing so, we hope to understand the biochemical properties of this highly conserved protein that allow for differential stimulation of the human and fly replicases, which themselves differ in oligomeric structure, and nearly ten fold in the level of stimulation by their cognate mtSSBs. We have generated three variants of Dm mtSSB that introduce changes in amino acid residues located on the protein surface, and that may participate in both physical and functional interactions with pol γ. We have examined their ability to bind ssDNA and to stimulate DNA polymerase activity. We expect to draw a structure‐based biochemical comparison between the interactions of Dm mtSSB and Hs mtSSB with their respective pol γ holoenzymes.

Grant Funding Source: NIH GM45295
Modes of mitochondrial DNA replication and its regulation in Drosophila melanogaster (573.3)
Authors: Lillsunde Paivi , Oliveira Marcos , Griffith Jack , Jacobs Howard , Kaguni Laurie S.
Published: June 2021

Although replication of the mitochondrial DNA genome has been studied extensively in mammalian and yeast models, the mechanisms of mitochondrial DNA (mtDNA) replication in the widely‐used model organism Drosophila melanogaster remain largely unknown. We employ a comparative approach to analyze mtDNA replication intermediates using two‐dimensional neutral agarose gel electrophoresis (2DNAGE), and to visualize their architecture by transmission electron microscopy (TEM). Our studies target the nuclear‐encoded replisome proteins (DNA polymerase γ, mtDNA helicase and single‐stranded DNA‐binding protein), as well as transcription factors (mTTF and mTerf5), and their roles in the regulation of mtDNA replication through overexpression of both wild type proteins and biochemical variants in cultured insect cells and fly strains. Whereas 2DNAGE allows the analysis of minimally manipulated DNA preparations, TEM provides molecular details regarding the length of Okazaki fragments and on the presence of double‐stranded DNA and/ or RNA loops at the replication fork. In addition to the evaluation of the modes of replication in normal and perturbed cells, biotin labeling of the replisome proteins enables the determination of their positions on the replicating mtDNA molecule. Together, these methods can be used to identify novel structures and proteins involved in the progression of the replication process.

Grant Funding Source: Supported by Academy of Finland and NIH GM45295
Peculiarities of oxidative metabolism in workers of uranoprocessing company suffering from arterial hypertension (575.3)
Authors: Bekenova Farida , Abduldayeva Aigul
Published: June 2021

The aim of the study was to investigate the oxidative metabolism of the uranium processing enterprise workers with hypertension. For the study of the working population with hypertension 102 patients were selected, corresponding to the inclusion criteria ‐ age from 35 to 50, labor experience in the UE at least 5 years, AH 1 and 2 degrees. Groups have been formed: I group consisted of workers of UE undergoing chronic toxic effects of radiation; II group‐workers at UP, not in contact with harmful factors of production; III group ‐ workers at ball bearing plant SBBP. Analysis of the data showed the most pronounced intensification of lipid peroxidation in workers with AG stage I of the group. Average concentrations of DC and MDA in this group exceeded the same indices of control in 3.3 and 1.7 times, respectively (p<0,01). The prevalence in the blood of workers UE mainly primary products of lipid peroxidation due to the continuous process of the generation of free radicals under the influence of chronic toxic effects of radiation and the initiation of lipid peroxidation chain reactions. CAT activity and antioxidant activity in hypertensive patients in group I was significantly different from those in healthy individuals, respectively, decreasing by 23.5 and 38.7% of control values. At the same time, in groups II and III showed increased activity of CAT and AOA by an average of 44.2 and 8.6 %, respectively, relative to control.
Elucidating the role of cyclin‐dependent kinase 5 in insulin exocytosis: studies toward understanding a possible link between neurodegeneration and type II diabetes (576.4)
Authors: Trye Alice , Aguanno Ann
Published: June 2021

Cyclin dependent kinase 5 (CDK5) is an atypical member of the cyclin dependent kinase family, as it does not play a role in regulation of the cell cycle. Our lab studies CDK5 in the development of mammalian tissues, and has previously shown that chemical inhibition of CDK5 results in an aberrant neuronal phenotype in nervous system cell models. CDK5’s role in the development and functioning of the nervous system is now well established. Recent reports suggest CDK5 plays a role in non‐neuronal tissues. This prompted us to investigate this enzyme in the pancreas; specifically, whether CDK5 is a positive or negative regulator of insulin exocytosis, since published reports are contradictory. We use the acinar cell line AR42J as a model for the pancreas and insulin‐producing cells and have attempted to verify insulin secretion from transdifferentiated AR42J cells using a novel protein assay. Our method, however, proved problematic. Here, we describe a revised method of detecting secreted insulin and the results of chemical inhibition of CDK5 on insulin secretion. Our findings address the effectiveness of this modified method and begin to uncover the role of CDK5 in insulin exocytosis. These results further suggest that this “neuronal” protein is also important in pancreatic development. We propose, therefore, that CDK5 may be a potential link between two correlated diseases, Alzheimer’s Disease (AD) and Type II diabetes.

Grant Funding Source: ASBMB UAN Undergraduate Summer Research Award