bluuebunny/crossref_metadata_2025_split huggingface dataset:
Size Matters? Evaluating the Effect of Size on Anatomy Learning
Authors: Hildebrand Alexandra Helene , Wainman Bruce , Fenesi Barbara , Brewer‐Deluce Danielle , Dong Angela , Xie Jim , Yang Jack 
Published: June 2021

Until recently, the size of an anatomical structure for learning was simply its natural size. Now, with increasing accessibility of 3D scanning and printing, a highly accurate model of virtually any size can be produced. But the question remains, what is the best size for learning? To investigate the effect of object size on learning, 3D models were printed from surface scans of bones using a structured light 3D scanner. A human thoracic vertebra and a human hemipelvis were chosen both for their variation in size and the fact that bones lend themselves well to 3D printing. The bones were each printed in PLA filament at 50%, 100%, and 400% scale. Undergraduate students from McMaster University (n=120) with no prior knowledge of anatomy were randomized into six groups according to 1) which size of model they would learn from and 2) which bone they would learn first. Each participant was asked to learn nominal anatomy from both a hemipelvis and a vertebra model of the same size. After the learning stage, participants were immediately tested on a real bony specimen. The learning and testing stages were then repeated with the other bone. Finally, participants completed a mental rotations test (MRT) and operation span task (OSPAN) to control for any effects of individual differences in spatial ability or working memory on anatomy learning, respectively. Participants also completed a short qualitative survey about their opinions on the size, labelling, colour, and handling of the 3D printed models. Data collection is underway. Test performance will be analyzed using a 3 (bone size: 0.5x, 1x, 4x) × 2 (Bone type: vertebra, hemipelvis) factorial ANOVA. This study has been approved by the Hamilton Integrated Research Ethics Board (HiREB). Insight into how model size affects anatomy learning will provide useful information for educators looking at printed 3D and virtual reality models used for educational purposes.Support or Funding InformationThis study was internally funded by the Education Program in Anatomy at McMaster University.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using Anatomy to Foster Competencies in the First Year of Medical School
Authors: Fahl Jeffrey C , Petersen Kyle , Pettit Alesha , Ralston Claira , Simanton Edward , Haycocks Neil , Cosgrove Ellen M 
Published: June 2021

ObjectiveTo prepare medical students for life‐long learning by using a competency‐based approach to anatomy.BackgroundSmall group learning affords an opportunity for students to learn from each other while they develop four competencies: fostering knowledge for practice, interpersonal and communication skills, professionalism, and personal and professional developmentThe virtual anatomy course is taught in 35 two‐hour sessions with teams of students moving through stations where they teach each other. This approach requires the students prepare by focused reading to teach to their peers.DesignBefore each anatomy session, the students receive an iBook that contains both pre‐readings and modules. The readings replace the traditional introductory lecture; the modules direct the students through the concepts of the lab experience. Students work in pre‐assigned groups of six; two students from each group are required to teach one of the three modules. The modules are 20 minutes in length.OutcomesOver the course of 1 year of study, students must learn to disengage from their pre‐med passive approach to learning and engage in a professional active learning mode.Strengths and LimitationsThere was widespread skepticism that students would not know how to teach each other. Students took many months to switch from a lecture‐based system to active learning. However, by the end of the 12 months of anatomy sessions, the students were engaged, worked effectively with each other, and demonstrated mastery of the anatomy content both in class and on standardized NBME exams.Feasibility and generalizabilityThis approach of learning by teaching makes the out‐of‐class preparation necessary for a ‘flipped classroom’ into a more active process and results in deeper understanding than reading alone. It is both feasible and generalizable to other courses.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acinetobacter and Moraxella OriCs are Functional as Chromosomal Replication Origin Transplants in E. coli
Authors: Alqahtani Abdulaziz , Kadam Rohit , Alfeneikh Asrar , Alhamdani Halah , Grimwade Julia , Leonard Alan 
Published: June 2021

Bacterial chromosome replication is triggered by the initiator protein, DnaA, which assembles into a multimeric complex (orisome) that unwinds the replication origin, oriC, and helps to load the replicative DNA helicase. DnaA is highly conserved and the oriCs from nearly all bacteria carry clusters of oriC‐encoded DnaA recognition sites that guide step‐wise and properly timed orisome assembly. However, the arrangement of these sites is widely divergent among bacterial types and prior studies show that heterologous oriCs are only functional in extremely close relatives with nearly identical origin nucleotide sequence. To investigate the basis for DnaA recognition site diversity and possibly identify shared steps in orisome assembly, we replaced E. coli's chromosomal oriC with heterologous donor oriCs from its distant Gammaproteobacterial relatives and measured replication activity in vivo. Despite carrying little sequence similarity to the host, oriCs from Acinetobacter baylyi (ADP1) and Moraxella catarrhalis (ATCC 25240) were active in E. coli and used alternative pathways to assemble E. coli DnaA into functional orisomes. Mapping studies revealed the transplanted oriCs lacked the distinctive DNA unwinding element comprising 13mer repeat motifs as well as the arrayed low affinity DnaA recognition sites that are both hallmarks of E. coli oriC. Using flow cytometry analysis, we found that ADP1 and M. catarrhalis oriCs were also unable to initiate DNA synthesis at the proper cell cycle time in their E. coli hosts. Thus, the mechanisms for initiation timing must be distinct from those that permit replication fork assembly on these origins. We propose that E. coli and ADP1‐type orisomes share a sub‐architecture required for mechanical function (unwinding and helicase loading), but also carry type‐specific variations in the arrangement and affinity of DnaA recognition sites that are required for proper initiation timing using their cognate DnaAs. We propose that replication origin diversity reflects the many varieties of cell cycle‐coupled regulatory mechanisms used to control the availability/accessibility of DnaA at the oriCs of different bacterial types. Further studies of DnaA‐oriC interactions on heterologous origins may also reveal novel targets for broad spectrum bacterial growth inhibitors as well as those for type‐specific inhibition.Support or Funding InformationNational Institutes of Health (GM54042 to AL) and Florida Institute of Technology Holzer Lequear Fund for Molecular Genetics (to JG).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Confirmation and Effects of VACM‐1/Cul5 Gene Knockout in T47D Breast Cancer Cells and HUVEC Cells Using CRISPR‐Cas9 Approach
Authors: Bonema Sarah , Wabel Emma Ann , Brunink Schuylar , Burnatowaska‐Hledin Maria 
Published: June 2021

The VACM‐1/Cul5 protein is a part of the ubiquitin E3 ligase system responsible for ubiquitin‐dependent protein degradation. The VACM‐1/Cul5 dependent E3 ligase decreases cellular proliferation, and lack of regulation in this pathway can lead to cancer. The CRISPR‐Cas9 system is a bacterial immune system that functions by targeting specific sequences of DNA. It can be programmed to target genes of interest and enables specific gene editing in eukaryotic cells. This system was previously used to knockout VACM‐1/Cul5 in a T47D breast cancer cell line and in a human umbilical vein endothelial cell line (HUVEC). The immunostaining results of control and CRISPR‐transfected HUVEC and T47D cells indicate knockout of VACM‐1. Confirmation of the knockout in the T47D cell line was achieved through a T7 Endonuclease 1 mismatch cleavage assay, proving a homozygous mutation of both alleles. AlamarBlue® growth assays in the T47D cell line and Matrigel® growth assays in the HUVEC cell lines show that VACM‐1/Cul5 knockouts in both cell lines increase proliferation. Together, these results suggest that VACM‐1/CUL5 is an important regulator of cellular growth in breast cancer and endothelial cells. Our current work focuses on determining the effects of the VACM‐1/Cul5 knockout on T47D and HUVEC cell signaling pathways.Support or Funding InformationThis work was supported by the Sherman Fairchild Research Grant to S. Bonema and Biology Department.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A regulatory interplay: hnRNP A1 and AUF1 compete for the same IRES domain to regulate viral translation in EV71
Authors: Davila‐Calderon Jesse , Penumutchu Srinivas , Tolbert Blanton 
Published: June 2021

Enterovirus 71 (EV71), a single‐stranded RNA virus, represents a persistent global health threat against which there is no FDA‐approved vaccine or treatment. Enteroviruses use a type I IRES to facilitate protein synthesis and genome replication by recruiting multiple host RNA‐binding proteins (ITAFs) that fine‐tune translation. HnRNP A1 and AUF1 are antagonistic ITAFs that interact with the EV71‐IRES to upregulate or downregulate viral translation, respectively. However, the molecular mechanisms driving the IRES‐ITAFs interactions to regulate EV71 translation remain poorly understood. Compelled by these challenges, we pursue to identify the nature of the specific recognition between IRES‐ITAFs interactions and its effect on modulating viral replication. Here, using isothermal titration calorimetry (ITC) and Nuclear Magnetic Resonance (1H‐13C HSQC), we found a competition between hnRNP A1 and AUF1 for the same binding site on stem loop II (SL‐II) within the EV71 IRES which has been proven to be a vital RNA element for the virus. Specifically, we observed that, at high concentrations, hnRNP A1 can disrupt the AUF1‐SLII complex while AUF1 cannot outcompete hnRNP A1 for SL‐II binding when the reverse experiment is performed. Furthermore, we also identify conditions to obtain both antagonistic ITAFs bound to the SL‐II at their respective binding sites. Our observations fill important knowledge gaps as the data upholds a regulatory mechanistic model in which hnRNP A1 and AUF1 compete for association to the same IRES domain (SL‐II). Hence, the interaction of hnRNP A1 with SL‐II blocks AUF1 binding thus, activating viral translation which is vital for the virus. In conclusion, our observations enhance our understanding of how an ITAF interplay plays a key role in modulating viral translation and replication, and how EV71 IRES structure adapts to hijack cellular proteins.Support or Funding InformationNIHR01GM126833This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Genetic dissection of endogenous siRNA involvement in the nonstop decay pathway in C. elegans
Authors: Mortezavi Evana , Wong Grace L , Youngman Elaine M 
Published: June 2021

In Caenorhabditis elegans, endogenous small interfering RNAs known as 22G RNAs regulate expression of thousands of mRNAs and other transcripts in developing sperm and egg cells, and loss of 22G RNAs leads to reduced fertility. We are working to understand the molecular triggers that control which genes are targeted for regulation by 22G RNAs. Previous research in the Youngman lab identified a gene of unknown function, F43E2.6 (F43) that is targeted by 22G RNAs only in polymorphic wild strains of worms that lack a stop codon at F43. Conversely, when the stop codon is present, 22G RNA production is low. While it is well documented that mRNAs without a stop codon enter the nonstop decay pathway, resulting in destruction of the readthrough transcript, nonstop decay has not previously been linked to small silencing RNA pathways.In addition to 22G RNAs, the F43 transcript is targeted by a Dicer product, and RDE‐1‐bound Dicer products trigger 22G RNA synthesis in the classical exogenous RNAi pathway. This leads to a critical question about how 22G RNA production is triggered at this locus: is it caused by the nonstop polymorphism, targeting by the Dicer product, or by both pathways acting in cooperation? Further, how does this novel biogenesis pathway integrate into known pathways for 22G RNA production in the C. elegans germline? To determine whether production of 22G RNAs at the F43 locus is independent of the Dicer product and dependent solely on the nonstop polymorphism, we first generated rde‐1; F43 nonstop(ns) homozygotes, which carry the nonstop allele in absence of the Dicer‐dependent RNAi pathway, and have completed deep sequencing of 22G RNAs from this strain. To further define the genetic requirements for production of 22G RNAs targeting this locus, we have sequenced 22G RNAs from strains that carry the nonstop polymorphism but lack either portions of the small RNA biogenesis machinery or of the known nonstop decay machinery (mut‐7; F43ns, prg‐1; F43ns, skih‐2; F43ns, and dom‐34; F43ns). Bioinformatic analyses to assess small RNA levels targeting F43 are underway and will be presented. Although it is well established that 22G RNAs serve to defend the genome against foreign or invasive nucleic acids, this work represents the first evidence that they may also serve to defend the transcriptome from the deleterious effects of low‐quality mRNAs, and suggests the intriguing possibility that quality control of mRNAs may play a role in animal fertility.Support or Funding InformationNSF/MRI #1531855This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of protein‐protein interactions between isoforms of malate dehydrogenase and citrate synthase
Authors: Rementeria Gabriel , Berquist Blaine , Provost Joseph 
Published: June 2021

Sequential enzymes involved in a metabolic pathway are often found in specific and dynamic protein‐protein interactions. The assembly of proteins into this metabolon allow the efficient transfer of substrate between adjacent proteins increasing the throughput and decreasing the potential for side metabolic reactions to occur between competing enzymes. One such pairing is the malate dehydrogenase (MDH) and citrate synthase (CS). In the forward direction of the tricarboxylic cycle, the oxidation of malate to OAA catalyzed by MDH is thermodynamically unfavorable. Coupling and potentially direct shuttling of product‐substrate affords the cycle to continue. The interaction site between mitochondrial MDH and CS has been partially identified via computational docking and cross‐linking studies. However, the domains and key residues of interaction and their impact on the actions and affinity of various isoforms of MDH and CS have not been identified. Using two biophysical techniques, protein thermal melts and surface plasminogen resonance, we have found both mitochondrial and cytosolic human MDH interact with CS (mitochondrial 3 fold greater than cytosolic), while plant MDH shows no interaction. Traditional pulldowns were used in addition to biophysical techniques. Using molecular crowding agents, we have identified the affinity and stoichiometry of interactions. This will allow us to probe the structures for key residues of interaction in the future.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Keto Acids React with Human Serum Albumin to Form Products Similar to Advanced Glycation Endproducts (SAGEs)
Authors: Cai Ang , Liu Weixi , Tang Xiaodan , Ma Hang , Seeram Navindra P. , Dain Joel A. 
Published: June 2021

Diabetic ketoacidosis in children may contribute to cognitive decline. We report that in the model system, keto acids (KAs) can react with human serum albumin (HSA) and one of the effects is responsible for brain injury with children who have the diabetic ketoacidosis. KAs are a group of organic compounds containing functional groups including a carboxylic acid group and a ketone group. Some KAs, namely, pyruvic acid, α‐ketoglutaric acid, levulinic acid, and oxaloacetic acid, play significant roles in biological pathways related to carbohydrates metabolism. Although it is known that reduced derivatives of KAs including methylglyoxal are highly reactive to form advanced glycation endproducts (AGEs), the reactivity of KAs to form products similar to advanced glycation endproducts (SAGEs) remains unclear. Herein, we evaluated four keto acids including pyruvic acid, α‐ketoglutaric acid, levulinic acid, and oxaloacetic acid for their reactivity to form SAGEs with HSA. KAs were incubated with HSA at 37 °C for seven days and the formation of SAGEs were measured by fluorescent assay with an excitation and emission wavelength of 360 nm and 435 nm, respectively. Pyruvic acid and α‐ketoglutaric acid did not induce the formation of SAGEs while levulinic acid and oxaloacetic increased the formation of SAGEs compared to the control group. Further characterizations of KAs induced formation of SAGEs using a series of biophysical methods are warranted as they may provide critical insights of KAs induced formation of SAGEs and related physiological conditions. This result may contribute to understand brain injuries in diabetic ketoacidosis in children.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
FLUORESCENCE SPECTROSCOPIC ANALYSIS OF TonB‐DEPENDENT TRANSPORT IN Klebsiella pneumoniae
Authors: Kumar Ashish , Chakravorty Somnath , Yang Taihao , Majumdar Aritri , Newton Sally , Klebba Phillip 
Published: June 2021

Bacterial iron acquisition systems are potential targets for new antibiotics, because iron plays a central role in energy production, DNA synthesis, intermediate metabolism, nitrogen fixation, and oxygen detoxification. Bacteria actively acquire iron by secreting siderophores that chelate Fe3+, and then transporting the ferric siderophore complexes. Pathogenic K. pneumoniae produces at least four siderophores: enterobactin, glycosylated enterobactin, yersiniabactin, and aerobactin. Like all Gram‐negative species, K. pneumoniae obtains iron with TonB dependent transporters (TBDT) in its outer membrane (OM). For example, aided by the inner membrane protein TonB, that provides energy, the OM protein FepA actively transports ferric enterobactin (FeEnt). K. pneumoniae strain Kp52.145 contains three chromosomal and one plasmid‐mediated fepA homologs, that we individually cloned in pHSG575 and genetically engineered to encode Cys residues at strategic sites for attachment of maleimide fluorophores. Any of these FepA homologs efficiently functioned in the spectroscopic assay: FeEnt binding quenched light emissions from fluoresceinated FepA, but intensity recovered as the bacteria transported and depleted the ferric siderophore from solution. Furthermore, inhibitors that blocked the uptake of FeEnt prevented fluorescence recovery. This individualized, “species specific” method was sensitive and accurate, but genetic and biochemical manipulations of ESKAPE pathogens are potentially hazardous and technically difficult. So, we modified the approach to permit measurements of iron transport in clinical isolates without genetically engineering them. A TonB‐deficient E. coli strain with fluorescently labeled FepA in its OM binds, but cannot transport FeEnt, and thereby constitutes a “fluorescent decoy” (FD) sensor of [FeEnt]. When cohabiting an environment with ESKAPE bacteria (A. baumannii, P. aeruginosa, or E. cloacae), the E. coli sensor monitored their TonB‐dependent uptake of FeEnt. Such sensor strains are broadly applicable, in that they allow direct, in vivo measurements of the high‐affinity transport of virtually any ligand, in any organism. FD sensors also allowed high‐throughput screening of chemical libraries to find antagonists of TonB‐dependent iron uptake by K. pneumoniae or other bacteria. The increasing antibiotic resistance of Gram‐negative pathogens calls for novel agents against new microbial targets, and chemicals that block TonB action may thwart pathogenesis by preventing bacterial iron acquisition in humans and animals.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Med12 Mutations Promote Castration Resistant Prostate Cancer through Hyperactivated GLI3/SHH Signaling
Authors: Muthutkumar Soundharrya , Boyer Thomas , Burleson Marieke Oldenbroek 
Published: June 2021

Prostate cancer is the highest diagnosed and second most common cause of cancer death among US men. The onset and progression of prostate cancer is critically dependent upon androgens through their interaction with the androgen receptor (AR). Accordingly, it has long been established that androgen deprivation therapy leads to prostate cancer regression. Though androgen deprivation therapies are initially highly successful, most patients eventually suffer from recurrence of the disease in the form of castration resistant prostate cancer (CRPC). Currently there is no effective treatment for CRPC thereby highlighting the compelling need to address resistance mechanisms for patients that suffer from this lethal disease. Sonic hedgehog (SHH) signaling is a stromal‐epithelial interacting pathway that is critical for prostate development and cell growth. SHH signaling is induced by androgen deprivation thereby leading to re‐activation of AR target genes to promote prostate cancer cell growth in the absence of androgens. Furthermore, AR interacts with GLI proteins, downstream effectors in the SHH pathway. Therefore, GLI3‐dependent SHH signaling in androgen deprived cells could promote re‐activation of AR target genes and consequently lead to CRPC. It is currently unknown how GLI3‐dependent SHH signaling is induced in CRPC. In this study we show that stabilized GLI3 leads to the induction of GLI3‐dependent SHH signaling in prostate cancers that harbor mutations in MED12. MED12 is an Xq13‐encoded 230 kDa subunit of the RNA pol II transcriptional Mediator that has been found to be mutated in ~5% of prostate cancers. Through stable knockdown and luciferase assays, we show that MED12 mutations disrupt a Mediator‐imposed constraint on GLI3‐dependent SHH signaling. Furthermore, through stable knockdown and xenograft studies, we show that stable GLI3 is critically dependent for CRPC growth. The results of our study therefore indicate that prostate cancer patients with MED12 mutations relapse to CRPC after androgen deprivation therapy by hyperactivating the GLI3 dependent SHH signaling pathway. Overall, this study provides critical insight into possible treatment strategies for prostate cancer patients with MED12 mutations by using the GLI3/SHH signaling axis as a therapeutic target.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.