bluuebunny/crossref_metadata_2025_split huggingface dataset:
NADPH Oxidase 2 and Reactive Oxygen Species Mediate Pulmonary Arterial Smooth Muscle Cell Phenotypic Modulation Following Chronic Hypoxia
Authors: Cross Laura Weise , Vigil Maria A. , Jernigan Nikki L. , Resta Thomas C. 
Published: June 2021

Chronic hypoxia (CH), which is associated with chronic obstructive pulmonary disease and high‐altitude exposure, increases pulmonary vascular resistance and promotes pulmonary hypertension through mechanisms that stimulate vasoconstriction and arterial remodeling. Vascular remodeling is characterized in part by abnormal migration of pulmonary arterial smooth muscle cells (PASMCs). Based upon previous studies implicating NADPH oxidase 2 (NOX‐2) in the development of CH‐induced pulmonary hypertension, we hypothesized that NOX‐2‐generated reactive oxygen species (ROS) enhance PASMC migration following CH. To test this hypothesis, we harvested primary PASMCs from control and chronically hypoxic (4 wk at 0.5 atm) rats, cultured them in 5% serum DMEM F12 growth media for 3 days and in 0.3% serum media for 24 hr, and then plated the cells onto 8 μm polycarbonate membrane Transwell inserts (Costar). After allowing the PASMCs to adhere for 1 hr, they were treated with the general NOX inhibitor apocynin (30 μM), the specific NOX‐2 inhibitor GSK2795039 (500 nM), the superoxide dismutase/catalase mimetic EUK‐134 (10 μM), or a vehicle control (0.1% dimethyl sulfoxide) for 24 hr. PASMCs were fixed for 20 min in 4% paraformaldehyde, washed with phosphate‐buffered saline (PBS), stained with 1% Coomassie, and washed again with PBS. Transwell inserts were imaged with an upright brightfield microscope (Nikon) before and after the top layer of PASMCs was removed with a cotton swab. Cell migration was expressed as the percent of total cells that migrated through the Transwell membrane. Our results indicate that CH significantly (p < 0.05) enhances PASMC migration, while either inhibition of NOX‐2 or scavenging of ROS significantly blunts this response. We conclude that NOX‐2‐derived ROS contribute to enhanced PASMC migration following CH.Support or Funding InformationThis work was supported by NIH grants R01 HL132883 and T32 HL‐007736 (to T.C. Resta) and K12‐GM‐ 088021 (to A. Wandinger‐Ness).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PhUn in the Snow: 3rd Graders Learn About Exercise Physiology
Authors: Halpin Patricia A. 
Published: June 2021

Physiology Understanding (PhUn) week is an ongoing effective outreach program of the American Physiological Society. This annual event for K‐12 students is held every November. During PhUn week, students get to meet a physiologist and perform science activities. This past year a small private parochial school contacted me and asked me to come into the third grade class in January. The event started with an introduction to the job of a physiologist and an explanation of heart function. The students created their hypothesis Exercise increases heart rate and planned their experiment. The correct way to measure heart rate via the carotid artery was demonstrated by the physiologist and students took their baseline heart rate. Then together with their partner the students went outside to perform their experiment. There was fresh snowfall on the playground yet the students were undaunted to perform the exercise portion of the activity. One member of the student pair ran for three minutes around the perimeter of the playground. Upon stopping they measured their heart rate. The second member of the pair repeated the exercise and took their post exercise heart rate. The whole class went back into the classroom to discuss their results. A discussion was held and students stated their data from before (baseline) and after exercise. The students agreed that their data supported their hypothesis. The physiologist explained why the heart rate needs to increase with exercise. It is always a thrill to be able to work with young students and be the first scientist they have ever met. This PhUn week event is certain to create a lasting memory for both physiologist and students alike.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Student Perception and Preference in Learning of Sensorimotor tracts in Neuroanatomy: Evidence for gender differences
Authors: Akle Veronica , Gutierrez‐Herrera Jaime Andres , Vela‐Salazar Flor Milena , Blanco‐Alvarado Miguel Angel , Pena‐Silva Ricardo A. 
Published: June 2021

Neuroanatomy learning and teaching continue to be a challenge in any curricula. One reason is that relations among different structures is difficult to visualize. Spatial abilities are useful in learning neuroanatomy, but students' skills differ widely in any given medical class, specially between genders. Thus, building and manipulating physical models has shown to be useful for learning anatomy, as it incorporates basic sensorimotor skills in the learning process. This study examines the spatial abilities and perceptions of undergraduate medical students toward learning sensorimotor tracts. It also proposes that following a self‐guided virtual module, in addition to “threading” the tracts over a physical model of spinal cord and brainstem, which allows visualization of the trajectories, would improve the consolidation of the information, enhance the learning experience of the topic, and contribute to student autonomy.In a cohort of 70 undergraduate medical students, the perception of difficulties and learning strategies and actual spatial proficiency were measured using a questionnaire and a standardized Mental Rotation Test (MRT) in 2017‐II. A virtual module consisting of interactive videos, equipped with formative quizzes and a training module to practice spinal cord segment identification were developed in Spanish. Construction of the scaffold model for tract threading, instruction video for assembly of the model and student outcomes will be tested in 2018‐I following a randomized experimental crossover design.Results of the pilot study showed that the strategies used by students range widely from plain repetition to coloring. Analysis of the answers by gender show a difference in the strategies prioritized. While female students prefer drawing the tracts in colors significantly more than men, male students prefer mental visualization of the tracts and dislike drawing with colors pencils. Also, there is a wide range of spatial abilities in the class, according to the MRT. Just as reported in the literature for the rest of the world, the results of women are significant lower than in men (P<0.05).Preliminary evidence shows that the perceptions toward the difficulty of content, self‐efficacy and motivation differ between male and female students. Also, they utilize different strategies to study the topic. Further study would allow to develop and test tools, such as a 3D model of the spinal cord and brainstem to “thread” the tracts, that would benefit students with different learning styles.Support or Funding InformationVicerrectoría Académica, Universidad de los Andes.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Age‐ and Occupation‐Based Public Health Considerations Related to Osteoarthritis of the Knee Joint: A Cadaveric Study.
Authors: Immonen Jessica , Siefring Chris 
Published: June 2021

ObjectiveOsteoarthritis (OA) literature makes minimal suggestion regarding age of disease onset or preventative strategies to reduce risk for onset in various populations. In 2005, the Center for Disease Control and Prevention estimated that 33.6% of Americans 65+ years old were affected by OA; this cadaveric analysis suggests this is largely underestimated. The objective of this assessment is to identify at‐risk populations for OA in the knee joint and make recommendations to prevent or delay disease onset.DesignMorphometric analyses of the articular cartilage of the tibial plateau were performed on cadaver specimens using Image ProÒ software on three age populations and surface area measurements for articular cartilage degradation were compared to donors' reported ages, clinical histories and occupations.ResultsData showed that by the 7th decade of life, when patients are in their 60s, articular cartilage degeneration on the tibial plateau had commenced in 100% of specimen. All “homemakers” displayed above average medial tibial plateau degeneration (32.33 ± 24.85%) for their age group while simultaneously reporting pathologies in their clinical history that encourage a sedentary lifestyle. Atrophy or degeneration of crucial ligaments supporting the knee can encouraging sheering and cartilage degeneration. Cadaver knee specimens in the 70–79 years old age group displayed a 10.5% decrease in average ACL circumference (mm) compared to the group of <70 years old specimens (P < 0.001).ConclusionsThis assessment identifies an occupational class with a propensity to develop disease and also identifies a more realistic time frame than previous advisory committees have produced regarding age of disease onset and initiation of preventative measures. Especially given the ACL thins with aging, it is recommended that strengthening of the hip abductors and the musculature supporting the knee commence early in adult life to avoid valgus collapse and shearing at the knee joint.Support or Funding InformationInternal Grant from Rocky Mountain University of Health ProfessionsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Facial Canal Dehiscence: Radiological incidence and surgical/clinical relevance
Authors: Guzman‐Lopez Santos , Hernandez‐Trejo Andrea Fabiola , Quiroga‐Garza Alejandro , Elizondo‐Omaña Rodrigo Enrique 
Published: June 2021

IntroductionThe facial nerve is the most vulnerable structure to injuries in otological surgeries. The dehiscence of the facial canal (Facial Canal Dehiscence FCD) is the main factor that exposes it to iatrogenic injuries. The reported incidence varies between 6–57%.Materials and methodsHigh resolution head and neck computed tomography (CT) from a 2 year period were evaluated. Those with previous surgery in the temporal region, trauma, neoplasm, colesteatoma, or pathologies that alter the anatomy, were excluded.ResultsA total of 184 healthy temporal bones were analyzed. FCD was observed in 94 (51.1%) of the bones analyzed. The tympanic portion was the most frequently affected site (96.8%). There were no significant differences in gender, age, and symmetryDiscussionAn imaging study will have superior sensibility for diagnosing FCD, tan dissection in cadaver, reason why incidence may vary from previous reports.ConclusionEstablishing a presurgical evaluation by means of high resolution CT in order to assess the risk of CDF, would reduce the incidence of surgical injuries.Support or Funding InformationThere was no external funding. All funding provided by the department and medical schoolThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Dynamics studies of DNA polymerase kappa, a human protein involved in translesion synthesis
Authors: Leong Eleanor Ruth , Benitez Benedetta Sampoli 
Published: June 2021

Y family DNA polymerases are a class of enzymes characterized by their ability to copy damaged DNA in a process known as translesion synthesis. They do not eliminate the damage per se, but at least they are able to bypass a stalled replication, avoiding cell death. These polymerases bypass a wide variety of DNA damage, including thymine‐thymine dimers, inter‐strand DNA crosslinks, and other damage caused by reactive oxygen species. Unfortunately, they do not replicate the DNA with high fidelity and some polymerases are more error prone than others. In addition, the kinds of lesions that this group of enzymes can bypass are dependent on the enzyme itself, despite the general architectural similarities of all these enzymes. Human DNA polymerase kappa (pol k) is a member of the Y‐family of DNA polymerases involved in translesion synthesis in humans. Kinetics students have shown that this enzyme can bypass minor groove lesions such as O6‐methylated guanine (O‐metG) but not major groove lesions such as N2‐furfuryl‐guanine (N‐fG). Hydrogen exchange experiments have suggested solvent accessibility changes in various systems, hinting at a mechanism of conformational change upon substrate binding. Through molecular dynamics simulations, we tested the validity of the conformational change hypothesis by comparing the structural changes undergone by pol k in the presence of different templating lesions and with correct and incorrect incoming nucleotides. In addition, binary systems, comprised of just the DNA and the protein without the incoming nucleotide, were examined. Our results show that pol k does not attain a close, catalytically competent state in both the binary and the O‐metG systems, as evidenced by the few contacts between the protein and the incoming nucleotides. We have also identified key residues important for the stabilization of the incoming nucleotide as well as formulated a hypothesis to why the O‐metG lesion does not get bypassed as efficiently as other damages.Support or Funding InformationThis work was supported in part by the Rose M. Badgeley Residuary Charitable Trust Award and the Colette Mahoney Award to BSB.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of split Cas6 for inducible dimerization modules
Authors: Ling Justin A. , Vontalge Emma J. , Sashital Dipali G. 
Published: June 2021

Cas6 endoribonucleases are essential for guide RNA biogenesis during CRISPR‐Cas (clustered regularly interspaced short palindromic repeats‐CRISPR associated) immunity, and have exquisite specificity for short structured RNAs that form in the repeating regions of CRISPR transcripts. The Cas6 protein from Thermus thermophilus contains two domains, an N‐terminal domain (NTD) containing the endoribonuclease active site, and a C‐terminal RNA‐binding domain (CTD). The modular nature of this protein makes it a potential platform for molecular scaffolding by splitting the two domains and inducing dimerization through RNA binding. Here we show that the Cas6 NTD and CTD can be split into two separate proteins, and that the domains retain their RNA binding and cleavage activity. Fusion of the domains to the C‐termini of cyan or yellow fluorescent protein (CFP or YFP) enabled measurement of dimerization in the absence or presence of RNA using fluorescence resonance energy transfer (FRET). We find that wild‐type Cas6 NTD and CTD dimerize even in the absence of RNA. However, introduction of mutations in the interface between the two domains disrupts this interaction. The interface mutations do not disrupt RNA binding or cleavage, and addition of RNA restores dimerization of the two domains. These results demonstrate that split Cas6 can be developed into a molecular scaffolding platform that can be controlled through the addition of RNA.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of Chromatin on Genome Accessibility and Cleavage by CRISPR‐Cas9 in vivo
Authors: Verma Surbhi , Yarrington Robert , Trautman Jon , Schwartz Shaina , Carroll Dana 
Published: June 2021

CRISPR‐Cas9 is a prokaryotic immune surveillance complex, which has been adapted for use in eukaryotes as a revolutionizing RNA‐guided genome editing system. Despite its widespread success, differences in organization and structure of the prokaryotic versus eukaryotic genomes may limit its full utility. The eukaryotic genome is highly compact and organized into chromatin with stretches of DNA wrapped around histone protein cores (nucleosome). Additionally, for proper gene regulation, chromatin is further divided into nucleosome bound (NB) and nucleosome depleted regions (NDR). To make genome editing more efficient, we need to understand how chromatin may impact in vivo target accessibility and editing by CRISPR‐Cas9. Nucleosomes have been demonstrated to limit Cas9 accessibility in vitro, and indirect evidence suggests this may also be true in cells. To address this question directly, we are using Saccharomyces cerevisiae as a model system, as it has well‐characterized nucleosome locations and well worked out chromatin regulation. In particular, we are studying the cell cycle‐regulated HO (homothallic switching endonuclease) promoter in its native state as well as in mutants that contain constitutive NDRs in place of native NB regions. We find that several in vivo target sites within strongly positioned nucleosomes (NBs) are cut less efficiently than nearby nucleosome‐depleted regions (NDRs). Furthermore, when nucleosomes are displaced from NB targets by genetic manipulations, Cas9 cleavage efficiency increases. This indicates that reduced Cas9 cutting efficiency at NB targets is due to reduced accessibility to DNA rather than problems with Cas9 cleavage activity or poor guide‐RNA design. Developing an understanding of how genomic factors limit Cas9 accessibility will empower us to perform more precise and regulated genome modifications and to potentially design Cas9 variants with better chromatin access. Findings from the current study will help in making future genome targeting by CRISPR‐Cas9 more effective.Support or Funding InformationNational Institutes of HealthThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating RNA Hairpin Stability and Protein Binding Using Optical Tweezers
Authors: Plachinski Sarah , Furman Leah , McCauley Micah J. , Williams Mark C. , Núñez Megan E. 
Published: June 2021

More than 1.2 million people in the US are living with HIV. This RNA virus must be reverse‐transcribed into DNA before it can be integrated into the host genome and subsequently replicated. Retroviral DNA synthesis is generally understood, but questions remain about many identified critical elements. The transactivation response (TAR) RNA hairpin, specifically, inhibits the minus‐strand transfer step in reverse transcription and thereby regulates the life cycle of the HIV‐1 retrovirus. Before proceeding with reverse transcription, the TAR hairpin must be destabilized: chaperone assistance from the nucleocapsid (NC) protein can stimulate this rate‐limiting step 3000‐fold. Recent interest in the folding landscape of the TAR hairpin has revealed in‐vitro thermodynamic properties in the presence and absence of NC. These discoveries were enabled by optical tweezers force spectroscopy, the nature of which requires a complex TAR hairpin construct. Unfortunately, design and synthesis of the necessary TAR hairpin construct has limited the quantity and diversity of thermodynamic analyses regarding this essential facet of HIV‐1. We aim to develop efficient protocols to produce TAR RNA hairpin constructs optimized for optical tweezer manipulations.In order to manipulate individual hairpin molecules, the optical tweezers require that the RNA hairpin be ligated to two long double‐stranded DNA “handles.” One handle of the final construct specifically binds a bead in the optical tweezer dual‐laser trap, while the other handle binds a second bead that is manipulated via a micropipette. Tiny displacements of the second bead in the laser trap are used to calculate the force required to unzip the hairpin, and the thermodynamic parameters for force‐induced unfolding. The two ~3,000 base pair handles were constructed by amplification of bacterial plasmid DNA by PCR, restricted to create specific sticky ends, and purified by gel electrophoresis. RNA hairpins were transcribed from short DNA templates that were themselves synthesized by automated solid‐phase synthesis with specific sequences for RNA pol binding and the hairpin structure of interest. The handles were then individually ligated via several short DNA and RNA linker sequences to the RNA hairpin. Gel purification between ligation steps removed alternative ligation products, allowing size‐based verification and isolation of intermediates and the complete hairpin construct. Initial unzipping data support the correct length and composition of the final construct. We anticipate that subsequent data will contribute to a broader understanding of RNA hairpin stability as well as provide useful insight about a potential HIV‐1 drug target.Support or Funding InformationNIH‐NIGMS (Grant 2R01‐GM072462‐10), NSF‐MCB (Grant 1213883), Camille and Henry Dreyfus FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthesis and Characterization of RNA Aptamers Targeted at Aspergillus Cell Surface Carbohydrates
Authors: Woodhead Nicole M. , Silvestri Sabrina S. , Engelke David R. , Rohlman Christopher E. 
Published: June 2021

Aspergillosis is a disease characterized by the degradation of the respiratory system due to Aspergillus fungi. The slow growth rate of the infection allows it to establish a strong foot‐hold within the body, making it difficult to treat, and making early detection vital to successful therapy and patient survival. Traditional methods for the detection of Aspergillus in the body can be extremely difficult and invasive. Additionally, these methods provide only minimal rates of detection and high mortality rates in immuno‐suppressed patients. RNA aptamers have the potential to bind selectively to cell surface carbohydrates of the fungus Aspergillus, and aid in the detection of Aspergillosis, where fluorescent detection could be used to increase the sensitivity of ligand binding and the identification of infection. In this study, RNA aptamers targeted at the Aspergillus cell surface carbohydrate Galactomannan were selected from a 40 nucleotide randomized sequence population. Aptamer template pools were synthesized using PCR amplification to generate the initial cDNA pool. RNA aptamers were synthesized via in vitro transcription of the template pool utilizing modified NTPs. Successful aptamers were selected through rounds of binding to Galactomannan, followed by repetition of the amplification and transcription process. We will describe the characterization of the sequence diversity of the aptamer pool as well as its binding characteristics. We continue to refine the current pool of aptamers in order to obtain aptamers with high affinity for the carbohydrate Galactomannan. This research will help the advancement of our project goal of developing medical diagnostics and analytical biosensors which can detect the presence of Aspergillus.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.