bluuebunny/crossref_metadata_2025 huggingface dataset:
Muscle Activation Response Using a Constant Resistance Sled Trainer Within Different Tasks
Authors: Mathis Monica , Timon Stepanie , Taylor Heather , Rosario Martin G.
Published: April 2020

The XPO Trainer used in this inquiry is a novel device which provides low rolling resistance at low speeds with an immediate and automatic proportional gain in resistance with increased speed. Resistance exercise (RE) has been shown to induce many positive benefits to the body and mind, including hypertrophy of muscles, increased muscle performance, cardiovascular benefits, decrease risk of mortality, and decrease the risk of functional limitations.

PURPOSE

Examine the impact of pushing a constant resistance on gait and neuromuscular activation at low and high speeds in young, seemingly healthy adults.

METHODS

The study consisted of 21 healthy adult males and females (age: 25.07±3.46 years, weight: 148.2±25.96 lbs, height: 66.57±4.14 inches). Each participant wore surface electromyography electrodes on their dominant leg for their quadriceps (QUAD), hamstring (HAM), anterior tibialis (TA), and gastrocnemius (GA). First, the maximal voluntary isometric contractions were gathered for each muscle. Then, participants performed 3 trials of 4 conditions: walking, walking and pushing, running, and running and pushing. After the data was normalized as a percentage (0–100%) for the gait tasks and the muscle activation amplitude, a repeated measures ANOVA was performed with the SPSS 24 system.

RESULTS

The QUAD muscle exhibited a significantly higher muscle activation (P≤0.05) between walk (45.39±24.43) and walk push (74.40±56.73) tasks. Additionally, there is a significantly higher muscle activation (p≤0.05) between GA (81.85±37.84) and QUAD (46.06±23.93) during the walking task. A significant difference (p≤.05) was found between GA (0.33±0.17) and HAM (0.47±0.17) muscle activation timing during walking activity. During the walking activity, maximal muscle activation occurred in the GA muscle earlier than the HAM muscle. Additionally, there was a significant difference (p≤.05) in timing of the TA (0.43±0.17) vs QUAD (0.55±0.18), as well as the GA (0.41±0.10) vs the QUAD(0.55±0.18). TA and GA activation occurred significantly earlier than the QUAD activation during the walk and push task.

CONCLUSION

With the constant resistance sled being a novel device, it is important to understand how it affects the activation and response of muscles during different activities. Based on the results, patients can benefit from utilizing the sled in combination of walking and running to either promote a higher or lower muscle activation. With patients with weak QUADs or in need of QUAD strength training, a walking task is advocated. Additionally, if the goal of the intervention is to focus on endurance of the TA and GA, two of the main muscles used in postural balance, walking with the sled is recommended. Knowing all of these effects on muscle activation utilizing a constant resistance sled trainer will allow for more effective exercise training.
Identification of a CTRP9 Fragment Capable of Enhancing Bone Marrow Mesenchymal Stem Cell Cardioprotection via Cardioreparative Exosome Production Promotion
Authors: Wang Yajing , Liu Demin , Zhu Di , Lau Wayne Bond , Lopez Bernard , Christopher Theodore , Xie Dina , Zhang Zhen , Gao Erhe , Koch Walter , Ma Xinliang
Published: April 2020

Background

The safety of bone marrow mesenchymal stem cell (BMSC) therapy in ischemic heart injury has been clinically established. However, the poor survival of engrafted stem cells in the ischemic environment limits their therapeutic efficacy for post‐infarction cardiac repair. CTRP9 is a protective cardiokine capable of improving the local microenvironment. The current study attempted to 1) identify a small biologically active CTRP9 fragment with lower immunogenicity, and 2) test a hypothesis that said CTRP9 fragment may promote a healthy microenvironment, and improve implanted stem cell survival and resultant cardioprotection.

Methods and Results

Globular CTRP9 (gCTRP9, the most biologically active form of CTRP9) and 2 fragments of gCTRP9 (computationally predicted) were cloned and purified. In vitro experiments demonstrated that CTRP9‐F2 possesses biological activity equal to or exceeding gCTRP9. Wild‐type C57BL/6 mice were subjected to myocardial infarction and treated with BMSCs, CTRP9‐F2, or their combination. Administration of CTRP9‐F2, but not BMSCs alone, attenuated MI/R injury. Administration of CTRP9‐F2 plus BMSCs further enhanced the cardioprotective effect of CTRP9‐F2, suggesting a synergistic effect. BMSCs retention/survival in the ischemic region was significantly improved in the CTRP9‐F2 treated group. Conversely, the engrafted BMSC population was significantly reduced in the CTRP9 knockout heart. Mechanistically, CTRP9‐F2 promoted BMSC proliferation and migration, and protected BMSCs against H2O2‐induced cellular death. Interestingly, CTRP9‐F2 significantly enhanced BMSC exosome production, upregulated the exosomal cytoprotective molecule contents, and facilitated cardiomyocyte uptake of BMSC‐derived exosomes. Blockade of ERK1/2 completely abolished the effects of CTRP9‐F2 upon BMSC biology.

Conclusion

We identify, for the first time, a small CTRP9 fragment capable of promoting BMSC cardioprotection via augmented exosome‐mediated BMSC‐cardiomyocyte communication.

Support or Funding Information

W.W. Smith Foundation (to YJW)
Alcohol‐induced damage and death to developing and differentiated neurons.
Authors: Carter Wayne , Wijayamunige Buddhika , Wijesekara Anusha
Published: April 2020

Alcohol intoxication can induce foetal alcohol spectrum disorder after prenatal exposures, as well as damage neurons and contribute to neurodegeneration and cognitive decline in adults. The effect of alcohol on driving altered cellular mechanisms and post‐translational modification of proteins was investigated in both undifferentiated and differentiated human neural progenitor cells and SH‐SY5Y neuronal cells to mimic alcohol exposures during neuronal development and in adults. Human neural progenitor cells were differentiated into neuronal cells upon growth factor withdrawal, while all‐trans retinoic acid and brain‐derived nutrient factor were applied to differentiate SH‐SY5Y cells prior to a challenge with 0–200 mM alcohol for 3, 6, 12, or 24 hours. Alcohol exposure induced cell cytotoxicity in a concentration and time‐dependent fashion, with cell viability assessed using MTT, ATP, and LDH assays. Reactive oxygen species (ROS) generated in response to alcohol treatment were quantified using a dichlorofluorescin diacetate assay. The accumulation and quantitation of oxidatively damaged proteins was assessed by quantitation of protein carbonyl content and specific carbonyl group immuno‐blotting. In summary, at commonly encountered alcohol exposures, alcohol triggered cellular damage and death in developing as well as differentiated neurons.

Support or Funding Information

Commonwealth Scholarship Commission
Unmasking cAMP signalling in adipocytes
Authors: Salisa Laia Reverte , Pfeifer Alexander
Published: April 2020

In recent years, the prevalence of obesity has reached pandemic proportions. In 2016, the World Health Organisation estimated that ca. 3 million people die each year due to complications from being overweight or obese. With more overweight population than ever, finding new targets to counteract obesity has become crucial. Recruitment and activation of brown adipose tissue (BAT) has been postulated as a potential target to increase thermogenic energy expenditure (EE) in order to treat or prevent obesity and its comorbidities.

Cyclic adenosine monophosphate (cAMP) is a second messenger important in many biological processes. BAT thermogenesis is primarily mediated by activation of Gs‐coupled receptors such as the beta‐3 adrenergic receptor and the adenosine A2A receptor, which both increase intracellular cAMP. In mature brown adipocytes, cAMP is well known to induce lipolysis and UCP1‐mediated mitochondrial uncoupling through activation of protein kinase A (PKA). However, the precise role of cAMP signaling in adipocyte precursor cells has so far not been well studied. Furthermore, the role of other cAMP effectors (e.g. Exchange proteins activated by cAMP (EPAC)) in adipose tissue metabolism is not clear.

Treatment of brown adipocyte precursor cells using analogs specific to different cAMP effectors induce distinct cellular responses. Here, we used genetic and pharmacological approaches to decipher the role of cAMP effectors, focusing on EPAC proteins, in adipocytes. We demonstrate that activation of mature adipocytes and proliferation of brown preadipocytes require the activation of distinct effectors and signaling pathways downstream of cAMP. Furthermore, browning of white adipocytes appears to follow similar patterns. Our data indicate that, depending on the effector, either PKA or EPAC, cAMP regulates browning of white adipocytes by different mechanisms. Moreover, in vivo deletion of EPAC leads to reduced oxygen consumption after high fat diet, demonstrating that the role of cAMP in adipocytes is still not fully understood. In conclusion, we show that the effects of cAMP in BAT and browning of WAT differ vastly depending on both the state of adipocyte differentiation as well as the downstream effector.

Support or Funding Information

Institute of Pharmacology and Toxicology, University of Bonn, 53127 Bonn, Germany

SFB1328, Universitätsklinikum Hamburg‐Eppendorf, 20246 Hamburg, Germany
The Effect of Different Dietary Fats on the Longevity and Muscle Strength in Drosophila
Authors: Chizhikov Daniel , Lin Jason , Persinger Aaron , Puppa Melissa
Published: April 2020

Fat is an essential source of energy and is important for human growth and development. A high fat diet, however, leads to pathological obesity and associated comorbidities, such as reduced lifespan and muscle strength in both humans and model organisms. In this study we analyzed the effects of four dietary fats on lifespan and overall health using a Drosophila model system, which shares the mechanisms of energy processing, storage, and utilization with humans. One day old wild type male Drosophila were collected and placed on a control diet or high fat diets containing 5%, 10% or 15% of coconut oil (medium chain‐saturated fat), vegetable oil, olive oil, or flax seed oil (all three contain unsaturated fats). Lifespan was determined for 100 flies on each diet. Skeletal muscle performance was evaluated by climbing and flying assays. Among all the groups, flies on a control diet had the greatest survival with a median lifespan of 30.5 days, while flies on a 5% coconut oil diet had the second longest survival time, yielding a median lifespan of 13 days. For all other diets, the median lifespan was 2 days. Flies on the control diet also performed significantly better (p<0.05) in the flying and climbing assays relative to flies on high fat diets. Flies on a 5% coconut oil diet had a significantly higher (p<0.05) muscle strength based on the flying assay compared to flies maintained on 5% diets with unsaturated fats. Interestingly, intestinal permeability measured using the Smurf assay revealed no difference between flies maintained on a control diet, 5% coconut oil diet, and 5% vegetable oil diet, suggesting that the adverse health effects of a high fat diet did not result from the compromised function of the intestine. Taken together, these data indicate that plant based medium chain saturated fats preserved longevity and muscle function better than plant‐based fats containing high amounts of polyunsaturated fatty acids.

Support or Funding Information

Funding Source: University of Memphis SHS Faculty Grant
Pharmacologic Modulation of Mitochondrial Pathogenic Changes in Alzheimer’s Aβ‐Challenged Neurons
Authors: Rostagno Agueda , Sotolongo Krystal , Ghiso Jorge
Published: April 2020

Mitochondria play essential roles in maintaining the high levels of brain energy demands required to maintain physiological ion gradients across membranes critical for the generation of action potentials and to sustain transport systems at endothelial barriers. About 90% of the energy requirement is produced through glucose oxidation, a tightly controlled mitochondrial process leading to ATP production. As the major consumers of oxygen, mitochondria are also the most important generators of reactive oxygen species (ROS) as by‐products of the electron transport chain. Thus, it is not surprising that mitochondrial dysfunction is emerging as a major contributor to the pathobiology of age‐associated neurodegenerative disorders and is being considered a crucial player in the pathophysiology of Alzheimer’s disease (AD). In spite of numerous studies, the mechanistic links among ROS homeostasis, cellular metabolic alterations, and changes in cell bioenergetics, particularly at the level of synapsis and in relation to oligomeric forms of amyloid‐β (oligAβ), still remain elusive. Through a combination of classical biochemical and immunocytochemical approaches together with the real‐time assessment of global energy metabolism our studies provide insights into the detrimental role of oligAβ in neuronal cell lines and murine primary cortical neurons. Our findings demonstrate that oligAβ induces detrimental changes in mitochondrial function with loss of mitochondrial membrane potential, release of cytochrome C, and enhanced ROS generation. Assessment of global energy metabolism in the Seahorse metabolic analyzer after sequential addition of different modulators of the mitochondrial metabolic pathways, demonstrated an oligAβ‐mediated reduction in oxygen consumption affecting basal and maximal respiration capacity and causing decreased ATP production. Pharmacologic targeting of Aβ‐challenged neurons with compounds of known antioxidant activity restored mitochondrial integrity/function, rescuing metabolic and bioenergetic changes induced by oligAβ. Overall, our studies provide insights into the complex molecular mechanisms triggered by oligAβ which profoundly affect mitochondrial performance and highlight the potential of antioxidant pharmacologic targeting to ameliorate the metabolic/bioenergetics alterations encompassing the complex and multifactorial pathways affected in AD.

Support or Funding Information

NIH grants AG059595 and AG051266, the BrightFocus Foundation grant A2015275S, and the Alzheimer’s Association ZEN‐14‐283969
Protein interactions studies of the small TbTims chaperons in the mitochondrion of Trypanosoma brucei
Authors: Quiñones Linda , Tripathi Anuj , Chadhuri Minu
Published: April 2020

Trypanosoma brucei causes a neglected tropical disease known as trypanosomiasis that causes death in population of Sub‐Sahara Africa. T. brucei possesses a single mitochondrion and like other eukaryotes it imports most of its proteins into the mitochondria, however mitochondrial protein import machinery is divergent in this parasite, which makes it a useful target to control this disease. Nucleus‐encoded proteins are imported into T. brucei mitochondrion via two major heteromeric protein complexes in the outer and inner membrane, ATOM and TbTIM, respectively. Besides the membrane integral protein components of these complexes, TbTIM is also peripherally associated with 6 small TbTims (Tim9, 10, 8/13, 11, 12, and 13). At least three of these small TbTims (TbTim8/13, 11, & 12) are unique for T. brucei. Knock‐down of any of these 6 small TbTims destabilized the TbTIM17 complex therefore these proteins are essential in T. brucei. In fungi and human, the small Tims are soluble proteins that form hetero‐hexameric complexes. Each small Tims possesses two pairs of CX3C motifs that are required for proper folding of the monomers by formation of the two intramolecular disulfide bonds. T. brucei small Tims also possess these conserved motifs, except for TbTim12, which has only one pair. However, it is not clear how small TbTims interact with each other and what type of complex structure they form. Thus, our goal for these studies is to find the direct interaction pattern for the small TbTims. To complete this goal, we use yeast two hybrid (Y2H) analysis. Results showed that although all small TbTims can interact with one another, some of these interactions are stronger than others. Overall, we found that TbTim8/13 interacts strongly with TbTim9, TbTim10, and TbTim8/13 while TbTim13 interacts strongly with TbTim13. Based on these results we proposed 3 possible model for small TbTim heterohexameric complexes; 1) random order, and 2) defined order, and 3) modular with different sub‐complexes. We are now testing these models by structure‐modeling and site‐directed mutagenesis studies.

Support or Funding Information

This work is supported by the following NIH grants: 5RO1AI125666, 2SC1GM081146, and R25 GM05994
Male Sprague Dawley Rats use Deep Breaths for Ultrasonic Vocal Production
Authors: Schaefer Charles , Stein Amy , Riede Tobias
Published: April 2020

Rodents are highly vocal organisms. Vocal production must integrate breathing movements and movements of the vocal organ. This study was designed to investigate the role of deep breaths, also known as sighing, for vocal production. Sighing is a physiologically necessary breathing behavior conserved across all mammals. In ten awake and spontaneously behaving male rats breathing and vocal behavior was monitored. A tracheal implant measured subglottal pressure. The implant was connected to a pressure transducer mounted to a rodent jacket. Animals were tethered and allowed to move freely in the home cage. Subglottal pressure and sound were simultaneously recorded during five different contexts: anesthetic sleep, rest, noxious stimuli, exposure to another male, and exposure to a female. On average, sighs were produced between 15 and 40 times per hour. Exposure to a female Sprague‐Dawley rat was the only context with a small but significant increase in the sighing rate. Vocal activity increased in three contexts (female, male, noxious stimulus). On average 24% of all sighs contained vocalizations during those contexts but the rate of vocal sighs increased with vocal activity. Those findings confirm that vocal sighs are not only a regular fixture of rodent vocal behavior, but that sighs are recruited for vocal production if vocal activity increases. Both 22 kHz and 50 kHz ultrasonic vocalizations were observed in deep breaths. Sometimes multiple vocalizations were produced during the same exhalation. Finally, the pressure signal of the inhalation phase was modified when a vocalization was produced in the subsequent exhalation. This suggests preparation leading up to vocal production. In sum, we show that awake and spontaneously behaving rats use deep breaths for vocal production, and we present evidence that vocal production uses multiple breathing patterns to generate vocal signals. These observations inform our understanding of the physiology of vocal production in mammals and illustrates the value of the rat as model system.

Support or Funding Information

Midwestern University
Peer Instruction Fosters Student Engagement During Anatomy Large Group Active Learning Sessions
Authors: McCarthy Sarah A. , Labranche Leah , Keller Christopher , Kulesza Randy J.
Published: April 2020

At Lake Erie College of Osteopathic Medicine (LECOM), each unit of the Medical Gross Anatomy course is reviewed through a large group active learning session that has been traditionally termed a “forum”. Medical students are given a list of 10–15 open‐ended questions covering clinically relevant anatomy for each regional area of anatomy prior to each session. During this two hour session, students are randomly selected to answer the open‐ended questions in front of their peers. Student surveys highly rate these sessions and students traditionally have requested more of these sessions throughout the 13 week course. However, a downfall of the forum is that only 10–15 students get to participate in each of these sessions while the other students passively listen to their peers. In order to involve more students in the session, Peer Instruction was utilized during the Pelvis and Perineum forum. Peer Instruction, as introduced by Eric Mazur in 1991, has been used in large undergraduate introductory courses to encourage student engagement and learning. Our modified Peer Instruction activity asked learners to independently answer multiple choice questions through the online classroom response system DirectPoll, which covered similar topics as asked in the open‐ended questions given out prior to the session. If student performance was below 80%, the class would be asked to converse with their classmates and then answer the question again. Regardless of class performance, a student was randomly selected to answer the question and provide a rationale for how they answered that question. Further, the student was then asked follow up questions that covered the material relevant to the question. Our study hypothesized that students that participated in the Peer Instruction Forum will perform better on the Pelvis and Perineum anatomy exam when compared to performance in the previous year. Overall, the average performance on the pelvis and perineum exam between 2018 and 2019 was unchanged. However, student survey data suggests the students were more engaged during the sessions and found the Peer Instruction activity helped solidify concepts. In conclusion, Peer Instruction was well received by our students and we hope to expand its use throughout the anatomy curriculum.
Deep tissue imaging of B16 melanoma in mice by Multispectral Optoacoustic Tomography
Authors: Bahauddin Ammar , Panizzi Peter
Published: April 2020

Melanoma is an extremely aggressive form of skin cancer that will be diagnosed approximately 192,000 times in 2019. Of these, more than half will be the invasive variety. The 5‐year survival rates for a patient with invasive disease drops from ~98% to ~23%. Although cancer aggressiveness is mostly a consequence of patient specific environmental factors and cancer cell mutations, early detection remains a key determinant for improved patient’s outcomes. In this study, we used Multispectral Optoacoustic Tomography (MSOT), a novel diagnostic imaging tool with improved sensitivity and selectivity. MSOT imaging uses the optoacoustic properties of host tissue to provide structural information and in case of melanoma, a potent contrast agent for the detection of metastatic disease. In brief, cells constitutively expression of melanin provides a signal in MSOT that can be monitored over days to weeks. We studied both intradermal and intravenous models of melanoma and performed MSOT imaging every five days to monitor tumor formation and psuedotumor metastases. In our intradermal experiment, tumors were visible starting at day seven, but no metastasis were detected. However, we did detect metastatic disease in intravenous injected B16‐BL6 mice by MSOT imaging. In several animals, we also detected bilateral adrenal tumor, which is similar to the end‐stage human disease. Hematoxylin and eosin staining verified the presence of melanoma cells in the mice at both primary and distal sites. These findings validate the use of MSOT for the preclinical detection of melanoma in vivo and demonstrates the feasibility of using MSOT in the diagnosis of melanoma.

Support or Funding Information

Auburn University, Auburn, AL USATaibah University, Madinah, KSA
Interplay of YY1 and CtBP in the recruitment of PcG complexes to DNA.
Authors: Derleth Madeline R. , Avila Abigail , Atchison Michael , Basu Arindam
Published: April 2020

Recruitment of Polycomb Group (PcG) proteins to DNA is vital for normal development. PcG proteins regulate expression of homeotic genes that are essential for axial body patterning during development. Earlier work showed that YAF2 is responsible for PcG recruitment to DNA, which is mediated by YY1 DNA binding. Our goal is to understand the role of CtBP, YAF2 and YY1 in context of recruitment of Polycomb complexes to DNA. Using a co‐immunoprecipitation (CoIP) approach, different protein‐protein interactions are studied using protein‐specific antibodies. This approach will allow us to recognize binding interactions between specific proteins. Moreover, our preliminary results show that CtBP might interact directly with the C‐terminal end of YY1 to recruit PcG proteins. CtBP represses transcription in a histone deacetylase‐dependent or ‐independent manner, depending on the promoter context. Based on this information, we are investigating the modulation of transcriptional activity of YY1 upon interaction with CtBP using luciferase‐based assays. In conclusion, our studies will provide information regarding the interplay between CtBP, YAF2, and YY1 and their overall relationship in recruitment of PcG proteins to DNA.

Support or Funding Information

Start up funds from Penn State Brandywine
Peer‐Evaluation in a Team‐Based Learning Curriculum: Investigating the Relationship between Peer‐Evaluation Scores and Team Rank
Authors: Robertson Kyle , Byram Jessica
Published: April 2020

Introduction

Many medical school and health care professional curricula are incorporating teaching methods along with learning and assessment approaches, which aim to foster communication skills, professionalism, teamwork, and leadership skills. Team‐based learning (TBL) is a commonly used pedagogical approach to accomplish the flipped classroom and to foster professionalism, teamwork, and communication skills. Two components of TBL curriculum are 1) the utilization of peer evaluations for assessment purposes and 2) a team competition as an incentive structure. No standardized instrument exists for TBL peer evaluation and some studies have questioned the reliability and validity of the instruments for assessment purposes. Therefore, the objective of this study was to evaluate the relationship between averaged peer‐evaluation scores and rank in the team competition.

Methods

Teams (n=5) within a TBL graduate histology course during the fall semester of 2018 were ranked weekly in a team competition following each of the 10 TBL sessions. Rank was calculated based on averaged individual readiness assurance test scores (iRAT), group readiness assurance test scores (gRAT), and group activity scores for each TBL and was updated throughout the semester. Students (n=31) conducted peer‐evaluations of their team members at two different points (mid‐ and end‐of‐semester). The instrument for the peer‐evaluation contained 10 items that assessed professionalism and team engagement (e.g., is well‐prepared for team activities) on a four‐point scale from never to always. This tool was developed based on recommendations for TBL peer evaluations in the literature. Spearman correlations were used to determine the relationship between peer‐evaluation scores and team rank at each time point. Alpha was set at p≤0.05.

Results

There was a statistically significant (p=0.037) negative correlation between peer‐evaluation scores and TBL team rank at mid‐semester (r=−0.375) and stronger negative correlation existed at the end‐of‐semester (r=−.591, p<0.001). In other words, students on teams with a lower rank tended to give their peers lower evaluation scores.

Discussion and Conclusion

The relationship between the peer‐evaluation scores and the TBL team rank demonstrate a negative relationship. Thus, within this graduate level histology course utilizing the peer‐evaluation as a summative assessment may not be warranted as it may be unduly penalizing poor performing teams. Other courses utilizing team rank incentives may consider evaluating the use of peer evaluations as summative assessments. Furthermore, this study demonstrates a need for a standardized and reliable instrument for peer evaluations in a TBL curriculum.
Resolution Mediators in Pulmonary Inflammation Induced by Multi‐walled Carbon Nanotubes and Fullerenes in Mice
Authors: Lim Chol Seung , Porter Dale W. , Orandle Marlene S. , Green Brett J. , Barnes Mark A. , Croston Tara , Wolfarth Michael G. , Beezhold Donald H. , Siegel Paul D. , Ma Qiang
Published: April 2020

Exposure to certain nanoparticles can cause fibrosis and cancer as a result of unresolved inflammation. Inflammation resolution is largely mediated through specialized pro‐resolving mediators (SPMs). To understand the mechanism by which nanoparticle‐induced pulmonary inflammation is resolved, we analyzed and compared the inflammatory and pro‐resolving responses to fibrogenic multi‐walled carbon nanotubes (MWCNTs) and low‐toxicity fullerenes (C60F). Pharyngeal aspiration of MWCNTs at 40 μg/mouse or C60F at doses larger than 640 μg/mouse elicited pulmonary effects in B6C3F1 mice. Histopathology showed that both particles stimulated acute inflammation, predominated by neutrophils and macrophages, in the lung on day 1, which transitioned to histiocytic inflammation by day 7, and to granuloma and interstitial fibrosis by day 28 in MWCNTs, and to a much lesser extent, C60F exposed mice. Flow cytometric profiling of bronchoalveolar lavage fluid (BALF) cells revealed that neutrophil infiltration peaked at day 1 and declined to 36.6% for MWCNTs‐ and 16.8% for C60F (1,280 μg/mouse)‐treated mice by day 7, and to basal levels by day 28 in both groups, suggesting a rapid initiation phase and an extended resolution phase in both groups. Additionally, MWCNTs stimulated dynamic changes in eosinophils, bone marrow‐derived inflammatory macrophages, monocyte‐derived dendritic cells, and T lymphocytes, indicating distinctive patterns of immune cell infiltration in the BAL in response to nanoparticles. MWCNTs stimulated high levels of pro‐inflammatory lipid mediators, including leukotriene B4 (LTB4) and prostaglandin E2 (PGE2), on day 1. At the same time, MWCNTs induced high levels of SPMs, including resolvin D1 (RvD1) and E1 (RvE1) and lipoxin A4 (LXA4), on day 1, which continued to rise and peaked on day 7, creating a high RvD1/LTB4 or RvD1/PGE2 ratio. C60F stimulated a similar pattern of changes in the mediators but to a much lesser extent. Together, these findings suggest that MWCNTs at a low dose and fullerenes at a high dose induce time‐dependent progression from type 1 to type 2 inflammation with an extended resolution phase mediated through SPMs.

Support or Funding Information

This work was funded by grant 09390BN5 (DWP and QM) and grant 09390BMX (QM) from the Health Effects Laboratory Division, the Nanotechnology Research Center, and the NORA Research Program at the National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, USA.
iDISCO Allows Complete Visualization and Analysis of Postnatal Enteric Nervous System Development in a Comparative Pig Model
Authors: Erwin Sara , Touvron Mélissa , Odle Jack , Van Landeghem Laurianne , Blikslager Anthony T. , Ziegler Amanda L.
Published: April 2020

The enteric nervous system consists of a dense, complex network of neurons and glia which are instrumental in the maturation of normal intestinal physiology after birth. Our lab uses a comparative pig model to study the postnatal development of the enteric glial network and its role in regulating intestinal barrier functions in neonates. Immunolabeling‐enabled three‐dimensional (3D) imaging of solvent‐cleared organs (iDISCO) is a method of preparing tissue samples for volume imaging with a light sheet microscope. iDISCO has been optimized for use in primarily mouse organs and embryos for the study of early development and offers a more complete picture of the tissue than traditional histological analysis. Our objective was to optimize the iDISCO protocol for use in porcine intestinal tissue to allow complete qualitative and quantitative analysis of postnatal development of the enteric glial network in our comparative pig model. Antibodies against glial cell markers S100b, Sox10, and glial fibrillary acidic protein (GFAP) were used to triple‐stain fixed full‐thickness 3mm by 5mm samples of porcine jejunum using the iDISCO protocol. Samples were imaged with a light‐sheet microscope (Ultra‐II, LaVision BioTec®) using three different fluorescent channels and datasets were visualized and analyzed in 3D with Imaris software (Oxford Instruments®). The percent volume of GFAP+ glial cells was quantified by manually masking individual intestinal villi and optimizing a surface algorithm to identify glial network structures within those villi. Antibodies against all three markers tested produced the predicted staining pattern with minimal non‐specific staining. Percent of jejunal villus volume occupied by GFAP+ glia is higher in 6‐week‐old versus 2‐week‐old pig (0.49% versus 0.23%, *P≤0.05). Ongoing work will optimize quantification techniques for S100b and Sox10, and assess co‐localization patterns of these glial markers in the jejunum at discrete timepoints postnatally. iDISCO is a powerful imaging modality which will allow our lab to directly assess the expansion, complexity, and localization of glial cell subtypes by marker co‐expression analysis at discrete postnatal timepoints, and will be utilized in future studies to explore effects of disease and external interventions on the enteric glial network.

Support or Funding Information

NIH P30 DK034987 UNC CGIBD Pilot Feasibility; NIH T32 5T32DK007737‐22 UNC Basic Science GI Research Training Fellowship; USDA National Institute of Food and Agriculture, Animal Health Projects 1007263 and 07985; 2017 NC State CMI TPP Seed Grant
Studies of the First Cyclization Domain (Cy1) of Yersiniabactin Biosynthesis and its Utility in Domain‐Swapping
Authors: Gonzalez Reyaz , Henriquez Bryan , Frueh Dominque , Dowling Daniel P.
Published: April 2020

Yersinia pestis the causative agent for the bubonic plague releases the virulence factor yersiniabactin (Ybt) in iron‐deficient environments in order to chelate iron from its host. The siderophore Ybt is synthesized by Yersinia p. through a hybrid assembly line of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) proteins. Ybt contains two thiazolines and one thiazolidine essential for its chelating function; investigation of the biosynthetic gene cluster shows there are three cyclization domains (Cy1, Cy2, Cy3) within the large high molecular weight proteins that introduce these moieties, making this an ideal system to better understand the Cy domain. Current work has been to structurally characterize Cy1 through X‐ray crystallography. This enzyme is air‐sensitive and aggregates rapidly when exposed to oxygen, likely due to interprotein disulfide bond formation. In order to optimize crystal growth conditions in an aerobic environment two cysteines predicted to cause aggregation were mutated. This engineered construct was verified to reduce aggregation through size exclusion chromatography (SEC) and is currently being used to optimize crystal growth conditions. To further probe the Cy domains, a chimeric construct of Cy1 was created with a linked docking domain from epothilone (Epo) biosynthesis. The Epo biosynthetic gene cluster contains a Cy domain that has been characterized. Functional assays were performed to probe whether “cross‐talk” between gene clusters after swapping Cy domains is possible by monitoring formation of product. These results will contribute towards our knowledge of the cyclization domain and protein‐protein interactions between various hybrid NRPS/PKS systems.

Support or Funding Information

The authors kindly acknowledge the MIT Structural Biology Core facility and Staff Scientist Robert Grant. This research was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number 1R15GM123425‐01 to D.P.D.
Disuse Atrophy Elevates the Release of Skeletal Muscle‐derived Extracellular Vesicles but Lowers Serum EV Concentration in Rats
Authors: Van Pelt Douglas W. , Butterfield Timothy A. , Dupont-Versteegden Esther E.
Published: April 2020

Extracellular vesicles (EVs) have gained appreciation as important mediators of intercellular communication and physiological functions across almost any cell type in which they have been studied. However, how muscle disuse and atrophy alter production of EVs is largely unknown, and may have a role in dictating the poor systemic outcomes that occur with muscle atrophy and physical inactivity. The purpose of this study was to determine how muscle disuse and atrophy alter the concentration of serum EVs and the production of muscle‐derived EVs. Adult 10 month old male Fisher 344/BN rats underwent hindlimb suspension (HS) for either 4 hours (4h HS; n=4), 24 hours (24h HS; n=4), or 7 days (7d HS; n=5). Serum was collected and EVs were isolated using ExoQuickTM Precipitation solution. For collection of muscle‐derived EVs, soleus muscles were excised and incubated in continuously gassed (95%O2/5% CO2) Kreb’s Henseleit Buffer (118.5mM NaCl, 1.2mM MgSO4, 4.7mM KCl, 1.2mM KH2PO4, 25mM NaHCO3, 2.5mM CaCl2; pH 7.4) supplemented with 5mM glucose at 37°C for 1 hour. Compared with weight bearing rats (WB; n=7), all HS groups exhibited a drastically lower (70–75%; p<0.05) serum EV concentration. The much lower EV concentration is not likely due to reduced production of muscle EVs however, as muscle‐derived EV release (vesicles/mg muscle) was 28% higher from 4h HS and 66% and 60% higher (p<0.05) from 24h and 7d HS muscle, respectively, compared with WB. Altogether, these data suggest that there are rapid systemic changes in EV biology occurring very early with muscle disuse and atrophy as evidenced by the robust and rapid changes in EV serum concentration. There is also a rapid increase in the release of EVs from muscle during disuse that is maintained as the muscle atrophies, which may be important for communication of muscle with other organs. Finally, the lack of association in serum EV concentration and the changes in the rate of EV release from muscle emphasizes the complications and pitfalls of assuming the contribution and behavior of muscle EVs based on serum measurements alone.

Support or Funding Information

Funded by NIH grant AT009268.
The unintentional effects on body donation programs of a competency‐based curriculum in post‐graduate medical education
Authors: Noel Geoffroy P.J.C , Dube Joseph , Venne Gabriel
Published: April 2020

A competency‐based curriculum shifts the focus from acquiring knowledge to using knowledge, placing emphasis upon the practices used by professionals and encouraging students to utilize their medical knowledge for specific tasks performed within realistic settings. As medical programs place increasing importance on competency‐based training and surgical simulations for residents, anatomy laboratories and body donation programs find themselves in a position of adapting to changing needs and demands. These skill‐centered curricula exhibit a growing demand for simulation facilities as well as specimens with more realistic properties, and institutions must assess how current or new teaching and embalming techniques respond to the demands of the residency programs they serve. Similarly, institutions’ body donation programs must adapt to a competency‐based curriculum’s greater need for cadaveric specimens, which presents many challenges to a body donation program, ranging from the logistical to the ethical.
Protein Kinase A‐mediated Regulation of SLC5A8 Surface Expression
Authors: Fong Peying , Li Yonghai
Published: April 2020

SLC5A8 (solute carrier gene family 5A, member 8) encodes the sodium monocarboxylate transporter, SMCT1. SMCT1 mediates cotransport of sodium and monocarboxylates, such as short chain fatty acids, in many tissues, including thyroid, kidney, and colon. Inspection of SMCT1’s primary amino acid sequence identifies not only a carboxyl terminus post‐synaptic density/discs large/zona occuldens 1 (PDZ) binding motif postulated to be important in macromolecular transport protein complexing, but also consensus sites for phosphorylation by multiple classes of kinases. Notably, SMCT1 contains a single protein kinase A (PKA) consensus site: RRMT (amino acids 50–53), with T53 potentially functioning as the site of phosphorylation. The present study considers the hypothesis that PKA regulates SMCT1 surface expression, as reported for the sodium‐glucose cotransporter (SGLT1, the product of the SLC5A1 gene). The following experiments test whether elimination of the phosphorylation site disrupts PKA‐mediated regulation of surface expression. Using site‐directed mutagenesis, we disrupted the consensus motif and concomitantly disabled phosphorylation by replacing T53 with an alanine residue (T53A). To facilitate downstream assays, we generated T53A on the background of a human SMCT1 expression construct containing an amino‐terminus, extracellular hemagglutinin (HA) tag. In transiently expressing COS‐7 cells, surface luminescence measurements confirmed T53A resides in the cell surface at levels similar to wild‐type SMCT1, and immunoblot measurements indicate similar total expression levels. Wild‐type SMCT1‐expressing cells displayed increased luminescence signal (~20%) subsequent to forskolin (10 μM) treatment to stimulate adenylate cyclase and activate PKA, consistent with augmented surface expression. In contrast, cells expressing T53A were indifferent to forskolin treatment. As an independent measure of surface expression, plans are underway to perform cell‐surface immunoprecipitation (CS‐IP) experiments. Taken together, these data suggest that, although constitutive plasma membrane residency does not depend on an intact PKA consensus site, forskolin‐activated, PKA‐dependent phosphorylation can increase SMCT1 levels in the plasma membrane, and thereby augment potential transport capacity.

Support or Funding Information

NIH/NIGMS Award 1R15GM101674‐01A1 (to PF)
Copper Oxide Nanoparticles Activate Nrf2, KEAP 1, and Downstream Target Genes in JB6 Cells possibly through ROS Generation and Antioxidant Response Element (ARE) Mechanisms
Authors: Barber Tabatha L. , Aldinger Joni , Arnold Joshua , Leonard Stephen , Ding Min
Published: April 2020

Occupational exposures to Copper Oxide (CuO) nanoparticles havebeen reported to be harmful to human health, with possible risk of cancer amongworkers from many sectors. CuOnanoparticles have not, to our knowledge, been extensively examined forpotential carcinogenic toxicological effects. To investigate the mechanisms ofCuO‐induced pathogenesis, the effect of metal oxide fine and nanoparticles on antioxidantresponse mechanisms and ROS generation were investigated. The results indicated that CuO caused a2‐fold increase in AP‐1 activity in JB6 cells. The induction of AP‐1 activityin cultured cell lines was time and dose‐dependent. The signal transductionpathways for AP‐1 activation were also investigated. Western Blot analysisdemonstrate that CuO nanoparticles stimulate phosphorylation of p38 MAPK andERKs. CuO also generated ROS whenincubated with the cells as measured by electron spin resonance (ESR). Nano‐sized CuO generated more ROS than thefine sized particles when incubated with the cells. COMET assay suggests thatexposure of the cells to CuO resulted in DNA damage. Cells pre‐treated with ROSinhibitors sodium formate and Polyvinylpyridine‐N‐Oxide (PVPNO) before exposureto CuO nanoparticles demonstrate a significant reduction in p‐38 and ERKactivity, suggesting the H2O2 may have a role in the ROSmechanism. CuO Nanoparticles induced Nrf2 translocation and activation in adose and time‐dependent manner. Glutathione S‐transferase and NAD(P)H:quinoneoxidoreductase 1(NQO1) were increased in a time‐dependent manner following JB6exposure to CuO nanoparticles. Unraveling the complex mechanisms associatedwith these events suggests that activation of Nrf2 and subsequent activation ofthe ARE by ROS generation may contribute to adverse health effects followingexposure to CuO nanoparticles.

Support or Funding Information

This project was supported by a grant from the Nanotechnology Research Center.
Implementing Pathology Peer Teaching Program in a Medical School
Authors: Lomiguen Christine M. , Gross Hannah , Terrell Mark A. , Ogunsakin Olalekan , Junsanto-Bahri Tipsuda
Published: April 2020

Purpose

Pathology intimidates many first‐year medical students because it requires interdisciplinary critical thinking required to diagnose and to treat disease, and often, students are uninterested in the subject. These two factors cause students to disengage in the educational setting. To combat this problem, a mutually‐beneficial pilot teaching assistant (TA) program was developed in a General Pathology course to facilitate first‐year medical students’ abilities to apply General Pathology to integrative, interdisciplinary questions early in undergraduate medical education. TAs were second‐year medical students who cognitively benefited from the experience of teaching first years through the educational principle “To teach is to learn something twice,” thus aiding in their preparations for licensure examinations.

Methods

Thirteen (13) medical students from the second‐year Systemic Pathology class at an urban osteophathic medical school volunteered to serve as TA’s for the first‐year General Pathology class. The first‐year General Pathology class consisted of 135 medical students, divided into 23 small groups composed of between two to eight students per group. The 23 groups were then divided into two lecture halls. At least one pathology faculty member was also present in each lecture hall. First‐year students were presented with an integrative pathology case, provided to second‐year students in advance, and given guiding questions with the support of their assigned second‐year TA to develop critical thinking.

Results

Results consisted of positive preliminary feedback in the program’s initial implementation in the Spring 2019 semester from both first‐year students interested in exposure to board‐relevant questions early in their education and second‐year medical students actively preparing for their board examinations and interested in reviewing first‐year material.

Conclusion

Pathology requires the integration of foundational concepts from anatomy, biochemistry, physiology, and immunology to understand the various aspects of the disease process. Training developed in digesting complex material is of utmost importance for success on national licensure examinations, such as the United States Medical Licensing Examinations (USMLE) and Comprehensive Osteopathic Medical Licensing Examinations of the United States of America (COMLEX‐USA), for board certification. The pilot teaching assisting program in the General Pathology course exposed first‐year medical students to challenging, integrative questions early in undergraduate medical education and re‐exposed second‐year medical students who were actively preparing for licensure examinations, benefiting both sets of students. Further studies will evaluate the effectiveness of the pathology teaching assistant program on academic performance and on licensure examinations.