bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationThis 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 InformationMidwestern University
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 InformationAuburn 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 InformationStart 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

IntroductionMany 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.MethodsTeams (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.ResultsThere 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 ConclusionThe 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 InformationThis 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 InformationNIH 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 InformationThe 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 InformationFunded by NIH grant AT009268.
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 InformationNIH/NIGMS Award 1R15GM101674‐01A1 (to PF)