bluuebunny/crossref_metadata_2025_split huggingface dataset:
Myocardial Oxygen Saturation Measured by Photoacoustic EKV Imaging
Authors: Hackfort Bryan T. , Chalise Upendra , Daseke Michael J. , Lindsey Merry L. 
Published: April 2020

Cardiac remodeling following myocardial infarction (MI) leads to structural and vascular changes in the myocardium. To better understand the mechanisms involved, we used photoacoustic ECG‐gated Kilohertz Visualization imaging (PA EKV) to measure oxygen saturation in the myocardium. Photoacoustic imaging is a noninvasive imaging technique that uses near‐infrared laser light to optically excite molecules in vivo. Oxygenated hemoglobin absorbs laser light at a different wavelength (850nm) from non‐oxygenated hemoglobin (750nm) which allows for the detection of the relative oxygen saturation (sO2) of a tissue and the amount of hemoglobin present (HbT). In this study, photoacoustic imaging was combined with EKV imaging for continuous measurement of sO2 and HbT throughout the contraction cycle.Imaging was done on C57Bl/6J mice (F, 4–5 months of age) to measure baseline sO2 levels in the anterior and posterior myocardium. An MI was induced by ligation of the left anterior descending coronary artery. Mice were imaged at days 1, 7, and 14 after MI to measure changes in myocardial sO2. All data presented here are from diastole of the long axis orientation and similar results were obtained from short axis measurements. Oxygen saturation on the anterior myocardial wall decreased from 74.7% pre‐MI to 19.9% 1 day after MI (p<0.0001). Oxygen saturation showed recovery at 7 (54.9%, p=0.023) and 14 (49.9%, p=0.007) days post infarct, yet remained significantly reduced from pre‐MI levels. Oxygen saturation was lower on the posterior myocardium (57.2%) compared to the anterior myocardium but did not change following MI.Our results demonstrate oxygen saturation begins to recover 7 days after MI as the heart adapts to ligation and neoangiogenesis is stimulated. This study validates the use of PA EKV for the measurement of oxygen saturation in the infarcted myocardium.Support or Funding InformationWe acknowledge funding from National Institutes of Health under Award Numbers HL075360, HL129823, and HL137319, and from the Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development under Award Numbers 5I01BX000505. The UNMC Ultrasound Core is supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institution of Health under grant P30 GM127200.
What is the Functional Role of Organelle Zinc finger 1 in the Plant C‐to‐U RNA Editing Complex?
Authors: Jimenez-Salinas Alfredo , Cortez Carlos Rodrigo , Hayes Michael Lloyd 
Published: April 2020

Higher plants utilize protein
complexes to precisely edit hundreds of RNA transcripts in organelles. The mechanistic model of RNA editing involves a C‐to‐U deamination reaction facilitated through the interaction of the RNA with a protein complex. Complex models have been proposed, but the minimal “editosome” capable of catalyzing C‐to‐U changes and the functions of many of the protein components have not been revealed. This research focuses on describing the functional role of one critical protein component belonging to the RAN binding protein 2 type zinc fingers protein family, Organelle Zinc finger 1 (OZ1). Zinc finger proteins have been associated with RNA binding; though RNA binding and site recognition in editosomes is known to occur via PPR proteins, OZ1 may be interacting with a scaffold RNA sequence. Recombinant OZ1 from Arabidopsis thaliana was used to analyze zinc coordination and RNA binding of this zinc finger protein. Spectrophotometric analysis of OZ1 after immobilized metal affinity chromatography (IMAC) suggests nucleic acid association. ICP‐MS and ESI‐MS analysis of a truncated variant of OZ1, only the sequence containing its two zinc finger domains (OZ1zf), suggests that OZ1 is coordinating only one zinc ion instead of the proposed two. Computational analysis of OZ1 dependent sites in Brachypodium distachyon returned a potential motif conserved among OZ1‐dependent editing sites. Electrophoretic mobility shift assays (EMSAs) are used to test RNA binding and the motif. Understanding this protein will bring us a step closer to manipulating this precise editing machine for use in biotechnology and may be used for purposes of crop improvement.Support or Funding InformationFunding for this work was provided by NIH SCORE SC2 ID: SC2 GM122718 awarded to Dr. Michael L. Hayes and the NIH MBRS‐RISE R25 GM 061331‐17 grant in support of Alfredo Jimenez‐Salinas.
Comparison of intrinsic chemosensitivity of acutely dissociated medullary serotonergic and retrotrapezoid nucleus neurons
Authors: Wu Yuanming , Bravo Eduardo , Richerson George Bradley 
Published: April 2020

ObjectiveSerotonergic neurons of the medullary raphe and Phox2b expressing neurons of the retrotrapezoid nucleus (RTN) are both putative respiratory chemoreceptors. Here we compare their intrinsic response to acidosis using patch clamp recordings after acute dissociation in vitro.MethodsThe midline medulla and rostral ventrolateral medulla were dissected from mice (>P10) that expressed fluorescent reporters: 1) Phox2b‐Cre::Floxed‐tdTomato:ChAT‐GFP for RTN, and; 2) ePet‐EYFP for 5‐HT. Cells were treated with papain, mechanically dissociated, plated on CELLocate coverslips and allowed to adhere for a variable period before recordings were made. RTN neurons were identified as those that expressed tdTomato but not GFP. In some cases, immunohistochemistry was used to ensure that RTN neurons did not express tyrosine hydroxylase. Patch clamp recordings were made with standard methods previously used to study chemosensitivity of these neurons in culture and slices. Neurons were considered “chemosensitive” if they increased their firing rate by more than 20% in response to a decrease in pH from 7.4 to 7.2 (due to an increase in PCO2 from 5% to 9%, balance O2). Neurons were chosen for recordings only if they were isolated from other neurons. In some cases a lack of synaptic connections was verified with immunohistochemistry for MAP2 using confocal microscopy. The methods used to dissociate and record 5‐HT and RTN neurons were identical.Results45% of 5‐HT neurons (n=84) were chemosensitive, with a mean increase in firing rate by 135%+/−115% (mean+/−SD). 38% of these neurons increased firing rate by more than 50% (i.e. to 150% of baseline firing rate. In contrast, only 29% of RTN neurons (n=21) increased their firing rate by more than 20%, and none of them increased their firing rate by more than 50%. Chemosensitive RTN neurons increased firing rate by a mean of 29%+/−8%. The mean firing rate of chemosensitive 5‐HT neurons was 1.1+/−0.6 Hz at pH 7.4 and 2.15+/−0.9 Hz at pH 7.2 (n=38; p<0.0001). The mean firing rate of chemosensitive RTN neurons was 3.3+/−2.3 Hz at pH 7.4 and 4.3+/−2.8 Hz at pH 7.2 (n=6; p<0.05). Recordings were performed in a separate cohort of 5‐HT neurons using a change in CO2 from 5% to 9% in 21% O2 (balance N2) and their response was not different than in 95% O2.Conclusions5‐HT neurons had a high degree of intrinsic (cell‐autonomous) chemosensitivity to small changes in pH that was similar to that reported for these neurons in acute brain slices and in cell culture. Intrinsic chemosensitivity of RTN neurons was uncommon. When present it was small, similar to that previously reported in a small percentage of neurons in the neocortex, hippocampus and cerebellum. RTN neurons may play a more important role either as “emergency chemoreceptors” (i.e. responding to very large pH changes) or as relays from other chemoreceptors than as pH sensors themselves.Support or Funding InformationNIH U01 NS090414
Chemotherapeutic drugs negatively affect myoblast proliferation and myotube anabolism by reducing rDNA transcription
Authors: Guo Bin , Belcher Daniel J. , Nader Gustavo A. 
Published: April 2020

Cancer cachexia negatively affects quality of life and contributes to increased morbidity and mortality in a wide variety of cancer types. Emerging evidence suggests that, in addition to tumor derived factors, chemotherapeutic drugs may also play a role in muscle wasting. The goal of this study was to determine the cellular mechanisms underpinning skeletal muscle depletion by chemotherapy drugs. Treatment of myoblasts or myotubes with Paclitaxel (PTX) or Doxorubicin (DXR) reduced cell viability in a dose dependent manner with myoblasts displaying more susceptibility than myotubes regardless of the drug. At non‐lethal doses, PTX or DXR induced significant reductions in myoblast proliferation (47%, p<0.01 and 43%, p<0.05 respectively). In myoblasts, PTX and DXR caused a reduction in rDNA transcription of 24% (p<0.0001) and 19% (p<0.0005) relative to control and a concomitant reduction in rRNA of 16% (p<0.0001) and 18% (p<0.0001), respectively. In myotubes, PTX or DXR reduced rDNA transcription by 21% (p<0.0001) and 30% (p<0.0001) compared to control, and consequently rRNA was 20% (p<0.01) and 17% (p<0.05) lower after the respective drug treatments. The reduction in rRNA reduced translational capacity in myotubes as evidenced by lower global protein synthesis of 52% (p<0.001) and 47% (p<0.001) following exposure to PTX or DXR. These results indicate that chemotherapeutic agents may affect muscle mass by impairing the ability of myogenic cells to proliferate, and by reducing the capacity for protein synthesis in myofibers via a reduction in rDNA transcription.Support or Funding InformationSupported by NIH grant AR073385
Evidence for Progressive Shifts Towards α‐Myosin Isoform with Paradoxical Increases in Cardiac Hemodynamic Loads in Post‐Natal Healthy Rats
Authors: Geist Gail Elizabeth , Combs Ariana C. , Leinwand Leslie , del Rio Carlos L. 
Published: April 2020

IntroductionThe balance of fast (α) and slow (β) cardiac myosin heavy chain (MYHC) isoforms is known to change with both aging and disease. While pathological states with increased hemodynamic loads are often accompanied by shifts toward energy‐sparing β isoforms, the underlying mechanism(s) of these changes remain poorly understood. Neonatal rats are a promising model to study such cardiac MYHC remodeling, as they show a well‐documented transition (β to α) during post‐natal development. In these experiments, MYHC‐isoform switches and in vivo cardiac structure/hemodynamic changes were evaluated during post‐natal development.MethodsCardiac echocardiographic and systemic/left ventricular (LV) hemodynamics were evaluated in Sprague‐Dawley rats (n = 61) ranging from post‐natal day (PND) 3 to adulthood. Hemodynamics were obtained before/after challenges with sodium nitroprusside (SNP, 25 μg/kg IV) and/or phenylephrine (PE, 10 μg/kg IV) to study cardiovascular responsiveness. LV’s were also collected (n = 48) for myosin isoform analysis via SDS‐PAGE MYHC separating gels.ResultsAs expected, post‐natal rats showed a progressive decline in β‐MYHC expression from PND3 (1.29 ± 0.05 α:β) to adulthood when α‐MYHC was dominant (12.78 ± 2.51 α:β). Concomitantly, postnatal rats showed progressive increases (P<0.05) in systemic/LV pressures (MAP: 46 ± 1 at <PND14 to 103 ± 2 mmHg, P<0.05), LV mass (0.16 ± 0.02g at <PND14 to 0.84 ± 017g) and diastolic dimensions (79 ± 6 at <PND14 to 288 ± 14 μL), with vastly preserved heart rates. Pressor responses to PE increased during maturation, while all age groups were sensitive to SNP‐mediated vaso‐relaxation. Ejection fraction was preserved during postnatal maturation with young pups showing lower rates of LV pressure development (dPdtmax 4052 ± 125 at <PND14 vs. 9478 ± 231 mmHg/s, P<0.05) consistent with the early expression of β‐MYHC.ConclusionsTaken together, these data provide evidence of a maturing cardiovascular system in post‐natal rats showing progressive increases in systemic/ventricular pressures and cardiac dimensions, with paradoxical shifts towards α‐MYHC isoforms. In short, in rats, the cardiac β‐MYHC isoform consistently declines over maturation despite sustained increases in systemic load.
Characterization of New Optical Imaging Biomarkers of Peripheral Nerve Overstimulation
Authors: Tao Michael , Monroy Guillermo , Erfanzadeh Mohsen , Rafi Harmain , Vakoc Benjamin , Vasudevan Srikanth , Hammer Daniel 
Published: April 2020

An animal study was designed to explore the potentially damaging effects of electrical overstimulation on peripheral nerves using a polarization‐sensitive optical coherence tomography (PS‐OCT) imaging and stimulation platform. Lewis rats (n = 15) were divided into 3 groups with 2 different levels of stimulation applied to the exposed sciatic nerve: Group 1 (sham control): 0.0 mA/0 Hz, Group 2: 3.4 mA/50 Hz, and Group 3: 6.8 mA/100 Hz. On day 1, the protocol contains Von Frey and walking track behavioral tests, followed by sciatic nerve exposure surgery (left leg). Electrical stimulation of the nerve was performed for one hour, with a one‐hour recovery period. PS‐OCT and optical coherence tomography angiography (OCT‐A) imaging were performed both prior to and during these periods. On day 7, this protocol was repeated without stimulation. OCT imaging was performed along a 5‐mm length of the sciatic nerve, including sites proximal and distal to injury. Both the stimulated and contralateral sciatic nerves were then resected and processed for histology.Image analysis quantified the effects of electrical stimulation at the two time points and allows comparison between stimulation levels. In all cohort groups, PS‐OCT image features remained consistent on day 1. On day 7, we observed relatively few PS‐OCT or OCT‐A changes compared to day 1 in the sham control animals. Groups 2 and 3 showed a clear increase in OCT‐A perfusion in the stimulated region, while there is a drop in PS‐OCT signal in the same area. The PS‐OCT signal drop‐out appears more pronounced in Group 2 compared to Group 3. It is not yet clear if the PS‐OCT signal drop‐out is related to demyelination or from shadowing from hyperperfused tissue. Walking track video was analyzed frame‐by‐frame for animal foot spread to determine sciatic and tibial function indices. This data, along with Von Frey withdrawal forces, were analyzed with statistical comparison tests (ANOVA) and indicated no significant differences between the groups nor significant interactions when compared across stimulation group, pre‐ and post‐exposure, and ipsilateral‐contralateral leg. Histological samples were processed for histomorphometry and axonal damage proximal and distal to the injury, as well as for immunohistochemistry of axons, macrophages, and nuclei in the stimulated and unstimulated regions in all groups.PS‐OCT and OCT‐A proffer real‐time observation of nerve changes with overstimulation that are undetectable by behavioral testing.Support or Funding InformationNIH SPARC
Identifying Metabolic Pathways of c‐MET Tyrosine Kinase Inhibitor Tepotinib in Human and Mouse Liver Microsomes
Authors: Li Feng , MacKenzie Kevin R. , Nyshadham Pranavanand , Kerlec Kimberly A. , Matzuk Martin M. 
Published: April 2020

Tepotinib (TPB) is a highly selective and potent c‐Met inhibitor that has shown promising activity in a phase I trial for non‐small cell lung cancer (NSCLC). Recent studies indicate that TPB can overcome resistance to epidermal growth factor receptor inhibitors driven by aberrant MET activation, and the Food and Drug Administration has granted a breakthrough therapy designation for tepotinib in patients with metastatic NSCLC harboring METex14 skipping alterations who have progressed on prior platinum‐based chemotherapy. The metabolism of TPB remains largely unknown, but is likely to play a critical role in both safety and efficacy: understanding the metabolism of TPB could improve safety and predict possible adverse effects and drug‐drug interactions. In the current study, we investigated the metabolism and bioactivation of TPB in human and mouse liver microsomes (HLM and MLM) using LC‐MS‐based metabolomic approaches. We identified over 20 TPB metabolites and adducts in liver microsomes. Monohydroxyl‐TPB (O+TPB) is the dominant metabolite in both HLM and MLM, and demethyl‐TPB is secondary to O+TPB. Using methoxyamine as a trapping agent, we infer that two aldehydes are generated in HLM; one of these was also observed in MLM. No adducts were observed in liver microsomes incubated with TPB in the presence of trapping agents glutathione or potassium cyanide. Using recombinant CYP450 isoenzymes, we showed that CYP3A4 and CYP3A5 are the primary contributors to the formation of both monohydroxyl‐TPB and the methoxyamine‐trapped TPB aldehydes. This study offers a comprehensive view of TPB metabolism and identifies metabolites that will be helpful to evaluate adverse effects of TPB and possible drug‐drug interactions in TPB‐treated hepatocytes and model organisms.Support or Funding InformationThis work was supported by Cancer Prevention & Research Institute of Texas (RP160805), the Eunice Kennedy Shriver National Institute of Child Health and Human Development (P01 HD087157‐01A1), Welch Foundation Grant (H‐Q‐0042), and Bill and Melinda Gates Foundation (INV‐001902) to Dr. Martin M. Matzuk and the National Institute of Diabetes and Digestive and Kidney (R01‐DK121970) to Dr. Feng Li.
Salivary Origins of Exogenous Cytokinins
Authors: Seng Stephannie , Connor Edward 
Published: April 2020

Cytokinins are a class of phytohormones which play an important role in a variety of plant processes, such as cell growth and differentiation, formation of roots and shoots, and the reallocation of nutrients to form mobilizing sinks. Exogenous cytokinins found in plant‐manipulating insects are known to contribute to many effects on host plants. Perhaps the most apparent effect is that cytokinins, in the presence of auxin, leads to cell division and the proliferation of plant tissue; consequently, plant galls or tumors are formed. Cytokinins have also been implicated in the reallocation of sugars and nutrients for the oviposition site to act as a mobilizing sink, where the intention may be gall induction or sustenance for the organism. There are two known pathways for the biosynthesis of isoprenoid cytokinins. The first is the tRNA‐ipt pathway, in which an adenine residue is modified by the addition of an isoprenoid chain by the tRNA‐ipt gene. In plant plastids and bacteria, the methyl erythritol (MEP) pathway is the main source of the prenyl group, but in the cytosol of plants and insects, the prenyl group is synthesized via the mevalonate (MVA) pathway. Cytokinins are produced upon the degradation of the prenylated tRNA. To further investigate the role of cytokinins in gall induction by insects, I performed gene expression experiments using the gall‐inducing Eurosta solidaginis and its bacterial symbiont Wolbachia sp. I also performed targeted gene expression analyses in order to examine the expression of genes in the MEP, MVA, and tRNA‐ipt pathways. Up‐regulation of these pathways in the salivary gland implies that cytokinins are being synthesized in the salivary glands by either the insect and/or its bacterial symbiont. Along with the known widespread distribution of cytokinins in insects, these further imply that cytokinin production and secretion has been amplified and is used by the insects to manipulate their host plants. However, the genes involved in cytokinin production have also been found in other insect groups, so this plant manipulation may not just be exclusive to gall‐inducing insects, suggesting widespread hostplant manipulation among plant‐eating insects.Support or Funding InformationNIH MBRS‐RISE: R25‐GM059298
Validation of Antibodies against Histone Modifications and Transcription Factors for ChIC/CUT&RUN Assays
Authors: Din Shabana , Saito Kan , Silverman Jennifer , MilliporeSigma Jeffrey Turner 
Published: April 2020

DNA‐protein interactions in the nuclei control a wide variety of biological processes such as development, differentiation, and cellular response to environmental changes. Chromatin immunoprecipitation (ChIP) has been used to analyze association of proteins with genomic DNA sequences. Although there have been several improvements with the technology over the decades, fundamental challenges remain with ChIP, including high background that limits sensitivity, high amounts of cellular input, and artifacts resulting from cross‐linking and solubilization.To overcome the challenges with ChIP, Cleavage Under Targets and Release Using Nuclease (CUT&RUN) was developed in Steven Henikoff’s lab by improving Chromatin Immune‐Cleavage (ChIC), which was originally developed in Ulrich Laemmli’s lab. In ChIC/CUT&RUN, binding sites of transcription factors in the genome were detected by targeting micrococcal nuclease conjugated with protein A (pA‐MN) through a specific antibody to detect genome‐wide transcription factor binding sites or histone modifications on native chromatin. Since the process doesn’t require cross‐linking and solubilization, and only the targeted fragments are released into solution and the majority of DNA is left behind, the results from ChIC/CUT&RUN assays have exceptionally low background levels compared with ChIP assays. As a result, ChIC/CUT&RUN provides high‐quality results using much smaller number of the cells (100 cells for a histone modification analysis and 1,000 cells for a transcription factor binding site analysis) than traditional ChIP assays.Here we performed ChIC/CUT&RUN assays with various antibodies against popular histone modifications including H3K4me3 and H3K27me3, and transcription factors such as CTCF. The sequencing data was compared with ChIP‐seq data in the ENCODE database. The sequencing data of ChIC/CUT&RUN assays with each of the antibodies tested showed very similar patters to the ENCODE ChIP‐seq data with the same targets. The results not only confirmed the power of the ChIC/CUT&RUN technology but also validated the suitability and comparability of these antibodies for use in a variety of studies.
Can Classrooms Have an Impact on Student Stress?
Authors: Oyejide Kafayat , Hanna James , Daniel Kristy , Huertas Mar 
Published: April 2020

Stress negatively impacts a person’s health, and stress in the school environment may negatively impact student performance. For the past five years, the graduation rate for undergraduate students has been falling. The main factors contributing to this decline may involve indirect or direct stress on the student. For instance, learning space and environment impact undergraduate students and their stress levels which impacts their graduation rates. We hypothesized that students in the SCALE UP (Students Centered Active Learning Environment for Undergraduate Programs) classroom have lower concentrations of salivary stress biomarkers (cortisol and α‐amylase) than students in traditional classrooms, and perform better academically than their counterparts. We screened students learning introductory biology in two different learning environments, a SCALE UP and a lecture hall, collected salivary samples twice a week, and asked the students to self rate their stress level from not stressed to very stressed. Saliva was collected at the beginning and end of each class period to measure a change in cortisol or α‐ amylase over the class period. We used an Enzyme‐Linked Immunosorbent Assay (ELISA) to quantify the concentration of salivary cortisol and a colorimetric method to determine the α‐ amylase. Students in the SCALE UP classroom had lower α‐amylase activity than those in a traditional lecture hall. Cortisol concentrations were not correlated with the classroom environment. The results of the α‐amylase assay aligned closer with the literature, indicating that salivary α‐amylase is better at identifying acute stressors, such as a classroom environment than cortisol. The setup of a SCALE UP class may have less of an impact on student stress than the pedagogy. The results of the study highlight the importance of pedagogy on student stress and the significance of salivary α‐amylase as an acute, noninvasive stress biomarker.Support or Funding InformationThis research was supported by the U.S. Department of Education HIS STEM program (841.031c), Award #P021C160036