bluuebunny/crossref_metadata_2025_split huggingface dataset:
Using Near Infrared Spectroscopy, Diffuse Correlation Spectroscopy, and Intravital Video Microscopy to Monitor the Skeletomuscular and Cerebral Microcirculation
Authors: Mawdsley Laura , Rajaram Ajay , Yip Lawrence , Ellis Christopher G. , Diop Mamadou 
Published: April 2020

IntroductionThe microcirculation is the primary facilitator of oxygen delivery in tissue, and dysfunction in the microvasculature is often the first indication of a disease state. The microcirculation can be studied using a variety of methods, both invasive and non‐invasive. Two non‐invasive optical methods are near infrared spectroscopy (NIRS) and diffuse correlation spectroscopy (DCS). NIRS is sensitive to changes in tissue hemoglobin concentration and oxygen, while DCS is sensitive to changes in blood flow. However, neither method can directly visualize changes on a capillary by capillary basis. Intravital Video Microscopy (IVVM) allows us to do so, but is an invasive technique that requires surgery to expose the microcirculation. By using IVVM we can compare a direct visualization of the microvasculature to the signal received by NIRS/DCS in an equivalent microvascular bed. In addition, we can compare the NIRS/DCS signal from the skeletal muscle to the signal from the brain, allowing us to simultaneously monitor change in these two organs in response to disease progression or in response to stimulus.MethodsData is collected from Sprague Dawley rats (n=4) using both a dual wavelength Olympus inverted microscope (with 2 Rolera XR cameras and a beam splitter) and an in‐house NIRS/DCS device. Rats are anaesthetized and have their right extensor digitorum longus (EDL) muscle, found in the hind limb, exposed and reflected over the objective. Probes for the NIRS/DCS device are located on the left hind limb and the top of the skull. IVVM is used to collect microvascular velocity, hematocrit, and oxygen saturation from the right EDL, and NIRS/DCS will be used to collect microvascular hematocrit, oxygen saturation, and blood flow data from the left hind limb and the brain. Microvascular blood flow and oxygen saturation data from the left and right hind limbs will be compared, then the NIRS/DCS‐gathered data from the left hind limb will be compared to NIRS/DCS‐gathered data from the brain.DiscussionThis study will be the first to make a direct comparison between NIRS/DCS and IVVM measurements of the microcirculation, and will provide insight into the sensitivity of NIRS/DCS and its ability to accurately monitor the microvasculature. In addition, this study will be the first to make a direct comparison between skeletomuscular and cerebral NIRS/DCS measurements. Future work includes using this approach to determine the differences in effect of a phenylephrine bolus or an inspired oxygen challenge on these two microvascular beds. Further studies will determine whether disease progression occurs on the same timeline in the brain as it does in skeletal muscle.Support or Funding InformationNSERC Discovery Grant to C.G. Ellis.
Mitochondria‐targeted scavenger of reactive isolevuglandins, mito2HOBA, protects from mitochondrial dysfunction and inflammatory injury
Authors: Mayorov Vladimir , Uchakin Peter , Amarnath Venkataraman , Panov Alexander , Bridges Christy , Uzhachenko Roman , Zackert Bill , Dikalova Anna , Moore Christy , Davies Sean , Dikalov Sergey 
Published: April 2020

Hypertension is linked to oxidative stress and inflammation producing highly reactive dicarbonyl lipid peroxidation products, isolevuglandins (IsoLG) which covalently modify and cross‐link proteins via lysine residues and promote end‐organ‐damage. Mitochondrial dysfunction has been associated with inflammation; however, its molecular mechanisms and pathophysiological role are still obscure. We hypothesized that inflammation‐induced isolevuglandins contribute to mitochondrial dysfunction and mortality. To test this hypothesis, we have (a) investigated the mitochondrial dysfunction in response to synthetic 15‐E2‐isolevuglandin (IsoLG) and its adducts; (b) developed a new mitochondria‐targeted scavenger of isolevuglandins by conjugating 2‐hydroxybenzylamine to the lipophilic cation triphenylphosphonium, (4‐(4‐aminomethyl)‐3‐hydroxyphenoxy)butyl)triphenylphosphonium (mito2HOBA); (c) tested if mito2HOBA protects from mitochondrial dysfunction and mortality using a lipopolysaccharide model of inflammation. Acute exposure to either IsoLG or IsoLG adducts with lysine, ethanolamine or phosphatidylethanolamine inhibits mitochondrial respiration and attenuates Complex I activity. Complex II function was much more resistant to IsoLG. We confirmed that mito2HOBA markedly accumulates in isolated mitochondria and it is highly reactive with IsoLGs. To test the role of mitochondrial IsoLGs, we studied the therapeutic potential of mito2HOBA in lipopolysaccharide mouse model of sepsis. Mito2HOBA supplementation in drinking water (0.1g/L) to lipopolysaccharide treated mice increased survival by 3‐fold, improved complex I‐mediated respiration, and histopathological analyses supported mito2HOBA‐mediated protection of renal cortex from cell injury. Depletion of Cyclophilin D partially attenuated IsoLG‐induced mitochondrial dysfunction suggesting that mitochondrial IsoLG can affect both Cyclophilin D and Complex I functions. These data support the role of mitochondrial IsoLG in mitochondrial dysfunction and inflammation. We conclude that reducing mitochondrial IsoLGs may be a promising therapeutic target in inflammation and conditions associated with mitochondrial oxidative stress and dysfunction.Support or Funding InformationThis work was supported by the National Institutes of Health grants (R01HL124116 and R01HL144943) and Navicent Health Foundation Research & Education Grant.
Genetic Inhibition of Nfatc2 Attenuates Asparaginase Hypersensitivity in Mice
Authors: Rathod Sanjay , Ramsey Manda , Finkelman Fred D. , Fernandez Christian A. 
Published: April 2020

IntroductionThe development of a T cell‐dependent antibody response to asparaginase (ASNase) is the most common adverse effect of treatment with this chemotherapeutic agent for pediatric acute lymphoblastic leukemia (ALL). Anti‐ASNase antibodies decreases ASNase levels and drug efficacy and can mediate anaphylaxis. Transcription factors of the nuclear factor of activated T cells (NFAT) family play a critical role in Th cell activation, differentiation, and effector function. Here we show that a member of this family, Nfatc2 (NFAT1), upregulates Th2 responses, with Nfatc2‐deficient ASNase‐immunized mice being completely protected from anaphylaxis. Consistent with this, our clinical data demonstrate increased risk of ASNase hypersensitivity in pediatric ALL patients who express the NFATC2 rs6021191 intronic variant, which increases Nfatc2 expression, whereas the risk of ASNase hypersensitivity is decreased in ALL Down syndrome patients, who overexpress NFAT pathway inhibitors.HypothesisNfatc2 deficiency protects mice against ASNase‐induced hypersensitivity by suppressing type 2 immunity.MethodsNfatc2‐deficient mice and WT littermates were immunized with native E. coli ASNase using aluminum hydroxide adjuvant, after which immune cell phenotype and levels of basophil activation, cytokines, and ASNase‐specific antibody were determined. Hypersensitivity reactions to ASNase were assessed by detecting hypothermia with rectal thermometry. β‐hexosaminidase release was used for in vitro analysis of the degranulation of peritoneal mast cells (PMCs) obtained from naïve and ASNase‐immunized mice, while passive cutaneous anaphylaxis, passive systemic anaphylaxis, and active systemic anaphylaxis models were used for in vivo determinations.ResultsNfatc2‐deficient mice were protected against ASNase hypersensitivity reactions. Naïve Nfatc2‐deficient mice had fewer CD4+IL‐4+ cells and lower IL‐4 and IL‐13 levels than wild‐type mice. ASNase‐immunized Nfatc2‐deficient mice had increased levels of Treg cells and the regulatory cytokines, IL‐10 and TGF‐β1, along with decreased plasma IgG and IgE anti‐ASNase and total IgE levels. Consistent with this, basophils/peritoneal mast cells from Nfatc2‐deficient mice had decreased FcɛRI expression and IgE‐mediated activation.ConclusionNfatc2 deficiency decreases ASNase hypersensitivity by increasing regulatory responses and attenuating type 2 immune responses to ASNase immunization. Nfatc2 may be a promising target for the prevention of ASNase‐induced hypersensitivity.Support or Funding InformationUniversity of Pittsburgh School of Pharmacy & NIH Grants RO1 CA216815
Regulators Of G‐Protein Signaling 2 And 4 Differentially Regulate Nicotine‐Induced Affective Behavioral Responses
Authors: DSouza Manoranjan S. , Snyder Madison , Ho Sheng-Ping , Yi Tsai , Seeley Sarah , Rorabaugh Boyd R. 
Published: April 2020

High rates of tobacco smoking are observed in individuals suffering from anxiety or depression. Nicotine is a major psychoactive component of tobacco smoke and previous work has shown that nicotine can produce both anxiolytic‐ and antidepressant‐like effects. Regulators of G protein (RGS) signalling proteins negatively regulate intracellular signaling pathways of neurotransmitters (e.g. adrenaline, dopamine and serotonin), which mediate the antidepressant‐ and anxiolytic‐like effects of nicotine. The objective of the study was to determine the role of the RGS2 and RGS4 in the anxiolytic‐ and antidepressant‐like effects of nicotine. Antidepressant‐ and anxiolytic‐like effects were assessed using the tail suspension model and elevated plus maze, respectively. We used both male and female mice lacking either RGS2 (i.e. RGS2 KO) or RGS4 9i.e. RGS4 KO) and their wildtype counterparts. Both male and female RGS2 KO mice showed anxiolytic effects to nicotine (0.1 mg/kg; i.p., base) compared to respective saline controls. In contrast, no nicotine‐induced (1 mg/kg; i.p., base) antidepressant‐like effects were observed in male and female RGS2 KO mice compared to respective saline controls. Together, these data suggest that RGS2 plays a role in nicotine‐induced anxiolytic effects but not nicotine‐induced antidepressant‐like effects. In contrast, nicotine‐induced antidepressant‐like effects were observed in male RGS4 KO mice, but not female RGS4 KO mice compared to respective saline controls. Also, nicotine‐induced anxiolytic effects were not observed in either male or female RGS4 KO mice. Together, these data suggest that RGS2 and RGS4 differentially mediate nicotine‐induced affective responses. Clinically, these data support a role for targeting these proteins in treating nicotine addiction in patients suffering from comorbid anxiety and/or depression.Support or Funding InformationThis work was supported by a Bower, Bennett and Bennett grant awarded to Dr. Manoranjan S. D’Souza by the Raabe College of Pharmacy, Ohio Northern University (ONU), Ada, Ohio.
Dietary Effects on Fiber Type Plasticity in the Masseter Muscle
Authors: LaRocque Holly , Rossiter Jeffrey A. , Menegaz Rachel A. 
Published: April 2020

Clinical and experimental studies have shown the masticatory complex (the skeletal and soft tissues associated with feeding) to be phenotypically plastic with regard to dietary material properties. Harder, tougher diets generally produce larger skeletal elements with greater bone density. However, less is known about the plasticity of the chewing musculature. Muscles are composed of two primary fiber types: slow‐twitch (type I) and fast‐twitch (type II). Fast‐twitch fibers produce rapid, powerful contractions but fatigue more quickly than slow‐twitch fibers. Here we use a rodent model to examine the plasticity of the masseter muscle fibers in response to inter‐individual variation in dietary material properties. We hypothesize that rats raised on hard, tough diets will have more fast‐twitch muscle fibers with greater cross‐sectional area, representing an increase in force production by the masseter in response to the dietary material properties.Male Sprague‐Dawley rats were raised from weaning (3 weeks) to adulthood (16 weeks) in two dietary treatment groups: a “hard/tough” pellet diet and a “soft” meal diet. Post‐sacrifice, fixed muscles were embedded in paraffin and divided into anterior, middle, and posterior regions. To visualize fast‐twitch fibers, 6 sections per region per individual were stained with a primary antibody for the fast isoforms of myosin heavy chain (MHC) and a fluorescent secondary antibody. Images were collected at 4× using a fluorescent confocal microscope. ImageJ was used to collect total muscle cell area, fast‐twitch cell area, and slow‐twitch and fast‐twitch cell counts. Mann‐Whitney U tests were used to compare cell count and area between the treatment groups (n=5/group, α = 0.05).Preliminary results indicate that hard/tough diets tend to be associated with a decrease in slow‐twitch fiber count and an increase in fast twitch fiber cross‐sectional area in the rat masseter. This allows for a greater proportion of fast‐twitch muscle fibers in a given muscle volume. We propose that this change reflects an increase in muscle force production during feeding on mechanically challenging food items. The results of this study allow for greater understanding of skeletal muscle growth postnatally through adulthood, and provides insight into the consequences of material properties of diet for the function and dysfunction of the human masticatory complex. Future studies are needed to address how muscle fiber plasticity changes with age and with intra‐individual variation in the material properties of diet.Support or Funding InformationFunding was provided by the NSF (BCS‐1061368), the Wenner‐Gren Foundation, the American Society of Mammalogists, and the American Association for Anatomy Innovations Program.
The Regulatory Role of Phosphorylation of Mitochondrial Transcription Factor B2 (TFB2M)
Authors: Moya Gonzalo , Bostwick Alicia M. , Senti Mackenna L. , Dittenhafer-Reed Kristin E. 
Published: April 2020

Mammalian cells contain genetic information in two compartments, the nucleus and the mitochondria. Mitochondrial DNA (mtDNA) encodes thirteen protein subunits required for oxidative phosphorylation. The remaining mitochondrial proteome is encoded by the nuclear genome; which includes additional oxidative phosphorylation subunits, as well as the proteins necessary for mtDNA replication, expression and stability. Therefore, to respond to metabolic changes, mitochondrial gene expression must be coordinated with nuclear gene expression. While the control of nuclear gene expression is widely studied, there is a need to understand the regulation of mtDNA transcription. Our central hypothesis is that reversible protein post‐translational modifications of the mtDNA transcriptional machinery is a means to tune mtDNA transcription in response to changes in cellular metabolism. To address this gap in knowledge, this research focuses on a member of the mtDNA transcription initiation complex, mitochondrial transcription factor B2 (TFB2M). TFB2M melts mtDNA at the promoter to allow for the RNA polymerase (POLRMT) to access the DNA template and initiate transcription. Three phosphorylation sites have been previously identified on TFB2M by mass spectrometry: threonine 184, threonine 313 and serine 197. To mimic the behavior of phosphorylation on these three sites, individual amino acids were mutated to glutamate, an amino acid that mimics the size and charge of the phosphoryl group. WT and phosphomimetic TFB2M were expressed in E. coli and purified. The purified proteins were analyzed for their ability to bind mtDNA through a pull‐down assay and fluorescence polarization was used to determine the binding dissociation constants for the light strand promoter. Interactions between TFB2M phosphomimics and POLRMT were also assessed. The data support the hypothesis that the phosphorylation at these sites may decrease the ability of TFB2M to bind to mtDNA. In vitro transcription assays were performed to determine the impact of these modifications on TFB2M’s role in transcription initiation. It is expected that these findings will lead to a more complete understanding of the dynamics and coordination of mitochondrial and nuclear gene transcription, as well as adding to the understanding of the importance of protein post‐translational modifications in regulating aspects of mitochondrial function.Support or Funding InformationThis material is based upon work supported by the National Science Foundation Division of Molecular and Cellular Biosciences under Grant No. 1814845.
Kv1.3 Constitutes a Valid Target for Reducing Neuroinflammation in the Wake of Ischemic Stroke in Male, Female and Aged Mice
Authors: Chen Yi-Je , Nguyen Hai M. , Cui Yanjun , Wulff Heike 
Published: April 2020

The voltage‐gated Kv1.3 channel was first discovered in human T cells in 1984 and has since then been pursued as a target for autoimmune diseases. Kv1.3 is further expressed on pro‐inflammatory microglia and our laboratory therefore evaluated the small molecule Kv1.3 blocker PAP‐1 in models of ischemic stroke. In both male C57BL/6J mice and male Wistar rats, treatment with PAP‐1 started 12 hours after reperfusion reduced infarction and inflammatory brain cytokine levels and improved neurological deficit on day‐8 after reperfusion MCAO (Y.‐J. Chen et al. 2018). In order to test the therapeutic hypothesis that Kv1.3 inhibition is equally effective in both sexes, we established 60‐min MCAO with reperfusion in female C57BL/6J mice. Females have smaller infarcts, but 4‐months old animals of both sexes showed qualitatively similar pathologies with similar IBA+‐microglia/macrophage and T cell densities and a GFAP+ glial scar. Based on patch‐clamp recordings performed on acutely isolated CD11+ cells Cx3cr1GFP/+ male and female mice exhibit similar Kv1.3 current densities on microglia/macrophages from the infarcted area. Immunofluorescence revealed that in both male and female Cx3cr1GFP/+ mice Kv1.3 expression increases with time after MCAO, peaks around day‐7 and is localized to GFP+ microglia/macrophages and CD3+ T cells. Since these findings suggest that both sexes would equally benefit from Kv1.3 inhibition, we subjected 4‐month and 20‐month old male and female WT and Kv1.3−/− mice to 60 min‐MCAO with reperfusion. T2‐weigthed MRI revealed that Kv1.3‐deletion produces a significant reduction in infarct volume on day‐8 in both sexes. The reduction in infarction was most effective in aged Kv1.3−/− females. Based on these observations and the finding that treatment with PAP‐1 significantly reduces infarction in 4‐months old WT female mice (n=14), we conclude that Kv1.3 inhibition effectively reduces stroke associated inflammation in both sexes independent of age.Support or Funding InformationSupported by NIH (NS100294).
Reframing Lower‐Level Bloom’s Taxonomy Exam Questions in Applied Anatomy Alters Statistical Outcomes for Item Performance: A Pilot Study
Authors: Stevens Kelsey , Immonen Jessica , Dykstra Emily 
Published: April 2020

BackgroundBloom’s Taxonomy is a hierarchical model designed to categorize course learning objectives based on the complexity of learning associated with the task. Little is known about performance and abilities of entry‐level allied health students related to Bloom’s Taxonomy within anatomy education.ObjectiveThe objective of this assessment was to use exam reviews from 2018’s PAS 501 Applied Anatomy course to re‐create exam items at higher levels of Bloom’s learning. Cohort performance at the “Remembering” level of Bloom’s was compared to 2019 reconstructed questions to determine if graduate‐level PA students are most capable of memorizing anatomy or interpreting more detailed applications.MethodsFour “Remembering” questions were selected from the 2018 PAS 501 Abdomen written exam. The regional concepts in these questions were reframed in clinical scenarios and questions where regional anatomy needed to be applied at higher levels of thinking. Statistics of cohort performance in the form of the DI, PBC and the DisI were used to compare the lower‐ and higher‐level Bloom’s questionsResultsTwo questions reframed as clinical scenarios and demonstrated decreases in the DI and an increase in the PBC. Two questions were initially written as basic definition questions and reconstructed to represent a discussion of regional anatomy that required the student to understand and reinterpret the information. One of these questions displayed an increase in the DI and the PBC.ConclusionsHigher level Bloom’s questions framed as clinical scenarios (N=2) displayed noted increases in the PBC implying these questions are quality discriminators for students who do well overall and those who do not. Reframing “Remembering,” definition questions to “Understanding” level questions may increase item success for clinically‐minded graduate‐level students. Additional work is required to draw meaningful conclusions from this pilot data.Support or Funding InformationN/A
Inositol polyphosphate multi‐kinase is a novel regulator of reverse‐transsulfuration pathway
Authors: Tyagi Richa , Snyder Solomon H. , Paul Bindu 
Published: April 2020

Inositol polyphosphate multikinase (IPMK) is one of the members of inositol phosphate kinase family that generates inositol polyphosphates. IPMK possesses inositol phosphate kinase (IP3‐kinase) as well as phosphatidylinositol kinase (PI3‐kinase) activities. IPMK is a pleiotropic protein and non‐catalytically regulates mammalian target of rapamycin complex 1 (mTORC1), serum response factor, p300, and tumor suppressor protein p53. We report that IPMK regulates expression of cystathionine γ‐lyase (CSE) and 3‐mercaptopyruvate sulfurtransferase (3‐MST), enzymes involved in Hydrogen sulfide production. Protein levels of CSE and MST are increased in IPMK null fibroblasts as compared to wild type fibroblast cells. Regulation of CSE and MST is independent of catalytic activity of IPMK. IPMK regulates CSE expression at the transcriptional level. Since cystathionine β‐synthase (CBS) is expressed at relatively lower levels as compare to CSE in fibroblasts, CSE is the major source of cysteine generation from cystathionine in mouse fibroblasts. Lysates prepared from IPMK null fibroblasts produced more cysteine as compared to wild type cells. Hydrogen sulfide levels are also increased in IPMK null fibroblasts. Expression of CSE is inducible upon condition such as lipopolysaccharides (LPS), and inflammation mediated by tumor necrosis factor α (TNF‐α). We propose that IPMK acts as a repressor of CSE expression.Support or Funding InformationThis work was supported by NIH grants MH18501 to S.H.S.