bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of various solvents on the cytotoxic activity and stability of Bleomycin for in vitro cell culture based assays
Authors: De La Paz Rosa , Gupta Dipankar , Shenoy Anitha , Kolli ChandraSekhar , Shenoy Vinayak 
Published: June 2021

BackgroundBleomycin sulfate is a metal‐complexing glycopeptide that is commonly used as an experimental model for pulmonary fibrosis. Pulmonary fibrosis induced by Bleomycin sulfate is mediated via DNA damage and cytotoxic actions on the alveolar epithelial cells. Among various factors, the in vitro actions of Bleomycin sulfate depend considerably on its chemical stability and the solvent used to dissolve the drug. In this study, we evaluated the effects of various solvents [water, dimethyl sulfoxide (DMSO), saline and Dulbecco's Modified Eagle Medium (DMEM)] on the in vitro cytotoxic activity and stability of Bleomycin.MethodsThe cytotoxic effect of Bleomycin sulfate was evaluated on alveolar epithelial cells (A549) using various concentration (10, 15 and 20 mcg/mL) of the drug dissolved in different solvents. A549 cells were seeded at a concentration of 5 × 103 cells per well in a 96 well‐plate and incubated for 24 hours. These cells were then exposed to various concentrations of Bleomycin sulfate in the different solvents for the next 24 hours. The cell viability was determined using the MTT (3‐(4, 5‐dimethylthiazolyl‐2)‐2, 5‐diphenyltetrazolium bromide) reduction assay.ResultsSignificant cytotoxic response was observed with all the doses of Bleomycin sulfate (10, 15 and 20 mcg/mL) when it was dissolved in either water or DMSO. However, this was not the case with DMEM or saline since lower concentration of Bleomycin sulfate (10 mcg/mL) failed to induce cell death. We noticed that treatment of A549 cells with any of the solvents by themselves (vehicle control) did not induce cell death. With regards to DMSO, we used a final concentration of 0.025% v/v in the cell culture media.ConclusionWe conclude that water or DMSO (at a final concentration of 0.025% v/v) may serve as suitable solvents to dissolve Bleomycin sulfate for carrying out in vitro cell culture assays. Also, it appears that higher concentrations of Bleomycin sulfate are required to show cytotoxic activity when DMEM or saline is used as a solvent. Further studies are warranted using high performance liquid chromatography (HPLC) to explore drug stability in the various solvents used.Support or Funding InformationSeed Funding from California Health Sciences UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deconstructing pulmonary fibrosis at single‐cell resolution
Authors: Kropski Jonathan A , Calvi Carla L , Habermann A. Chris , Guitierrez Austin , Winters Nichelle , Rodenberry John C , McDonnell Wyatt , Shaver Ciara M , Ware Lorraine B , Mallal Simon , Blackwell Timothy S , Banovich Nicholas E 
Published: June 2021

RationaleDespite years of research, the fundamental mechanisms driving the pathogenesis of pulmonary fibrosis remain unclear. Numerous histopathologic patterns of pulmonary fibrosis have been identified in association with different patterns of risk factors, but to date it remains unclear as to what mechanisms are shared across different forms of pulmonary fibrosis and which drive distinct pathologies and outcomes. We hypothesize that using single‐cell transcriptomic approaches, we can decipher both the conserved and distinct mechanisms driving pulmonary fibrosis phenotypes.MethodsAt the time of lung transplantation, single‐cell suspensions were generated from the lung parenchyma of pulmonary fibrosis patients and from declined donor lungs (controls). Unsorted single‐cell suspensions and CD45 depleted fractions were used for scRNA‐seq. Single‐cell RNA‐sequencing library preparation was performed using the 10X Genomics 3′ or 5′ kit, and sequencing was performed on an Illumina hiSeq4000 or Novaseq. Following alignment, demultiplexing was performed using Cell Ranger. Graph‐based clustering and scRNA‐seq analysis was performed using the Seurat package in R. Developmental lineage reconstruction was performing using Monocle and p‐Creode. Diagnoses were assigned based on clinical interpretation of explant pathology.ResultsJoint graph‐based clustering and canonical correlation analysis of scRNA‐seq profiles from >30,000 cells from control (n=7), IPF (n=7), chronic hypersensitivity pneumonitis (cHP, n=4), and nonspecific interstitial pneumonia (NSIP, n=3) identified 21 distinct clusters, representing the major known subtypes in the lung, as well as numerous intermediate/transitional cell types and/or states. Strikingly, across pulmonary fibrosis phenotypes, collagen and ECM gene expression was highly enriched in ACTA2low, PDGFRa+ fibroblasts, while collagen and ECM gene expression were lower in ACTA2hi myofibroblasts; few collagen‐expressing inflammatory or epithelial cells were identified. A series of cell types co‐expressing AT1 and AT2 markers were identified in both fibrotic and control lungs. Compared to NSIP and cHP, IPF epithelial cells demonstrated increased senescence markers.ConclusionsAcross different histopathologic forms of pulmonary fibrosis, both shared and divergent pathologic gene expression programs can be identified. Further study is needed to more comprehensively define the common gene expression programs that drive progressive pulmonary fibrosis.Support or Funding InformationThis work was supported by NIH/NHLBI, the Doris Duke Charitable Foundation, and Boehringer Ingleheim.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Delta‐like 1 Homolog (DLK1) Identifies a Specific Subset of Orexin Neurons and Contributes to Anxiety Behavior
Authors: Harris Tatiyana , Bugescu Raluca , Kelly Jaylyn R. , Leinninger Gina 
Published: June 2021

Lateral hypothalamic area (LHA) neurons expressing the neuropeptide orexin (OX) are implicated in diverse physiology including energy homeostasis, arousal, reward processing and anxiety. It has long been speculated that there may be separate populations of OX neurons to control distinct functions. Indeed, slice electrophysiology studies identified two functionally distinct subtypes of OX neurons, but there has been no molecular “marker” to differentiate them as needed to parse their respective functions. We examined whether the transmembrane protein Delta‐like 1 homolog (DLK1) distinguishes subsets of OX neurons, since it is expressed in the hypothalamus and has been implicated in at least two physiologic processes regulated by OX neurons: energy balance and anxio‐depression. Using immunofluorescence, we found that DLK1 is selectively expressed in ~50% of OX neuronsin mice: we refer to these to as OXDLK1 neurons to differentiate them from the OX neurons that lack DLK1 (OXOnly neurons). We also observed OXDLK1 and OXOnly subsets in human hypothalami, indicating that both populations are relevant to understanding human physiology. Intriguingly, DLK1 protein is present in the cleaved biologically active form in the adult mouse LHA. Moreover, DLK1 expression in the LHA is regulated by circadian timing, but not by alterations in energy status (e.g. fasting, leptin), suggesting that DLK1 protein might contribute to specific physiologic processes. While DLK1 is vital for normal development, its role in the established nervous system, and in OX neurons, is unclear. We therefore used an “adult‐onset deletion” method to explore the contribution of DLK1 to behavior. Briefly, adult DLK1flox/flox mice were injected in the LHA with either AAV‐GFP (Control mice) or AAV‐Cre‐GFP to induce Cre‐mediated deletion of DLK1 selectively from the OX‐DLK1 neurons (DLK1‐Null mice). Intriguingly, the DLK1‐Null mice exhibited increased locomotor activity, but decreased anxiety compared to Control mice via the forced swim and elevated plus maze tests. Collectively, our data identify DLK1 as a protein marker that can be used to distinguish OX subpopulations and suggest a role for DLK1 protein in regulating anxio‐depressive behavior. Going forward, understanding the function of DLK1, and the OXDLK1 neurons may provide insight into how to modulate specific aspects of OX neuron‐regulated physiology.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cardiorespiratory and Cerebrovascular Responses to Leg Cycling Exercise during Muscle Metaboreflex Activation with Blood Flow Restriction in Moderate Normobaric Hypoxia
Authors: Alsalahi Sultan , Huynh Richard , Oldham Cleo , Pitman Ben , Thomas Callum , Lucas Rebekah , Fisher James P 
Published: June 2021

Skeletal muscle blood flow restriction and normobaric hypoxia are strategies adopted during exercise training because the associated reductions in local or systemic oxygenation may enhance athletic performance. Both manoeuvres evoke cardiorespiratory and cerebrovascular responses but have not been studied in combination. Blood flow restriction during exercise activates metabolically sensitive group III and IV skeletal muscle afferents (i.e., muscle metaboreflex activation), while systemic hypoxia activates the peripheral chemoreflex. We sought to determine whether the cardiorespiratory and cerebrovascular response to muscle metaboreflex activation with blood flow restriction during leg cycling exercise is augmented in moderate normobaric hypoxia.Nine healthy men (age 23±2 years; mean±SE) participated in two experimental trials performed under normoxic (fraction of inspired O2 [FiO2] = 21 %) and hypoxic (FiO2 = 13.5 %; equivalent of altitude of 3500 m) conditions whilst seated in a semi‐recumbent position in an environmental chamber. Trials consisted of a resting baseline period (3 min), followed by low intensity leg cycling exercise (64±7 Watts) under free‐flow conditions (~3½ min), and then bilateral thigh cuff inflation to 100 mmHg to induce blood flow restriction and muscle metaboreflex activation (~3 ½ min). Peripheral capillary oxygen saturation (SpO2; finger pulse oximetry), mean arterial pressure (MAP; Finometer and SunTech), heart rate (HR; electrocardiogram), the partial pressure of end‐tidal carbon dioxide (PETCO2; capnography), minute ventilation (VE; pneumotachometer) and middle cerebral artery mean blood flow velocity (MCAV; transcranial Doppler) were recorded.At baseline, SpO2 was lower during hypoxia (87±1 %) than normoxia (97±1 %; P<0.05), but MAP, HR, PETCO2, VE and MCAv were not different between trials (P>0.05). Leg cycling under free‐flow conditions significantly increased MAP, HR, PETCO2, VE and MCAv (P<0.05 vs. baseline), with no differences observed between hypoxic and normoxic trials (P>0.05). MAP (Δ14±2 vs. Δ16±2 mmHg; P=0.215), HR (Δ10±2 vs. Δ8±3 b·min−1; P=0.251), PETCO2 (Δ−1.1±0.3 vs. Δ−3.4±1.8 mmHg; P=0.596), VE (Δ4±1 vs. Δ4±1 L·min−1; P=0.446) and MCAv (Δ4±2 vs. Δ4±1 cm·s−1; P=0.408) responses to blood flow restriction during leg cycling (i.e., muscle metaboreflex activation) were not different in the hypoxic and normoxic trials.These preliminary findings suggest that moderate normobaric hypoxia (FiO2 = 13.5 %) does not augment the cardiorespiratory and cerebrovascular responses to muscle metaboreflex activation with blood flow restriction during low intensity leg cycling.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Electrical stimulation of the cervical Sympathetic Chains decreases upper airway resistance in conscious Zucker‐Fat Rats
Authors: Sun Melissa H , Zenebe Woineshet J , Lewis Stephen J , Hsieh Yee‐Hsee 
Published: June 2021

BACKGROUNDThe cervical sympathetic chains (CSC) provide sympathetic drive to carotid sinus baroreceptor afferents and chemoreceptive glomus cells within the carotid bodies. Zucker‐fat rats are a model of sleep apnea as they present with both obstructive and central apneas. We have previously demonstrated that CSC stimulation improves airway patency in anesthetized Zucker‐fat rats.AIMWe sought to investigate (1) whether electrical stimulation of the cervical sympathetic chain in conscious Zucker‐fat rats will lead to decrease in specific airway resistance, and (2) whether intermittent or constant stimulation was more effective.METHODSIn Zuckerfat rats (n=6), both CSC were implanted with cuff electrodes, which were trocared to a headcap cemented on the skull. Two weeks after recovery, the rats were placed in double chambered plethysmography (DCP) chamber where specific airway resistance was measured. Two protocols (intermittent (30s on/30s off for 15 min) or constant (3 min every 10 min) were performed using the stimulation parameters of 0.8 mA, 7.5 Hz, and 0.2 ms to assess efficacy on airway resistance.RESULTSWe present two novel findings. First, bilateral CSC stimulation reduced upper airway resistance by 12 ± 5%. Second, intermittent stimulation progressively decreased upper airway resistance by 30 ± 8% whereas sustained stimulation progressively increased upper airway resistance by 34 ± 11%.CONCLUSIONFor the first time, we have demonstrated that CSC stimulation reduces upper airway resistance in conscious Zucker‐fat rats and the pattern of stimulation is critical to its efficacy.Support or Funding InformationSupported by NIH1OT2OD023860‐01, Galvani BioelectronicsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Symptom Severity Impacts Inflammation and Sympathetic Over‐activity in Post‐Traumatic Stress Disorder (PTSD)
Authors: Fonkoue Ida T , Jones Toure N , DaCosta Dana , Kankam Melanie , Park Jeanie 
Published: June 2021

Patients with post‐traumatic stress disorder (PTSD) are at significantly higher risk of developing hypertension and cardiovascular disease. Low‐grade inflammation and overactive sympathetic nervous system (SNS) activity are suggested as contributing mechanisms. Our prior studies showed that while SNS reactivity during stress is heightened in PTSD, resting MSNA and inflammation was not elevated in young combat Veterans with PTSD as a group; however, prior results did not take into account the potential influence of PTSD symptom severity on neural cardiovascular function. The aim of the current study was to determine if PTSD patients with greater PTSD symptoms measured via the gold‐standard clinician administered PTSD scale (CAPS) have higher SNS activity and inflammation compared to PTSD patients with less severe symptoms. To answer this question, we recruited 21 veterans clinically diagnosed with PTSD (mean CAPS score, 68±4). We measured muscle sympathetic nerve activity (MSNA) via microneurography and blood pressure using standard technique. We collected blood samples to measure serum levels of two inflammatory biomarkers: highly sensitive C‐reactive protein (hsCRP) and interleukin‐6 (IL‐6). For data analysis, the participants were grouped based on the median CAPS score of 70, with the low CAPS group (L‐CAPS) having a score ≤ 70 and the high CAPS group (H‐CAPS) having a score > 70. The two groups (L‐CAPS, n=11; H‐CAPS, n=10) were matched for age, body mass index (BMI) and selective serotonin reuptake inhibitor (SSRI) prescription. Resting blood pressure and heart rate were similar between the two groups. However, the H‐CAPS group had a significantly greater MSNA at baseline when quantified as both burst frequency (26±5 vs 13±2 bursts/min, p= 0.011) and burst incidence (35±6 vs 21±3 bursts/100hb, p= 0.022) compared to the L‐CAPS group. Similarly, hsCRP (2.02±0.71 vs 0.74±0.18, p= 0.036) and IL‐6 (5.44±1.86 vs 2.35±0.73, p= 0.055) levels were higher in the H‐CAPS group compared to the L‐CAPS group. Furthermore, the levels of hsCRP (r= 0.766, p< 0.001) and IL‐6 (r= 0.594, p=0.007) were positively correlated with MSNA. When the groups were analyzed separately, the correlation between MSNA and hsCRP was stronger in the H‐CAPS group (r=0.822, p=0.012) than the L‐CAPS group (r=0.604, p=0.065). These results suggest that the severity of PTSD is associated with chronically increased MSNA and inflammation, and SNS overactivity may be mechanistically linked to inflammation in severe PTSD.Support or Funding InformationSupported by NIH HL‐098744, NIH DK‐00756, VA Merit I01CX001065, AHA 15CSA24340001This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of Bacterial Endotoxin (LPS) on Cardiac and Neural Function in Various Animal Models
Authors: Cooper Robin Lewis , McNabb Micaiah , Saelinger Christa , Greenhalgh Abigail , Istas Oscar , Stanback Maddie , Stanback Stanback , Anyagaligbo Ogechi , Bernard Jate , Danley Melody L , Bierbower Sonya M , Ghoweri Adam , Thibault Olivier 
Published: June 2021

The endotoxic effect from gram negative bacteria is primarily due to the lipopolysaccharides (LPS). LPS activates the innate immune response through a Toll‐like receptor 4 (TLR4) known as the CD14/TLR4/MD2 receptor complex in mammals. The Toll receptors are conserved from primates to insects. However, in insects the peptidoglycan recognition proteins (PGRPs) are the receptors which respond to LPS from gram negative bacteria. These PGRPs activate the Immune Deficient (IMD) signaling cascade. There is a family of these receptors known in Drosophila melanogaster but their expression profiles in different tissues has yet to be fully elucidated. We examined a variety of model preparations to better understand the acute actions of exposure to LPS of Serratia marcescens to better understand the varied mechanisms of action. There is a differential, dose dependent effect of LPS in increasing and decreasing HR heart rate (HR) in the larval medicinal blow fly (Phaenicia sericata) and a fruit fly (Drosophila melanogaster). LPS depressed evoked and miniature (quantal) EJPs while hyperpolarizing the skeletal muscle in larval Drosophila, but increased EJPs with no effect on muscle membrane potential at the crayfish NMJ. Both NMJs are glutamatergic. LPS had no effect on sensory transduction of proprioceptive sensory neurons in crayfish and blue crab. LPS at the cholinergic frog NMJ depressed synaptic transmission with no effect on muscle membrane potential. LPS depressed sensory‐CNS‐motor nerve circuits in both crayfish and larval Drosophila. Synaptically‐evoked population spikes in field CA1 of the mouse hippocampus were also significantly reduced by acute LPS applications. The varied effects of LPS in different model systems paves the way to examining differential cellular mechanisms induced by acute exposure to LPS.Support or Funding InformationDept of Biology, Univ. of KY laboratory funds and personal funds (RLC)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Central Command Increases Muscle Sympathetic Nerve Activity More to Contracting than Non‐Contracting Muscle during Unloaded Rhythmic Exercise in Humans
Authors: Taylor Chloe E , Boulton Daniel , Howden Erin , Siebenmann Christoph , Macefield Vaughan G 
Published: June 2021

We have previously shown that the increase in muscle sympathetic nerve activity (MSNA) to contracting muscle during sustained isometric exercise is due primarily to central command, and that the contracting muscle does not exhibit a metaboreflex. Here, we tested the hypothesis that MSNA increases to the contracting muscle also during rhythmic isotonic exercise, in which muscle metabolites will not accumulate because the contraction is performed without external load. MSNA was recorded from the common peroneal nerve in 10 participants and negative‐going sympathetic spikes extracted during 50 cycles of sinusoidal (0.15 Hz) isotonic dorsiflexions of the ipsilateral or contralateral ankle. Electromyographic activity (EMG) was recorded from the tibialis anterior muscle on both sides. Cross‐correlation analysis between the MSNA and EMG revealed a marked cyclic modulation of MSNA to the contracting (ipsilateral) muscle. This modulation, in which MSNA increased during the contraction phase, was three times greater than that to the non‐contracting muscle (modulation index = 27.4 ± 3.2 vs 9.2 ± 1.5 %; P<0.001). There were no differences in either the intensity or magnitude of modulation of EMG during ipsilateral and contralateral contractions. We conclude that, in addition to static isometric exercise, central command increases MSNA to the contracting muscle during rhythmic isotonic exercise.Support or Funding InformationThis work was supported by the National Health & Medical Research Council (Grants 1029782 and 1100040).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tandem Requirement for Renal D1R and D5R Functionality
Authors: Rozyyev Selim , Crusan Annabelle P , Tiu Andrew C , Jurgens Julie A , Quion Justin Michael B , Asico Laureano D , Felder Robin A , Jose Pedro A , Villar Van Anthony M 
Published: June 2021

The peripheral dopaminergic system assists in maintaining blood pressure homeostasis by engendering natriuresis mainly through the D1R and D5R receptors. These receptors share structural features and pharmacological profiles; there is no specific agonist that can discriminate between these two receptors. Genetic ablation of one receptor in the kidney results in the loss of activity of the other. Therefore, we evaluated the mechanisms for the cooperative and co‐dependent interaction between these two receptors. Both receptors share the same anatomical distribution, specifically at the brush border and luminal membranes of renal proximal tubules and collecting ducts, respectively. These receptors dimerized (~110 kDa) and coimmunoprecipitated each other, basally and after fenoldopam (D1R/D5R agonist, 1 μM/15 min) treatment. Bimolecular fluorescence complementation showed that heterologously expressed D1R and D5R tagged with the C and N termini of the fluorescent protein EYFP, respectively, heterodimerized basally in HEK‐293 cells. This was corroborated by confocal microscopy of HEK‐293 cells that were transiently transfected with D1R::RFP and D5R::GFP, which showed colocalization basally at the plasma membrane, most abundantly at the apical membrane in polarized cells. The D1R and D5R partitioned to lipid and non‐lipid rafts; agonist treatment enhanced the lipid raft segregation of D1R and D5R. Both receptors were abundantly expressed in renal proximal tubules of human and C57Bl/6 mouse kidneys, where they colocalized basally that was increased by fenoldopam (1 μM/15 min). D1R/D5R stimulation is known to increase cAMP production and inhibit renal sodium transport. Receptor gene silencing via antisense technology in human renal proximal tubule cells (hRPTCs) decreased the cAMP response to fenoldopam (−71±2% for D1R, vs. −38±7% for D5R, vs. −90±2% for both, *P<0.05). In hRPTCs grown in Transwells, fenoldopam or ouabain (500 nM/30 min) given at the basolateral compartment inhibited Na+/K+‐ATPase (NKA) activity and increased intracellular Na+. siRNA‐mediated gene silencing of DRD1/DRD5 prevented the inhibition of NKA activity by fenoldopam (*P<0.05). Our observations demonstrate the requirement for D1R/D5R to form heterodimers to achieve their full functionality.Support or Funding InformationR01DK039308, R01HL092196, and P01HL074940This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.