bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ex Vivo Elastase Treatment Disrupts Parenchymal Structure And Enhances Airway Narrowing in Precision Cut Lung Slices
Authors: van Dijk Eline Margaretha , Culha Sule , Bidan Cecile , Menzen Mark , Gosens Reinoud 
Published: November 2023

BackgroundChronic obstructive pulmonary disease (COPD) is a chronic lung disease caused primarily by smoking. COPD patients suffer from a loss of lung function due to airway inflammation, airway remodelling and in some patients emphysema development. Alterations in the parenchymal structure, due to the presence of elastolytic enzymes and oxidative stress may affect airway mechanics in COPD. Here, we hypothesize that ex vivo treatment of lung slices with elastase or H2O2 enhances bronchoconstriction caused by a disrupted parenchymal structure.AimTo investigate the relationship between the parenchymal structure and airway narrowing in an ex vivo precision cut lung slice (PCLS) model after treatment with elastase or H2O2.MethodsLungs of C57Bl/6J mice (n=8) were sliced (250 μm) and treated ex vivo with elastase (2.5 μg/ml) or H2O2 (200 μM) for 16 hours. Following treatment, slices were washed twice with medium and incubated for another 24 hours after which the slices were collected. To determine airway narrowing, methacholine‐induced contraction studies were performed. Parenchymal structure was assessed by confocal microscopy after which mean linear intercept (Lmi) was determined. Gene expression levels of markers of alveolar type I and II cell repair, known to be decreased in COPD, were assessed using RT‐PCR.ResultsFollowing elastase, but not H2O2 treatment, slices showed a significant increase in Lmi (basal 75.91 μm vs elastase 90.18 μm (p=0.04) and H2O2 84.95 μm (p=0.76)), reflective of an altered parenchymal structure. In addition, elastase enhanced methacholine‐induced airway narrowing as shown by a significant decrease in pEC50 (−5.78 vs −6.68 log M, p=0.01). H2O2 did not change pEC50 (−5.78 vs −5.99 log M, p=0.64), but showed a trend for increasing Emax (44.12 vs 67.35 % contraction, p=0.06). Elastase did not alter Emax significantly (44.12 vs 61.11 % contraction, p=0.12) Expression levels of alveolar type I and type II markers Aqp5 and RAGE were decreased (p<0.05) following elastase treatment, whereas H2O2 –treatment decreased the expression of RAGE, Con43, T1α, PAI1 and SFTPC mRNA (p<0.05).ConclusionTreatment of PCLS with elastase, but not with H2O2 enhances airway narrowing in response to methacholine. This is associated with decreased gene expression levels of several markers of alveolar repair and a larger Lmi. These findings support our hypothesis that the link between increased bronchoconstriction and a disrupted parenchymal structure can be mimicked using PCLS ex vivo. PCLS may be a promising model to study therapies aimed at lung repair.Support or Funding InformationThis study was sponsored by a Vidi grant by NWO (016.116.309)
Regulation of Kv7.5 Potassium Channels by Protein Kinase C in Human Airway Smooth Muscle Cells
Authors: Haick Jennifer M , Brueggemann Lioubov I , Byron Kenneth L 
Published: November 2023

Kv7.5 potassium (K+) channels were recently found to be expressed in airway smooth muscle cells (ASMCs). Bronchoconstrictor agonists inhibit the outward flux of K+ through these channels, an effect which has been implicated as a mediator of their bronchoconstrictor actions in human airways. To test the hypothesis that Kv7.5 channels in ASMCs are regulated by bronchoconstrictor agonists via protein kinase C‐ (PKC‐) mediated phosphorylation, FLAG‐tagged human Kv7.5 channels were overexpressed in human trachealis smooth muscle cells (hASMCs). Function of the channels was assessed by measuring outward Kv7.5 currents using whole cell patch clamp electrophysiology. Indirect activation of PKC by the bronchoconstrictor agonist histamine (1 μM) reduced Kv7.5 currents by 34.6 ± 7% at −20 mV (n=6, p<0.05). This effect was abolished upon blockade of the histamine H1 receptor with chlorpheniramine (30 μM). The PKC activator phorbol 12‐myristate 13‐acetate (PMA, 1 nM), but not its inactive analog 4α‐PMA (1 nM), also reduced Kv7.5 currents (75.5 ± 7.6% at −20 mV, n=4, p<0.05). Phosphorylation of the channel protein was assessed by immunoprecipitating the channels (using anti‐FLAG antibodies) and Western blotting with anti‐phosphoserine PKC substrate antibodies. Both histamine and PMA significantly increased Kv7.5 channel phosphorylation and this effect was reduced by the PKC inhibitor Ro‐31‐8220 (1–10 μM). Two putative PKC‐phosphorylation sites on Kv7.5, serine 441 (S441) and threonine 505 (T505), were mutated so that they can no longer be phosphorylated. Neither Kv7.5‐T505A or Kv7.5‐S441A currents were inhibited by histamine (<8% inhibition at −20 mV, n=5, p>0.05, and <14% inhibition at −20 mV, n=7, p>0.05, respectively), however both mutant channels remained sensitive to PMA (58.3 ± 16.8% at −20mV, n=5, p<0.05, and 63 ± 7.5% at −20 mV, n=6, p<0.05, respectively). Despite the insensitivity of Kv7.5‐S441A currents to histamine treatment, Western blot analyses indicated that histamine still induced serine phosphorylation of Kv7.5‐S441A channels. Determining the mechanism by which Kv7.5 channels are regulated in hASMCs will aid in the understanding of how these channels contribute to the regulation of airway diameter and how Kv7 channels in ASMCs may be effectively targeted for treatment of diseases associated with airway obstruction, such as asthma.Support or Funding InformationLoyola University Chicago
Effects of PACAP administration in the extended amygdala on acoustic startle reflex
Authors: Seiglie Mariel , Cottone Pietro , Sabino Valentina 
Published: November 2023

Anxiety‐related disorders are the most common forms of mental disorders; characterized by feelings of excessive worry in the absence of specific external stimuli, they are accompanied by physical, affective and behavioral symptoms.Pituitary adenylate cyclase‐activating polypeptide (PACAP) is a 38‐amino acid peptide expressed not only in the hypothalamus but also in various extra‐hypothalamic regions including the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST). PACAP and its receptor PAC1 (PAC1R) have been proposed to play a key role in mediating the behavioral and endocrine responses to stress; however, few studies have examined the involvement of the extrahypothalamic PACAP system in the modulation of stress. Our aim was to elucidate the role of the PACAP/PAC1R system of the CeA and BNST in the context of anxiety‐like behaviors.Acoustic startle response (ASR) is a behavioral test sensitive to states of anxiety and fear. The effects of PACAP bilateral microinfusion into either the CeA or the BNST of male rats on ASR were assessed in this study. In addition, to test the role of the endogenous system, the effects of intra‐CeA and intra‐BNST PACAP antagonist PACAP(6–38) on ASR were assessed, both on basal and on footshock‐induced sensitization of ASR.Infusion of PACAP into both the CeA and BNST increased ASR. Importantly, doses of the antagonist PACAP(6–38) which per se did not affect ASR, were able to prevent the sensitization of ASR induced by footshock, when infused into either the CeA or BNST.These data prove an anxiogenic role for the PACAP/PAC1 system of the extended amygdala and suggest that hyperactivity of this system may underlie the anxiogenic effects of stress.Support or Funding InformationNIH grants # MH093650, MH091945, DA030425; T32 grant GM008541
TDP‐43 interacts with cannabinoid type 1 receptor modulating its subcellular localization and function
Authors: Filipeanu Catalin , Deshotels Matthew , Weed Peter , Ogunlade Blessing , Robinson Anastasia G , Winsauer Peter , Guidry Jessie J. 
Published: November 2023

To identify new molecular chaperones of cannabinoid type 1 receptor (CB1R) we used a proteomic approach in HEK293T cells, transfected with two spicing variants of this receptor, CB1R and CB1AR. These two isoforms differ only in the composition of the N‐terminus domain. Surprisingly, the only protein found to interact differently with CB1R and CB1AR was transactive response DNA‐Binding Protein 43 (TDP‐43), which is known to be involved in the pathology of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The specific interactions between CB1R and TDP‐43 were also confirmed by co‐immunoprecipitation not only in transfected HEK293 cells, but also at the endogenous level in rat crude spinal cord. TDP‐43 overexpression decreased CB1R plasma membrane levels by stimulating receptor internalization, but it did not change the total receptor levels. Further, TDP‐43 enhanced CB1R ubiquitination to similar levels, as induced by the full CB1R agonist, CP55940. Also, overexpression of TDP‐43 resulted in reduced effects of CB1R effects on cAMP, P‐ERK1/2, and P‐CREB responses. Present results revealed TDP‐43 as an unexpected molecular chaperone of CB1R and these findings may have significance not only in the regulation of CB1R intracellular trafficking, but also in the treatment of ALS and FTLD.Support or Funding InformationThis work was supported by Startup Funds from Howard University (CF) and USPHS grant DA019625 (PW) from the National Institute on Drug Abuse
MAPK Signaling Plays an Important Role in Differentiating Paraquat‐ and Diquat‐Induced Hepatocellular Injury
Authors: Tong Chris , Fry Maddie , Stamper Brendan D 
Published: November 2023

ObjectiveThe objective of this study was to utilize the structure‐toxicity relationship between two quaternary ammonium‐containing herbicides, paraquat and diquat, in order to pinpoint key mechanisms propagating diquat‐induced liver injury.MethodsBoth dose‐ and time‐dependent toxicity screens were performed in an in vitro liver model (TGF‐alpha transgenic mouse hepatocyte cell line; TAMH) to determine toxicologically relevant concentrations of paraquat and diquat. TAMH cells were then exposed to the respective median lethal dose (LD50) for each herbicide, harvested and processed for RNA isolation. Microarrays were then performed using Affymetrix Mouse Gene 2.1 ST array strips and analyzed using DAVID (https://david.ncifcrf.gov). Results from the whole genome expression studies were compared with the MicroArray Quality Control (MAQC)‐II dataset (GSE24363) to validate robust markers for diquat‐induced liver injury.ResultsLD50 values for paraquat and diquat were calculated to be 126 μM and 18 μM, respectively. Time‐based toxicity screening indicated that significant differences in toxicity between paraquat and diquat manifested after 9 hours of exposure at LD50 concentrations. Whole genome expression analysis at this time point identified mitogen activated protein kinase (MAPK) signaling to be significantly upregulated following diquat exposure when compared to either paraquat exposure (p < 1E‐03) or control (p < 1E‐06). Mining of the MAQC‐II dataset identified 42 upregulated transcripts specifically associated with diquat toxicity, of which 19 were shared in our TAMH experiments. All 19 of these shared transcripts were associated with MAPK signaling pathways.ConclusionParaquat and diquat are structurally‐related herbicides currently in use that have been shown to be toxic in multiple human organ systems. Studies reporting direct comparisons and potential genotoxicity between these herbicides are lacking. The work presented herein found diquat to be seven times more hepatotoxic than paraquat. Based on the gene expression array studies, the differential toxicity between the two herbicides appears to be at least in part MAPK‐mediated. The identification of MAPK signaling as an important contributor to diquat‐induced hepatocellular injury was validated through MAQC‐II dataset mining. Future work is currently underway to investigate how phosphoprotein targets within MAPK signaling cascades are altered between paraquat and diquat, and to subsequently integrate results from the protein‐based experiments with gene expression data.Support or Funding InformationThis work was supported by the Pharmacy Research Incentive Grant Program through the Pacific University School of Pharmacy.
Changes in Hydrogen Sulfide Concentrations in Porcine Ocular Anterior Segment Explants Under Elevated Perfusion Pressures: Role Of Prostaglandins
Authors: Robinson Jenaye , Mitchell Leah , Nguyen Olivia , Opere Catherine , Ohia Sunny , Njie‐Mbye Ya Fatou 
Published: November 2023

We have evidence that H2S donors can reduce intraocular pressure in rabbits presumably by increasing aqueous humor (AH) outflow. The aim of the present study was to assess the contribution of H2S present in tissues of the trabecular meshwork in the observed increase in AH outflow facility in an ex vivo porcine anterior segment model.MethodsPorcine ocular anterior segment explants were perfused with Dulbecco's Modified Eagle's Medium maintained at 37° C and gassed with 5% CO2 and 95% air, under an elevated pressure of 15 mm Hg for four hours. Perfusates from the anterior segment explants were collected and immediately assayed for their H2S content using the well‐established Methylene Blue assay. In some experiments, explants were perfused with cyclooxygenase (COX) inhibitors [flurbiprofen (30 μM) and indomethacin (10 μM)]. The concentration of H2S was also measured in the AH (as positive control), and at normal perfusion pressure of 7.35 mmHg in the absence of COX inhibitors.ResultsIncreasing perfusion pressure from 7.35 to 15 mm Hg significantly (p < 0.001) increased H2S concentrations from 0.4 ± 0.1 to 67.6 ± 3.6 nM/μg protein. As a reference value, the H2S concentration in the AH was 1.5 ± 0.2 nM/μg protein. In the presence of the COX inhibitors, flurbiprofen (30 μM) and indomethacin (10 μM), the effects of elevated pressure on H2S levels were significantly (p < 0.001) reduced from 67.6 ± 3.6 to 0.8 ± 0.1 and 4.2 ± 0.68 nM/μg protein, respectively.ConclusionWe conclude that the elevated perfusion pressure‐induced increase in H2S concentrations in the perfusate is dependent upon the endogenous production of prostaglandins. However, the exact mechanism whereby elevated perfusion pressures leads to changes in basal H2S is unknown and merits further investigation.
The Therapeutic Potential of mGlu3 Antagonism in Correcting Hippocampal Long‐term Depression Impairments in a Mouse Model of MECP2 Duplication Syndrome
Authors: Stansley Branden , Gogliotti Rocco , Senter Rebecca , Fisher Nicole , Emmitte Kyle , Lindsley Craig , Conn P. Jeffrey , Niswender Colleen 
Published: November 2023

Precise levels of the transcriptional regulator Methyl CpG Binding Protein 2 (MeCP2) are known to be a key determinant for proper neurological function. Mutations in the MECP2 gene results in a disease known as Rett syndrome; in contrast, genetic duplication of MECP2 leads to a disorder known as MECP2 Duplication syndrome. The latter genetic neurodevelopmental disorder has a wide symptomology including stereotypy, seizures, cognitive deficits, and autism. In rodent models of MECP2 Duplication Syndrome in which MeCP2 is overexpressed, increases in glutamatergic synapse formation in hippocampal neurons occur, as well as impairments in hippocampal synaptic plasticity, a neurological phenomenon thought to be involved in memory and cognition. In accordance with this notion, MeCP2 overexpression causes memory and cognitive deficits in mice. Interestingly, the metabotropic glutamate receptor 3 (mGlu3) has been shown to be involved in regulating synaptic plasticity and cognitive behaviors in mice, and mGlu3 expression appears to correlate with MeCP2 expression levels in the brain. We hypothesized that that an upregulation of mGlu3 leads to an impairment in synaptic plasticity and cognitive deficits in MeCP2 overexpressing mice. To investigate the mechanisms by which MeCP2 overexpression induces neurophysiological impairments, a MeCP2‐Tg1 mouse model was used. Our results indicate that mGlu3 is upregulated the hippocampus of MeCP2‐Tg1 mice, correlating with an increase in MeCP2 protein expression. Electrophysiological experiments found that paired‐pulse facilitation was enhanced and input‐output intensity was reduced at the SC‐CA1 hippocampal synapse in the MeCP2‐Tg1 mice compared to wild‐type controls. Furthermore, (S)‐3,5‐Dihydroxyphenylglycine (DHPG) induced‐long term depression (LTD) was impaired in MeCP2‐Tg1 mice. Interestingly, an mGlu3 negative allosteric modulator, VU0650786, was capable of normalizing DHPG‐induced LTD to the level of that observed in wild‐type controls. These results suggest that overexpression of mGlu3 may contribute to synaptic plasticity impairments within the hippocampus of MeCP2‐Tg1 mice. Future experiments are aimed at elucidating behavioral correlates of these neurophysiological abnormalities and their underlying mechanisms.Support or Funding InformationT32NS007491 (BJS), rettsyndrome.org postdoctoral fellowship (RGG), Weatherstone predoctoral fellowship (BKS), T32 GM07628 (NF), R01 MH099269 (KAE), U54 MH084659 (CWL), R01 NS031373 (PJC), R21 MH102548 (CMN), Autism Speaks Treatment Award (CMN).
Kir4.1 (KCNJ10) Inhibition with a Newly Developed Antagonist Induces Diuresis in Rats
Authors: Kharade Sujay V. , Kurata Haruto , Hopkins Corey R. , Denton Jerod S. 
Published: November 2023

Inward rectifier potassium (Kir) channels play fundamental roles in diverse physiological processes, and in some cases, represent novel drug targets for human diseases. Outside of the nervous system, Kir4.1 (encoded by KCNJ10) is prominently expressed in the basolateral membrane of the thick ascending limb of Henle, distal convoluted tubule, and collecting duct of nephron. Kir4.1 can form homotetrameric channels or heterotetrameric channels with Kir5.1, although the most common channel subtype in the kidney is the heteromeric Kir4.1/5.1 form. Genetic ablation of Kir4.1 in mice or humans causes severe salt and water wasting resembling clinical phenotypes associated with diuretic use, suggesting that Kir4.1 might represent a novel diuretic target. Therefore, the aim of this study was to develop a selective small‐molecule inhibitor of Kir4.1 with which to evaluate its therapeutic potential. From a high‐throughput screen of ~ 75,000 compounds from the Vanderbilt Institute of Chemical Biology library, we identified an inhibitor, termed VU992, which inhibits homotetrameric Kir4.1 with an IC50 of 760 nM and heterotetrameric Kir4.1/5.1 with an IC50 of approximately 25 μM (33‐fold selectivity). In addition, VU992 is at least 65‐fold selective for Kir4.1 over other renal Kir channels Kir1.1 and Kir7.1. SAR exploration revealed VU992 bears fundamental parts to inhibit efflux through Kir4.1 channel. Finally, oral administration of VU992 dose‐dependently increases urine output in volume‐loaded, freely behaving rats. This study shows proof‐of‐concept that homotetrameric Kir4.1 might represent a molecular target for a novel class of diuretic for the treatment of hypertension. In addition, its basolateral localization might offer advantages over other diuretic targets that are located on the luminal membrane of the nephron.Support or Funding InformationNIH 5R21 NS073097‐01S1
Mitochondrial Topoisomerase Dysfunction promotes Mitochondrial DNA (mtDNA) damage leading to Myocardial Mitochondrial Dysfunction
Authors: Edwards John , Mitry Maria , Soler Amanda , Laurent Dimtri 
Published: November 2023

Topoisomerases are highly conserved proteins that resolve the topological difficulties of DNA replication. In post mitotic cells, the mitochondrial genome continues to replicate about once a month and resolving the topology of mtDNA replication would be almost insurmountable in the absence of the three unique mitochondrial topoisomerases. We have previously reported that diabetes or alcoholism induces mitochondrial topoisomerase dysfunction to promote mtDNA degradation as an antecedent event to cell death. It remains unclear how chronic hyperglycemia causes this shift in the mitochondrial topoisomerase function from the physiologic to the pathological. Work by others has suggested that nuclear topoisomerases are sensitive to the oxidant stress molecules. To that end, we hypothesize that low levels of ROS alter mitochondrial topoisomerase function to accelerate DNA strand breakage. DNA relaxation of supercoiled DNA is a hallmark measurement of topoisomerase function. Supercoiled DNA incubated with isolated mitochondria generated the same isopane pattern of relaxation as compared to purified topoisomerase1 (Top1). Isolated mitochondria from cell lines in which Top1 or Topo‐2beta were knockout retained DNA relaxation function suggesting the presence of the three unique functional mitochondrial topoisomerases. In the absence of mitochondrial extracts; 125–1000 μM H2O2 did not alter DNA relaxation compared to vehicle controls. Myocardial mitochondrial extracts alone increased DNA relaxation compared to controls and this was significantly elevated (p<.05) in the presence of increasing concentrations of H2O2. Similar findings were obtained using a DNA cleavage assay; alone 60 μM H2O2 did not cause DNA cleavage but exacerbated DNA cleavage in the presence of mitochondrial extracts. Using the iron chelators, EDTA or deferoxamine, we determined that this is increase was not due to a Fenton effect. Using intact isolated intact mitochondria; incubation with H2O2 significantly increased mitochondrial superoxide levels indicating that H2O2 ready enters the mitochondria and is able to elevate intramitochondrial concentrations of superoxide. Previously we have reported that chronic hyperglycemia transiently elevated intramitochondrial superoxide levels in the H9c2 cell line. Collectively these results indicate that ROS alters mitochondrial topoisomerase function to increase DNA strand breakage. Separate from a direct impact of oxidative stress, our studies find that alteration of mitochondrial topoisomerase reaction mechanism accelerates and propagates an increase in mtDNA damage. These findings indicate that the functional state of the mitochondrial topoisomerases have important consequences for the myocardium and participate in the pathophysiology leading to the development of cardiomyopathy.Support or Funding InformationSupported in part by NIH RO3HD065551