bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lipopolysaccharide Induces TLR4 Dependent Activation of VRAC (LRRC8) in Nodose Neurons and May thereby Impair Satiety Signal
Authors: Wang Runping , Zhang Yanhui , Cicha Michael Z , Sah Rajan , Chapleau Mark W , Abboud Francois M 
Published: November 2023

Recent reports indicate that lipopolysaccharide (LPS) reduces the response of vagal afferent nodose neurons to CCK and leptin and thereby reduces satiety signaling and contributes to hyperphagia (de La Serre et. al. 2015). Here we defined the specific ion channel involved in the response of nodose neurons to LPS. We suspected that a Chloride (Cl−) channel may be responsible based on our earlier finding that high fat diet reduces the CCK‐sensitive Ca2+‐dependent Cl− current in intestinal vagal afferent nodose neurons (FASEB J 2015. 29: 818.4). We used whole‐cell voltage‐clamp on dissociated nodose neurons from C57BL/6 mice, and found that application of 10μg/ml LPS induced a large, slowly developing inward Cl− current in 14 of 18 cells (17.2±3.0 pA/pF, n=14). The current was attenuated (4.0±1.1 pA/pF, n=14; p<0.01) when intracellular Cl− concentration was reduced from 133mM to 4mM. We concluded that this LPS‐induced Cl− channel is the ubiquitous Volume‐Regulated Anion Channel (VRAC) with Leucine Rich Repeats Containing protein 8a (LRRC8a or SWELL1) for two reasons: 1) the current was significantly reduced (6.6±2.0 pA/pF, n=6, p<0.01) by DCPIB (4‐(2‐butyl‐6,7‐dichloro‐2‐cyclopentylindan‐1‐on‐5‐yl)oxybutyric acid), a selective blocker of VRAC; and 2) the current was reduced from 18.1±2.9 pA/pF (n=7) to 4.3±1.3 pA/pF in neurons exposed to LRRC8a‐shRNA (n=5, p<0.01) compared to a scrambled shRNA control. Our data also suggests that the effect of LPS on the channel is not a direct one but is mediated by TLR4 (toll‐like receptor 4) since HEK293 cells, which express SWELL1/LRRC8a but not TLR4, rarely responded to LPS (1 out of 6 cells), and the response was not significant (7.8±4.7 pA/pF, n=6, p>0.05) compared to the positive control response to hypoosmolarity (30.9±7.9 pA/pF, n=5, p<0.01). We conclude that LPS activates LRRC8 in nodose neurons. Excessive TLR4 mediated activation of this channel by LPS may inhibit the CCK and leptin induced satiety signal, leading to hyperphagia in high fat diet and LPS induced obesity.Support or Funding InformationHL014388‐43
Possible role of myosin 2 and myosin 5C in the anterograde trafficking of KCa3.1 in a polarized epithelium
Authors: Farquhar Rachel , Rodrigues Ely , Hamilton Kirk L. 
Published: November 2023

Recently, we reported that KCa3.1 is trafficked to the basolateral membrane (BLM) of epithelial cells by a Rab1‐ and Rab8‐dependent mechanism (Bertuccio et al. PlosONE 9:e92013, 2014). Here, we extend our work on the role of microtubule/microfilament function in the trafficking of KCa3.1 to the BLM of Fischer rat thyroid (FRT) epithelial cells. We used our FRT epithelial cell line stably expressing KCa3.1‐BLAP/Bir‐A‐KDEL (cells grown on filters), immunoblot and Ussing chamber experiments in this study. Our immunoblot approach was to streptavidin label channels that arrive at the BLM to determine the effects of inhibitors of microtubule/microfilament function on the trafficking of the KCa3.1. We examined the effect colchicine, a microtubule inhibitor (10 μM for 0, 3 and 5 hr); ML‐9 an inhibitor of myosin light chain kinase (10 μM for 0, 3, and 5 hr) and 2,3‐butanedione monoxime (BDM) an inhibitor of muscle and non‐muscle myosin 2 and myosin 5 (10 mM for 0, 3, 5 hr) on trafficking of KCa3.1 to the BLM. As determined by immunoblot, colchicine significantly reduced trafficking of KCa3.1 by 70±8% (at 5 hr, P≤0.05, n=5); ML‐9 decreased trafficking of the channel by 70±3% (5 hr, n=5) and BDM reduced trafficking of the channel by 47±11% (5 hr, P≤0.05, n=5) compared to controls. We examined the effects of the inhibitors on the functional expression of KCa3.1 by Ussing chamber experiments. By using 1‐EBIO (100 μM, an activator of KCa3.1) to stimulate current via KCa3.1, we determined that colchicine (5 hr treatment) decreased 1‐EBIO‐stimulated current by 40±20% (P≤0.05, n=5); ML‐9 (5 hr) reduced stimulated current by 60±10% (P≤0.05, n=5) and BDM (5 hr) decreased stimulated current by 46±8% (P≤0.05, n=5) compared the controls. These data suggest that myosin 2 and myosin 5 may be critical for the trafficking of KCa3.1 to BLM of polarized epithelial cells.Support or Funding InformationThis work was supported by UORG and Dean's Fund grants from the Univ. of Otago (KLH).
Identification of ANO1 (TMEM16A) activator purified from Cucumis sativus: Its role for treatment of cystic fibrosis and gastrointestinal disorders
Authors: Kazi Mirajul Hoque , Saha Tultul , Woodward Owen M , Guggino William B 
Published: November 2023

BACKGROUNDCFTR functions as a chloride (Cl−) channel. Its dysfunction limits Cl− secretion leading to viscous mucus in the airway and intestinal lumen. Increases in cytosolic Ca2+ activate the epithelial Calcium activate Chloride Channel (CaCC), which provides an alternative Cl− secretory pathway in CF. In the present study, we purified a positive fraction from Cucumis sativus and isolated compounds that activated ANO1, member of the CaCC family.METHODSThe crushed fruit extracted was collected as aqueous solution by a fruit crusher followed by fractionation using High Performance Liquid Chromatography (HPLC). Short‐circuit current (Isc) and chloride (Cl−) conductance were measured in T84 cells by Ussing chamber. ANO1 current was measured in xenopus expression system.RESULTSAdministration of crude as well as purified compound to the apical side of epithelial cells activates Isc in intact as well as basolaterally permeabilized colonic T84 cells in a dose dependent manner. The stimulated Cl− secretion can be inhibited by DIDS, a calcium activated chloride channel blocker and BAPTA‐AM, an intracellular calcium channel blocker. The cAMP stimulated Cl− secretion was not altered by administration of this compound demonstrating it does not activate the CFTR mediated Cl− secretion, only the Ca2+ activated Cl− secretion. Using a Xenopus oocyte expression system, where the oocytes express the ANO1 (TMEM16A) CaCC channel endogenously, we confirmed that both the crude and purified fractions of the C. sativus extract activate the ANO1 channel. The molecular size and fragmentation pattern of three compounds were identified by mass spectrometry. To discern the mechanism of the ANO1 activation we measured intracellular Ca2+ levels in human bronchial epithelial cells (IB3‐1) derived from a cystic fibrosis patient, and found the active fraction of the C. sativus extract increases intracellular Ca2+, thereby activating ANO1 channels and Cl− secretion. Together, our data provides evidence that purified extract from C. sativus has potential therapeutic value for treatment of cystic fibrosis and gastrointestinal disorders like constipation by activating ANO1 mediated Cl− secretion in epithelial cells.Support or Funding InformationIndian Council of Medical Research and Dept. of Biotechnology, Govt. of India
Sarcolipin Ablation Impairs Muscle Regeneration After Acute Injury
Authors: Bellissimo Catherine Anne , Bombardier Eric , Chambers Paige , Fajardo Val Andrew , Tupling Russ 
Published: November 2023

Sarcolipin (SLN) is a small transmembrane protein that regulates the sarco(endo)plasmic reticulum calcium ATPase (SERCA) pump in muscle. SLN is highly expressed in embryonic muscle and is involved in the regulation of myogenesis. SLN is also upregulated in mouse dystrophic muscles but the role of SLN in muscular dystrophy is unknown. Since muscular dystrophy is characterized by high rates of muscle degeneration/regeneration, one hypothesis is that SLN may be important in the regeneration process but this has not been examined specifically. Here, we used an acute cardiotoxin injury model and Sln‐null mice to determine SLN's role in muscle regeneration. The tibialis anterior of 30 mice, 15 wild type (WT) and 15 Sln‐null mice were injected with cardiotoxin and sacrificed at day 7, 14 and 21 post injury. Muscle homogenates and sections were prepared and used for immunoblotting and immunofluorescence analyses, respectively. Interestingly, ectopic expression of SLN was seen in WT animals only at 21 days post injury. Correspondingly, Sln‐null mice showed elevated centralized nuclei, a marker of regenerating fibers, specifically at day 21 post injury compared to their WT counterparts (2.7 ± 1.0 ×10−4 vs. 3.8 ± 0.6 ×10−4, per regenerating area (μm2), p=0.03). In addition, the average cross‐sectional area of the regenerating myofibers was significantly smaller in the Sln‐null mice only at day 21 post injury compared to WT (1925 ± 168 μm2 vs. 1471 ± 57 μm2, p =0.01 ). Taken together, the failure to clear central nuclei and increase myofiber size at day 21 post injury indicates that SLN ablation delays the later stages of muscle regeneration.Support or Funding InformationThis work was supported by the Canadian Institutes of Health Research (CIHR; MOP 86618 and MOP 47296 to A.R.T).
Viscoelastic Model for Microglial Oscillations
Authors: Park Eun Young , Cho Youngbin , Ko Ung Hyun , Park Jin‐Sung , Shin Jennifer 
Published: November 2023

IntroductionMotility is a critical physiological process in wound healing, cancer metastasis, and immune responses. It involves complex orchestration of cytoskeletal proteins and adhesion molecules to generate protrusive forces, contractility, and directionality. Motile traits often represent the functional or physiological state of the cells. For example, some types of monocytes feature the patrolling mode and transform into directional migratory mode upon the inflammatory conditions. In this study, we present microglia, a type of immune cells in the central nervous system, which exhibit several distinct morphological phenotypes, namely ramified, withdrawal, transitional, motile, and locomotory, depending on its physiological state. Especially the microglial cells in the motile state show striking oscillatory behavior in the absence of external cues, which can be induced to transform into persistent migration upon stimulation. This rapid and reversible transformation was found to involve dynamic rearrangements of cytoskeletal proteins and redistribution of forces, evidenced by traction force microscopy (TFM). To unravel the complexity in the multiple involvement of cytoskeletal proteins in force generation, here we focus on the oscillatory behavior of the microglia and propose a simple lumped‐parameter viscoelastic model with mass, spring and damper components each of which represents elastic contractility and viscous adhesive drag induced by cytoskeletons and associated proteins. Through this study, we aim to investigate the roles of the cytoskeletal proteins and adhesions in each of the elements in the model.MethodsMicroglia were extracted from the cortex in the brain of rats and seeded in a fibronectin coated microchannel (3×0.242×14 mm, W×H×L) for real time imaging. To measure the cellular physical forces, we performed TFM using a soft PA gel. For the simulations of models and analysis, we used MATLAB.Results and DiscussionsWe studied the roles of the cytoskeletal proteins and adhesions by chemically inhibiting them. Moreover, we employed TFM to visualize the redistribution of stresses during oscillation. Based on the obtained results of temporal and spatial changes in cellular positions and associated forces, we found the mechanical correlations between cellular components and viscoelastic elements by the simulation. We propose that the oscillatory phenotypes of microglia are regulated by the microtubules in terms of maintenance of morphologies, and by the actin filaments and myosin II in terms of contractility.Support or Funding InformationThis work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (NRF‐2013S1A2A2035518)
AD‐20 Inhibits Prostate Cancer Cell Migration and Tumor Metastasis by Suppressing NRP1 Expression
Authors: Li Xin 
Published: November 2023

Metastasis, a complex, multi‐step process, is the leading cause of death in patients diagnosed with cancer. Interaction between tumor cells and extracellular matrix (ECM) plays a critical role in the regulation of metastasis. Natural products have been a source of novel drugs for decades owing to their versatile biological activities and comparatively low toxicity. In our study we screened a natural product database for anti‐migration effect on prostate cancer PC‐3 cell line and eventually acquired a natural product AD‐20 exhibiting inhibitory effects in both in vitro and in vivo intravenous injection xenograft model.To understand how AD‐20 suppresses cell migration and tumor metastasis, LC‐MS/MS analysis was employed to identify significant different proteins (SDP) in AD‐20 treated PC‐3 cells. Then a SDP‐based protein‐protein interaction network was generated and integrated into Gene ontology (GO) enrichment network containing three cell movement associated GO terms. Consequently, two SDPs, neuropilin 1 (NRP1) and fibronectin (FN), were annotated with all three cell movement associated GO terms, suggesting that NRP1 and FN may be the key proteins accounting for the functions of AD‐20.As a co‐receptor for VEGF and several other growth factors (GFs), NRP1 is largely involved in angiogenesis and tumor progression. Besides functions relevant to GFs, NRP1 also regulates the interaction between cells and ECM by interacting with active integrin α5β1 and facilitating its turnover. FN is a high molecular weight ECM protein, whose assembly is largely mediated by the function of integrin α5β1. By Western Blot we confirmed that AD‐20 inhibits expression of both NRP1 and FN in PC‐3 cells, consistent with our results in proteomic study. Cell motility is attenuated in NRP1−/− PC‐3 cells, while the effect of AD‐20 is milder in NRP1−/− cells than in wild type cells. These results indicate that AD‐20 functions on cell migration partly, if not solely, through impairing NRP1 expression. Besides cell motility, cell adhesion to FN is also compromised in AD‐20 treated wild type cells and NRP1−/− PC‐3 cells. Further, in fibroblast cell model AD‐20 treatment also suppresses NRP1 and FN expression and FN filaments assembly. The results in both cell types suggest that AD‐20 plays a comprehensive role in modulating the interaction between cancer cells and ECM.In summary, we found that AD‐20 inhibits prostate cancer PC‐3 cell migration and tumor metastasis by inhibiting NRP1 expression. Besides, AD‐20 may influence the interaction between cells and ECM by impacting NRP1 function, which is critical in metastasis process. Concerning the potential of NRP1 as a target for anti‐cancer drug development and the clinical trials of NRP1 monoclonal antibody, AD‐20 may have a bright prospect to be developed into a chemical based anti‐cancer drug targeting NRP1 and relevant pathways.Support or Funding InformationNational Natural Science Foundation of China
The Role of Taurine in the Anoxia‐Tolerant Western Painted Turtle Brain
Authors: Miles Ashley , Buck Leslie 
Published: November 2023

Unlike the anoxia‐intolerant mammalian brain, which undergoes hyperexcitability and neuronal death in response to anoxia, the western painted turtle (Chrysemys picta) can survive extended periods of anoxia without significant neuronal damage. This is in part accomplished by a large increase in the concentration of the inhibitory neurotransmitter gamma‐aminobutyric acid (GABA) resulting in suppression of spontaneous electrical activity (i.e. spike arrest) through a mechanism known as shunting inhibition. In addition to GABA, levels of the amino acid taurine also increase in the anoxic turtle brain, but its function is not known. It is speculated that taurine can function like a neurotransmitter by acting through GABA receptors. Given the important role of GABA in the anoxic turtle brain, we wanted to investigate the role of taurine by determing whether turtle cortical neurons are responsive to taurine and what receptor(s) taurine may be acting through. Using the whole cell patch clamp technique, we measured the electophysiological properties of neurons in response to taurine. We found that 10mM taurine caused the membrane potential of cortical neurons to depolarize approximately 8mV, similar to the effects of GABA and anoxia. Additionally taurine increased the whole cell conductance (Gw) >2‐fold. These effects were reduced but not completely abolished when strychnine, a glycine receptor antagonist, and/or gabazine, a GABAA receptor antagonist were applied. Together these results suggest that taurine can act through both glycine and GABA receptors to effect turtle cortical neurons and therefore may also play a signaling role during anoxia in the turtle cortex.Support or Funding InformationThis research is supported by the Ontario Graduate Scholarship (OGS) and Canadian Graduate Scholarship‐Master's (CGS‐M NSERC) awarded to Ashley Miles and the Discovery Accelerator Supplements Grant awarded to Les Buck.
Dysfunction of Astrocytes in the Nucleus Tractus Solitarii Leads to Cardiorespiratory Compromise
Authors: Talman William T. , Lin Li‐Hsien , Jones Susan , Dragon Deidre Nitschke , Wu Yuanming , Richerson George B. 
Published: November 2023

We have previously shown that saporin (SAP) microinjected into the nucleus tractus solitarii (NTS) causes loss of astrocytes while producing no immunohistochemical evidence of damage to NTS neurons. Loss of NTS astrocytes leads to loss of cardiovascular reflex control and, in some animals, sudden death as a result of asystole. Knowing that respiratory dysfunction and apnea may lead to asystole, we hypothesized that astrocytic dysfunction compromises respiratory control and may lead to apnea and sudden death. We sought to test that hypothesis in rats treated with bilateral NTS injections of the ribosomal toxin SAP, a chronic model of astrocytic loss, or fluorocitrate, which produces an acute model of astrocytic dysfunction. We first sought to determine if the loss of astrocytes due to SAP was due to necrosis or apoptosis by injection of SAP unilaterally into the NTS. We found loss of immunoreactivity for glial fibrillary acidic protein (GFAP), a marker of astrocytes, two hours, six hours, one day and seven days after SAP injection and increased immunoreactivity for the necrosis marker, receptor interacting protein (RIP), 6 hours and one day after SAP injection. In contrast, SAP treatment did not lead to apoptosis as documented by absence of positive cells in TUNEL reaction in the NTS. Injection of SAP into the NTS of neonatal rats (P13) led to loss of GFAP immunoreactivity seven days later as it also does in the adult rat brain. Therefore, in brain slices from P20–23 rats that had been treated unilaterally with SAP seven days previously, we performed patch clamp recording from neurons in the NTS and found that their intrinsic electrophysiological properties were unremarkable compared to neurons on the control side. In adult animals we monitored breathing chronically in a plethysmographic chamber after bilateral NTS microinjection of SAP. Seven days after injection, awake, treated rats, in contrast to control rats, began to manifest slow, irregular breathing patterns during eupneic breathing. During acute studies of astrocytic dysfunction, rats were anesthetized with isoflurane and respiration was monitored by intercostal electromyographic recording concurrent with recording arterial blood pressure and heart rate. Bilateral injections of fluorocitrate were made into NTS at 15 minute intervals for 2 hours. Fluorocitrate treated rats, but not rats treated with vehicle or PBS, developed slowed breathing followed by periodic apnea. These studies suggest that SAP causes necrosis of astrocytes and spares neuronal function. Either astrocytic necrosis or transient astrocytic dysfunction in the NTS interferes with normal cardiorespiratory regulation and leads to hypopnea and apnea that may contribute to death.Support or Funding InformationNIH R01HL088090 and VA Merit Review
Comparing the Location of Medullary Dorsal Horn Neuronal Activation during Voluntary Diving with the Central Terminations of Nerves that Innervate the Nose and External Nares
Authors: DelPrete Benjamin , Lahrman Kenneth , Warren Erik , DiNovo Karyn , McCulloch Paul 
Published: November 2023

When animals dive underwater there is a reflex initiation of the diving response. This cardiorespiratory response includes parasympathetically‐mediated bradycardia, a sympathetically‐mediated increase in total peripheral resistance, and apnea. The afferent limb of this reflex includes the medullary dorsal horn (MDH), specifically the superficial laminae of the MDH between the pyramidal decussation caudally and obex rostrally. This is also the location of the central terminations of trigeminal nerves that innervate the nose and external nares, such as the anterior ethmoidal nerve (AEN) and nasal branch of the infraorbital nerve (ION). The objective of this research was to compare the pattern of neuronal activation of secondary neurons activated by voluntary diving, as determined by neuronal Fos production, with the central terminations of the AEN and ION, as determined by the transganglionic tracer Wheat Germ Agglutinin (WGA). Sprague‐Dawley rats (N=12) were trained to voluntarily dive underwater through a 5 m maze. Six rats had 24 dive trials over 2 hours to activate brainstem neurons to produce Fos, while the remaining rats were cage controls. Other rats had 1 μl of 100% WGA injected into the AEN (N=4) or ION (N=9), 4 μl of 100% WGA injected under the skin of the hairy (N=6) or glabrous (N=3) nose, or 60 μl of 50% WGA injected into the nasal passages (N=4) or nasal vestibule (N=3). The location of Fos positive neurons within the MDH of the diving rats was then compared to the MDH labeling pattern from the WGA injected rats. Results indicate that WGA central termination sites showed varying degrees of overlap with the location of Fos‐positive neurons, depending upon which nerve or nasal area received the WGA injection. We conclude that the ION, in addition to the AEN, could possibly be an anatomical pathway that provides the afferent information necessary to initiate the mammalian diving response.Support or Funding InformationSupported by CCOM Suarez Summer Research Fellowships (BDP and KAL), CHS MBS program (EAW), and MWU Intramural funding (PFM).