Targeting SWI/SNF ATPases in enhancer-addicted prostate cancer.

The switch/sucrose non-fermentable (SWI/SNF) complex has a crucial role in chromatin remodelling1 and is altered in over 20% of cancers2,3. Here we developed a proteolysis-targeting chimera (PROTAC) degrader of the SWI/SNF ATPase subunits, SMARCA2 and SMARCA4, called AU-15330. Androgen receptor (AR)+ forkhead box A1 (FOXA1)+ prostate cancer cells are exquisitely sensitive to dual SMARCA2 and SMARCA4 degradation relative to normal and other cancer cell lines. SWI/SNF ATPase degradation rapidly compacts cis-regulatory elements bound by transcription factors that drive prostate cancer cell proliferation, namely AR, FOXA1, ERG and MYC, which dislodges them from chromatin, disables their core enhancer circuitry, and abolishes the downstream oncogenic gene programs. SWI/SNF ATPase degradation also disrupts super-enhancer and promoter looping interactions that wire supra-physiologic expression of the AR, FOXA1 and MYC oncogenes themselves. AU-15330 induces potent inhibition of tumour growth in xenograft models of prostate cancer and synergizes with the AR antagonist enzalutamide, even inducing disease remission in castration-resistant prostate cancer (CRPC) models without toxicity. Thus, impeding SWI/SNF-mediated enhancer accessibility represents a promising therapeutic approach for enhancer-addicted cancers.

Development of an orally bioavailable mSWI/SNF ATPase degrader and acquired mechanisms of resistance in prostate cancer.

Mammalian switch/sucrose nonfermentable (mSWI/SNF) ATPase degraders have been shown to be effective in enhancer-driven cancers by functioning to impede oncogenic transcription factor chromatin accessibility. Here, we developed AU-24118, an orally bioavailable proteolysis-targeting chimera (PROTAC) degrader of mSWI/SNF ATPases (SMARCA2 and SMARCA4) and PBRM1. AU-24118 demonstrated tumor regression in a model of castration-resistant prostate cancer (CRPC) which was further enhanced with combination enzalutamide treatment, a standard of care androgen receptor (AR) antagonist used in CRPC patients. Importantly, AU-24118 exhibited favorable pharmacokinetic profiles in preclinical analyses in mice and rats, and further toxicity testing in mice showed a favorable safety profile. As acquired resistance is common with targeted cancer therapeutics, experiments were designed to explore potential mechanisms of resistance that may arise with long-term mSWI/SNF ATPase PROTAC treatment. Prostate cancer cell lines exposed to long-term treatment with high doses of a mSWI/SNF ATPase degrader developed SMARCA4 bromodomain mutations and ABCB1 (ATP binding cassette subfamily B member 1) overexpression as acquired mechanisms of resistance. Intriguingly, while SMARCA4 mutations provided specific resistance to mSWI/SNF degraders, ABCB1 overexpression provided broader resistance to other potent PROTAC degraders targeting bromodomain-containing protein 4 and AR. The ABCB1 inhibitor, zosuquidar, reversed resistance to all three PROTAC degraders tested. Combined, these findings position mSWI/SNF degraders for clinical translation for patients with enhancer-driven cancers and define strategies to overcome resistance mechanisms that may arise.

Local magnetic delivery of adeno-associated virus AAV2(quad Y-F)-mediated BDNF gene therapy restores hearing after noise injury.

Moderate noise exposure may cause acute loss of cochlear synapses without affecting the cochlear hair cells and hearing threshold; thus, it remains "hidden" to standard clinical tests. This cochlear synaptopathy is one of the main pathologies of noise-induced hearing loss (NIHL). There is no effective treatment for NIHL, mainly because of the lack of a proper drug-delivery technique. We hypothesized that local magnetic delivery of gene therapy into the inner ear could be beneficial for NIHL. In this study, we used superparamagnetic iron oxide nanoparticles (SPIONs) and a recombinant adeno-associated virus (AAV) vector (AAV2(quad Y-F)) to deliver brain-derived neurotrophic factor (BDNF) gene therapy into the rat inner ear via minimally invasive magnetic targeting. We found that the magnetic targeting effectively accumulates and distributes the SPION-tagged AAV2(quad Y-F)-BDNF vector into the inner ear. We also found that AAV2(quad Y-F) efficiently transfects cochlear hair cells and enhances BDNF gene expression. Enhanced BDNF gene expression substantially recovers noise-induced BDNF gene downregulation, auditory brainstem response (ABR) wave I amplitude reduction, and synapse loss. These results suggest that magnetic targeting of AAV2(quad Y-F)-mediated BDNF gene therapy could reverse cochlear synaptopathy after NIHL.

Design, synthesis, and biological evaluation of phenyl thiazole-based AR-V7 degraders.

Multiple Splice variants of AR have been reported in the past few years. These splice variants are upregulated in most cases of CRPC resulting in poor prognosis. Most of these variants lack the ligand binding domain (LBD) but still bind to DNA resulting in constitutive activation of downstream targets. The AR-V7 splice variant has been characterized extensively and current clinical trials in CRPC are exploring the use of AR-V7 as a biomarker. New therapeutic molecules that selectively target AR-V7 are also being explored. However, there is a dearth of information available on the selectivity, phenotypic responses in AR-V7 dependent cell lines and pharmacokinetic properties of such molecules. Using our proprietary computational algorithms and rational SAR optimization, we have developed a potent and selective AR-V7 degrader from a known AR DNA binding domain (DBD) binder. This molecule effectively degraded AR-V7 in a CRPC cell line and demonstrated good oral bioavailability in mouse PK studies. This tool compound can be used to evaluate the pharmacological effects of AR-V7 degraders. Further exploration of SAR can be pursued to develop more optimized lead compounds.

Prostaglandin E2 inhibits profibrotic function of human pulmonary fibroblasts by disrupting Ca2+ signaling.

We have shown that calcium (Ca2+) oscillations in human pulmonary fibroblasts (HPFs) contribute to profibrotic effects of transforming growth factor-ß (TGF-ß) and that disruption of these oscillations blunts features of pulmonary fibrosis. Prostaglandin E2 (PGE2) exerts antifibrotic effects in the lung, but the mechanisms for this action are not well defined. We thus sought to explore interactions between PGE2 and the profibrotic agent TGF-ß in pulmonary fibroblasts (PFs) isolated from patients with or without idiopathic pulmonary fibrosis (IPF). PGE2 inhibited TGF-ß-promoted [Ca2+] oscillations and prevented the activation of Akt and Ca2+/calmodulin-dependent protein kinase-II (CaMK-II) but did not prevent activation of Smad-2 or ERK. PGE2 also eliminated TGF-ß-stimulated expression of collagen A1, fibronectin, and α-smooth muscle actin and reduced stress fiber formation in the HPFs. RNA sequencing revealed that HPFs preferentially express EP2 receptors relative to other prostanoid receptor subtypes: EP2 expression is ~10-fold higher than that of EP4 receptors; EP1 and EP3 receptors are barely detectable; and EP2-receptor expression is ~3.5-fold lower in PFs from IPF patients than in normal HPFs. The inhibitory effects of PGE2 on synthetic function and stress fiber formation were blocked by selective EP2 or EP4 antagonists and mimicked by selective EP2 or EP4 agonists, the phosphodiesterase inhibitor isobutylmethylxanthine and forskolin, all of which elevate cellular cAMP concentrations. We conclude that PGE2, likely predominantly via EP2 receptors, interferes with Ca2+ signaling, CaMK-II activation, and Akt activation in IPF-HPFs and HPFs treated with TGF-ß. Moreover, a decreased expression of EP2 receptors in pulmonary fibroblasts from IPF patients may contribute to the pathophysiology of this disease.

TRPV4 Stimulation Releases ATP via Pannexin Channels in Human Pulmonary Fibroblasts.

We previously described several ionic conductances in human pulmonary fibroblasts, including one activated by two structurally distinct TRPV4 (transient receptor potential, vanilloid-type, subtype 4)-channel agonists: 4αPDD (4α-phorbol-12,13-didecanoate) and GSK1016790A. However, the TRPV4-activated current exhibited peculiar properties: it developed slowly over many minutes, exhibited reversal potentials that could vary by tens of millivolts even within a given cell, and was not easily reversed by subsequent addition of two distinct TRPV4-selective blockers (RN-1734 and HC-067047). In this study, we characterized that conductance more carefully. We found that 4αPDD stimulated a delayed release of ATP into the extracellular space, which was reduced by genetic silencing of pannexin expression, and that the 4αPDD-evoked current could be blocked by apyrase (which rapidly degrades ATP) or by the P2Y purinergic receptor/channel blocker pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), and could be mimicked by exogenous addition of ATP. In addition, we found that the 4αPDD-evoked current was blocked by pretreatment with RN-1734 or HC-067047, by Gd3+ or La3+, or by two distinct blockers of pannexin channels (carbenoxolone and probenecid), but not by a blocker of connexin hemichannels (flufenamic acid). We also found expression of TRPV4- and pannexin-channel proteins. 4αPDD markedly increased calcium flashing in our cells. The latter was abrogated by the P2Y channel blocker PPADS, and the 4αPDD-evoked current was eliminated by loading the cytosol with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or by inhibiting Ca2+/calmodulin-sensitive kinase II using KN93. Altogether, we interpret these findings as suggesting that 4αPDD triggers the release of ATP via pannexin channels, which in turn acts in an autocrine and/or paracrine fashion to stimulate PPADS-sensitive purinergic receptors on human pulmonary fibroblasts.

Ca2+/calmodulin-dependent protein kinase IIß and IIδ mediate TGFß-induced transduction of fibronectin and collagen in human pulmonary fibroblasts.

It is now clear that in addition to activating several complex kinase pathways (Smad, MAP kinase, PI3 kinase), TGFß also acts by elevating cytosolic Ca2+ concentration within human pulmonary fibroblasts. Ca2+/calmodulin-dependent protein kinase II (CamK II) is also known to regulate gene expression in fibroblasts. In this study, we examined the interactions between calcium signaling, activation of CamK and other kinases, and extracellular matrix (ECM) gene expression. Human pulmonary fibroblasts were cultured and stimulated with artificially generated Ca2+ pulses in the absence of TGFß, or with TGFß (1 nM) or vehicle in the presence of various blockers of Ca2+ signaling. PCR and Western blotting were used to measure gene expression and protein levels, respectively. We found that Ca2+ pulses in the absence of TGFß increased ECM gene expression in a pulse frequency-dependent manner, and that blocking Ca2+ signaling and the CamK II pathway significantly reduced TGFß-mediated ECM gene expression, without having any effects on other kinase pathways (Smad, PI3 kinase, or MAP kinase). We also found that TGFß elevated the expression of CamK IIß and CamK IIδ, while siRNA silencing of those two subtypes significantly reduced TGFß-mediated expression of collagen A1 and fibronectin 1. Our data suggest that TGFß induces the expression of CamK IIß and CamK IIδ, which in turn are activated by TGFß-evoked Ca2+ waves in a frequency-dependent manner, leading to increased expression of ECM proteins.

Electrophysiological characterization of voltage-dependent calcium currents and TRPV4 currents in human pulmonary fibroblasts.

We have presented indirect evidence of a key role for voltage-dependent Ca(2+) currents in TGFß-induced synthetic function in human pulmonary fibroblast (HPF), as well as in bleomycin-induced pulmonary fibrosis in mice. Others, however, have provided indirect evidence for transient receptor potential vanilloid 4 (TRPV4) channels in both of those effects. Unfortunately, definitive electrophysiological descriptions of both currents in HPFs have been entirely lacking. In this study, we provide the first direct electrophysiological and pharmacological evidence of the currents in HPFs at rest and during overnight stimulation with TGFß. These currents include a Ca(2+)-dependent K(+) current, a TRPV4 current, a chloride current, and an L-type voltage-dependent Ca(2+) current. Evidence for the TRPV4 current include activation of a large-conductance change by two putatively TRPV4-selective agonists (4α-phorbol-12,13-didecanoate; GSK1016790A), with a reversal potential near 0 mV, partial sensitivity to two different TRPV4-selective blockers (RN1734; HC067047), and partial reduction following removal of external Na(+) Substantial reduction of the evoked current was seen following the coapplication of RN1734, DIDS, and niflumic acid, suggesting that a chloride current is also involved. The voltage-dependent Ca(2+) current is found to be "L-type" in nature, as indicated by the voltage and time dependence of its activation, deactivation, and inactivation properties, and by its pharmacology (sensitivity to replacement with barium and inhibition by nifedipine, verapamil, or mibefradil). We also found that overnight treatment with TGFß evoked a periodic current (inward at negative holding potentials, with reversal potential near 0 mV), which is sufficient to trigger the voltage-dependent Ca(2+) currents and, thereby, account for the rhythmic Ca(2+) oscillations, which we have described previously in these cells.

Membrane Perturbation-Associated Ca2+ Signaling and Incoming Genome Sensing Are Required for the Host Response to Low-Level Enveloped Virus Particle Entry.

UNLABELLED: The type I interferon (IFN) response is an important aspect of innate antiviral defense, and the transcription factor IRF3 plays an important role in its induction. Membrane perturbation during fusion, a necessary step for enveloped virus particle entry, appears sufficient to induce transcription of a subset of IFN-stimulated genes (ISGs) in an IRF3-dependent, IFN-independent fashion. IRF3 is emerging as a central node in host cell stress responses, although it remains unclear how different forms of stress activate IRF3. Here, we investigated the minimum number of Sendai virus (SeV) and human cytomegalovirus (HCMV) particles required to activate IRF3 and trigger an antiviral response. We found that Ca(2+) signaling associated with membrane perturbation and recognition of incoming viral genomes by cytosolic nucleic acid receptors are required to activate IRF3 in response to fewer than 13 particles of SeV and 84 particles of HCMV per cell. Moreover, it appears that Ca(2+) signaling is important for activation of STING and IRF3 following HCMV particle entry, suggesting that Ca(2+) signaling sensitizes cells to recognize genomes within incoming virus particles. To our knowledge, this is the first evidence that cytosolic nucleic acid sensors recognize genomes within incoming virus particles prior to virus replication. These studies highlight the exquisite sensitivity of the cellular response to low-level stimuli and suggest that virus particle entry is sensed as a stress signal. IMPORTANCE: The mechanism by which replicating viruses trigger IRF3 activation and type I IFN induction through the generation and accumulation of viral pathogen-associated molecular patterns has been well characterized. However, the mechanism by which enveloped virus particle entry mediates a stress response, leading to IRF3 activation and the IFN-independent response, remained elusive. Here, we find that Ca(2+) signaling associated with membrane perturbation appears to sensitize cells to recognize genomes within incoming virus particles. To our knowledge, this is the first study to show that cytosolic receptors recognize genomes within incoming virus particles prior to virus replication. These findings not only highlight the sensitivity of cellular responses to low-level virus particle stimulation, but provide important insights into how nonreplicating virus vectors or synthetic lipid-based carriers used as clinical delivery vehicles activate innate immune responses.

Calcium Homeostasis and Ionic Mechanisms in Pulmonary Fibroblasts.

Fibroblasts are key cellular mediators of many chronic interstitial lung diseases, including idiopathic pulmonary fibrosis, scleroderma, sarcoidosis, drug-induced interstitial lung disease, and interstitial lung disease in connective tissue disease. A great deal of effort has been expended to understand the signaling mechanisms underlying the various cellular functions of fibroblasts. Recently, it has been shown that Ca(2+) oscillations play a central role in the regulation of gene expression in human pulmonary fibroblasts. However, the mechanisms whereby cytosolic [Ca(2+)] are regulated and [Ca(2+)] oscillations transduced are both poorly understood. In this review, we present the general concepts of [Ca(2+)] homeostasis, of ionic mechanisms responsible for various Ca(2+) fluxes, and of regulation of gene expression by [Ca(2+)]. In each case, we then also summarize the original findings that pertain specifically to pulmonary fibroblasts. From these data, we propose an overall signaling cascade by which excitation of the fibroblasts triggers pulsatile release of internally sequestered Ca(2+), which, in turn, activates membrane conductances, including voltage-dependent Ca(2+) influx pathways. Collectively, these events produce recurring Ca(2+) oscillations, the frequency of which is transduced by Ca(2+)-dependent transcription factors, which, in turn, orchestrate a variety of cellular events, including proliferation, synthesis/secretion of extracellular matrix proteins, autoactivation (production of transforming growth factor-ß), and transformation into myofibroblasts. That unifying hypothesis, in turn, allows us to highlight several specific cellular targets and therapeutic intervention strategies aimed at controlling unwanted pulmonary fibrosis. The relationships between Ca(2+) signaling events and the unfolded protein response and apoptosis are also explored.

Disruption of Calcium Signaling in Fibroblasts and Attenuation of Bleomycin-Induced Fibrosis by Nifedipine.

Fibrotic lung disease afflicts millions of people; the central problem is progressive lung destruction and remodeling. We have shown that external growth factors regulate fibroblast function not only through canonical signaling pathways but also through propagation of periodic oscillations in Ca(2+). In this study, we characterized the pharmacological sensitivity of the Ca(2+)oscillations and determined whether a blocker of those oscillations can prevent the progression of fibrosis in vivo. We found Ca(2+) oscillations evoked by exogenously applied transforming growth factor ß in normal human fibroblasts were substantially reduced by 1 µM nifedipine or 1 µM verapamil (both L-type blockers), by 2.7 µM mibefradil (a mixed L-/T-type blocker), by 40 µM NiCl2 (selective at this concentration against T-type current), by 30 mM KCl (which partially depolarizes the membrane and thereby fully inactivates T-type current but leaves L-type current intact), or by 1 mM NiCl2 (blocks both L- and T-type currents). In our in vivo study in mice, nifedipine prevented bleomycin-induced fibrotic changes (increased lung stiffness, overexpression of smooth muscle actin, increased extracellular matrix deposition, and increased soluble collagen and hydroxyproline content). Nifedipine had little or no effect on lung inflammation, suggesting its protective effect on lung fibrosis was not due to an antiinflammatory effect but rather was due to altering the profibrotic response to bleomycin. Collectively, these data show that nifedipine disrupts Ca(2+) oscillations in fibroblasts and prevents the impairment of lung function in the bleomycin model of pulmonary fibrosis. Our results provide compelling proof-of-principle that interfering with Ca(2+) signaling may be beneficial against pulmonary fibrosis.

Discovery of O-(3-carbamimidoylphenyl)-l-serine amides as matriptase inhibitors using a fragment-linking approach.

Matriptase is a cell-surface trypsin-like serine protease of epithelial origin, which cleaves and activates proteins including hepatocyte growth factor/scatter factor and proteases such as uPA, which are involved in the progression of various cancers. Here we report a fragment-linking approach, which led to the discovery of O-(3-carbamimidoylphenyl)-l-serine amides as potent matriptase inhibitors. The co-crystal structure of one of the potent inhibitors, 6 in complex with matriptase catalytic domain validated the working hypothesis guiding the development of this congeneric series and revealed the structural basis for matriptase inhibition. Replacement of a naphthyl group in 6 with 2,4,6-tri-isopropyl phenyl resulted in 10 with improved matriptase inhibition, which exhibited significant primary tumor growth inhibition in a mouse model of prostate cancer. Compounds such as 10, identified using a fragment-linking approach, can be explored further to understand the role of matriptase as a drug target in cancer and inflammation.

Structure-guided discovery of 2-aryl/pyridin-2-yl-1H-indole derivatives as potent and selective hepsin inhibitors.

Hepsin, a type II transmembrane serine protease, is upregulated in prostate cancer and known to be involved in the progression of metastasis. Here we report a structure-guided approach, which resulted in the discovery of 2-aryl/pyridin-2-yl-1H-indole derivatives as potent and selective inhibitors of hepsin. Potent and selective inhibition of hepsin by compound 8 is likely due to interactions of the amidine group at the S1 site with the cyclohexyl ring from the 2-aryl group projecting towards the S1' site and the tert-hydroxyl group interacting with His57 side-chain as revealed by X-ray crystallography. Compounds 8 and 10, showed Ki of 0.1 µM for hepsin, and exhibited inhibition of invasion and migration of hepsin-overexpressing cell line. Compounds described here could serve as useful tool reagents to investigate the role of hepsin as a potential therapeutic target in cancer.

Structure-guided discovery of 1,3,5 tri-substituted benzenes as potent and selective matriptase inhibitors exhibiting in vivo antitumor efficacy.

Matriptase is a serine protease implicated in cancer invasion and metastasis. Expression of matriptase is frequently dysregulated in human cancers and matriptase has been reported to activate latent growth factors such as hepatocyte growth factor/scatter factor, and proteases such as urokinase plasminogen activator suggesting that matriptase inhibitors could have therapeutic potential in treatment of cancer. Here we report a structure-based approach which led to the discovery of selective and potent matriptase inhibitors with benzene as central core having 1,3,5 tri-substitution pattern. X-ray crystallography of one of the potent analogs in complex with matriptase revealed strong hydrogen bonding and salt-bridge interactions in the S1 pocket, as well as strong CH-π contacts between the P2/P4 cyclohexyl and Trp215 side-chain. An additional interaction of the pendant amine at cyclohexyl with Gln175 side-chain results in substantial improvement in matriptase inhibition and selectivity against other related serine proteases. Compounds 15 and 26 showed tumor growth inhibition in a subcutaneous DU-145 prostate cancer mouse model. These compounds could be useful as tools to further explore the biology of matriptase as a drug target.

Development of an orally bioavailable mSWI/SNF ATPase degrader and acquired mechanisms of resistance in prostate cancer.

Mammalian switch/sucrose non-fermentable (mSWI/SNF) ATPase degraders have been shown to be effective in enhancer-driven cancers by functioning to impede oncogenic transcription factor chromatin accessibility. Here, we developed AU-24118, a first-in-class, orally bioavailable proteolysis targeting chimera (PROTAC) degrader of mSWI/SNF ATPases (SMARCA2 and SMARCA4) and PBRM1. AU-24118 demonstrated tumor regression in a model of castration-resistant prostate cancer (CRPC) which was further enhanced with combination enzalutamide treatment, a standard of care androgen receptor (AR) antagonist used in CRPC patients. Importantly, AU-24118 exhibited favorable pharmacokinetic profiles in preclinical analyses in mice and rats, and further toxicity testing in mice showed a favorable safety profile. As acquired resistance is common with targeted cancer therapeutics, experiments were designed to explore potential mechanisms of resistance that may arise with long-term mSWI/SNF ATPase PROTAC treatment. Prostate cancer cell lines exposed to long-term treatment with high doses of a mSWI/SNF ATPase degrader developed SMARCA4 bromodomain mutations and ABCB1 overexpression as acquired mechanisms of resistance. Intriguingly, while SMARCA4 mutations provided specific resistance to mSWI/SNF degraders, ABCB1 overexpression provided broader resistance to other potent PROTAC degraders targeting bromodomain-containing protein 4 (BRD4) and AR. The ABCB1 inhibitor, zosuquidar, reversed resistance to all three PROTAC degraders tested. Combined, these findings position mSWI/SNF degraders for clinical translation for patients with enhancer-driven cancers and define strategies to overcome resistance mechanisms that may arise.

Novel non-canonical TGF-ß signaling networks: emerging roles in airway smooth muscle phenotype and function.

The airway smooth muscle (ASM) plays an important role in the pathophysiology of asthma and chronic obstructive pulmonary disease (COPD). ASM cells express a wide range of receptors involved in contraction, growth, matrix protein production and the secretion of cytokines and chemokines. Transforming growth factor beta (TGF-ß) is one of the major players in determining the structural and functional abnormalities of the ASM in asthma and COPD. It is increasingly evident that TGF-ß functions as a master switch, controlling a network of intracellular and autocrine signaling loops that effect ASM phenotype and function. In this review, the various elements that participate in non-canonical TGF-ß signaling, including MAPK, PI3K, WNT/ß-catenin, and Ca(2+), are discussed, focusing on their effect on ASM phenotype and function. In addition, new aspects of ASM biology and their possible association with non-canonical TGF-ß signaling will be discussed.

Magnetic Targeting of Gadolinium Contrast to Enhance MRI of the Inner Ear in Endolymphatic Hydrops.

OBJECTIVES: 1. Determine the feasibility and efficiency of local magnetic targeting delivery of gadolinium (Gad) contrast to the inner ear in rodents. 2. Assess any potential ototoxicity of magnetic targeting delivery of Gad in the inner ear. 3. Study the utility of magnetic targeting delivery of Gad to visualize and quantify endolymphatic hydrops (EH) in a transgenic mouse model. STUDY DESIGN: Controlled in vivo animal model study. METHODS: Paramagnetic Gad was locally delivered to the inner ear using the magnetic targeting technique in both rat and mouse models. Efficiency of contrast delivery was assessed using magnetic resonance imaging (MRI). Ototoxicity of Gad was examined with histology of the cochlea and functional audiological tests. The Phex mouse model was used to study EH, hearing loss, and balance dysfunction. Magnetic targeting delivery of Gad contrast was used in the Phex mouse model to visualize the effects of EH using MRI. RESULTS: Magnetic targeting improved the delivery of Gad to the inner ear and the technique was reproducible in both rat and mouse models. The delivery method did not result in microstructural damage or any significant hearing loss in a normal animal. Magnetic targeting of Gad in the Phex mouse model allowed detailed visualization and quantification of EH. CONCLUSION: This study provided the first evidence of the effectiveness and efficiency of the local magnetic targeting delivery of gadolinium contrast to the inner ear and its application to the visualization and quantification of EH. Laryngoscope, 133:914-923, 2023.

Differential Cellular Sensing of Fusion from within and Fusion from without during Virus Infection.

The physical entry of virus particles into cells triggers an innate immune response that is dependent on both calcium and nucleic acid sensors, with particles containing RNA or DNA genomes detected by RNA or DNA sensors, respectively. While membrane fusion in the absence of viral nucleic acid causes an innate immune response that is dependent on calcium, the involvement of nucleic acid sensors is poorly understood. Here, we used lipoplexes containing purified reovirus p14 fusion protein as a model of exogenous or fusion from without and a cell line expressing inducible p14 protein as a model of endogenous or fusion from within to examine cellular membrane fusion sensing events. We show that the cellular response to membrane fusion in both models is dependent on calcium, IRF3 and IFN. The method of sensing fusion, however, differs between fusion from without and fusion from within. Exogenous p14 lipoplexes are detected by RIG-I-like RNA sensors, whereas fusion by endogenous p14 requires both RIG-I and STING to trigger an IFN response. The source of nucleic acid that is sensed appears to be cellular in origin. Future studies will investigate the source of endogenous nucleic acids recognized following membrane fusion events.

Transforming growth factor-ß evokes Ca2+ waves and enhances gene expression in human pulmonary fibroblasts.

Fibroblasts maintain the structural framework of animal tissue by synthesizing extracellular matrix molecules. Chronic lung diseases are characterized in part by changes in fibroblast numbers, properties, and more. Fibroblasts respond to a variety of growth factors, cytokines, and proinflammatory mediators. However, the signaling mechanisms behind these responses have not been fully explored. We sought to determine the role of Ca(2+) waves in transforming growth factor-ß (TGF-ß)-mediated gene expression in human pulmonary fibroblasts. Primary human pulmonary fibroblasts were cultured and treated with TGF-ß and different blockers under various conditions. Cells were then loaded with the Ca(2+) indicator dye Oregon green, and Ca(2+) waves were monitored by confocal [Ca(2+)](i) fluorimetry. Real-time PCR was used to probe gene expression. TGF-ß (1 nM) evoked recurring Ca(2+) waves. A 30-minute pretreatment of SD 208, a TGF-ß receptor-1 kinase inhibitor, prevented Ca(2+) waves from being evoked by TGF-ß. The removal of external Ca(2+) completely occluded TGF-ß-evoked Ca(2+) waves. Cyclopiazonic acid, an inhibitor of the internal Ca(2+) pump, evoked a relatively slowly developing rise in Ca(2+) waves compared with the rapid changes evoked by TGF-ß, but the baseline fluorescence was increased. Ryanodine (10(-5) M) also blocked TGF-ß-mediated Ca(2+) wave activity. Real-time PCR showed that TGF-ß rapidly and dramatically increased the gene expression of collagen A1 and fibronectin. This increase was blocked by ryanodine treatment and cyclopiazonic acid. We conclude that, in human pulmonary fibroblasts, TGF-ß acts on ryanodine-sensitive channels, leading to Ca(2+) wave activity, which in turn amplifies extracellular matrix gene expression.