bluuebunny/crossref_metadata_2025 huggingface dataset:
Garden cress (Lepidium sativum L.) Seed Oil: Alternative Source for ALA
Authors: Shetty Umesha Shankara , Akhilender Naidu K.
Published: November 2023

Garden cress (Lepidium sativum L.) is an edible, herb belonging to Cruciferae family. The seeds of garden cress claimed to possess medicinal properties (galactogogue, diuretic, demulcent, aphrodisiac, carminative and emmenagogue). Garden cress seeds contain 21–24 % oil. Garden cress seed oil(GCO) nutritionally enrich with Alpha‐linolenic acid (30–34%), Antioxidants (Tocopherols‐1799 mg/kg oil, Phenolics‐124 mg GAE/kg of oil) and Phytosterols (14.33 mg/g) and it is also relatively stable oil due to the presence these minor molecules. Despite its great medicinal and nutritional value, garden cress has not received the attention it deserves. The United Nation Organization's. FAO has classified Cress is one of the underutilized or neglected crop among age old crops. If these neglected crops not paid attention, they may be extinct near future.

The present study was part of Ph.D. thesis, the objective of thesis was aimed to explore & utilize the garden cress seed oil as a new source of health oil. Garden cress oil extracted from seeds and charterized for physicochemical properties, toxicants, safety and nutritional evaluations. It is also demonstrated utilization of GCO by using edible oil blending and microencapsulation technologies for successful application in foods system.

Our research findings show that GCO is free from of any unusual fatty acid, it has good natural antioxidants with potential radical scavenging activity. Rats fed with GCO observed there are no significant changes in food intake, body weight gain, organ weights, clinical enzymes, histological changes in vital organs. GCO significantly modulated fatty acid and lipid profile. ALA from GCO and its blended oil was well absorbed, accumulated and metabolized to LCPUFAs (n‐6/n‐3 PUFA ratio of 2.3 to 2.6. and in native oils 50.2–157). By feeding rats with GCO significantly increased the tocopherols levels (12.2–21.6 %) and activity of antioxidant enzymes, and also significantly lowered platelet aggregation by increasing n‐3 PUFA levels in platelets and decreasing proaggregatroy prostaglandin thromboxane and prostacyclin.

GCO was microencapsuled with Whey protein by using spray drying technique. Microencapsulation offered good oxidative protection during storage and also during baking, when GCO microcapsules incorporated in the cookies. These studies clearly indicated that GCO has potential qualities to become alternative source of omega‐3 PUFA for vegetarians. Some of the above findings were published food & nutrition journals, unpublished data will be discussed during the conference.
Influence of Omega‐3 Fatty Acids Intake on Human Responsiveness to Ambient Air Pollution Exposure
Authors: Shen Wan , Samet James , Diaz‐Sanchez David , Tong Haiyan
Published: November 2023

Exposure to air pollution, including particulate matter and gaseous pollutants, is associated with respiratory and cardiovascular morbidity, such as vascular dysfunction, high blood pressure, cardiac dysrhythmia, and impaired pulmonary function. Eicosapentaenoic acid (20:5, n‐3; EPA) and docosahexaenoic acid (22:6, n‐3; DHA) are the primary beneficial omega‐3 fatty acids found in seafood and fish oil. Dietary consumption of EPA and DHA has been shown to confer protection to the cardiovascular system. This registered and IRB approved panel study is designed to investigate the relationship between dietary consumption of EPA and DHA and ambient air pollution‐induced adverse cardiovascular and pulmonary responses. Cardiovascular and pulmonary indices in healthy 25–55 year‐old subjects consuming at least 3 g/week of EPA and DHA from supplements and dietary sources for a period of 6 months prior to enrollment (High Group) will be compared to a control group (Low Group) with an intake of no more than 1 pill/month of fish oil supplement and 1 serving (4–6 oz)/month of ocean fish/shellfish during the previous 6 months prior to enrollment. Subjects will undergo a battery of tests over 5 2‐day sessions at the U.S. Environmental Protection Agency Human Studies Facility, with each 7 or more days apart. The following endpoints will be measured: blood pressure, heart rate variability, blood fatty acid levels, endothelial cell function, retinal microvascular diameter, and spirometry. Subjects will wear GPS and activity monitors overnight during visits in order to permit a quantitative assessment of individual exposure to pollutants measured using area air quality monitoring stations and satellite imaging data. We hypothesize that dietary consumption of EPA and DHA influences adverse cardiopulmonary responses to ambient air pollution exposure.

Support or Funding Information

U.S. Environmental Protection Agency
Overexpression of MCPIP1 attenuates IL‐6‐induced vascular smooth muscle cell migration by suppressing IL‐6‐stimulated STAT3 signaling
Authors: Chang Yingzi
Published: November 2023

Vascular injury‐induced inflammation is the initial step of angioplasty‐associated vascular restenosis. As a major pro‐inflammatory cytokine, interleukin‐6 (IL‐6)‐induced motogenic effects on vascular smooth muscle cells is considered as the major contributor. Attenuation of vascular injury‐induced neointima thickening was observed by blocking STAT3 tyrosine phosphorylation, a key component of IL‐6 signaling. MCPIP1 (monocyte chemotactic protein [MCP]–induced protein 1; also known as ZC3H12A), an immune response modifier expressed in mice macrophage in response to inflammatory stimulation, plays a negative role in the process of inflammation. Our previous in vivo results revealed that MCPIP1 expression levels are increased following balloon‐catheter‐induced rat carotid injury at 3, 7 and 14 days. Our current study was designed to test if upregulation of MCPIP1 plays a negative role in injury‐induced vascular stenosis by suppressing IL‐6‐induced vascular smooth muscle migration. Using subcultured (passages 5–9) smooth muscle cells isolated from rat aortae, we found that: 1) Overexpression of wild‐type MCPIP1 abolished IL‐6‐induced smooth muscle cell migration; 2) expression of wild‐type MCPIP1 reduced IL‐6‐induced STAT3 phosphorylation but not the expression level of total STAT3. The aforementioned results suggest that the upregulation of MCPIP1 may serve as a negative regulator in vascular injury‐induced stenosis by inhibition of IL‐6 signaling and subsequent induction of smooth muscle cell migration.

Support or Funding Information

A.T. Still University Warner's grant
Latest reference genome rat strain‐specific variants provide new insight into human diseases
Authors: Tutaj Monika , Smith Jennifer R , De Pons Jeffrey L , Laulederkind Stanley J , Tutaj Marek A , Hayman G Thomas , Wang Shur‐Jen , Petri Victoria , Dwinell Melinda R , Shimoyama Mary E
Published: November 2023

Popularization of DNA sequencing technologies and increasing amount of data generated by researchers demand efficient methods to search and analyze sequence data. As part of a large corpus of genomic, genetic, phenotype and disease data, the Rat Genome Database (RGD) provides information about strain‐specific variants, including copy number variations, single nucleotide variants (SNVs), indels and mutations, generated from whole genome sequencing (WGS) of rat strains used as models for a variety of common human diseases. These variants were originally analyzed against previous rat genomic assemblies (RGSC 3.4 and RGSC 5.0). To bring this up to date, RGD is re‐analyzing available rat strain whole‐genome sequences using the newest version of the rat reference assembly (RGSC 6.0, released in 2014). Difficulties with correct variant identification and developing reliable calling algorithms require extensive validation. To accomplish this, we are developing an analysis pipeline which utilizes state‐of‐the‐art tools for variant calling and effect prediction. In addition, we are expanding our current search tools to include the ability to use the combination of a variant position in the genome with the functional annotations assigned to RGD genes and strains. The combination of improved variant data and disease, phenotype, pathway, function and chemical interaction information will help researchers find appropriate models for disease research and drug testing.

Support or Funding Information

The National Heart, Lung, and Blood Institute of the National Institutes of Health, Grant #R01HL064541
The sigma‐1 receptor modulates methamphetamine‐dysregulated dopamine neurotransmission
Authors: Sambo Danielle , Lin Min , Richardson Ben , Jagnarine Darin , Madhurt Shetty , Owens Anthony , Febo Marcelo , Henry Keith , Daws Lynette , Bruijnzeel Adriaan , Khoshbouei Habibeh
Published: November 2023

Dopamine neurotransmission is highly compromised by psychostimulants such as methamphetamine, a substrate for the dopamine transporter (DAT). The mechanistic aspects of methamphetamine's interactions with DAT are well understood; however, little is known about the molecular actions of methamphetamine once it enters dopaminergic neurons. Methamphetamine is also known to interact with the sigma‐1 receptor (σ1R), an inter‐organelle signaling modulator located at the endoplasmic reticulum. Using complimentary experimental strategies we have identified a novel mechanism for σ1R regulation of dopamine neurotransmission in response to methamphetamine. Single neuron physiology revealed that treatment with the selective σ1R agonist PRE‐084 prevented methamphetamine‐induced, DAT‐mediated increases in the spontaneous firing activity of dopamine neurons. In addition, σ1R activation reduces methamphetamine‐induced DAT‐mediated currents, which have previously been linked to the actions of methamphetamine. Simultaneous patch‐clamp and amperometry and in vivo chronoamperomtry revealed σ1R agonist treatment decreased methamphetamine‐stimulated dopamine efflux without affecting basal dopamine neurotransmission. In vivo studies indicate that σ1R agonist treatment decreased methamphetamine‐stimulation of acute psychomotor responses, motivated behavior as measured by conditioned place preference, and methamphetamine‐induced reductions in reward thresholds as measured by intracranial self‐stimulation. Notably, this study revealed that the σ1R interacts with DAT. This interaction provides evidence for a functional link between endoplasmic reticulum and plasma membrane via a Ca2+‐dependent mechanism. Broadly, these data provide evidence for σ1R regulation of dopamine neurotransmission and the potential therapeutic use of drugs that target the σ1R for the treatment of methamphetamine addiction.

Support or Funding Information

DA026947, NS071122, OD020026, DA026947S1, University of Florida McKnight Brain Institute Fellowship
Systemic Oxytocin Attenuates Methylphenidate Self‐Administration and Potentiates Its Effects on Dopamine in the Nucleus Accumbens Shell in Rats
Authors: Tanda Gianluigi , Rohn Matthew C.H. , Newman Amy H. , Coggiano Mark , Zanettini Claudio , Katz Jonathan L. , Leggio Lorenzo , Lee Mary R.
Published: November 2023

Clinical and preclinical reports have shown that the hypothalamic neurohormone oxytocin plays a role in social bonding, a behavior that is also controlled by the neurotransmitter dopamine. Dopamine also plays an important role in both natural and pathologic behaviors related to reward and reinforcement, including substance use disorders. Dopamine is especially relevant for psychostimulant used is orders, and early reports have shown that oxytocin treatments might interfere with psychostimulant behavioral effects. Several recent studies have focused on oxytocin effects in models of reward or drug abuse in order to understand whether this neuropeptide interferes with pathological compulsive behaviors. Thus, oxytocin has been tested as a potential therapy for alcohol and psychostimulant use disorders. In the present study, the effects of oxytocin were assessed in Sprague‐Dawley rats: 1) trained to self‐administer methylphenidate intravenously (1 mg/kg, fixed ratio 5) during single daily 1‐hr sessions, 2) implanted with microdialysis probes to assess methylphenidate(0.1–1 mg/kg, i.v.) effects on extracellular levels of dopamine in the nucleus accumbens, a brain area related to reward and reinforced behavior, and 3)placed in infrared activity monitors to measure behavioral activation.

Injections of oxytocin (0.1–2mg/kg, i.p.) 10 min before the sessions dose‐dependently decreased maximal self‐administration of methylphenidate (0.03–1.0 mg/kg/injection, i.v.). Though, oxytocin (0.2–2 mg/kg, i.p.) had little to no significant effects on extracellular levels of dopamine in the accumbens shell, oxytocin dose‐dependently enhanced the dopamine levels stimulated by intravenous methylphenidate (0.1–1 mg/kg, i.v.). Notably, this enhancement of dopamine levels did not result in stimulation of behavioral activities at levels higher than those produced by methylphenidate alone.

The present results suggest that oxytocin attenuates the psychostimulant‐like reinforcing effects of methylphenidate, likely acting through altered dopamine neurotransmission in the terminals of the mesolimbic system. Thus, these results confirm and extend the potential translational therapeutic value of oxytocin for psychostimulant use disorders.

Support or Funding Information

Research Funded by:NIDA‐IRP, NIH/DHHS; Bench‐to‐Bedside Grant by the OBSSR/NIH; Intramural Grant ZIAAA000218‐01 (CPN Section) DICBR/NIAAA/NIH/DHHS.
Roles for Type‐2 Cannabinoid Receptors in the Retrieval and Retention of Fear Memory in Mice
Authors: Duphare Chandni , Li Yong , Kim Jimok
Published: November 2023

Demand for medical marijuana is rising, but psychotropic harm is the major problem for its clinical use. Harnessing only beneficial effects of cannabis is critical for its therapeutic application, but has been difficult because only one type of cannabinoid receptor—CB1R—is known to be expressed in the brain and mediate both beneficial and detrimental effects of cannabis. Recent discoveries of type‐2 cannabinoid receptors (CB2Rs) in the brain challenge the status of CB1Rs as the only cannabinoid receptor in the nervous system, and require the determination of medical benefits, if any, of CB2Rs. CB2R knockout mice display reduction in fear memory, suggesting that CB2Rs might have therapeutic potential for fear memory management. We hypothesize that pharmacological blockade of CB2Rs might modulate aversive fear memory in mice. First, the effect of CB2R blockade on fear memory retrieval was tested. C57BL/6J mice were injected with the CB2R antagonist SR144528 (1 mg/kg; i.p.) or vehicle once a day for 2 weeks. After the drug treatment, the mice were fear‐conditioned with foot shocks and tones. Contextual and cued fear memory was assayed from freezing behavior 24 hours after the conditioning. SR144528 had little effect on the retrieval of contextual or cued fear memory. Second, the effect of CB2R blockade on fear memory retention was examined. Drug‐naive C57BL/6J mice were fear‐conditioned, and then, daily treated with SR144528 (1 mg/kg; i.p.) or vehicle for 1 week. After the 1‐week treatment, the mice were tested for fear memory. The post‐conditioning treatment with SR144528 increased cued, but not contextual, fear memory. In summary, our data indicate that pharmacological blockade of CB2Rs after, but not before, adverse experience elevates the retention of hippocampus‐independent fear memory. Unveiling more detailed roles of CB2Rs in modulating fear memory will provide insights into the cannabinoid‐mediated treatment of aversive memory in conflict‐affect individuals.

Support or Funding Information

This work was supported by the Medical Scholars Program of Medical College of Georgia, Augusta University.
Regulation of α2A Adrenergic Receptor Trafficking and Signaling by Amyloid Precursor Protein
Authors: Gannon Mary , Zhang Fang , Chen Yunjia , Wang Qin
Published: November 2023

The pathogenic effect of the amyloid precursor protein (APP) cleavage product Aβ in Alzheimer's disease is well established. However, less is known about the physiological function of APP outside of the disease state. While it has been implicated in several functions, including neuronal development, intracellular transport, and neuronal homeostasis, less is known about its ability to modulate GPCRs. In this study, we identified a novel direct interaction between APP and the α2A Adrenergic Receptor (α2AAR) that occurred at the intracellular domains of both proteins. The APP interaction with α2AAR was promoted by agonist binding and competed with arrestin binding to the receptor. Consistent with reduced arrestin interaction with α2AAR in the presence of APP, agonist driven internalization of α2AAR was reduced, and desensitization of α2AAR‐induced ERK1/2 activation was also attenuated. Furthermore, in cells overexpressing APP, a lesser extent of receptor downregulation was observed at 4, 12, and 24 hours following treatment with norepinephrine. Our study suggests that APP, as a novel interacting partner of α2AAR, stabilizes α2AAR at the cell surface and prolongs its signaling. Given the important role of α2AAR in the noradrenergic system, this novel regulation of α2AAR by APP may have an impact on modulation of noradrenergic tone in the brain.
Improving Cellular Uptake and Endosomal Targeting of Neurokinin 1 Receptor Antagonists
Authors: Veldhuis Nicholas A , Mai Quynh N , Conner Josh N , Graham Bim , Halls Michelle , Lieu TinaMarie , Bunnett Nigel W
Published: November 2023

Intro

G protein‐coupled receptors (GPCRs) are cell‐surface sensors that control most pathophysiological processes. Following stimulation, many activated receptors redistribute to endosomes and evidence suggests that endosome‐localized receptors generate signals that are distinct from cell surface signaling outputs. Importantly, these endosomal signals underlie physiologically relevant cellular processes. We have recently observed that Substance P‐mediated endosomal signalling from the Neurokinin 1 Receptor (NK1R) is associated with central pain transmission and hence, the selective inhibition of NK1R within endosomes could be therapeutically advantageous.

Methods and Results

We have synthesized a series of membrane anchored drug conjugates including Cyanine‐5 fluorophore conjugated to spantide (Span‐Cy5), cholestanol (Cy5‐Chol), spantide and cholestanol (Cy5‐Span‐Chol), or a PEG‐ethyl ester control (Cy5‐EE). Cellular uptake, membrane association and ligand‐receptor interactions were determined by live cell confocal microscopy and fluorescence correlation spectroscopy. Biological activity of these constructs was determined by ERK functional assays. These data demonstrate that cholestanol membrane anchors enhance cellular uptake, membrane‐localized drug concentrations and endosomal distribution of fluorescently labelled, lipid‐conjugated compounds. Furthermore, these compounds can interact with the NK1R and demonstrate improved analgesic potential in pre‐clinical pain models, relative to their non‐targeted counterparts.

Support or Funding Information

Funding support for the National Health and Medical Research Institute and the ARC Centre of Excellence for Bio‐Nano Science and Technology
Selective Activation of T cell S1P Receptor 1 Attenuates the Expression of TGF‐beta1 from Fibroblasts
Authors: Lor Paia , Le Helen , Talbott George , Jin Zhuqiu
Published: November 2023

Infiltration of T cells in patients with myocardial infarction is associated with increased heart attack and fibrosis. Our previous work indicated that conditional knockout of T cell S1P receptor 1 (S1P1) renders mouse hearts vulnerable to fibrosis. It suggests that activation of S1P1 in T cells may attenuate the fibrotic pathway in fibroblasts. In this study, SEW2871 (1 μM), a selective S1P1 agonist, was added into medium of NIH3T3 fibroblasts alone and medium of co‐cultured Jurkat CD4 T cells and NIH3T3 fibroblasts for 48 hours. Both cell number counting and cell survival ability were determined. The expression of TGF‐beta 1 in fibroblasts was determined with Western blotting. After 48 hours, SEW2871 enhanced the expression of TGF‐beta 1 in fibroblasts. However, SEW2871 decreased the expression of TGF‐beta1 in fibroblasts co‐cultured with CD4 T cells. After pretreatment with W146, the selective antagonist of S1P1, the expression of TGF‐beta1 in fibroblasts and fibroblasts co‐cultured with T cells remained no changes. To explore the mechanism, S1P (1 μM) was added into CD4 T cell‐containing RPMI 1640 medium supplemented with charcoal‐stripped albumin for 1 hour. Cytoplasmic, membrane, nuclear (soluble and chromatin –bound), and cytoskeleton fractions of T cells were isolated and purified. S1P induced translocation of S1P1 from membrane to nuclear fraction. W146 abolished this translocation. These results suggest that activation of fibroblast S1P1 plays a pro‐fibrotic effects whereas activation of CD4 T cell S1P1 is anti‐fibrotic. Translocation of S1P1 from plasma membrane to nucleus in T cells may have effect in reduction of expression of TGF‐beta1.
Regulation of the Sigma‐1 Receptor Quaternary Structure by Ligand Binding
Authors: Hong W. Conrad
Published: November 2023

Since the initial proposal of sigma receptors four decades ago, extensive studies have shown that the sigma‐1 receptor (σ1R) is a unique membrane protein that may serve as a molecular chaperone. In earlier attempts to purify sigma receptors from native tissues using [3H](+)SKF10047 or [3H]azido‐DTG as affinity ligands, the labeled protein complexes appeared to be > 150 kDa under non‐denaturing conditions. Recent breakthrough on the crystal structure of σ1R revealed a homotrimer, with each protomer containing a single transmembrane domain and a cytoplasmic ligand‐binding domain. Whether ligand binding can affect the quaternary structure of σ1R has not been fully investigated. We devised a novel biochemical method to examine the multimeric status of σ1R. Epitope‐tagged σ1R from transfected cells was solubilized using mild detergents, electrophoretically separated under non‐denaturing conditions, and detected by immunoblotting. The majority of σ1R appeared as high molecular weight (MW) bands (apparent MW > 132 kDa), suggesting a high‐order quaternary organization of multimers. Pretreatment with σ1R agonists, such as (+)‐pentazocine and DTG, decreased σ1R high‐MW bands, while increased σ1R lower‐MW bands that were presumably dimers and monomers. Conversely, pretreatment with σ1R antagonists, such as BD1063 and haloperidol, exhibited opposite effects on σ1R multimerization. These results demonstrate that the quaternary structure of σ1R is regulated by binding of agonists or antagonists in a distinct manner. As σ1R has been shown to alter the function of various membrane proteins through dynamic interactions, we hypothesize that σ1R agonists promote the formation of σ1R monomers and facilitate their modulation of interacting partners.

Support or Funding Information

Butler University Faculty Startup Fund and Holcomb Research Award.
The small GTPase Rab43 is a novel regulator of G protein‐coupled receptor trafficking
Authors: Li Chunman , Huang Wei , Wu Guangyu
Published: November 2023

Ras‐like small Rab GTPases are master regulators of vesicle‐mediated membrane traffic. Recent studies have identified several Rab GTPases involved in the anterograde cell surface transport of newly synthesized G protein‐coupled receptors (GPCRs). Here, we have screened the Rab GTPase family using stable cells expressing α2B‐adrenergic receptor (α2B‐AR), a prototypic GPCR, and demonstrated that Rab43 is a novel regulator of GPCR trafficking. Attenuation of Rab43 function, via expression of its dominant‐negative mutants, siRNA‐mediated depletion, and expression of its GTPase‐activating protein RN‐tre, significantly attenuated the cell surface expression of α2B‐AR and induced an extensive accumulation of the receptor in the endoplasmic reticulum (ER). The studies using inducible α2B‐AR‐expressing cell lines and a previously characterized Golgi‐localized α2B‐AR mutant, in combination with bredeldin A treatment/washout assay, further showed that Rab43 controlled the ER export rate and content of nascent receptors. More interestingly, in addition to α2B‐AR, the cell surface transport of a number of other GPCRs, but not VSVG and EGF receptor, was regulated by Rab43. Furthermore, Rab43 was shown to directly interact with α2B‐AR, specifically its third intracellular loop, in an activation‐dependent fashion, but it was unable to associate with the tethering factor protein p115, a downstream effector of Rab1 GTPase which is well‐known to control the ER‐Golgi traffic. Altogether, these data demonstrate a novel Rab43‐mediated, as yet undefined pathway for members of the GPCR family to move forward from the ER to the Golgi.

Support or Funding Information

R01GM118915
When the Tail Wags the Dog: Phosphodiesterase 4D7 Regulation of a cAMP‐signalosome Located at the Rear of Migrating Smooth Muscle Cells Regulates Localized RhoA/ROCK Signaling and Tail Retraction During Migration
Authors: Maurice Donald H , Truong Tammy , Wudwud Alie
Published: November 2023

Inhibiting the maladaptive migration of human arterial smooth muscle cells (HASMCs) in injured blood vessels may help reduce the development of intimal lesions in vascular diseases, including atherosclerosis or restenosis. Agents that act to increase intracellular levels of cyclic adenosine monophosphate (cAMP) impact numerous individual events involved in coordinating HASMC migration. Signaling via cAMP is ubiquitous and dynamic, and requires control through signaling termination mechanisms. cAMP Phosphodiesterase (PDE) enzymes catalyze the hydrolysis of cAMP and are critical in the control of cAMP signaling, most specifically when they regulate cAMP levels within numerous discrete subcellular domains. Through spatial restriction, these enzymes are thought to allow cAMP‐elevating agents to selectively regulate numerous distinct responses. The PDE4D gene family plays a dominant role in cAMP hydrolysis in HASMCs. Considerable interest exists in the idea that the unique N‐terminal domains of the numerous distinct PDE4D isoform variants promotes their spatial restriction in cells, and that this is coordinated through selective interactions of these PDE4Ds with other proteins. This study examined the targeting of individual PDE4D isoforms in HASMCs through the overexpression of GFP constructs containing the unique N‐terminal domains of the PDE4D isoforms (NT‐PDE4D/GFP) known to be expressed endogenously in these cells (PDE4D8, 9, 5, 7). Although transient over‐expression of NT/PDE4D8‐GFP, NT/PDE4D9‐GFP or NT/PDE4D5‐GFP did not significantly impact HASMC morphology or migration character or speed, over‐expression of NT/PDE4D7‐GFP was observed to alter HASMC morphology of migrating HASMCs as well as their migratory velocity. By combining these observations with the effects of selective siRNA‐mediated knockdown of PDE4D7, we established that PDE4D7 is involved in controlling rear retraction in migrating HASMCs. We show that altering PDE4D7 targeting and expression in these cells affects rear retraction largely through its ability to impact RhoA‐ROCK signaling. We also report that PDE4D7 interacts with AKAP5, PKA and EPAC1 in migrating and immotile HASMCs. Our results suggest a functional role for localized PDE4D7 activity in regulating cAMP‐mediated rear retraction in migrating HASMCs, and identify PDE4D7 as a potential therapeutic target in controlling HASMC migration.

Support or Funding Information

Canadian Institutes of Health Sciences
Role of MitoBK Channels in Renal Cold Preservation
Authors: Shrum Stephen , Parajuli Nirmala , Rusch Nancy J. , MacMillan‐Crow Lee‐Ann
Published: November 2023

Patients with end‐stage renal failure require kidney transplantation to avoid life‐long hemodialysis associated with high mortality. Kidneys from brain‐dead or deceased donors are placed in cold storage (CS) preservation solutions until a recipient is identified. However, kidneys exposed to CS solutions are subjected to extended ischemia, and our laboratory reported that renal CS induces oxidative stress and subsequent mitochondrial damage. Thus, there is a critical need to develop strategies to protect donor kidneys from CS ‐induced damage to improve long‐term renal function in the transplant recipients. In this regard, the high‐conductance Ca2+‐activated K+ (BK) channel recently was discovered in the mitochondrial inner membrane (mitoBK) of cardiomyocytes and other cell types. When activated, the mitoBK channel reportedly protects against ischemia‐reperfusion injury in the heart by reducing mitochondrial damage and reactive oxygen species (ROS). We show, for the first time, that the mitoBK channel is expressed in the mitochondria of rat renal proximal tubular cells in vitro and in rat kidney in vivo. Our preliminary evidence suggests that CS reduces expression of the pore‐forming a subunit of the mitoBK channel in rat renal proximal tubular cell (n=3) and rat kidney (n=4) whole‐cell lysates. Thus, we hypothesize that: MitoBK channels down‐regulate in response to CS‐induced mitochondrial damage, contributing to donor kidney injury. We also propose that restoring mitoBK channel function will provide protection from CS ‐induced injury and improve transplanted kidney function. Our preliminary studies using High Resolution Respirometry (HRR) with fresh kidney biopsies show that Complex I, II, and III (but not Complex IV) activity was significantly impaired in rat kidneys exposed to CS for 18 hours. Twenty‐four hours after renal transplant, recipients of kidneys exposed to 18 hours of CS showed elevated serum creatinine (4.4 mg/dl, n=6) compared to rats having undergone only sham surgery (0.3 mg/dl, n=5). Blood urea nitrogen also was elevated in transplant recipients of CS kidneys (CS + transplant, ~140 mg/dl; sham, ~20 mg/dl). These indicators of impaired renal function correlated with loss of mitoBK protein in CS kidneys. Ongoing patch‐clamp studies are evaluating the properties and function of mitoBK channels in our cell and animal models of renal CS, and evaluating whether pharmacological activation of mitoBK channels during CS can attenuate ROS, limit mitochondrial injury, and improve the function of transplanted kidneys.

Support or Funding Information

Funded by AHA Predoctoral Fellowship #16PRE30830010 and UAMS pilot grant G1‐51454‐99.
Muscle Nicotinic Acetylcholine Receptors containing α1‐V275 or α1‐Y233 Mutants found in a functional Screen reveal enhanced Agonist Potencies
Authors: Siehler Sandra , Ferrand Sandrine , Kelley Nicholas , Bill Anke , Freuler Felix , Imbert Pierre‐Eloi , Marelli Anthony , Gerwin Nicole , Sivilotti Lucia G , Miraglia Loren , Orth Anthony P , Oakeley Edward J , Schopfer Ulrich , Groot‐Kormelink Paul J
Published: November 2023

The adult form of the human muscle nicotinicacetylcholine receptor (nAChR) consists of four different subunits forming apentamer around the channel pore, (α1)2β1δɛ. The two orthosteric binding sites are located at the α1‐ɛ and α1‐δ interfaces. A library of 2816α1 subunit mutants was created using high throughput mutagenesis by error‐prone PCR and sequences of all mutants were determined using single molecule real timesequencing. Each α1 mutant was transiently transfected with wildtype β1, δ, and ɛ subunits into HEK293 cells, and the channel function examined in an ion flux assay. Mutant hits were selected that responded to the nicotinic agonistepibatidine, but were resistant to the orthosteric blockers α‐bungarotoxin ortubocurarine. Eight α1 subunit mutant hits were discovered, six of which contained mutations at Y233 or V275 in the transmembrane domain. Three single point mutations (Y233N, Y233H, and V275M) were functionally characterized, and found to enhance the potencies of five channel agonists tested. This suggests that the mutations do not impair blocker binding, but rather evoke again‐of‐function phenotype, e.g. increased agonist sensitivity. Immunocytochemical staining showed that the surface expression of mutated α1subunits was comparable to the wildtype protein. Functional analysis of the mutant library containing alterations for each of the 457 amino acids of the human α1 subunit therefore yielded structure‐function insights for the nAChR.

Support or Funding Information

This work was supported by the Novartis Institutes for BioMedical Research (NIBR).
Computational approaches to understanding activation of the angiotensin II type 1 receptor
Authors: Anderson Shane D , Botello‐Smith Wesley M. , Luo Yun Lyna , Andresen Bradley T.
Published: November 2023

The angiotensin II type 1 receptor (AT1R) is a major player in the cardiovascular system and is involved in volume and salt homeostasis. Multiple models of the AT1R have been made to predict molecular function; however, the previous models were invalidated by the crystallization of the AT1R bound to ZD 7155 (4ZUD) as well as the inverse agonist olmesartan (4YAY). Molecular dynamics models of receptors have a benefit as they allow dynamic interrogation of structure‐activity relationships and aid in designing novel therapeutics. Therefore, the aim of this project is to generate models of the empty and active human AT1R to determine structural changes involved in receptor activation. To produce a model of the empty AT1R, olmesartan and BRIL were removed from 4YAY, and the missing loops and the amino‐terminus were added to the structure. This model was inserted into an 87% POPC and 13% cholesterol membrane and simulated using AMBER16 for 150 ns. Atomic fluctuation measurements from the 150ns simulation revealed extensive movements within the helices as well as the loops; generally, the empty AT1R relaxed resulting in increased distances between helixes. Residue alpha carbons were measured between 4YAY and the stable empty AT1R structure. There were multiple changes including mean changes at Tyr351.39 (1.9 Å), Arg167ECL2 (2.1 Å), Ser189ECL2 (3.7 Å), Val2646.59 (4.4 Å), Gln2676.62 (4.6 Å), Iso2884.38 (1.9 Å), and Phe3047.55 (4.7 Å) and Phe3138.54 (9.2 Å). These changes are presumed to reflect the induced fit structure of olmesartan‐bound AT1R. A constitutively active AT1R (CA‐AT1R) was constructed from the stable empty AT1R by generating N111G AT1R followed by a 300 ns MD simulation. Major changes between the empty and CA‐AT1R occurred with residues Tyr351.39 (1.4 Å), Trp2195.62 (2.7 Å), Leu2977.48 (2.9 Å), and Phe3138.54 (9.5 Å). Intramolecular alpha carbon measurements between the models resulted in displaying changes specific to olmesartan binding such as Ser189ECL2 and Ala1634.61 to Gln2676.62: 6.0 Å and 18.2 Å, respectively, for olmesartan bound compared to 10.7 Å and 14.5 Å for the empty AT1R and 10.4 Å and 14.6 Å for the CA‐AT1R. Conversely, there are changes unique to CA‐AT1R structure deeper into the AT1R such as Ser2526.47 to Iso2907.40 and the salt bridge between NH1 Arg1263.50 and O− Arg2346.29: 8.6 Å and 10.4 Å for CA‐AT1R, respectively, compared to 6.7 Å and 8.6 Å for olmesartan bound and 6.7 Å and 2.7 Å for the empty AT1R. Future directions of this project include docking angiotensin II to the empty and CA AT1R as well as the biased agonist [Sar1‐Ile4‐Ile8]‐angiotensin II to the empty AT1R and comparing the conformational changes.

Support or Funding Information

This project was funded by internal WesternU funds.
A novel regulatory role of RGS4 in δ‐opioid receptor mediated neuronal outgrowth and differentiation
Authors: Georgoussi Zafiroula‐Iro , Pallaki Paschalina , Serafimidis Ioannis , Symeonof Alexandra , Sofia Koutloglou , Papakonstantinou Maria , Georganta Eirini , Thomaidou Dimitra , Gaitanou Maria
Published: November 2023

Regulators of G proteins signaling (RGS) despite their initial role as GTPase activating proteins, serve several cellular functions varying from tolerance, dependence, neuroprotection, transcription to tumorgenesis. Evidence suggests the cytoplasmic/nuclear distribution and translocation of RGS proteins is mediated by interactions with other proteins which mediate cross talk between G proteins and other cellular processes. RGS4 a member of the B/R4 family of RGS proteins has been found to be a universal negative regulator of μ, δ and κ‐opioid receptor signaling that confers selectivity for G protein coupling to these receptors by directly interacting with them (Georgoussi et al., 2006, 2012; Leontiadis et al. 2009; Papakonstantinou et al., 2015). It was also shown that δ‐opioid receptor (δ‐OR) forms a multicomponent signaling complex, consisting of Gi/Go proteins and the Signal Transducer and Activator of Transcription STAT5B, that leads to neurite outgrowth and neuronal differentiation via a phosphoryalted‐STAT5B‐Gαi/o pathway (Georganta et al., 2010, 2013). Knowing that RGS4 is a multitask protein we questioned whether RGS4 could be implicated in neuronal differentiation and neurite outgrowth upon δ‐OR activation through a similar signaling pathway. We demonstrate that RGS4 directly interacts with STAT5B in vitro and in living cells independently of δ‐OR presence. This interaction involves the N‐terminal portion of RGS4 and the DNA‐binding‐SH3 domain of STAT5B. Expression of RGS4 in HEK293 cells expressing δ‐OR and/or erythropoietin receptor results in inhibition of [D‐Ser2, Leu5, Thr6]‐enkephalin (DSLET) and erythropoietin‐dependent STAT5B phosphorylation and subsequent transcriptional activation. Measurements of DSLET‐dependent neurite outgrowth of neuroblastoma cells is also blocked by RGS4 expression, whereas primary cortical cultures of RGS4 knock out mice (RGS4−/−) exhibit enhanced neuronal sprouting after δ‐OR activation. Finally, neuronal progenitor cultures from RGS4−/− mice exhibit enhanced proliferation with concomitant increases in the mRNA levels of the anti‐apoptotic genes Bcl2 and Bcl‐xl STAT5B target genes. These observations suggest that RGS4 plays a significant regulatory role in opioid dependent neuronal differentiation and neurite outgrowth via a “non‐canonical” signaling pathway involving STAT5B‐directed responses.

Support or Funding Information

This work was supported by the Excellence II‐3722, (“NO‐ALGOS”) of the Hellenic General Secretariat of Research and Technology and was carried out in facilities of the OPENSCREEN‐GR National (Greece) Research Infrastructure. Z.G participates in the EU COST Action CM1207 (GLISTEN).
The Role of Bitter Taste Receptors (T2Rs) in Cystic Fibrosis
Authors: Jaggupilli Appalaraju , Singh Nisha , Robert Renaud Sylvain , Hanrahan John , Duan Kangmin , Chelikani Prashen
Published: November 2023

Introduction

Bitter taste receptors (T2Rs) belong to the G protein‐coupled receptor superfamily. In humans, 25 T2Rs perform a chemosensory function, however very little is known regarding their expression and function in extraoral tissues. Recent studies showed that T2Rs recognize quorum‐sensing molecules (QSMs) secreted by bacteria. Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). The opportunistic pathogen Pseudomonas aeruginosa colonizes CF airways and secretes QSMs, which increase intracellular calcium and modulate innate immune responses through unidentified cell surface receptors. We hypothesized that T2Rs are expressed in epithelial cells of human airways, where they mediate QSM responses and modulate immune responses in CF.

Methods and Results

We monitored the expression of T2Rs in primary CF and non‐CF bronchial epithelial cells. Cells were studied at both the mRNA and protein levels using qPCR and FACS analysis, respectively. We found similar expression of T2Rs in non‐CF and CF cells. Next we treated these cells with a number of QSMs and measured the intracellular calcium mobilized. We found that the calcium mobilized was sensitive to the PLC inhibitor U‐73122 suggesting that the signaling in these cells occurs predominantly through the QSM‐T2R‐Gαβγ‐PLC pathway. To further characterize the candidate T2Rs that may recognize specific QSMs and modulate signaling in CF, a heterologous expression system was used to stably express selected T2Rs and study the effects of QSMs. We found that a single QSM (for example, 3‐oxo‐C12‐AHL) can activate more than one T2R, and that multiple QSMs can activate the same T2R.

Conclusions

T2Rs are expressed in bronchial epithelial cells and are functional. Although expression was not altered in CF cells, these results suggest there is a synergistic effect of QSMs on T2R signaling in CF airways and provides a rationale for further research on the pathophysiological function of T2Rs in extraoral tissues. These mechanistic studies should improve our understanding of host response mechanisms and may lead to new therapeutic approaches for CF that target T2Rs.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada (NSERC)
Inhibition of soluble and membrane‐bound neutrophil elastase by inhibitor POL6014
Authors: Korkmaz Brice , Hamon Yveline , Guarino Carla , Epinette Christophe , Gauthier Francis , Sellier‐Kessler Odile , Chevalier Eric
Published: November 2023

Neutrophil elastase (NE) is involved in the degradation of extracellular matrix molecules making it an attractive target for the development of anti‐inflammatory compounds in neutrophilic pulmonary diseases. It is mainly secreted extracellularly by activated neutrophils at inflammatory sites but a minor fraction is translocated to the membrane where it remains proteolytically active. We investigated the properties of the novel Protein Epitope Mimetic (PEM) POL6014, a medium sized fully synthetic macrocycle, towards soluble and membrane‐bound NE. Circulating human neutrophils from healthy volunteers were purified and used within 3 hours after activation from Calcium ionophore (A23187) as source of membrane bound NE. The enzymatic reaction using the Fluorescence Resonance Energy Transfer (FRET) Technology was started by addition of a specific human NE substrate (ABZ‐APEEIMRRQ‐EDDnp, 13 μM final) 5 minutes after incubation of POL6014 with human NE. We found that POL6014 inhibits membrane‐bound human NE and soluble human NE with similar Ki values in the sub‐nanomolar range (0.69 ± 0.31 nM (n=7) and 0.53 ± 0.37 nM (n=3), respectively). This means that POL6014 could inhibit human NE stoichiometrically in pathophysiological conditions so that no molar excess of inhibitor would be required to control NE activity. In addition we evaluated POL6014 activity on rat and non‐human primate (NHP) NE in order to qualify these species for in vivo testing. Lysates of circulating neutrophils from both species were used as source of NE. Species selective FRET substrates (ABZ‐QPMAVVQSVPQ‐Yno2 for rodent, ABZ‐APQQIMDDQ‐EDDnp for primate) were used to determine the inhibitory potency of POL6014. We found that POL6014 is also a potent low nanomolar inhibitor with IC50 = 1.0 ± 0.5 nM (n=4) against rat NE and IC50 = 2.2 ± 1.4 nM (n=3) against Cynomolgus (Macaca fascicularis) NHP NE, which makes these species appropriate to test the in vivo efficacy and toxicology of POL6014. In conclusion, POL6014 showed potent inhibition of soluble and membrane‐bound human NE and similar potency across human, rat and NHP.

Support or Funding Information

This work was supported by Polyphor Ltd.
Investigating novel small compounds targeting the Laminin Receptor for their effect on modifying immune and stem cell viability and differentiation
Authors: Loperena‐Medina Sue Heidi , Neto Manoel Figueiredo , Umbaugh Charles Samuel , Haffner Abigail , Figueiredo Marxa Leao
Published: November 2023

Osteoarthritis affects over 27 million Americans age 25 and older and is a debilitating disease in which articular cartilage degradation leads to inflammation and pain in the joint. Medical management has not been very successful, as cartilage is avascular and has a difficult time regenerating itself. In joints, there are laminin receptors (LR) which bind to PEDF (pigment epithelial derived factor). When PEDF is bound to a LR, it has anti‐inflammatory, pro‐chondrogenic and anti‐angiogenic characteristics. However, the relatively large size and low stability of PEDF at 25°C indicates that smaller, more stable molecules could be an alternative for therapeutic purposes. We developed seven small compounds that bind to the PEDF docking zone at the LR, with the goal to mimic the effects of PEDF. In order to test our drugs in a pro‐inflammatory environment, THP‐1 macrophage cultures were stimulated with lipopolysaccharide. Quantitative real‐time polymerase chain reaction (qPCR) was used to assess up‐ and down‐regulation of inflammatory genes, including IL‐1β. Compound 3 (C3) appears to be the best PEDF mimic as it reduced IL‐1β expression by ~8‐fold. A cell pellet chondrogenesis assay using adipose‐derived stem cells (ASC) was used to examine the effect of compounds on upregulating cartilage‐specific genes at day 8 of differentiation. In the future, ASC pellets will be harvested at differentiation days 7, 14 and 21 to determine the timing of cartilage‐specific gene expression following treatment with the small compounds. From our results, C3 appears to be the most promising drug for future development since it is able to reduce expression of pro‐inflammatory gene IL‐1β and promote ASC to express cartilage‐specific genes. Additionally, C3 also appears to alter phenotype of cancer cells towards antitumor activity in several assays including scratch, endothelial tube, and proteomic assays. Future studies will continue to investigate C3 and a series of small molecule analogs designed and synthesized in collaboration with a medicinal chemistry laboratory.

Support or Funding Information

CTSI Core Facility Award, Showalter Research Foundation Full Proposal Award, Merial Veterinary Scholars Program, and start up funds from the BMS department.
Defining the role of different MAPK signaling pathways following APAP and AMAP exposure
Authors: Nguyen Ngoc Uy , Valeu Lynn , Stamper Brendan
Published: November 2023

Objective

The objective of this study was to better characterize the role of mitogen‐activated protein kinase (MAPK) activation in acetaminophen (APAP) toxicity using structure‐toxicity relationships with acetyl‐m‐aminophenol (AMAP). Both c‐Jun N‐terminal kinase (JNK) and extracellular signal regulated kinase (ERK) signaling were investigated.

Methods

Dose‐dependent toxicity assays were performed in an in vitro liver model (TGF‐a transgenic mouse hepatocytes; TAMH) to determine non‐toxic concentrations of the following MAPK modulators: ERK inhibitor (FR‐180204), ERK inducer (Menadione – MEN), JNK inhibitor (JNK‐IN‐8, SP600125, Leflunomide ‐ LEF), and JNK inducer (cycloheximide – CHX). Protein immunoblotting was then used to confirm MAPK activation. Pretreatment with modulators was then coupled to toxicity assays with APAP and AMAP to determine their effect on cell viability. Quantification of additional protein biomarkers related to MAPK signaling was also assessed in an attempt to elucidate mechanistic details.

Results

Measured non‐toxic concentrations of modulators were as follows: 10mm for FR‐190204, 2mM for MEN, 2nM for CHX, 50mM for LEF, 1mM for JNK‐IN‐8, and 1mM for SP600125. Time‐based immunoblotting results demonstrated peak modulation by MEN, LEF, JNK‐IN‐8, and SP600125 to occur after two hours, whereas peak CHX‐mediated JNK induction occurred after 24 hours. Induction of either JNK or ERK decreased cell viability when dosed in conjunction with APAP, with no observable effect on AMAP.

Conclusion

Previous studies have established the role of JNK during APAP‐induced hepatocellular injury, whereas the role of ERK is poorly defined. As expected, when dosed with APAP, inducing JNK promoted cell death; however, the induction of ERK also promoted cell death. AMAP, a relatively non‐toxic regioisomer of AMAP was not affected by pretreatment with MAPK modulators suggesting that additional work is needed to clarify the role of ERK in APAP‐induced injury.

Support or Funding Information

Source of Research Support: This work was supported by a Pacific University Research Incentive Grant