bluuebunny/crossref_metadata_2025 huggingface dataset:
Activity transcription factor 3 suppressed LPS‐induced adipocyte inflammation in experimental sepsis
Authors: Wang Yu‐Hsin , Cheng Ching‐Feng , Lai Pei‐Fang
Published: November 2023

Endotoxemia, a state of excessive endotoxin (lipopolysaccharide, LPS) in the circulation, frequently results in fatal sepsis. Activating transcription factor (ATF)‐3 is a member of the ATF/cAMP‐response element‐binding protein family of transcriptional factors. It is also induced by stress condition in a variety of tissues, including the adipose tissue. The aim of this study is to assess the possible protective role of ATF3 in adipose tissue inflammation of LPS‐induced polymorphonuclear leukocyte recruitment in experimental sepsis. ATF3‐knockout (KO) and wild‐type mice (WT) were injected with LPS (5 mg/kg, ip) to induce endotoxemia. Multifaceted approaches including the in vivo animal endotoxemic mice model for examining biochemical analysis, the expression of inflammatory mediators, and pathophysiological examinmation in the adipocytes were investigated. Results demonstrated that LPS enhanced ATF3 protein expression in WT control mice, but not in ATF3‐KO group in the adipocytes. We found that the protein expression of iNOS and the numbers of polymorphonuclear leukocyte were significantly increased in the adipose tissue of KO mice, but not observed in WT mice after LPS induction. Furthermore, restored ATF3 gene expression by transfection of adeno‐associated virus (AAV) in the KO mice, the increased WBC levels, iNOS expression, and LPS‐mediated polymorphonuclear leukocyte recruitment were significantliy suppressed that comparing to the WT mice. These results suggested that ATF3 gene may play a critical role to overcome the adipose tissue inflammation in experimental sepsis.

Support or Funding Information

supported by MOST and Tzu Chi Medical center
Splenic Natural Killer Cell Phenotypes Are Heritable and Correlate with Lifespan
Authors: Bumgardner Sara A , Zhou Yang , Jiang Zixuan , Coe Erica J , Pazdro Robert
Published: November 2023

Natural Killer (NK) cells are cytotoxic innate immune cells integral to the host defense against viruses and neoplastic cells. With age, NK cells undergo immunosenescence, yielding functional impairments in immune surveillance. While numerous mechanisms have been explored to explain NK cell dysfunction with age, it is currently unknown if the aged NK cell phenotype is heritable. In this study, we evaluated conventional splenic NK cell maturation phenotypes, as defined by CD27 and CD11b surface expression, from 15 classical inbred and 4 wild‐derived aged murine strains. We found that both total and mature NK cell proportions, along with their median surface density of CD27 and CD11b, are highly heritable across strains. We additionally identified total splenic NK cell proportions as an independent predictor of median lifespan in mice. When select strains were compared to younger controls, NK cell phenotypes were variably stable across age, with C57BL/10J and A/J strains demonstrating phenotypic evidence of NK cell immunosenescence. These results reveal strong narrow‐sense heritability of murine NK cell phenotypes and intriguing models for future mechanistic and functional NK cell studies.

Support or Funding Information

Research was funded by the University of Georgia Office for the Vice President for Research and the College of Family and Consumer Sciences.
Silica‐Triggered Multi‐Organ Autoimmune Gene Expression in Lupus‐Prone Mice is Ablated by Docosahexaenoic Acid Consumption
Authors: Bates Melissa A. , Gilley Kristen N. , Jackson‐Humbles Daven N. , Harkema Jack R. , Holian Andrij , Pestka James J.
Published: November 2023

While heredity is a primary predisposing factor for autoimmunity, the exposome can critically influence latency and severity of autoimmune diseases such as systemic lupus erythematosus (SLE). We have previously demonstrated that airway exposure to the toxicant crystalline silica (cSiO2) triggers early loss of self‐tolerance in the lung thereby accelerating onset of systemic autoimmunity and exacerbates glomerulonephritis in the female NZBWF1 mouse, a widely used SLE model. Remarkably, dietary supplementation with the omega‐3 polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA), abrogates cSiO2‐induced lung inflammation, systemic autoimmunity and nephritis in this model. We hypothesized here that cSiO2‐triggered inflammation in the lung precedes systemic autoimmunity and nephritis in NZBWF1 mice and is a primary target for the prophylactic action of DHA. Beginning at 6 wk of age, female NZBWF1 mice were fed an isocaloric AIN‐93G diet containing 0.0, 0.4, or 1.0 % DHA. Two wk later, mice were intranasally instilled with 1.0 mg cSiO2 once per week for 4 wk and maintained on experimental diets until sacrifice. At 13, 16, 19 and 22 wk of age, cohorts were sacrificed and lung, spleen, and kidney were collected. Total RNA from these tissues for each cohort was isolated and purified, and gene expression levels were determined with the nCounter® PanCancer Immune Profiling for mouse, a multiplexed method for immune profiling of 800 target genes. cSiO2‐induced elevations in gene expression were first evident in the lung in cohorts from 13 wk and later whereas in spleen and kidney elevations were seen at 16 wk and thereafter. Genes observed to upregulated by cSiO2 included chemotactic factors, cytokines, B cell receptors, and remarkably, genes involved in interferon regulation. Importantly, cSiO2‐triggered expression of these genes was dose‐dependently suppressed by DHA in all three tissues over time. Taken together, marked attenuation cSiO2‐induced elevations of inflammatory and immune genes by DHA in the lung, spleen and kidney suggests that supplementation with this omega‐3 PUFA might be used to mitigate environmental triggering of autoimmune disease.

Support or Funding Information

Lupus Foundation of America, Inc.
Transcriptomic And Proteomic Analysis To Delineate The Mechanism Of Antibiofilm Activity Of 3‐Furancarboxaldehyde On Group A Streptococcus
Authors: Pandian Shunmugiah Karutha , Subramenium Ganapathy Ashwinkumar
Published: November 2023

Group A Streptococcus (GAS, Streptococcus pyogenes), an uncompromising human exclusive pathogen is responsible for various invasive and non‐invasive diseases. The biofilm forming ability of the multi‐virulent pathogen is considered as an important factor for its growth and persistence in the human host. The current study demonstrates the antibiofilm activity of 3Furancarboxaldehyde (3FCA), a compound present in floral honey, against GAS biofilm, and analyses its effect on multiple aspects of GAS (morphology, virulence, aggregation, gene expression and proteomic analysis) at its minimal biofilm inhibitory concentration (132μg/ml). 3FCA was found to modify the growth pattern of GAS in solid and liquid media and enhance the rate of auto‐aggregation. Real Time gene expression analysis showed downregulation of covR gene, which is speculated to be the prime target for the antibiofilm activity. The gel based proteomic and Matrix‐assisted laser desorption/ionization (MALDI) analysis of GAS upon treatment with 3FCA revealed upregulation of proteins such as ABC transporter (ATP‐binding protein) MtsB SPy_0454, putative cell envelope proteinase PrtS SPy_0416, which are speculated to be involved in increased hyaluronic acid capsule production and increased virulence of GAS respectively. Few other proteins which are involved in TCA cycle were also found to be differentially regulated. This study reveals the mechanism of antibiofilm activity of 3FCA at both genomic and proteomic levels.
Inhibition Of Biofilm And Virulence Of Candida albicans By A Marine Bacterial Isolate From Palk Bay
Authors: Subramenium Ganapathy Ashwinkumar , Swetha Thirukkannamangai Krishnan , Pandian Shunmugiah Karutha
Published: November 2023

The yeast Candida albicans is an opportunistic pathogen in the commensal human micro flora. The pathogen is considered as a chief causative agent of candidiasis, a very common fungal infection in human. The yeast to hyphal transition and ability to form hypoxic biofilm on medical devices is well allied with virulence and antifungal resistance of C. albicans. Antagonistic agents targeting biofilm formation is the current necessity to turn down the recalcitrant biofilm cells, vulnerable to antifungal treatment. Thus, the present study is aimed at inhibiting biofilm formation and other virulence of C. albicans with a compound of microbial source. Initially, the biofilm inhibitory potential of Bacillus subtilis (Genbank Acc. No. KC433737), a marine bacterial isolate from Palk Bay was assessed. The ethyl acetate (EA) extract of B. subtilis showed potent anti‐biofilm activity, inhibiting 90% of biofilm cells approximately. Mass‐spectrometric analysis of EA extract of B. subtilis unveiled, a furan class compound as a major component of the extract. In furtherance, the anti‐biofilm potential of pure compound was evaluated at increasing concentrations, which showed 85% biofilm inhibition at 400 μg/mL concentration without affecting the growth. The non‐antifungal activity of both, the extract as well as compound was determined using broth micro dilution method. Light microscopic, confocal laser scanning microscopic and scanning electron microscopic images of control, EA extract and compound treated samples evidenced significant reduction in the thickness of biofilm. In vitro bioassays revealed the potential of compound to reduce other virulence factors of C. albicans such as production of lipase, protease, ergosterol and yeast to hyphal transition. The increase in susceptibility of compound treated C. albicans to antifungals was demonstrated through well‐diffusion assay. In addition, Fourier transform infrared analysis delineated the difference in carbohydrate moiety of exopolysaccharide (EPS) upon treatment. qPCR analysis showed differential regulation of genes responsible for ergosterol production and other virulence of C. albicans. Thus, the obtained results suggest that a furan class compound could act as a potent therapeutic medication for biofilm associated C. albicans infections.
APOE genotype influences glial activity in MS
Authors: Langston Zachary , Lahey Matthew , Affaneh Amira , Cudaback Eiron
Published: November 2023

Multiple sclerosis (MS) is an autoimmune demyelinating disease characterized by chronic inflammatory foci within the central nervous system (CNS). Resident immunocompetent cells of the CNS, namely microglia and astrocytes, respond to a variety of sterile and non‐sterile inflammagens by initiating neuroinflammatory cascades. Although these inflammatory processes are necessary for pathogen destruction, clearance, and tissue repair, disproportionate or protracted responses are thought to contribute to the development and progression of many brain disorders, including MS. While the molecular determinants influencing sensitivity and responsiveness of the affected brain to these inflammatory insults remain poorly understood, genetic studies have identified many potential disease risk alleles. Although the precise mechanisms responsible for this influence remain unclear, APOE represents a key modulator of microglial and astrocyte function. Therefore, we decided to examine how APOE genotype modulates glial function in response to general inflammagens. Our results further elucidate the CNS inflammatory mechanisms underlying MS, allowing for identification of potential therapeutic agents to mitigate disease progression, severity, and improve clinical outcomes.
Pathological Role of Two Chemokines RANTES and MIF in Ischemic Stroke
Authors: Liu Yu‐Chuan , Tang Sung‐Chun , Liou Houng‐Chi , Tu Huang‐Ju , Kang Kai‐Hsiang , Liou Horng‐Huei , Jeng Jiann‐Shing , Fu Wen‐Mei
Published: November 2023

WHO reported that around 15 million people have a stroke worldwide every year. Among them, ischemic stroke accounts for about 80 percent in all cases, but the treatment of ischemic stroke is still limited to tPA injection. Therefore, developing the new strategy to treat stroke is important and urgent. Cytokines and chemokines were found to contribute to the infarct size in stroke. This study aimed to find out the pathological roles of two chemokines, Macrophage migration inhibitory factor (MIF) and Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES) in stroke.

In our study, the human blood samples from stroke patients before and after tPA injection were evaluated by human cytokine array and ELISA kits. Wistar rats were subjected to transient middle cerebral artery occlusion (tMCAo) and treated with MIF or RANTES via femoral vein injection right after tMCAo. Twenty‐four hours after tMCAo, the infarct volume was evaluated by TTC staining and the neurological function was determined by the modified set of the mNSS. In addition, the blood‐brain barrier (BBB) of tMCAo rat was evaluated by Evans blue dye. Primary neuron and adult rat brain endothelial cell (ARBEC) were also subjected to oxygen‐glucose deprivation (OGD)/reoxygenation to examine the cell viability or BBB integrity after treatment of MIF or RANTES.

In patients, the concentrations of MIF and RANTES were significantly increased following ischemic stroke before tPA injection. In Wistar rats, the exogenous administration of MIF (3.3 μg/kg) after tMCAo significantly increased the infarct volume; however, the RANTES‐treated (10 μg/kg) rats decreased infarct size after ischemia/reperfusion. To investigate how MIF and RANTES influence the outcome after ischemic stroke, we examined the BBB permeability after tMCAo. It was found that MIF increased the BBB permeability; however, RANTES reduced the BBB leakage after ischemia/reperfusion. In ARBEC culture also showed that MIF increased, but RANTES reduced the BBB leakage after OGD/reoxygenation.

In conclusion, the levels of MIF and RANTES were elevated after ischemia. MIF increased the neuron damage in tMCAo rats; in contrast, RANTES exerts a neuroprotective effect after tMCAo. Our study indicates that MIF and RANTES may be a good drug target for developing drugs for the treatment of ischemic stroke.

Support or Funding Information

This work was supported by the Ministry of Science and Technology grants, 104‐2321‐B‐002‐033, Taiwan.
Divergent Influence of Binge Drinking and Subsequent Intermittent Stress on Drinking Behaviors and Select Brain Protein Levels in Male and Female Mice
Authors: Devaud Leslie L , Garcia Andres , Perius Megan , Finn Deborah A
Published: November 2023

Alcohol misuse and abuse are serious problems in our society and stress may increase the risk for escalation of drinking from recreational to problematic. The present study explored the effect of prior binge drinking on subsequent drinking behaviors following intermittent predator odor stress exposure, which is a model of traumatic stress. Adult male and female C57BL/6 mice had either 7 binge ethanol sessions (binge EtOH) or drank water (water group). Following a 30‐day abstinence period, all mice were provided free access to 10% ethanol (10E) and water via lickometers for 4 weeks, with 30 min exposure to predator odor during the middle 2 weeks (2X/week). At 24 hrs after the final drinking session, mice were euthanized, and brains were harvested and frozen for later Western Blot analyses vs separate naïve controls. Dissected prefrontal cortex (PFC) and hippocampus tissues were assayed for levels of CRF‐R1 (corticotropin releasing factor receptor 1), P450scc (an enzyme involved in steroidogenesis) and the GABAA receptor (GABAAR) alpha1 subunit. Whereas intermittent predator odor significantly increased anxiety‐like behaviors in male and female mice, measured on the elevated plus maze, it significantly increased 10E licks over baseline to a greater extent (78%) in the binge EtOH males versus the water males (27%). In contrast, females had no change in drinking following stress, regardless of prior binge drinking experience or not. Western blot analyses showed divergent responses across proteins and sex. In PFC, there were no significant differences between the binge EtOH and water groups. CRF‐R1 levels were unchanged across male or female groups, but there was a > 25% decrease in P450scc levels in males and a 20% increase in females, vs naïve controls. Levels for the GABAAR alpha1 subunit did not change in males but decreased by 20% in stressed females vs naïve. In contrast, male hippocampal tissue showed different responses between the stressed binge EtOH and water groups, vs naïve. CRF‐R1 levels were increased by 25% in water males with no change in binge EtOH animals. P450scc and GABAAR alpha1 subunit levels were unchanged in water groups but decreased by 13% and increased by 22% in the binge EtOH groups, respectively. Taken together, these data reinforce the importance of assessing sex differences as the synergistic effect of binge drinking and stress on alcohol intake occurred only in male mice. Additionally, the divergent neurochemical responses in males and females suggest that stress exerted the greatest effect in the PFC while binge EtOH exerted the greatest effect in the hippocampus.

Support or Funding Information

Supported by BX001070 and BX002966 (DF) from the Department of Veteran Affairs
Discriminative stimulus effects of novel carbonic anhydrase activator BD117
Authors: Spoon Taylor Alexis , Sanku Rajesh Kishore Kumar , Draghici Bogdan , Ilies Marc A , Walker Ellen
Published: November 2023

Carbonic anhydrase is a zinc enzyme that catalyzes the reversible hydration of carbon dioxide to bicarbonate and protons and is involved in brain homeostasis and regular cerebral function. Previous research from our laboratory has suggested that activation of carbonic anhydrase may protect against scopolamine, ketamine, and acetazolamide induced recognition memory deficits. However, the underlying pharmacology of carbonic anhydrase activation is unknown and what neurotransmitter systems are modified by a general activation of brain carbonic anhydrase isozymes. The purpose of the current project was to train our lead, novel carbonic anhydrase activator BD117 as a discriminative stimulus. Our in vitro and ex vivo studies indicate BD117 is brain penetrant and will activate key carbonic anhydrase isozymes in the brain such as hCA‐IV, hCA‐VA, and hCA‐IX. We hypothesize that BD117 can serve as a discriminative stimulus and other carbonic anhydrase activators with a similar profile will substitute for BD117. In addition, we predict the discriminative stimulus effects of BD117 will be antagonized by carbonic anhydrase inhibitor acetazolamide. Initial experiments examined doses of BD117 on response rates in Swiss‐Webster, male mice (n=11). We found doses above 3 mg/kg reduced responding for food reinforcement. Therefore, a lower dose of 0.3 mg/kg BD117 and saline were initially chosen as the discriminative stimuli for food‐reinforced responding using a two‐choice operant training procedure. After 20 training sessions, no evidence of discriminative control was obtained and no evidence of rate‐decreasing effects was observed. Therefore, the training dose was increased to 1.0 mg/kg BD117. After meeting testing criterion, six consecutive days of correct nose‐poke appropriate responding, mice were tested with additional doses of BD117. BD117 produced dose‐dependent substitution without decreasing response rates at the doses tested. In summary, BD117 was established as a discriminative stimulus. To the best of our knowledge, this is the first example of a central nervous system enzyme activator trained as a discriminative stimulus. These results and additional future studies will further our understanding of the role carbonic anhydrase in functional processes in the brain.
Structure‐Guided Design for Agonist Selectivity with α7‐nAChRs
Authors: Camacho‐Hernandez Gisela Andrea , Kaczanowska Katarzyna , Harel Michal , Beck Christina , Doan Lisa , Binding Hanna , Taylor Palmer
Published: November 2023

Nicotinic acetylcholine receptors (nAChRs) are ligand‐gated ion channels present in the peripheral and central nervous systems. Pre‐ and post‐synaptic nAChRs modulate CNS functions, and ligands selective for nAChR subtype may form useful therapies for disorders associated with development and aging of the nervous system. We have developed a small sub‐group of 4,6 di‐substituted 2‐aminopyrimidines that interact with the acetylcholine binding protein (AChBP) and activate α7‐nAChRs. These agents show Kd and Ka values for both AChBP binding and α7‐nAChR activation between 10 and 70nM. To examine the structural basis of agonist selectivity, we employ measurements of ligand binding to AChBP, and analysis of ligand binding poses from X‐ray crystal structures, along with α7‐nAChR activation on intact cells in culture. Crystal structures suggest a distinct binding pose is required for α7‐nAChR activation enabling us to define the molecular determinants giving rise to activation and selectivity for the α7 subtype. By combining the picolyl substitution at the 4‐position of the pyrimidine and departing from the symmetry of the molecule, we are able to discriminate between α7 activation and generalized binding at the subunit interface. The 4,6‐disubstituted 2‐aminopyrimidines have high selectivity for α7‐nAChR. Crystallography studies using the soluble acetylcholine binding protein (AChBP), a surrogate protein of the extracellular domain of α7‐nAChR, reveal three distinctive binding interactions and poses of nicotinic agonists: 1) cation‐π interactions for quaternary amines and an aromatic nest of side chains at the subunit interface, 2) stabilization of secondary amines and imines by a hydrogen bond from its protonated basic nitrogen to the backbone carbonyl of a conserved tryptophan on the principal subunit face, 3) interaction of less basic multi‐ring pyrimidine‐pyridine heterocycles at the binding interface. This new family of 2‐aminopyrimidines enables us to define receptor subtype selectivity and achieve a structural landscape for α7 selective activation amongst a plethora of CNS nAChR subtypes.

Support or Funding Information

NIH Grant GM 18360‐44
Identifying Receptor‐Ligand Interactions That Drive Gβγ‐Dependent Signaling
Authors: Cameron Robert Bruce , Beeson Craig Bruce , Schnellmann Rick
Published: November 2023

Understanding differences in ligand bias enable more targeted application of pharmacotherapy to various disease states. Our laboratory has previously demonstrated that in renal proximal tubule cells (RPTC), the beta‐2 adrenergic receptor agonist formoterol induces mitochondrial biogenesis, while the beta‐2 adrenergic receptor agonist clenbuterol does not. We hypothesized that formoterol and clenbuterol engage distinct signaling programs by engaging in different interaction profiles with the beta‐2 adrenergic receptor. We examined Gαs‐ and Gβγ‐dependent signaling in RPTC following stimulation with 30 nM formoterol and 30 nM clenbuterol and found that both ligands caused significant accumulation of cAMP but that only formoterol caused a significant increase in Akt phosphorylation. To understand the structural basis of these differences in signaling, formoterol and clenbuterol were docked to the orthosteric binding site of both inactive (3NYA, 3NY8, 5D5B) and active (4DLE, 4LDL, 4LDO) crystal structures of the beta‐2 adrenergic receptor. For each pose, receptor‐ligand interactions were tabulated at each structural feature. Each interaction was assigned a value of +1 for formoterol and −1 for clenbuterol, and interactions were summed for all six crystal structures. The methoxyphenyl group of formoterol showed a greater likelihood of aromatic interactions with W109 and V114 and contact interactions with F193. Interestingly, the formamide and hydroxyl groups of formoterol exhibited a greater interaction frequency with the backbone of C191, but the amino group of clenbuterol was more likely to engage in strong interactions at that site. Similarly, the secondary amino groups of both formoterol and clenbuterol interacted with D113, but formoterol was more likely to do so with strong hydrogen bond or ionic interactions. Overall, the longer and larger functional groups of formoterol allowed it to stretch across the binding pocket of the beta‐2 adrenergic receptor to simultaneously interact with TM3, TM5, and ECL2 in a manner distinct from clenbuterol and may drive the differences in signaling between formoterol and clenbuterol. These interaction profiles provide key structural data for the development of beta‐2 adrenergic receptor agonists that selectively activate specific signaling programs.

Support or Funding Information

R.B.C. is supported by F30 DK104550 and T32 GM008716 (National Institutes of Health). C.C.B. is supported by P20 GM103542 (National Institutes of Health). R.G.S. is supported by R01 GM084147 (National Institutes of Health) and 1BX000851 (Department of Veterans Affairs).
Identification of Binding Sites for Small Molecule Modulators of Gβγ Signaling
Authors: Fisher Isaac James , Connelly Sara , Dumont Mark , Smrcka Alan
Published: November 2023

G‐protein coupled receptors (GPCRs) transduce extracellular stimuli to intracellular signaling events via G‐proteins and β‐arrestins and are targeted by up to 40% of FDA approved drugs. Targeting proteins downstream of the GPCR, such as G‐protein β, allows for selective modulation of specific pathways downstream of GPCRs. Inhibition of interactions between Gβγ and specific effectors has shown to have therapeutic potential in diseases such as heart failure and inflammation (Lehmann et al., 2008. and Casey et al., 2010.)

Previously, our laboratory identified small molecules that bind to a common site for protein‐protein interactions on Gβ. These compounds selectively modulate Gβγ‐dependent activity of various downstream effectors. We hypothesize that these compounds differentially modulate Gβγ‐mediated signals due to binding to specific subsets of amino acids on Gβ. To find the binding site for these compounds, we used a random mutagenesis screen in yeast. The yeast mating response to α‐factor is mediated by the Gβγ homolog, Ste4p. This pathway when activated can inhibit yeast growth making it an attractive tool for examining the inhibition of βγ and homologs using small molecules.

Gallein, a well‐characterized Gβγ inhibitor, inhibited the α‐factor/Ste4P mediated mating response pathway and it suppressed α‐factor induced growth arrest. To screen for residues on Ste4p that bind gallein, we set up a yeast strain where α‐factor stimulates growth on media lacking histidine and showed that gallein inhibits growth of this strain in the presence of α‐factor. We randomly mutagenized ste4p and 500,000 variants were screened for colonies that grew in the presence of gallein. Two classes of mutations were found. Class I mutants had high basal activity suggesting that the mutations decreased the affinity of Ste4P for Gpa1p. Indeed, these mutations were located at positions known to be important for mammalian Gα binding to Gβγ. Class II mutants did not exhibit high basal activity. To test whether these mutants were resistant to gallein inhibition because they lack a gallein binding site, we examined the concentration dependence for gallein inhibition in a reporter assay. The IC50's for the Class I mutants were not different from wild‐type suggesting that resistance was not conferred by a loss of gallein binding. We are in the course of examining class II mutants. One double mutant, G182R, C282E, showed an increased IC50 for gallein while other remain to be examined. All of these mutations map to a region on a critical Gβγ protein‐protein interaction surface.

Finding gallein binding specific residues will allow us to define an inhibitor binding site on Gβ, and may allow for rational design of novel therapeutic inhibitors. In addition, it will help us understand the mechanism for selective inhibition of signaling downstream of Gβγ by small molecules.
Critical Role of R7 Binding Protein in the Sensation of Acute and Pathologic Itch
Authors: Pandey Mritunjay , Zhang Jian‐hua , Adikaram Poorni , Mishra Santosh K , Harris Benjamin , Kahler John F , Loshakov Anna , Sholevar Roxanne , Genis Allison , Hoon Mark , Neubig Richard , Simonds William F
Published: November 2023

Itch is a common irritating sensation, which triggers a desire to scratch. Itch sensation could be triggered due to potentially harmful agents such as insects, parasites and certain plant alkaloids or in a variety of diseases conditions such as eczema, dry skin itch or jaundice or peripheral neuropathies.

In the present study we identified R7BP, a palmitoylated membrane anchoring protein that facilitates Gαi/o –mediated GTPase activating protein (GAP) activity, diminishes scratching in response to a number of pruritogens administerd either intradermally in the nape of the neck or in the central nervous system by lumbar puncture. However, when tested to a number of sensory nociceptive modalities which included mechanical, heat and chemically induced pain, the R7BP KO mice did not show any significant changes except in response to the heat induced pain. The pruriceptive defect in R7bp KO was reversed in a double knock out mice also lacking OPRK1, encoding the Gαi/o coupled kappa opioid receptor.

A similar pruriceptive phenotype was observed in a knock‐in of GAP‐insensitive mutant mice with a Gαo (Gnao1 G184s/+) mutation. Further in a model of atopic dermatitis, R7BP KO mice showed diminished scratching behavior and enhanced sensitivity to kappa opioid agonists as compared to wild type littermates.

Based on our observations, R7BP has an important role to play in somatosensory itch sensation and probably it functions by modulating the kappa opioid receptor. Our findings also suggest that Gαo has a significant role in itch perception and it could be involved regulation of kappa opioid receptor activity to modulate itch sensation.

Taken together, our results indicate that R7BP is a key regulator of itch sensation and suggest that the potential targeting of R7BP – dependent GAP activity as a novel therapeutic strategy for pathologic itch.

Support or Funding Information

This research was supported by the Intramural Research Programs of the National Institute of Diabetes and Digestive and Kidney Diseases and the National Institute of Dental and Craniofacial Research
Identification of Novel Small Molecule Stabilizers of RGS4 Protein Levels
Authors: Karaj Behirda , Sjogren Benita , Neubig Richard R.
Published: November 2023

G protein‐coupled receptors (GPCRs) are one of the largest membrane protein families and represent one of the most common class of drug targets, with 30% of the currently approved drugs on the market. Regulators of G protein Signaling (RGS) are crucial regulators of GPCRs that act as GTPase accelerating proteins (GAPs) on active, GTP‐bound, Gα subunits of heterotrimeric G proteins, thereby attenuating downstream signaling. RGS4 protein levels has been shown to be decreased in conditions such as breast cancer, non‐small cell lung cancer, and schizophrenia. Given the implications that low RGS4 protein levels can have in many diseased states, our study intends to identify small compounds that stabilize the levels of RGS4 protein.

To identify small molecules that enhance the RGS4 protein levels, we screened library collections of over 100,000 compounds in a cell based high‐throughput assay. 29 hits identified from the screen were further confirmed in dose response with fresh powder. Nine compounds were selected for follow up and their effects on RGS4 mRNA, protein half‐life and endogenous protein levels were tested. Our current data shows that all nine compounds enhance RGS4 protein through a post‐transcriptional mechanism as they do not significantly increase the RGS4 mRNA levels, but do extend RGS4 protein half‐life. Representative compound 10 and 11 (numbered based on the original 29 hits) are the most potent enhancers of RGS4 protein levels with EC50 values of around 200 nM. These, as well as all other hits tested, also showed a significant and dose‐dependent increase in endogenous RGS4 protein levels in SH‐SY5Y cells.

Of high interest are two structurally related groups that show effects on RGS4 protein levels when tested in both a recombinant system and endogenous system (SH‐SY5Y cells). Their effect on the proteasomal degradation as well as mechanism of action is under further investigation. Further development of these compounds could lead to development of novel drugs that can help stabilize RGS4 protein levels.

Support or Funding Information

Supported by: The National Institutes of Health (R01 GM110195)
RGS2 modulates the selection of Gαi/o subtype involved in dopamine D2 receptor signaling
Authors: Luessen Deborah J , Hinshaw Tyler P , Sun Haiguo , Chen Rong
Published: November 2023

Introduction

Regulator of G‐protein signaling (RGS) proteins modulate GPCR activity by terminating G protein signaling through acceleration of GTP hydrolysis. It is believed that RGS proteins are recruited by and bind to activated Gα subunits to exert GTPase‐activating (GAP) activity. However, recent literature has indicated that certain RGS proteins bind to GPCRs and this binding is critical for receptor‐mediated G protein signaling. Previously, we reported that RGS2 is a negative modulator of dopamine D2 receptor‐mediated Gαi/o signaling. The present study aimed to investigate the mechanism underlying the interaction of RGS2 with D2Rs and Gαi/o proteins.

Methods

In the present study, we utilized a neuroblastoma 2a cell line stably expressing the short form of dopamine D2 receptors (N2A‐D2S). RGS2 N‐terminus truncation (partial and full truncation) mutants, RGS2 siRNA and corresponding controls were transiently transfected into N2A‐D2S cells. Immunoprecipitation was performed to evaluate the coupling between D2Rs and RGS2. Immunocytochemistry and confocal microscopy were utilized to study agonist (quinpirole) stimulated recruitment of Gαi/o subtypes to surface D2Rs. [35S] GTPγS binding was performed to measure D2R‐stimulated Gαi/o activation.

Results

Our data indicate that RGS2 interacts with D2Rs via its N‐terminus and modulates D2R‐stimulated recruitment of Gαi/o subtypes. Specifically, we found that: 1) RGS2 couples to D2Rs via its N‐terminus. RGS2 N‐terminus truncation abolishes the co‐localization of RGS2 with surface D2Rs 2) RGS2 N‐terminus truncation attenuated D2R‐stimulated Gαi/o activation; and 3) RGS2 knockdown alters the selection of Gα subtype recruited to activated D2Rs. D2R stimulation by quinpirole (5 min, 1 μM) induced translocation of Gαi2, not Gαo, towards the plasma membrane in the control cells. However, RGS2 knockdown by siRNA resulted in the translocation of Gαo, not Gαi2, towards the plasma membrane after D2R stimulation.

These results suggest that the interaction between RGS2 and D2Rs mediates the selection of the Gαi/o subtype engaged in D2R‐mediated signaling. The finding of this study highlights a novel mechanistic action of RGS2 in regulation of D2R‐mediated Gαi/o signaling.

Support or Funding Information

This work is supported by NIH NIAAA training grant T32 AA007565, DA006634 and a pilot grant from Center for Molecular Communication and Signaling at Wake Forest University.
Stable Gastric Pentadecapeptide BPC 157 Antagonizes Hydrogen Peroxide Induced Oxidative Stress in Drosophila melanogaster
Authors: Filosevic Ana , Waldowski Rozi Andretic , Vidovic Tinka , Sikiric Predrag , Drmic Domagoj
Published: November 2023

In this work we hypothesize novel anti‐oxidative role for the stable gastric pentadecapeptide BPC 157 and we test this using a model organism, Drosophila melanogaster. BPC 157 was originally an anti‐ulcer peptide used in trials for ulcerative colitis and now is in trials for the treatment of multiple sclerosis where it largely interacts with NO‐system (Curr Pharm Des 2014;20(7):1126–35). BPC 157 is also thought to be a novel mediator of Robert's cytoprotection in rat studies, where its endothelium and mucosal protection is believed to be related to oxidative stress injury (Curr Pharm Des 2014;20(7):1126–35).

To explore anti‐oxidative properties of BPC 157 we used the animal model of hydrogen peroxide (H2O2) induced oxidative stress in Drosophila melanogaster. Addition of increasing concentrations of H2O2 to regular Drosophila medium dose‐dependently shortened the lifespan of wild type (wt) Canton S Drosophila males (maximal survival on H2O2 is 8 days, compared to average lifespan of 80 days on regular fly food medium). Longevity of H2O2 fed males increased when they were grown on H2O2 medium supplemented with BPC 157. At 7 days, 54% of H2O2 wt males survived, compared to 84% of 0.6% H2O2 + 3.5 ng/ml BPC 157; at 12 days there was no survival on 0.6% H2O2, and 4% survival on 0.6% H2O2 + 3.5 ng/ml BPC 157.

These results shows that; first, BPC 157 is biologically active in this invertebrate model system, and second, that that BPC 157 and H2O2 likely act on similar molecular pathways regulating longevity. Doses effective in promoting increased survival on H2O2 were extrapolated from biologically active doses in mammals. In conclusion, this suggests likely conservation of mechanisms by which BPC 157 antagonizes Reactive Oxidative Species (ROS) in invertebrates and vertebrates. Based on these intriguing observations we will investigate mechanisms by which BPC 157 exerts its anti‐oxidant actions. Because of the significant genetic and mechanistic conservation between Drosophila and vertebrates, discoveries made in this simple model organism will have significant translational potential and relevance for human health.

Support or Funding Information

University of Zagreb, Zagreb, Croatia (Grant number BM099)
EVALUATION OF EMBRYOTOXICITY AND TERATOGENICITY OF MALTODEXTRIN‐COATED CADMIUM SULFIDE QUANTUM DOTS
Authors: Sánchez Janet , Reyes‐Esparza Jorge , Rodriguez‐Fragoso Lourdes
Published: November 2023

The wide and frequent use of engineered nanomaterials (NMs) raises serious concerns about their safety for human health. Particularly quantum dots (QDs), have also shown that can reach the embryo through the placenta, a natural barrier for a large variety of organic substances with diverse molecular structures, and may cause developmental deformities. Our aim is to evaluate the embryotoxic potential of maltodextrin‐coated cadmium sulfide (CdS) quantum dots (QDs). We studied the effects of CdS‐maltodextrin QDs on embryo and fetus development using an embryotoxicity and teratogenicity assay on chicken embryos. Six groups received CdS‐maltodextrin QDs in different concentrations (0.001, 0.01, 0.1,1 μg/Kg). Test solutions (1 mL) were added to the air sac under sterile conditions. Each solution was injected after drilling into the shell at the blunt end of the egg; after injection, the holes were immediately sealed with melted paraffin wax. The eggs were then transferred to and maintained in a forced draft incubator at 37.5 °C with a relative humidity of 65% until the desired stage of development was reached (72 h). Embryos in each group were fixed in buffered formal saline (pH 7.4), dehydrated, and stained with acetocarmine for routine histological examination. The embryo was examined and staged according to morphological criteria previously outlined by Hamburger and Hamilton. Our data showed that chicken embryos exposed to CdS‐maltodextrin QDs (0.001, 0.01, 0.1 and 1 μg/Kg) in ovo for 72h showed growth and developmental alterations during the early stage and at the end of their development in a dose‐dependent manner. Decreased development was observed during early stages (Stages 9/10 on the Hamburger‐Hamilton scale) when compared with untreated eggs (Stage 13). Chicken embryos exposed to lower CdS‐maltodextrin QDs doses (0.01, 0.1 and 1 ng/Kg) and incubated in ovo for 21h also showed growth and development alterations during the early stages and at the end of their development in a dose‐dependent manner. Current studies have also shown that CdS‐dextrin induces embryotoxicity and teratogenicity, affecting mainly the CNS, the neural tube and somites in chicken embryos. The nature of the observed abnormalities suggests that these effects could be directly associated with nanoparticle concentrations affecting somitogenesis. Therefore, according to the results, there is a high probability that the prolonged accumulation of QDs in the maternal organism may be potentially harmful on embryo and fetus development.
Regulation of Hepatic CYP3A4 by 3‐Methylcholanthrene in Humanized PXR‐CAR‐CYP3A4/3A7 Mice
Authors: Crosby Michael , Riddick David S.
Published: November 2023

Polycyclic aromatic hydrocarbons (PAHs) are a broad class of priority environmental pollutants. These chemicals exert a wide array of health effects through activation of the aryl hydrocarbon receptor (AHR). A well‐documented response to AHR activation is the pronounced induction of cytochrome P450 1A1 (CYP1A1), a drug‐metabolizing enzyme involved in phase I drug biotransformation. Interestingly, less is known about the regulation of human cytochrome P450 3A4 (CYP3A4) by PAHs. Alterations of CYP3A4 expression and activity are of tremendous importance to drug metabolism, since this enzyme's abundance and central role in drug metabolism rank it among the most important human cytochromes P450. As such, CYP3A4 is a common target for drug interactions. Our laboratory has previously shown that treatment of male C57BL/6 mice with 3‐methylcholanthrene (MC), a model PAH, suppressed the activity and expression of hepatic Cyp3a11, the mouse counterpart of human CYP3A4. Controversially, studies in primary human hepatocytes and liver‐derived continuous cell lines report both inductive and suppressive effects of PAHs on human CYP3A4 expression. The current study used MC to study the effects of PAHs on CYP3A4 activity and expression in a novel humanized PXR‐CAR‐CYP3A4/3A7 mouse model [Hasegawa et al. Mol Pharmacol 80: 518–28, 2011]. Adult male and female mice were treated by intraperitoneal injection with MC (80 mg/kg), or corn oil vehicle, and euthanized 24 or 72 hours after injection. Livers were subsequently harvested for analysis of CYP3A4 catalytic activity, protein expression and mRNA expression. Marked induction of mouse hepatic Cyp1a1 expression by MC at both 24 and 72 hours was confirmed as a positive control response. Basal hepatic CYP3A4 expression at the catalytic activity, protein, and mRNA levels was significantly higher in female vs. male mice, a sexual dimorphism not reported in the original published characterization of this humanized mouse strain. A similar sex difference in hepatic CYP3A4 expression was documented previously in a CYP3A4/3A7‐transgenic mouse line [Cheung et al. J Pharmacol Exp Ther 316: 1328–34, 2006]. Further, MC decreased CYP3A4 activity and expression in female mice 72 hours after injection. Despite much lower basal hepatic expression of CYP3A4 compared to females, similar trends for CYP3A4 suppression by MC were observed in male mice; these trends did not achieve statistical significance. Our results suggest that this humanized mouse model will be useful for further investigations of the underlying mechanisms and functional consequences of hepatic CYP3A4 suppression by PAHs in an in vivo system.

Support or Funding Information

Canadian Institutes of Health Research
Tipping the Balance of Hepatic CYP3A4 Activity Through Integrin‐Mediated Outside‐In and Inside‐Out Signaling
Authors: Jonsson‐Schmunk Kristina , Croyle Maria
Published: November 2023

Study objective

We have previously shown that engagement of integrin receptors by adenovirus infection or an uninfectious RGD peptide significantly suppresses hepatic cytochrome P450 3A4 (CYP3A4) and that removal of RGD epitopes from the adenovirus capsid reverses this effect. Integrin receptors are activated by two different signaling pathways. “Outside‐in” signaling is initiated when a ligand binds to the extracellular domain of the integrin receptor and “inside‐out” signaling is activated by intracellular proteins binding to the cytoplasmic domain of the β subunit of the integrin receptor. Because both pathways could be impacted by virus infection, the objective of this study is to determine how key moderators of each pathway impacts hepatic CYP3A4 expression and function.

Methods

The impact of outside‐in signaling was assessed in the human hepatocyte cell line HC‐04 through transfection of siRNA specific for the alpha and β subunits of the integrin receptor while impact of inside‐out signaling was assessed by transfection of siRNA for talin, an intracellular protein that connects the integrin β subunit to actin. CYP3A4 activity (P450‐Glo™), gene expression (qRT‐PCR) and protein levels (Western Blot) were determined after silencing was confirmed. Changes in protein levels of talin were determined by Western Blot after 24–72 hours of infection with a recombinant adenovirus (AdlacZ). Changes in gene expression pathways associated with integrins and CYP3A4 in response to silencing of integrins or talin was assessed by PCR.

Results

Silencing of the β3 subunit reversed the suppression induced by adenovirus infection. This was not observed when the alpha subunit was silenced. In contrast, silencing of talin 1 resulted in a 2.3 fold increase in CYP3A4 activity in the absence of virus infection. However, talin protein levels were reduced by 80% 48 hours after infection with AdlacZ. Changes in gene expression pathways were detected for pathways associated with integrins, talin and CYP3A4.

Conclusions

Our results suggest that integrins play an important role in the regulation of hepatic CYP3A4 through the inside‐out singling pathway. Silencing of talin alone resulted in an increase in CYP3A4 activity, suggesting that the inside‐out pathway may play a role in the maintenance of baseline hepatic CYP3A4 activity and may contribute to variation in hepatic CYP3A4 activity in the human population. Understanding the interplay between integrins, talin and CYP3A4 could be used to modulate drug metabolism to improve therapeutic outcomes in healthy patients and those with disease states influenced by integrin expression such as osteoporosis, ulcerative colitis and cancer.

Support or Funding Information

This project was supported by Grant U01AI078045 (M.A.C.), a special research grant from the University of Texas Vice President for Research (M.A.C.) and a University of Texas Continuing Graduate Fellowship (K.J.‐S.).
In Vitro Assessment of Drug Interaction Potential of Licorice Extracts
Authors: Tonsing‐Carter Alyssa , Li Guannan , van Breemen Richard
Published: November 2023

Although hormone therapy remains the standard of care for women experiencing vasomotor symptoms related to menopause, concerns over an increased risk of stroke and breast cancer have women considering botanical dietary supplements for management of these symptoms. Licorice (Glycyrrhiza sp.) is popular among women as an alternative for hormone therapy, and it has a long history of use in Traditional Chinese Medicine. To ensure the safe use of licorice dietary supplements, we investigated their potential for drug interactions. Standardized licorice extracts of Glycyrrhiza glabra, G. inflata, and G. uralensis, the three most common species used in dietary supplements, were studied for possible inhibition or induction of cytochrome P450 enzymes involved in drug metabolism. In studies using human liver microsomes, and a cocktail probe substrate assay, all three licorice species showed inhibition of CYP2B6, CYP2C8, CYP2C9 and CYP2C19. G. inflata also inhibited CYP1A2, CYP2D6 and CYP3A4. Induction qualified cryopreserved human hepatocytes were used to predict effects of the extracts on cytochrome P450 enzyme induction. CYP3A4 was unaffected by all Glycyrrhiza sp, and CYP1A2 and CYP2B6 were induced in hepatocytes by G. uralensis and G. inflata. Due to these data, dietary supplements containing G. uralensis and G. inflata are more likely than G. glabra to cause clinically relevant drug interactions.

Support or Funding Information

National Institutes of Health grant P50 AT000155 from the Office of Dietary Supplements and the National Center for Complementary and Integrative Health and training grant T32 AT007533 from the National Center for Complementary and Integrative Health.