bluuebunny/crossref_metadata_2025_split huggingface dataset:
A series of substituted isoindolinones as selective small molecule modulators of the Gβγ‐SNARE interaction
Authors: Zurawski Zack P , Temple Kayla Jo , Han Changho , Delbove Claire , Senter Timothy J , Hyde Karren , Lindsley Craig W , Zhang Qi , Stauffer Shaun R , Hamm Heidi E 
Published: November 2023

The interaction of G betagamma (Gβγ) subunits with its effectors is a key mechanism for G protein coupled receptor‐mediated signal transduction. One such effector is the soluble N‐ethylmaleimide‐sensitive attachment protein receptor (SNARE) complex, to which Gβγ binds to inhibit exocytosis downstream of calcium release. Starting from a single hit from a focused high‐throughput screen of a library of small molecules designed to block protein‐protein interactions, we have generated relatively potent and selective modulators of Gβγ‐effector interactions, in particular, the interaction of Gβγ with the SNARE protein SNAP25. From this hit, a series of substituted isoindolinones was developed. It was then determined that these compounds were also able to inhibit a second effector of Gβγ subunits, the G‐protein coupled inwardly rectifying potassium channel (GIRK). Utilizing structure‐activity relationships, we were able to further refine the potency and selectivity of these compounds, generating Gβγ‐binding small molecules that were selective for blocking interaction with an individual effector. Finally, we show that these compounds are able to abolish the inhibitory effect of the α2 adrenergic receptor on vesicle release in neurons. Together, these studies demonstrate the ability to generate selective small molecule modulators of Gβγ‐effector interactions and the suitability of such molecules for investigating mechanisms of Gi/o‐coupled GPCR‐mediated inhibition of exocytosis.Support or Funding InformationThis work was supported by the National Institutes of Health National Eye Institute [R01‐EY010291]; and the National Institutes of Health National Institute of Mental Health [MH101679‐ 01A1]. ZZ was supported by the T32 Training in Pharmacological Sciences, T32 GM07628.
Structure‐Dependent β2 Adrenergic Receptor Agonists Induce Mitochondrial Biogenesis through the Gβγ‐Akt‐eNOS‐cGMP Pathway
Authors: Cameron Robert , Beeson Craig , Schnellmann Rick 
Published: November 2023

Acute kidney injury (AKI) occurs in hospitalized patients and carries a high morbidity and mortality with no treatments beyond renal replacement therapy. AKI is characterized by mitochondrial dysfunction, particularly in the renal proximal tubule cell (RPTC). The induction of mitochondrial biogenesis (MB) is a therapeutic target for AKI. Our group has shown that formoterol, a βγ2 adrenoceptor agonist, can induce MB in vitro and in vivo, and stimulate recovery of mitochondrial and renal function following AKI in mice. In contrast the β2 adenoceptor agonist clenbuterol does not induce MB. Our goal was to elucidate the signaling pathway by which formoterol induces MB and examine why clenbuterol does not. RPTC were treated with 30 nM formoterol or 30 nM clenbuterol following pretreatment with DMSO, 100 nM gallein, 10 mM L‐NAME, 100 nM MK2206, or 5 mM ODQ for 30 min. Oxygen consumption rates (OCR) were measured using the Seahorse XF‐96 analyzer. Protein phosphorylation was assessed by immunoblot analsis. Formoterol, but not clenbuterol increased Akt phosphorylation in RPTC at 30 min, and this increase was attenuated by pretreatment with the Gβγ inhibitor gallein. Pretreatment of RPTC with gallein also attenuated formoterol‐induced increases in FCCP‐uncoupled oxygen consumption rate (FCCP‐OCR), a biomarker of MB. eNOS is a downstream target of Akt and was measured following formoterol or clenbuterol treatment with and without the Akt inhibitor MK2206 (100 nM). Formoterol, but not clenbuterol increased eNOS phosphorylation at 1 hr, and this increase was attenuated by pretreatment with MK2206. Additionally, pretreatment with either the NOS‐inhibitor L‐NAME or the guanylate cyclase inhibitor ODQ attenuated formoterol‐induced increases in FCCP‐OCR. These results show that formoterol induces MB through a Gβγ‐Akt‐eNOS‐cGMP‐dependent pathway, while clenbuterol does not activate Akt to initiate this pathway. This study is the first to associate Gβγ‐and cGMP‐dependent signaling with β2‐adrenoceptor induced MB.Support or Funding InformationThis study was supported by R01 GM084147 to RGS, F30 DK104550 to RBC, and by the Biomedical Laboratory Research and Development Program of the Department of Veterans Affairs (BX‐000851).
Crystallographic Studies of Human Acetylcholinesterase Reactivation by Oximes: Towards a Neutron Structure
Authors: Gerlits Oksana , Fajer Mikolai , Cheng Xiaolin , Blumenthal Donald , Taylor Palmer , Radic Zoran , Kovalevsky Andrey 
Published: November 2023

Human acetylcholinesterase (hAChE) is responsible for degrading neurotransmitter acetylcholine at synapses of the nervous system. Organophosphate (OP) nerve agents and pesticides inactivate hAChE through chemical modifications of the enzyme's active site leading to a life‐threatening increase of acetylcholine concentration. Exposure to OPs is fatal if not treated with antidotes. However, the current generation of antidotes is not highly efficient, and the rates of reactivation are far slower than the catalytic rate of acetylcholine hydrolysis.Insights into the molecular structures of Torpedo (1) and mammalian AChEs (2) as well as other cholinesterases reveal the underlying limitations in enhancing reactivation rates but are limited in their ability to resolve positions of protons and hydrogens important for understanding of proton transfer in reactivation of organophosphate inhibited cholinesterases. The only experimental method that can provide this information is macromolecular neutron crystallography. In combination with X‐ray crystallography it is capable of providing insights into the active sites of enzymes with the atomic resolution.In preparation for the neutron diffraction studies we have obtained several new high resolution X‐ray structures of hAChE at room and low temperatures in the apo form and in complex with oxime reactivators and other ligands. The enzyme crystallizes in a novel crystal form with the unit cell amenable to neutron crystallography (space group P31, a=b=124.3, c=129.1 Å). Detailed analysis of the reactivators and ligand interactions at the enzyme active will be presented.Support or Funding InformationThis work was supported by the CounterACT Program, National Institutes of Health Office of the Director, Grant 1U01NS083451 from NINDS.
Role of Blueberry in modulating suicide gene SKA2 in an animal model of Posttraumatic stress disorder (PTSD)
Authors: Ebenezer Philip J , Nair Anand R , Francis Joseph 
Published: November 2023

Background and RationalePost‐traumatic stress disorder (PTSD) has been related to suicidal thoughts and behavior. The principal origin and the mechanism contributing to the suicidal thoughts in PTSD is not known. Understanding molecular mechanisms and identifying novel genes that might contribute to suicide in the context of PTSD might be vital in the prevention of suicide. Recently, SKA 2 (spindle and kinetochore associated complex subunit 2) a novel genetic and epigenetic target was shown to decrease in patients that attempted or committed suicide. In addition, it has been shown that SKA2 genes chaperone the glucocorticoid receptors from the cytoplasm to the nucleus, thereby possibly assimilating and protecting against stress. It has been well established that blueberries (BB) attenuate stress factors and inflammation. In addition, we recently showed that BB modulates serotonin levels in the brain of PTSD animals. Due to these beneficial properties of BB, in this study we explored the possible role played by BB in modulating SKA2 genes in an animal model of PTSDMethodsRats were fed with a blueberry‐enriched (2%) or a control diet. Rats were exposed to cats for one hour on days 1 and 11 of a 31‐day stress schedule to simulate traumatic conditions. At the end of the study, the rats were euthanized and prefrontal cortex (PFC), hippocampus (HC) and plasma were isolated. We measured plasma SKA2 levels in the experimental animals by ELISA. In addition SKA2 level was also measured in PFC and HC regions of the brain were quantified by western blotting and RT‐PCR.ResultsWe found that our PTSD animals had a decreased levels of SKA2 in the plasma, hippocampus and prefrontal cortex compared to control (non‐PTSD) animals. In contrast, treatment with blueberry in the PTSD animals increased SKA2 levels both in the blood and in the brain regions.ConclusionsThe finding that SKA2 gene is down regulated in the blood and brain indicates a possible target for further investigating into suicide prevention using our animal model of PTSD. Furthermore, the finding that non pharmacological agents like blueberry is able to modulate SKA2 gene in context of PTSD is novel. Further studies to understand the molecular mechanism of protection by BB might provide a potential therapeutic target for the treatment of PTSD.Support or Funding InformationThis work was supported by U.S. Highbush blueberry council (USHBC) grant to Dr. Francis.
Arsenic silences liver PDK4 expression through activation of histone H3K9 methylatransferase G9a
Authors: Zhang Xi , Choiniere Jonathan , Bennett Jason , Wang Li 
Published: November 2023

It is well established that the increased liver cancer incidence is strongly associated with epigenetic silencing of tumor suppressor genes; the latter is contributed by the environmental exposure to arsenic. The pyruvate dehydrogenase kinase 4 (PDK4) is a mitochondrial protein that regulates TCA cycle. However, little is known about its role in arsenic‐mediated tumor progression. In the present study, we show that the inactivation of PDK4 in hepatocellular carcinoma (HCC) cells is resulted from aberrant repressive histone modification. Histone methyltrasferase G9a‐ and Suv39H‐mediated histone H3 lysine 9 (H3K9) methylations contribute to the PDK4 silencing in HCC cells. The PDK4 expression is induced by G9a inhibitor BRD4770 (BRD) and Suv39H inhibitor Chaetocin. In contrast, arsenic exposure increases G9a protein expression and the global levels of H3K9 di‐ and tri‐methylations (H3K9me2/3). Compared to BRD treated cells, arsenic exposure restores the decreased H3K9me2/3 enrichment in the PKD4 promoter. Our results also show that the PDK4 expression is refractory to BRD‐mediated transcriptional activation upon arsenic exposure. When C57Bl/6 mice receive arsenic in drinking water alone or in combination with BRD treatment, the hepatic expression of PDK4 is induced by BRD which is markedly reversed by arsenic. Based on these observations, we conclude that arsenic disrupts the epigenetic restoration of PDK4 expression in both HCC cells and in mouse liver.Support or Funding InformationNIEHS 1R01ES025909
Anti‐invasive activity of capsaicin and natural capsaicin‐like compounds in human NSCLC
Authors: Nolan Nicholas A , Hurley John D , Williams Cathryn , Rollyson William D , Brown Kathleen C , Akers Austin T , Eskew Matthew N , Perry Haley E , Egleton Richard D , Dasgupta Piyali 
Published: November 2023

Non small lung cancer (NSCLC) accounts of 80% of all lung cancer cases. About 30–40% of NSCLC patients present with metastatic disease at the time of their diagnosis. The average survival time of such NSCLC patients is about 7 months. The invasion of neoplastic cells to the surrounding stroma is a vital step of cancer metastasis. Recent studies have shown that nutritional compounds (by themselves or in combination with standard chemotherapy) can suppress metastasis of several human cancers. Capsaicin is the spicy ingredient of chili peppers. Recent studies have shown that capsaicin inhibits the invasion and metastasis of several types of human cancers including melanoma, prostate cancer and cholangiosarcoma. We investigated the anti‐invasive properties of capsaicin and two capsaicin‐like compounds, capsiate and capsiconiate, found in select variety of chili peppers on NSCLC. We measured the anti‐invasive activity of these compounds by the Boyden chamber assay. We found that capsaicin and capsiate displayed equivalent anti‐invasive activity in NSCLC cells. In contrast, capsiconiate did not suppress the invasion of NSCLC cells. The anti‐invasive activity of capsaicin, capsiate and capsiconiate in human NSCLC was found to be independent of their growth‐inhibitory activity. Currently, we are testing the anti‐metastatic activity of capsaicin in syngenic mouse models of metastasis. The results of our studies may foster the hope of nutrition‐based interventions in NSCLC metastasis.Support or Funding InformationFunding for our study was supported by the an NIH R15‐AREA Grant (1R15HL113681‐01A1), AICR Investigator Grant, and a NASA Undergraduate Fellowship to NAN
Effect of Acidic Tumor Microenvironment on Motility and Stemness of Non‐Small Cell Lung Cancer Cells
Authors: Arikatla Swetha , Livesey John C 
Published: November 2023

Ineffective tumor angiogenesis and uncontrolled proliferation of cancer cells leads to hypoxia and an acidic tumor microenvironment (TME). Acidosis, caused by the accumulation of lactic acid in solid tumors, is due to the induction of glycolysis in cancer cells by hypoxia. Tumor cells also maintain a high rate of aerobic glycolysis, also known as the Warburg effect. A better understanding of the effect of an acidic TME on motility and stemness of non‐small cell lung cancer (NSCLC) cells may allow the development of new treatments targeting this TME. A substantial alteration in phenotype was observed in NCI‐H292 and NCI‐H1650 cells propagated in media at acidic pH (a‐pH; pH ~ 6.5 to 6.7) compared to cells propagated in media at neutral pH (n‐pH; pH ~ 7.2 to 7.4). H292 cells propagated at a‐pH showed a significant decrease in motility compared to these cells propagated in n‐pH while no difference in motility was observed when H1650 cells were propagated in a‐pH or n‐pH. A significant decrease in cell proliferation was observed in H292 cells and H1650 cells propagated in a‐pH media. No significant difference was observed in mRNA and protein expression of E‐cadherin when either H292 or H1650 cells were propagated in a‐pH and n‐pH media. Vimentin mRNA expression was not observed in the H292 cell line while vimentin mRNA expression was observed in the H1650 cell line but no protein expression was observed in either cell line. Minimal Sox2 and OCT4 mRNA expression was observed in H292 cells and H1650 cells and was unaffected by acidity. These data indicate that as a single TME variable, a‐pH has little effect on EMT and stemness while reducing motility in only one of these two NSCLC cell lines.Support or Funding InformationSupported by University of the Pacific's Holmok Cancer Research Fund.
MITO‐APOCYNIN, A NOVEL MITOCHONDRIA‐TARGETED DERIVATIVE OF APOCYNIN, INHIBITS NLRP3 INFLAMMASOME ACTIVATION IN PRIMARY MURINE MICROGLIA IN DOPAMINERGIC NEUROTOXIC MODELS
Authors: Sarkar Souvarish , Panicker Nikhil , Neal Matthew Layne , Harischandra Dilshan , Jin Huajun , anantharam Vellareddy , Kalyanaraman Balaraman , Kanthasamy Arthi , Kanthasamy Anumantha 
Published: November 2023

The NLRP3 inflammasome signaling pathway has recently been recognized as a major player in neuro‐inflammation in the CNS. Oxidative stress and mitochondrial dysfunction are known to play key roles in the pathophysiological processes of many neurodegenerative diseases, including Parkinson's disease (PD). To date, the relationship between mitochondrial defects and neuro‐inflammation is not well understood. In the present study, we show that neurotoxic pesticide‐induced mitochondrial impairment induces the NLRP3 inflammasome's pro‐inflammatory cascade in primary microglia. Treating primary mouse microglia with LPS induced NLRP3 and pro‐IL1β expression. Interestingly, exposing LPS‐primed microglial cells to the mitochondrial complex I inhibitory pesticides rotenone and tebufenpyrad potentiated activation of the NLRP3 inflammasome and processing of pro‐IL‐1β in a time‐ and dose‐dependent manner, indicating that mitochondrial impairment heightened the pro‐inflammatory response in microglia. Morphological analysis revealed that rotenone and tebufenpyrad significantly increased mitochondrial circularity and solidity in LPS‐primed microglia as well as decreased mitochondrial perimeter relative to unprimed microglia, thereby augmenting mitochondrial fragmentation during neurotoxic pesticide‐induced inflammasome activation. Immunocytochemistry (ICC) revealed enhanced mitochondrial superoxide production in rotenone‐ and tebufenpyrad‐treated LPS‐primed microglia via the co‐localization of NLRP3 and mitochondrial superoxide in these cells. Finally, mito‐apocynin, a novel mitochondria‐targeted derivative of apocynin, was able to significantly reduce neurotoxic pesticide‐induced NLRP3 inflammasome activation through attenuation of mitochondrial superoxide generation from damaged mitochondria. Collectively, our studies demonstrate for the first time that mitochondrial impairment resulting from neurotoxic pesticide exposure can activate NLRP3 inflammasome signaling in microglia, further augmenting pro‐inflammatory events in the brain. Also, for the first time, we show that reducing mitochondrial superoxide generation by mitochondria‐targeted pharmacological agents can attenuate the inflammasome activation.Support or Funding InformationSupported by NS088206 and ES19267
Preclinical In‐vivo Safety Evaluation of Concanavalin A‐based Nanomicrobicide in Female Mouse Reproductive Tract
Authors: Ezoulin Miezan JM , Coulibaly Fohona , Dim Daniel 
Published: November 2023

ObjectiveTenofovir (TFV) loaded Concanavalin A nanoparticles (ConA‐NPs) was recently synthesized as mannose sensitive nanomicrobicide template for the prevention of HIV/AIDS transmission. To advance into clinical trials, such nanomicrobicide must be evaluated in preclinical safety and efficacy models. This study aims at examining the mucosal safety of these specific ConA‐NPs in the female mouse reproductive tract.MethodTFV loaded ConA‐NPs (~ 670 ± 234 nm, −15 ± 0.55 mV) were prepared via the “layer‐by‐layer” technology by coating a core template, calcium carbonate (CaCO3) crystal, with Con A crosslinked with glycogen. Pre‐clinical safety studies of intravaginally administered TFV loaded ConA‐NPs (100 mg/kg) were performed using a female mouse model C57Bl6. Mouse cervicovaginal lavage (CVL) and tissues (CVT) were collected 24h after a single application. Changes in the levels of inflammatory cytokines (IL‐1l, IL‐1β, IL‐6, IL‐7, IP‐10, MKC, and TNF‐l) were quantitated by a high‐sensitivity multiplexed bead‐based immunoassay. The histopathology of the genital tract (vagina, cervix, uterus, and ovary), rectum and other major organs (spleen, lung, liver, kidney, heart, brain) was studied using Hematoxylin & Eosin staining. The vaginal tissues were evaluated for localized immune cells (lymphocytes) infiltration by immunohistochemical staining for CD45.ResultsAfter 24h exposure to these NPs, the basal levels of proinflammatory cytokines (IL‐1l, IL‐1β, IL‐6, IL‐7, IP‐10, MKC, and TNF‐l) in mouse CVL and CVT increased 3 – 56 fold and 1.2 – 5.2 fold, respectively when compared to phosphate buffered saline (PBS) used as a negative control. The induced inflammation was associated with the infiltration of immune cells to the vaginal epithelium. The histological analysis of the reproductive tract revealed signs of epithelial disruption in the vagina. However, all other tissues were histologically similar to control tissue at 24 h post application.ConclusionThese experiments showed that the present ConA‐NPs are cytotoxic to the genital tract epithelial cells at 100 mg/kg. These data suggest the need for further preclinical studies to determine the maximum tolerable dose of the nano formulation for vaginal application.Support or Funding InformationThe project was supported by Award Number RO1 AI087304 from the National Institutes of Allergy and Infectious Diseases (Bethesda, MD, USA). The content is the sole responsibility of the authors and does not necessarily represent the official views of the National Institutes of Allergy and Infectious Diseases or the National Institutes of Health.
In Vivo Treatment with Natriuretic Peptide Receptor Type C Agonist Attenuates Overexpression of Cell Cycles Proteins and Hyperproliferation of Vascular Smooth Muscle Cells from SHR : Molecular Mechanisms
Authors: RAHALI Sofiane , ANAND‐SRIVASTAVA Madhu B 
Published: November 2023

Vascular remodeling due to hyper‐proliferation of vascular smooth muscle cells (VSMCs) is associated with hypertension. We previously showed that in vivo treatment of spontaneously hypertensive rats (SHR) with NPR‐C specific agonist C‐ANP4–23 attenuates the hyper‐proliferation of VSMC. We undertook the present study to investigate if the anti‐proliferative effect of C‐ANP4–23 treatment is mediated through the inhibition of the over‐expression of cell cycle proteins and explore the signaling molecules contributing to this effect. For this study, one‐week‐old SHR and age‐matched WKY rats were injected intraperitoneally with C‐ANP4–23 twice per week for 6 weeks and sacrificed at the end of the 8th week, blood pressure measurements were done by tail cuff method, proliferation of VSMC was determined by thymidine incorporation and MTT assay, and Western blotting was used to measure the levels of proteins. VSMC from SHR exhibited enhanced proliferation as compared to Wistar Kyoto rats (WKY) and C‐ANP4–23 treatment attenuated the hyper‐proliferation to control levels. In addition the overexpression of cyclin D1, cyclin A, cyclin E, cyclin dependent kinase 2 and 4 (cdk2, cdk4), phosphorylated retinoblastoma protein (pRb) and Giα proteins exhibited by VSMC from SHR was attenuated to control levels. Furthermore, the enhanced phosphorylation of ERK1/2, AKT, EGF‐R, PDGF‐R, IGF‐R and c‐Src was significantly decreased by C‐ANP4–23 treatment. Moreover, the enhanced levels of superoxide anion (O2 −), NADPH oxydase activity, and the enhanced expression of NOX4 and P47phox in SHRs were attenuated by C‐ANP4–23. These results indicate that in vivo NPR‐C activation attenuates the over‐expression of cell cycle proteins through the inhibition of the enhanced oxidative stress, c‐Src and EGF‐R, PDGF‐R, IGF‐R activation, MAPK signaling and over‐expression of Giα proteins resulting in the inhibition of the hyper‐proliferation of VSMC from SHR. Thus, it can be suggested that C‐ANP4–23 may be used as a therapeutic agent for the treatment of vascular complications associated with hypertension and atherosclerosis.Support or Funding InformationSupported by grants from Canadian Institutes of Health Research (CIHR) and Heart and Stroke Foundation of Canada (HSFC).