bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mechanisms of Tolerance to delta‐9‐THC in Rodent Models of Pathological Pain
Authors: Morgan Daniel , Yuill Matthew , Zee Michael L , LaFleur Rebecca , Guindon Josee 
Published: November 2023

The use of cannabinoids in pain management is of significant interest due to the antinociceptive efficacy of Δ9‐THC. However, tolerance to the antinociceptive effects of Δ9‐THC develops with repeated treatment. The objective of this study was to examine tolerance to daily administration of Δ9‐THC in clinically relevant rodent models of pathological pain. The effects of Δ9‐THC in pathological pain were assessed in male wild‐type and cannabinoid receptor 1 (CB1) desensitization‐resistant S426A/S430A mutant mice. First, we assessed tolerance to Δ9‐THC in mice subjected to the formalin test of inflammatory pain. Intraplantar formalin injection (10 μl at 2.5 %) produces a biphasic nociceptive response consisting of acute and inflammatory pain phases. Experimental groups were subjected to the formalin test after receiving daily intraperitoneal injections of Δ9‐THC (6 mg/kg) ranging from zero to twelve days. Wild‐type mice exhibited complete tolerance to the antinociceptive effects of Δ9‐THC in the formalin test after eight days of daily Δ9‐THC. Interestingly, we find that tolerance to the antinociceptive effects of Δ9‐THC is attenuated in S426A/S430A mutants for the inflammatory pain phase but not for the acute pain phase. The effect of the c‐jun N‐terminal kinase (JNK) inhibitor, SP600125, on Δ9‐THC tolerance was also examined. Pre‐treatment with SP600125 (3 mg/kg) attenuates tolerance for the antinociceptive effects of Δ9‐THC in both the acute and inflammatory pain phases. Tolerance to the anti‐allodynic effects of Δ9‐THC was examined in a mouse model of post‐surgical pain. Injection of Δ9‐THC completely alleviates thermal and mechanical allodynia following a 5 mm hindpaw surgical incision. Consistent with our previous work, tolerance to the anti‐allodynic effects of Δ9‐THC is attenuated in S426A/S430A mutant mice. These results demonstrate that Δ9‐THC exhibits robust antinociceptive effects in two separate rodent models of clinically relevant, pathological pain and are consistent with previous studies showing the attenuation of rapid tolerance to the antinociceptive effects of Δ9‐THC in S426A/S430A mutants using the tail‐flick and hotplate tests.Support or Funding InformationFunded by NIH grants DA036385 (DJM), DA037355 (DJM), and Texas Tech University Health Sciences Center School of Medicine 121035 (JG).
Increases in Locomotor Activity during Spontaneous Cannabinoid Withdrawal in Mice
Authors: Bergman Jack A , Reingold Curtis G , Barkin Claire E , Bergman Nora R , Nikas Spyros P , Makriyannis Alex , Paronis Carol A. 
Published: November 2023

Spontaneous cannabinoid (CB) withdrawal following exposure to Δ9‐tetrahydrocannabinol (THC) has been described man but has been inadequately characterized in mice. To address this issue, radiotelemetry was used to measure body temperature and locomotor activity in different groups of CD1 or C57 mice treated for 5–60 days with saline, 10–20 mg/kg/day of the CB partial agonist THC or 0.03–0.1 mg/kg/day of the CB high‐efficacy agonist AM2389. The effects of treatment were assessed throughout the regimen and for up to 10 days after the last injection to capture withdrawal‐related perturbations in thermoregulation or motor activity. Results show that acute exposure to AM2389 or THC decreased both temperature and activity. Rapid tolerance occurred to the hypothermic effects of the cannabinoids, whereas locomotor activity continued to be suppressed following each drug injection. In contrast, increases in locomotor activity were evident 12–72 hrs after discontinuing daily injections of AM2389, but not THC. Measurable effects following discontinuation of AM2389 injections were mild but reproducible across different groups of mice. These results indicate that the discontinuation of daily treatment with a CB high‐efficacy agonist will yield evidence of spontaneous withdrawal that may reflect prior dependence, but that the degree of cannabinoid dependence may vary in relation to the dose or efficacy of the agonist injected daily.Support or Funding InformationAcknowledgements: Funded by NIH/NIDA: DA035411, DA031020
PPAR‐α Modulates The Anti‐Inflammatory Effect Of Melatonin In The Secondary Events Of Spinal Cord Injury
Authors: Paterniti Irene , Campolo Michela , Cordaro Marika , Impellizzeri Daniela , Siracusa Rosalba , Crupi Rosalia , Esposito Emanuela , Cuzzocrea Salvatore 
Published: November 2023

Melatonin is the principal secretory product of the pineal gland and its role as an immumo‐modulator is well established. Recent evidence shows that melatonin is a scavenger of oxyradicals and peroxynitrite and reduces the development of inflammation and tissue injury events associated with spinal cord trauma. Previous results suggest that Peroxisome proliferator activated receptor alpha‐ α (PPAR‐α), an intracellular transcription factor activated by fatty acids, plays a role in control of secondary inflammatory process associated with spinal cord injury (SCI).With the aim to characterize the role of PPAR‐α in melatonin mediated anti‐inflammatory activity, we tested the efficacy of melatonin (30 mg/kg) in an experimental model of spinal cord trauma induced in mice by the application of vascular clips (force of 24 g) to the dura via a four‐level T5‐T8 laminectomy, and comparing mice lacking PPAR‐α (PPAR‐αKO) with wild type (WT) mice.The results obtained indicate that melatonin ‐mediated anti‐inflammatory activity is weakened in PPAR‐α KO mice, as compared to WT controls. In particular, melatonin was less effective in PPAR‐α KO, compared to WT mice, as evaluated by inhibition of the degree of spinal cord inflammation and tissue injury, neutrophil infiltration, pro‐inflammatory cytokine expression, NF‐κB activation, inducible nitric‐oxide synthase (iNOS) expression. This study indicates that PPAR‐a can contribute to the anti‐inflammatory activity of melatonin in SCI.
Astrocyte mitochondrial trifunctional protein mediates the neuroprotective effect of thyroid hormone after stroke in mice
Authors: Sifuentes Mikaela 
Published: November 2023

Stroke is a leading cause of death and disability in the United States, however few treatments options exist for patients. Thyroid hormone is observed to reduce stroke lesion size in mice, but the mechanism for this neuroprotection is still unknown. Astrocytes are critical support cells in the brain that our lab has shown readily respond to thyroid hormone by increasing energy production through the mitochondrial trifunctional protein (MTP), a protein complex that facilitates fatty acid oxidation (FAO). As increased energy metabolism is protective against brain injury, we hypothesized that thyroid hormone reduces lesion size by stimulating astrocyte MTP. We observe that treatment with the active form of thyroid hormone (T3) stimulates an increase in ATP production in human astrocytes, which can be blocked by pretreatment with an irreversible FAO inhibitor. Astrocytes in the presence of T3 exhibit an increased survival under in vitro stroke conditions, while this effect is not seen in astrocytes lacking MTP. Pharmacological and genetic inhibition of MTP in vivo also reduces thyroid hormone reduction of lesion size, suggesting that MTP activity is a critical component in brain damage from focal ischemia. Finally, we demonstrate that thyroid hormone protection against ischemia is attenuated in the presence of low concentrations of fluoroacetate, an astrocyte‐specific mitochondrial inhibitor. Taken together, our research indicates that thyroid hormone reduces stroke damage by stimulating astrocyte metabolism in an MTP‐dependent manner.Support or Funding InformationSupported in part by the William & Ella Owens Medical Research Foundation.
GPCRs expressed by cancer‐associated fibroblasts are potential therapeutic targets in pancreatic cancer
Authors: Zhou Shu , Chang Sarah , McCann Thalia , Nishihara Hiroshi , French Randall , Lowy Andrew , Insel Paul 
Published: November 2023

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense fibrotic stromal matrix (desmoplasia) composed of activated fibroblasts (PFs) /stellate cells (PSCs), immune/ inflammatory cells and other cell types. Considerable evidence implicates this unique tumor microenvironment, and in particular its stromal cells, as a key mediator of PDAC progression and drug resistance. The tumor stroma may thus be a target for the treatment of PDAC. Pancreatic cancer‐associated fibroblasts (CAFs), myofibroblast‐like cells, thought to be derived from PSCs and PFs, produce extracellular matrix proteins and, are responsible for the desmoplasia in PDAC. Blocking the activity of CAFs may thus be a means to improve the therapy and prognosis of PDAC. We hypothesized that G protein‐coupled receptors (GPCRs) expressed by pancreatic CAFs may be potential therapeutic targets for PDAC. As an initial effort to test this hypothesis, we used an unbiased approach, Taqman GPCR arrays that can assess ~360 non‐chemosensory GPCRs, as a means to identify and quantify the GPCRs expressed by pancreatic CAFs. Using CAFs isolated from the primary tumors of 5 patients, we discovered that CAFs share in their expression of 104 GPCRs, 35 of which had at least a 2‐fold increase in expression in CAFs compared to PSCs and PFs. An orphan GPCR, Orphan 1, is one of the most highly up‐regulated GPCRs in CAFs. We found that: 1) co‐culture of PSCs with a PDAC cell line (ASPC‐1) increases expression of fibrotic markers and Orphan 1 GPCR; 2) Transfection of Orphan 1 GPCR in PSCs increases the expression of fibrotic markers; 3) siRNA knockdown of Orphan 1 in CAFs decreases fibrotic marker expression and cytokine secretion. We conclude that a GPCRomic strategy can identify GPCRs that regulate pancreatic CAFs. One such GPCR, Orphan 1, contributes to pro‐fibrotic activities in CAFs and is a potential therapeutic target to blunt the fibrosis associated with pancreatic cancer.Support or Funding InformationNIH 5T32CA121938 and R21 CA189477
The GPCR CXCR4 and Tyrosine Phosphatase SHP2 Interact and Signal via a Novel Consensus Motif
Authors: Belcastro Lili T. , Paulukinas Ryan D. , Jancina Anastasia , Moore Catherine C. 
Published: November 2023

CXCR4, a chemokine G protein‐coupled receptor (GPCR), is essential for migration of neuronal and hematopoietic cells, as well as breast cancer cells during metastasis whereby CXCR4 dysregulation promotes a migratory and invasive phenotype. Normally following activation, CXCR4 is rapidly phosphorylated on Ser/Thr residues by GRKs, a key step in initiating adaptor recruitment, which promotes receptor desensitization and receptor trafficking to endocytic sites. Previously we identified that CXCR4 Ser/Thr phosphorylation is delayed upon SDF gradient sensing, and correlates with sustained signaling to a novel CXCR4‐SHP2‐ERK pathway. SHP2 is a protein tyrosine phosphatase (PTP) implicated in HER2(+) and triple‐negative breast cancers, whereby SHP2 transduces both mitogenic and migratory signals, driving hyperproliferation and invasion. SHP2 is also known to be recruited to tyrosine phosphorylated ITIM motifs (immmunoreceptor tyrosine‐based inhibitory consensus motifs), a hallmark found in inhibitory immune receptors with little to no evidence for its presence and function in GPCRs. We have identified a putative ITIM motif in CXCR4 that regulates SHP2 binding and signaling. Specifically, we assessed 1) if CXCR4 is tyrosine phosphorylated within the putative ITIM motif, using a rSHP2 phosphatase assay, pAb development, and mutagenesis, 2) if SHP2 interaction with CXCR4 is disrupted upon ITIM Tyr mutation, by co‐immunoprecipitation, 3) if CXCR4 signaling to SHP2 is disrupted upon ITIM Tyr mutation, and 4) if differences in signaling are due to improper localization and/or function of the mutant, by ELISA. Our results demonstrate that rSHP2 phosphatase is active on CXCR4, suggesting pTyr of CXCR4. Specific pTyr antibodies are under development to confirm these results. Additionally, Tyr mutation within the ITIM alters CXCR4 phosphorylation, suggesting pTyr of CXCR4 occurs within this motif. Furthermore, Tyr mutation disrupts CXCR4 immunocomplex formation with SHP2. Finally, sustained signaling to the SHP2‐ERK pathway is attenuated upon tyrosine mutation within the ITIM motif, and this effect is not due to improper receptor localization or function as demonstrated by cell surface expression and agonist‐dependent internalization. To date, our data support a working model that CXCR4 contains a functional ITIM motif capable of binding SHP2 and transducing sustained signaling to SHP2 upon SDF gradient sensing. These data have implications on aggressive breast cancers with dysregulated CXCR4 and SHP2.Support or Funding InformationSupported by NIH GM‐097718 and PDH SAP4100057688 grants.
RNA‐seq based transcriptome profiling of human and mouse dorsal root ganglion reveals a potential role for Protease Activated Receptor 3 (PAR3) in pain processing
Authors: Price Theodore , Torck Andrew , Ray Pradipta , Hassler Shayne , Hoffman Justin , Boitano Scott , Dussor Gregory , Vagner Josef 
Published: November 2023

The dorsal root ganglion (DRG) contains sensory neurons that innervate the surface of the body and many visceral organs. Included amongst these neurons are nociceptors, specialized neurons that detect damaging or potentially damaging stimuli, which are required for the detection of acute pain and play a key role in the development and maintenance of chronic pain states. RNA‐seq has recently been used to elucidate the transcriptome of this tissue in mouse and rat but the transcriptome of human DRG has not been explored. We obtained fresh, lumbar DRG tissue from female human donors and performed 75bp paired‐end polyA+ RNA‐sequencing on the Illumina platform. The sequenced fragments were mapped to the Gencode v14 reference transcriptome / hg19 reference genome to yield 80M mapped fragments, and relative transcript abundance in FPKM (Fragments Per Kilobase per Million mapped fragments) was quantified using the Tophat‐Cufflinks toolkit. We compared our RNA‐seq dataset to publicly available mouse DRG RNA‐seq data and performed integrative analysis with RNA‐seq data from several tissues associated with drug side effects (e.g. heart, small intestine, whole brain) to perform an unbiased search for conserved gene expression in DRG across both species. We find that there is broad conservation of known DRG and/or nociceptor enriched genes (e.g. P2XR3, SCN10A, SCN11A, NTRK1, MRGPRD) across mouse and human DRGs. Information theory approaches were used to identify tissue‐specific genes in human and mouse DRG compared to tissues 13 other tissues in human and mouse. We find strong correlation of expression across tissues between species for a few hundred DRG‐enriched transcripts, including known genes enriched in the DRG and previously unidentified ones. A conspicuous DRG enriched gene is F2RL2 which encodes PAR3. PAR3 expression in DRG is amongst the highest for all G protein coupled receptors (GPCRs) in human and mouse and mapping to existing cellular expression databases suggests neuronal expression in a population of nociceptors. To test the potential role of PAR3 in pain, we developed a novel ligand for this receptor using our synthetic tethered ligand discovery platform for PARs and show that it robustly activates calcium signaling in trigeminal ganglion neurons and causes mechanical hypersensitivity after hindpaw injection in mice but is devoid of activity at PAR2. Ongoing experiments are further evaluating the specificity of this compound. Our unbiased, human transcriptome approach to target discovery reveals PAR3 as a novel pain target.Support or Funding InformationNIH grants NS073664 and NS065926
The Anti‐viral Agent Tenofovir Induces Oxidative Stress and Apoptosis in HK‐2 Cells
Authors: Murphy Rachel , Stafford Reagan , Petrasovits Brooke , Boone Megan , Valentovic Monica 
Published: November 2023

Tenofovir disproxil fumarate (Viread) is a highly effective HIV antiviral drug approved for treating infections with Human Immunodeficiency Virus (HIV) and Hepatitis B. It is one of the first line drugs used to treat HIV and is efficacious in both novel and treatment‐experienced HIV patients. Tenofovir is administered orally as the prodrug tenofovir disoproxil fumarate (TDF), which is deesterified to the active drug tenofovir. However, renal damage is a major adverse effect associated with its use. Tenofovir can induce decreased glomerular filtration rate (GFR), renal failure, and Fanconi Syndrome. The exact mechanism of this toxicity remains unknown, largely due to limited experimental models. Our laboratory has established that clinically relevant concentrations of tenofovir are toxic within 72 h. The purpose of this study was to investigate the cellular mechanism of cytotoxicity in a human renal proximal tubular epithelial cell line (HK‐2). Tenofovir (TFV) is the active form and was used for all studies. HK‐2 cells were seeded and grown to confluency for 48 h followed by a 72 h exposure to 0–30 uM tenofovir. The vehicle was phosphate buffered saline (PBS). Cell viability was assessed using the MTT assay. Tenofovir induced a loss of cell viability when compared to vehicle within 48–72h but did not alter MTT following a 24 h exposure. Mitochondrial dysfunction was assessed by measuring ATP and ADP levels. A 72 h exposure to tenofovir drastically reduced (p<0.05) ATP levels compared to vehicle control. Oxidative stress was assessed using OxyBlot and showed an increase in protein carbonylation after 72 h exposure to 5–30 uM tenofovir. Oxidative stress induced by tenofovir was sufficient to induce an increase in 4‐hydroxynonenal (4‐HNE) adducted proteins and 3‐nitrotyrosine (3‐NT) addition to proteins. Tenofovir also activated cleavage of Caspase 3 and 9 relative to vehicle. These studies suggest that mitochondrial stress and apoptosis occur in HK‐2 cells treated with tenofovir.Support or Funding InformationSupported by NASA West Virginia Space Grant Consortium Graduate Fellowship to Rachel Murphy.
Fenofibrate upregulates SHP expression but does not alter CYP2D6 expression
Authors: Kent Rebecca , Jeong Hyunyoung 
Published: November 2023

Cytochrome P450 (CYP) 2D6 is a major drug‐metabolizing enzyme, responsible for eliminating 25% of marketed drugs. Factors involved in transcriptional regulation of CYP2D6 are poorly understood, and we recently identified SHP as a negative regulator of CYP2D6 expression (1). Factors that alter SHP expression could influence CYP2D6 expression, and therefore the extent of CYP2D6‐mediated drug metabolism. Fenofibrate has been previously reported to upregulate SHP expression in mouse liver (2). Our objective was to determine whether fenofibrate negativley alters CYP2D6 expression via upregulating SHP expression. CYP2D6‐humanized transgenic mice were administered with fenofibrate (100 mg/kg/day intraperitoneally for 5 days) or vehicle control. Hepatic mRNA and protein expression levels of CYP2D6 and SHP were measured. Results showed that while mRNA levels of SHP did not differ between the groups, protein levels of SHP increased by 2‐fold in fenofibrate‐treated mice. Despite the increased SHP protein levels, CYP2D6 expression did not decrease at the mRNA or protein levels. Similar results were observed in human hepatocytes treated with fenofibrate (at 200 μM for 48 hr). These results indicate that has minimal effects on CYP2D6 expression although it enhances SHP expression.Support or Funding InformationThis study was supported by NIH (GM112746 and HD065532).
Identification of Novel Probiotics to Modify Appetite and Satiety Directly Targeting the Ghrelin Receptor
Authors: Fuentes Cristine Torres , Schellekens Harriët , Hoevenaars Naomi , Ross Paul , Roy Bernard , Stanton Catherine , Dinan Timothy G. , Cryan John F. 
Published: November 2023

Ghrelin is a stomach‐derived peptide that acts as orexigenic hormone in the hypothalamus stimulating food intake through the growth hormone secretagogue receptor (GHS‐R1a). Dysregulated ghrelin signalling may contribute to the development of metabolic disorders such as obesity. Current pharmacologic anti‐obesity treatments lack efficacy and have shown severe side effects, highlighting the urgent need for novel strategies contributing to the maintenance of a healthy weight.Natural products are receiving special consideration as sources of bioactives with beneficial health effects which may be safer for consumers than synthetic therapeutics. One of the most investigated natural bioactives are live microorganisms with health benefit effects, also known as probiotics. Thus, food products containing probiotics are receiving increased scientific and commercial focus in recent years. These probiotic‐mediated beneficial effects on human health are due to changes in the gut microbiota via oral delivery of viable strains of bacteria that are then integrated into the gut system. Gut microbiota ferment nutrients and secrete bioactive compounds inducing several responses within the intestinal mucosa and controlling the gut's barrier and endocrine functions.In this study we investigate the ability of different probiotic bacteria to modulate the GHS‐R1a receptor. Bacteria were cultured overnight under anaerobic conditions at 37°C. Culture supernatants were collected by centrifugation, resuspended in 1× Hanks balanced salt solution containing 20mM HEPES, and incubated in anaerobiosis for 4 h. Afterwards, supernatants were used to investigate the probiotics ghrelin receptor activation potential by calcium mobilization and receptor internalization assays in human embryonic kidney cells (Hek) stably expressing the GHS‐R1a receptor.Different Lactobacillus and Bifidobacterium strains with ability to modulate the ghrelin receptor have been identified. Future investigations, including identification of the bioactives present in the probiotic supernatants as well as in vivo studies supporting these findings, are needed in order to confirm their potential as anti‐obesity therapeutics.Support or Funding InformationWork funded by CREMO SA