bluuebunny/crossref_metadata_2025 huggingface dataset:
Sepsis‐Associated Elastase and Proteinase 3 Induce Endothelial Permeability
Authors: Patterson Eric K , Cepinskas Gediminas K , Inoue Ken , Fraser Douglas D
Published: November 2023

Sepsis is a systemic inflammatory response to known or suspected infection, and associated with microvascular dysfunction and capillary leak. The cause of capillary leak may be due to substances released by activated polymorphonuclear neutrophils (PMNs) that adhere to microvascular endothelial cells. Following binding, PMN often degranulate releasing azurophilic enzymes (e.g. elastase (HLE) and proteinase 3 (PR3)) at the endothelial surface and into plasma. We hypothesized that the actions of these enzymes, particularly HLE and PR3 contribute to the loss of endothelial integrity.

Patients with severe sepsis (sepsis with evidence of at least one organ dysfunctioning), were recruited on admission to the intensive care unit. Patient samples were paired with age and sex‐matched controls. The mean age was 54 ± 12 and 52 ± 11 years for sepsis and controls respectively while both groups had 6/15 males. Septic patient pathogens were determined to be 4 gram positive, 7 gram negative, 1 fungal and 3 culture negative. We employed ELISAs to determine plasma HLE and PR3 concentrations and in vitro assays using human umbilical vein endothelial cells (HUVEC) treated with HLE or PR3. HUVEC monolayer resistance and Texas Red‐Dextran flow‐through were used to determine the enzyme's effect on monolayer integrity.

The sepsis plasma concentrations were significantly higher (p < 0.0001) compared to healthy volunteers in both HLE (224 ± 66.0 vs 13.9 ± 0.899 ng/mL) and PR3 (52.2 ± 7.63 vs 8.02 ± 2.18 ng/mL). For in vitro assays, we employed concentrations of HLE and PR3 higher than the plasma measurements in order to more closely mimic conditions at the site of degranulation. Treatment with 1, 2 or 5 μg/mL of either enzyme significantly decreased HUVEC monolayer resistance over the course of 90 minutes compared to control. Further, 1 hour of HLE or PR3 treatment significantly increased HUVEC permeability to Texas Red‐Dextran compared to controls.

The loss of endothelial integrity secondary to azurophilic enzyme release by adhered PMNs could result in microvascular dysfunction and capillary leak. Endothelial injury could also exacerbate further PMN recruitment. We conclude that neutrophil degranulation, as evidenced by increased plasma concentrations, can lead to increased endothelial permeability through a mechanism which in part involves the actions of HLE and PR3.

Support or Funding Information

Funded by the Lawson Health Research Institute Internal Research Fund.
Activation of AXL Receptor via Gas6 induces Preeclampsia in Rats
Authors: Dunaway Todd M , Lewis Joshua B , Hirschi Kelsey M , Mejia Camilo A , Mejia Juan F , Hall Parker D , Reynolds Paul R , Arroyo Juan A
Published: November 2023

Preeclampsia (PE) is a complicated obstetric complication characterized by increased blood pressure and decreased trophoblast invasion. The growth arrest‐specific 6 (Gas6) protein is known to induce dynamic cellular responses and is elevated in PE. This discovery suggests a mechanistic role for the Gas6 signaling pathway during PE progression. Gas6 binds to the AXL tyrosine kinase receptor and AXL‐mediated signaling is implicated in proliferation and migratory mechanisms in several tissues. Our objective was to determine the role of Gas6 and AXL signaling in the development of PE in the rat.

We utilized Gas6 to induce preeclamptic conditions in pregnant rats. Briefly, pregnant rats were divided into 3 groups that received daily ip injections of PBS, Gas6, or Gas6 + R428 (an AXL inhibitor). Treatment was done from embryonic day 7.5 (E7.5) to E18.5, at which time they were euthanized and tissues were harvested for histological and molecular characterization. Fetal and placental weights were recorded. Blood pressure, proteinuria and cell invasion was determined. We additionally characterized inflammatory conditions in the maternal lung due to links between respiratory sufficiency, placentation, and fetal health. Bronchoalveolar lavage fluid (BALF) was procured from maternal lungs for the assessment of inflammatory cells and cytokine secretion. Western blot was performed to determine AXL protein expression. We observed: increased proteinuria and elevated blood pressure in Gas6 treated animals, but each were inhibited by R428; placental and fetal weights revealed no significant changes among groups; immunohistochemical analysis revealed decreased trophoblast invasion in animals treated with Gas6. BALF revealed elevated cell diapedesis in Gas6 animals, which was absent in animals given R428. Quantitative RT‐PCR and immunoblotting revealed increased expression of RAGE (a pattern recognition receptor implicated in inflammation), AXL, and pro‐inflammatory cytokines TNF‐α and IL‐1β. We conclude that Gas6 signaling is capable of inducing PE and that inhibition of AXL receptor prevents disease progression in pregnant rats. These data suggest roles for Gas6/AXL in the development of PE and offer possible treatment avenues that could help in alleviating PE symptoms.
Revealing Age‐Associated Genetic Alterations in Mouse Oocytes by Single‐Cell Exome Sequencing
Authors: Lee Tin‐Lap , Qian Yan , Liao Jinyue , Zeng Xi , Tsui Kwok‐Wing Stephen , Li Tin‐Chiu , Chan Wai‐Yee
Published: November 2023

Advanced maternal age refers to women giving birth at the age of over 35. It has established that the condition links to inferior fertilization rate and higher genetic risks in the offspring. Despite external factors like hormones and follicular cells have been reported to associate with maternal aging, the genetic contribution remains largely elusive.

Due to the limitation of experimental technologies and the limited amount of sample materials, oocyte per se is seldom studied previously, especially in the field of genetic alterations. In this study, we aimed to identify natural and accelerated aging‐related genetic alterations, including single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) in mouse oocyte through novel single‐cell based whole exome sequencing. Four groups of female mice consisted of a young group (6‐weeks‐old), a natural aging group (36‐weeks‐old), an accelerated aging group (treated by 4‐Vinycyclohexene diepoxide, VCD) and a vehicle group (treated by sesame oil) were mated with an adult male. The DNA samples derived from maternal oocyte and blood, and those from offspring and male mice were collected, followed by whole exome sequencing and bioinformatics analyses. De novo DNA variations (DVs) were identified in both natural and accelerated aging groups. Genes associated with aging‐related diseases (Ttn, Macf1) were identified. We also detected de novo germline variations (DNGVs) that may contribute to implantation and embryonic development. Finally, we identified an aberrant CNV region associated natural aging oocytes, where it contained genes involved in cell apoptosis, aging diseases and reproduction.

Taken together, our study highlighted the key genetic targets associated with oocyte aging. The data allows dissecting the aging mechanisms that contribute to subfertility in women with advanced maternal age.
An Integrated Study of Gene Expression Profile Uncovers Similarity between Embryogenesis, Bone Development, Wound Healing, and Prostate Cancer
Authors: Rayburn Tyeler S , Mukherjee Angana , Byrd William A , Jones Jacqueline
Published: November 2023

Embryogenesis, bone development, wound healing and cancer are intersecting processes that share common metabolic processes, growth factors and transcription factors. While solid tumors are controllable, bone metastases remain a death sentence, which is due to the complex area of the bone. Interestingly, studies have shown that 100% of prostate cancer deaths involve bone metastases. Although the role of transcription factors in embryogenesis, bone development, wound healing, and prostate cancer are studied separately, the overlapping of factors or genes across the processes remains unexplored. This study aims to develop an approach for identifying the overlapping gene signatures and analyzing their expression and correlation with embryogenesis, bone development, wound healing, and prostate cancer. We extracted data for the 4 processes from Gene Expression Omnibus (GEO); accession numbers: GSE18290 (n=9), GSE51812 (n=12), GSE24742 (n=12) and GSE3325 (n=19). Twenty overlapping genes were randomly identified and statistical analysis performed to compare the gene expressions across all the processes. Most genes in a cluster analysis exhibited common expression patterns in all the processes. Interestingly, a PCA analysis revealed a clear positive correlation between prostate cancer and bone development. Furthermore RUNX2, FGFR1, TWIST1, FOXP1, FOXA1, NKFB1, ELF1, PDGFA, IL6, JAG1, JAG2, and PDGFA were significantly up‐regulated in prostate cancer. Thus, we concluded that the transcription factors that play a role in the fundamental processes of early bone development, embryogenesis, and wound healing also play a critical role in prostate cancer bone metastases. We have identified, using a novel gene expression profiling together with a meta‐analysis validation approach, a set of overlapping gene signatures in embryogenesis, bone development, wound healing and prostate cancer, which can be used as prognostic markers for prostate cancer bone metastasis.

Support or Funding Information

This work was supported by the Department of Environmental and Biological Sciences, Troy University.
Identification of Key Transcription Factor Target Interactions That Regulate Prostate Cancer Metastasis
Authors: Sharma Nitya , Pellegrini Kathryn L. , Giuste Felipe O. , Ouellet Veronique , Trudel Dominique , Mes‐Masson Anne‐Marie , Saad Fred , Osunkoya Adeboye O. , Petros John , Moreno Carlos S.
Published: November 2023

Prostate cancer remains the most commonly diagnosed cancer in U.S. males, and ranks second in mortality with over 28,000 deaths per year. The standard of care for patients with recurrent, aggressive prostate cancer is androgen deprivation therapy (ADT), but the benefits from ADT are typically short‐lived. Recurrent disease following ADT treatment is termed castration‐resistant prostate cancer (CRPC), and is generally incurable after progression to metastatic disease. Therefore, understanding the mechanisms underlying CRPC and subsequent progression to metastatic disease is critical. To gain insights into how transcriptional networks change in response to ADT and lead to metastasis, we have identified the relationships between transcription factors and corresponding gene targets in matched pre‐ADT and post‐ADT tissue samples, as well as matched primary and metastatic lesions. First, we generated gene expression data by sequencing RNA from 24 formalin‐fixed paraffin‐embedded patient‐matched pre‐ADT needle core biopsies and corresponding post‐ADT radical prostatectomy prostate cancer samples. For generating metastatic networks, we used publicly available expression data from matched primary prostate and metastatic tumors. Next, we integrated mRNA expression, protein‐protein interaction, and DNA binding motif data using the PANDA algorithm to reverse engineer transcriptional networks. We identified key transcription factors with significant gains or losses of interactions with target genes specifically in metastatic networks. We also identified putative novel key prostate cancer specific transcription factor interactions that share multiple gene targets, such as ETS1 and GATA2, possibly revealing relationships that directly contribute to increased metastatic potential. By comparing changes in post‐ADT and metastatic transcriptional networks, we may identify critical transcription factor‐target gene interactions that are essential for the progression of CRPC to metastatic disease. This study may thus provide insights into novel therapeutic approaches for treatment of CRPC and prevention of metastasis, as well as prognosis of patients with poor outcomes.

Support or Funding Information

Movember Foundation
Beneficial effect of exogenously applied melatonin on wound healing in skin injured animal model
Authors: Choi Jeonghyun , Won Jinyoung , Hong Yonggeun
Published: November 2023

Skin injury (SI) is generated by various situations such as surgery, burning etc.. Many factors are participated in wound healing processes. The present study was conducted to investigate the effect of exogenously applied melatonin (MT) on the wound healing. In this study, subjected Sprague‐Dawley rats were subdivided into three groups; Con (control), Veh (SI+Saline), MT (SI+MT). Several genes related to inflammation were analyzed in mRNA using RT‐PCR. The expression of TNF‐α was significantly increased in Veh compared to MT (p<.05), whereas, TNF‐R2 expression was higher than in MT (p<.05). Also, in MT group, the expression of p53 and CHOP as apoptosis marker was significantly increased compared to Veh (p<.05). IGF‐1, IGF‐1R, TGF‐β1, and COL1 expression related to collagen synthesis showed similar pattern. Several markers that related to angiogenesis were analysed from two wound margins. HIF‐1α and VEGF expression was significantly increased in MT compared to Veh (p<.05). Also, exogenous melatonin impacted on the upregulated expression of GPx in MT. We observed fibroblast like fibers, adipocytes, blood vessels, collagen like fibers, and hair follicles in H&E staining. Also, wound closing rate was significantly increased in MT compared to Veh at 2, 7, 10, and 21 day (p<.05). Taken together, it is suggested that exogenously applied melatonin might contribute to the skin restoration via regulation of various factors related to inflammation, collagen synthesis, and angiogenesis.

Support or Funding Information

Funds: NRF‐2013R1A2A2A01067169 to Y.H., 2014R1A1A3051724 to S.P., KRIBB Research Initiative Program (KGM4611613 to Y.H.), Republic of Korea
Cytoprotective Chaperone Proteins Are Novel Anti‐Inflammatory Targets in Sickle Cell Disease
Authors: Anea Ciprian , Kumar Sanjiv , Lee Itia , Brittain Julia
Published: November 2023

Sickle cell disease (SCD) is a pro‐inflammatory condition. Levels of TNF‐α, IL‐6, IL‐8, and IL‐10 are elevated. There is clear evidence of endothelial cells (EC) dysfunction, and increased leukocyte, and erythrocyte adhesion in patients even in the non‐crisis “steady state” condition. Additional insult, either via infection or vaso‐occlusive ischemia, induce a dramatic increase in inflammation and EC dysfunction in SCD. Furthermore, there is a kindling of coagulation activation in patients with SCD. We, and others, have reported elevated levels of thrombin and monocyte tissue factor (TF) expression in patients. Both thrombin and monocyte TF expression increase during acute clinical events. In addition to the chronic impairment of lung function, acute chest syndrome (ACS) adds further insult to lung and cardiovascular impairment. In fact, ACS is the leading cause of sudden death in patients with SCD. Although there are multiple etiologies for ACS, infection/sepsis and the dramatic innate immune and coagulation response to it remain a major contributor to morbidity and mortality during ACS. Novel methods to reduce the inflammatory response during infection are needed as are methods that normalize the chronic pro‐inflammatory state. Chaperone proteins, namely HSP90 and HSP70, are known agents that participate in inflammation and thus have significant potential to influence the inflammatory, pro‐coagulant burden. Therefore, in this study, we wanted to evaluate the novel anti‐inflammatory, anti‐coagulatory properties of the chaperone proteins in SCD. We had previously determined that inhibition of HSP90 using the drug AUY‐922 could block the bacterial toxin lipopolysaccharide (LPS) – induced TF expression and pro‐inflammatory cytokine release from monocytes. Therefore, we used the Townes mouse model of SCD to evaluate AUY‐922 in a pre‐clinical study. Townes mice with SCD or without were administered AUY‐922 intraperitoneal (IP) for 4 days prior to a 6 hour LPS‐mediated induction of the inflammatory response and coagulation activation. Notably, the dose of LPS failed to induce any pro‐inflammatory response in the AA mice (n=24). However, LPS‐induced an exaggerated response in the SS mice. Levels of TNF‐α, IL‐6, IL‐8, and IL‐10 were elevated up to 40,000 fold over control treated SS mice. Pre‐treatment with AUY‐922 either completely ablated, or significantly attenuated the inflammatory cytokine response and normalized EC function. Furthermore, the treatment with AUY‐922 doubled the amount of the anti‐inflammatory chaperone molecule HSP70 in the livers of the SS mice. Thus, taken together, our data demonstrate in both a pre‐clinical and an in vitro proof of principle study, that the chaperone proteins HSP90 and HSP70 are attractive targets at reducing the inflammatory burden and associated acute lung injury in SCD.
Inactivation of the glyoxalase anti‐glycation system in porcine brain post‐cardiac arrest and its preservation by pyruvate
Authors: Scott Gary Frank , Cherry Brandon H , Williams Arthur , Ryou Myoung‐Gwi , Nguyen Anh , Mallet Robert T
Published: November 2023

Background

Glyoxalase‐1 (GLO1) degrades the toxic glycolytic byproduct methylglyoxal, thereby preventing irreversible glycation of proteins and nucleic acids. However, the glyoxalase system may be inactivated by oxidative stress. Cardiac arrest (CA) and cardiocerebral resuscitation (CCR) provokes intense oxidative stress in the brain. However, the impact of CA/CCR on the brain's anti‐glycation machinery is unknown.

Purpose

To analyze the impact of ischemia‐reperfusion on the cerebrocortical glyoxalase system in a porcine model of CA/CCR, and to determine the ability of the natural antioxidant pyruvate to protect this crucial anti‐glycation system.

Methods

Domestic pigs (25–35 kg) were subjected to 10 min CA, followed by 4 min closed‐chest CCR, defibrillation and 4 h recovery. Sodium pyruvate (Figures: Pyr) or NaCl control was infused iv to 3.5–4 mM during CA/CCR and the first 60 min recovery. GLO1 content, activities of GLO1 and glutathione reductase (GR), and protein glycation (Arg‐pyrimidine immunoblot) were analyzed in frontal cortical homogenates collected at 4 h recovery.

Results

Compared with non‐arrested sham pigs (n=4), GLO1 content and GLO1 and GR activities (Figures 1–3) fell sharply after CA/CCR (n=4), but were preserved by pyruvate (n=4). Also, CA/CCR caused protein glycation, particularly at 65 kDa, the MW of the blood‐brain‐barrier component occludin, while pyruvate reversed this glycation.

Conclusions

Cardiac arrest‐resuscitation impaired the brain's GLO1/GR anti‐glycation defenses in a manner prevented by pyruvate treatment. Thus, the brain's GLO1 system is disabled by acute ischemia‐reperfusion, yet pyruvate intervention protected this pivotal, cerebroprotective system.

Support or Funding Information

NS076975 National Institute of Neurological Disorders and Stroke
Comparison of Topical PDGF and Naltrexone on Full‐Thickness Diabetic Wound Healing
Authors: Titunick Michelle Beth , Cain Jarrett , Zagon Ian , McLaughlin Patricia
Published: November 2023

Diabetes affects more than 29 million individuals in the US, and approximately 15% will develop chronic, non‐healing foot ulcers (DFU) that may result in amputation if left untreated. The current FDA‐approved DFU treatment is Regranex® which increases platelet derived growth factor (PDGF), but is expensive and has significant side‐effects. A new topical treatment utilizing naltrexone (NTX) to block classical opioid receptors as well as Opioid Growth Factor Receptor (OGFr) has been shown to accelerate full‐thickness cutaneous wounds in preclinical models of type1 and type 2 diabetes by increasing cell replication and angiogenesis. NTX blocks interaction of the inhibitory peptide OGF and OGFr; OGF is elevated in serum of diabetic humans and animals. To compare the efficacy of NTX and Regranex®, type 1 diabetes was induced in male Sprague Dawley rats (n=14) by injection of streptozotocin (40mg/kg i.p. on two consecutive days); control rats received vehicle only (n=13). Five weeks later, four 6 mm wounds were produced on the dorsum of each animal; wounds were randomly assigned treatment of PDGF (Regranex®), 0.03% NTX dissolved in moisturizing cream, or cream containing sterile saline (control). Residual wound size was photographically monitored over a 2 week period. DNA synthesis (BrdU integration), as well as PDGF and vascular epithelial growth factor (VEGF) expression were measured in wound tissue (3–4 rats per group) collected 1,2 and 4 days following surgery. Both NTX and Regranex® significantly accelerated wound closure in comparison to the vehicle‐treated wounds. By day 4 following surgery, there was a 1.2‐fold decrease in residual wound size in rats treated with either NTX or Regranex® relative to vehicle‐treated wounds, and by day 12, NTX‐treated wounds were reduced in size by 5‐fold relative to vehicle‐treated wounds. All wounds were <1% the original size by day 14.

Four days post‐wounding, DNA synthesis was elevated 43% in diabetic rats receiving NTX relative to diabetic vehicle‐treated rats (p≤0.01). Immunohistochemical examination of wounds one day after surgery revealed that Regranex®‐treated diabetic rats had 139% more PDGF positive cells than vehicle‐treated diabetic rats (p≤0.01), whereas NTX‐treated diabetic rats exhibited approximately 78% increase in PDGF+ cells compared to vehicle‐treated rats (p≤0.05). At 2 days, NTX‐treatment resulted in a 55% increase in PDGF+ cells relative to controls, whereas the Regranex and controls were comparable. Four days after surgery there was a 44% increase in VEGF+ vessels in NTX‐treated diabetic rats (p≤0.001) relative to those receiving vehicle only and a 69% increase when compared to diabetic Regranex®‐treated rats (p≤0.001). FGF immunohistochemistry did not indicated significant differences in expression at wound sites between any treatment groups. The data suggest that blockade of the OGF‐OGFr regulatory pathway utilizing NTX may be a safe and effective alternative treatment to Regranex® for DFUs.
Induction of Transformation by Freshly Fractured Silica in JB6 and BEAS‐2B Cells
Authors: Ding Min , Barber Tabatha , Aldinger1 Joni , Bowman Linda , Meighan Terence , Zhao Jinshun
Published: November 2023

Silica particles are considered to be fibrogenic agents and established carcinogens, but the mechanisms for disease initiation and progression are not well understood. Earlier studies demonstrated that the tumor suppressor gene, PDCD4, and its upstream regulator, miR‐21, may be considered as oncogenes for novel cancer prevention or anti‐cancer therapies. The present study examined whether freshly fractured silica particles induce cell transformation. The data indicated that chronically exposed human lung epithelial BEAS‐2B or JB6 cells to low‐dose silica resulted in neoplastic transformation assayed by soft agar. The alterations of miR‐21‐PDCD4 signaling in JB6 cells after exposure to freshly fractured silica particles were also tested. The results demonstrate that silica caused PDCD4 inhibition in JB6 cells, exposure of cells to silica caused a significant increase of miR‐21 expression and decrease of PDCD4 expression, inhibition of phosphorylated ERKs or phosphorylated p38 with U0126 or SB 203580 reversed silica‐induced PDCD4 inhibition; and, ROS scavengers, N‐acetyl‐L‐cysteine, reversed the inhibitory effect of silica on PDCD4 expression. These findings demonstrate that freshly fractured silica particles induce miR‐21 expression and PDCD4 inhibition, which may be mediated through ROS and ERK pathways. Unraveling the complex mechanisms associated with these events may provide insights into the initiation and progression of silica‐induced carcinogenesis.
Protective Role of Metallothioneins in Human Endothethial Cells in Acute Response to High Dose Cadmium Chloride
Authors: Zhang Yanmin , Kucynda Theresa T. , Marsboom Glenn , Kajdacsy‐Balla Andre
Published: November 2023

Introduction

Cadmium is a well‐recognized environmental pollutant with numerous adverse health effects. Recent research has demonstrated the association of cadmium with cardiovascular disease through endothelial dysfunction at substantially low levels of chronic exposure. However, little is know about the effect of high concentration cadmium effect on human endothelial cells after acute exposure.

Methods

Primary human umbilical vein endothelial cells (HUVECs) were treated with varying doses of cadmium chloride (CdCl2). Flow cytometry was used to evaluate the cells reactive oxygen species (ROS) with cell‐permeant 2′,7′‐dichlorodihydrofluorescein diacetate (H2DCFDA) probe. The oxygen consumption rate (OCR) of adherent cells was measured at 37°C using a Seahorse XF24 Extracellular Flux Analyzer. Gene expression was measured with real‐time RT‐PCR.

Results

The HUVECs cells were treated with varying dose of CdCl2 for 24 hours and cells viability was not changed at any of the dose used. Real time RT‐PCR data revealed both metallothionein‐1A (MT1A) and metallothionein‐2A (MT2A) gene levels were significant increased by 50 μM CdCl2 compared to the control levels (13.8‐fold and 5.4‐fold increase, respectively, P<0.05). OCR of HUVECs was decreased by 50 μM CdCl2 treatments from 66.97±2.41 to 42.00±5.22 pMoles/min (P<0.005), and H2DCFDA fluorescence intensity was decreased from 2989±87.75 in control to 2165±54.75 (P<0.005). However, with lower concentrations, short‐term exposure did not cause significant changes in ROS level and oxygen consumption rate. We are currently exposing cells to lower concentrations for longer period of time.

Conclusions

It is generally agreed that oxidative stress plays important roles in acute cadmium poisoning. Interestingly, our data demonstrated decreased oxygen consumption and lower ROS level upon acute high dose cadmium exposure. The possible mechanism could be that during acute response to high dose cadmium, metallothioneins (MTs) are induced and act as a potent sequestration mechanism, thus protecting against oxidative stress.
Mode of the inhibition of venom collagenase by actinonin
Authors: Price Joseph A. , Sun Jing
Published: November 2023

Snake envenomation remains an endemic health problem in much of the world. A new approach to controlling its pathology may be the use of enzyme inhibitors. A preliminary characterization was made of the mode of C. atrox collagenase inhibition by the protease inhibitor actinonin. The reaction progresses nearly linearly for close to an hour at room temperature. Reaction data for combinations of seven levels of inhibitor and six levels of substrate were fit to Michaelis‐Menten equations by nonlinear regression to determine values of apparent Km and Vmax. In these dose response experiments apparent Vmax, Km, and Vmax/Km decreased as the concentration of the inhibitor was increased. This pattern is most consistent with noncompetitive enzyme inhibition. These results better characterize this agonist in reactions with whole venom, and make actinonin a compound of interest in developing new anti‐venom therapeutics.

Support or Funding Information

No extramural funding supported this work.
A novel gene therapy in hemophilia A mouse model by using lipid‐coated Fe3O4 nanoparticles
Authors: Chen Yen‐Ting , Tsai Tung‐Chou , Lai Ping‐Shan , Chen Chuan‐Mu
Published: November 2023

Hemophilia A is a bleeding disorder disease caused by missing or defective coagulation factor VIII (FVIII), a clotting protein. It's a X‐linked recessive genetic disease, mainly due to gene mutation. Magnetic nanoparticles have been used for various biomedical applications, including tumor‐targeting drug carriers, magnetic resonance imaging etc., but was rarely utilized in the field of inherited disease. Dipalmitoylphosphatidylcholine (DPPC) formulate with iron oxide nanoparticles is concerned as a potential material in biomedical therapy with highly biocompatible. The aim of this research is to establish a non‐viral gene therapy approach to cure hemophilia A. In this study, B6;129S‐F8tm1Kaz/J knockout mice were used as the animal model which mimic sever hemophilia A patients. The biocompatible material‐ IO@DPPC (DPPC‐Fe3O4) was used as a DNA carrier to bring human B‐domain deleted FVIII cDNA (10 μg/mice) into hemophilia A mice by tail vein injection. C57BL/6 mice were used as a normal control and untreated hemophilia A mice as a negative control. Activated partial thromboplastin time results revealed that the blood clotting activity significantly improved in the treatment group when compared to negative control (P<0.05). Furthermore, approximately 75% of IO@DPPC and the FVIII‐eGFP plasmids entered the hepatocyte nuclear and expressed green fluorescent protein. Moreover, this therapy did not cause any liver injury from the histology examination in the safety test. In this research, injecting 10 μg/mice of FVIII‐eGFP plasmids showed a promising therapeutic effects on hemophilia A mice, but the therapeutic levels only maintain until two weeks. To prolong the therapeutic efficacy for more than 6 months, we intend to use higher dosage of FVIII‐eGFP plasmids (20 to 50 μg/mice). Taken together, we have successfully improved the bleeding disorder in hemophilia A knockout mice, that provide a potential novel gene therapy approach in the clinical use of hemophilia A patients.
Location, Location, Location: The Effect of Lorcaserin via Different Routes of Administration on Acute Oxycodone Analgesia
Authors: Lippold Kumiko , Dewey William L.
Published: November 2023

Pain is an unpleasant, subjective experience that serves as an important defense mechanism to noxious stimuli. There are several classes of analgesic drugs prescribed for the treatment of pain, with the most well‐known being opioids. Opioids are almost exclusively indicated for the treatment of moderate to severe pain but with their repeated use, there is a development of tolerance and dependence. With that known, there has been great interest to develop new analgesics or drug combinations that may reduce the incidence of side‐effects and facilitate the analgesic effects. Lorcaserin is a 5‐HT2C agonist that was originally indicated as a treatment for obesity but hypothesized as a potential treatment for drug dependence. Several preclinical studies have shown that lorcaserin reduced the self‐administration of several classes of drugs of abuse, including opiates. This observation prompted us to investigate whether lorcaserin would alter the basic pharmacology of opioids. To test this, we used a preclinical model (tail immersion test) that utilizes an acute, noxious thermal stimulus and is a heavily validated method for testing the antinociceptive properties of opiates. In the first round of studies we investigated the effect of subcutaneous lorcaserin pretreatment on the acute antinociceptive effect of oral oxycodone. All doses of lorcaserin tested produced a reversed dose‐response shift in the oxycodone curve (ED50 = 6.11mg/kg) to the left, with significant ED50 shifts occurring at 0.25mg/kg (ED50 =1.3mg/kg), 0.5mg/kg (ED50 = 1.21mg/kg), 1mg/kg (ED50= 2.08mg/kg), and 2mg/kg of lorcaserin (ED50=3.77mg/kg). It is important to note that none of the subcutaneous doses of lorcaserin tested had an antinociceptive effect on their own. Next, we administered lorcaserin via different routes of administration and found that its effects vary depending upon route of administration. Lorcaserin, administered via the intracerebroventricular route at 32, 64 or 128ug did not demonstrate antinociceptive activity but intrathecal lorcaserin (8, 16, 32, 64, 128ug) demonstrated a full dose‐responsive effect, with an ED50 of 54ug. Interestingly, we observed that ICV lorcaserin attenuated oral oxycodone's antinociceptive effects by 40%. Our results indicate that there are significant regional differences in the effect of lorcaserin and the 5‐HT2c receptor plays a differential role depending on the region of the central nervous system in the antinociception elicited by oxycodone. Further studies will investigate the role of potential off‐target effects of lorcaserin that may be mediated through the 5‐HT2A receptor.

Support or Funding Information

Funding provided by USPHS grant number DA036975.
Regulation of Cytoskeletal Remodeling Proteins in the Ventral Tegmental Area by Morphine, Stress, and TORC2
Authors: Kaska Sophia , Brunk Rebecca , Kechner Megan , Mazei‐Robison Michelle
Published: November 2023

Drug addiction and depression are two co‐morbid diseases that have a substantial burden on society. Increasing evidence suggests that dysfunction of the reward circuit could underlie both disorders. In support of this, chronic morphine and chronic social defeat stress (CSDS) have been shown to induce similar changes in ventral tegmental area (VTA) DA neuronal morphology. Specifically, our previous work demonstrated a morphine‐induced decrease in VTA DA size that was mammalian target of rapamycin complex 2 (TORC2) signaling‐dependent. Similarly, preliminary evidence from our lab suggests that mice that are susceptible to CSDS also have a decrease in VTA DA soma size. Our current objective is to identify the molecular mechanisms underlying these morphological changes. Given our data that morphine‐induced changes in VTA DA soma size are TORC2‐dependent and recent evidence that TORC2 signaling modulates cytoskeletal remodeling in the hippocampus, we hypothesize morphine and stress may alter the actin cytoskeleton in VTA DA neurons through alteration of TORC2 activity. We completed western blot analyses on micro‐dissected VTA tissue from mice exposed to chronic morphine or CSDS and found a significant decrease in the phosphorylation of cofilin, a protein involved in severing actin filaments. We are now examining molecules upstream of cofilin, such as Rac1 and p‐21 activated kinase (PAK) to determine if exposure to chronic morphine or stress similarly alters activity of these proteins. Additionally, to further investigate the potential role of TORC2 on mediating changes in the actin cytoskeleton, we are performing western blot analysis using VTA tissue from mice that either overexpress or knockout Rictor, a critical component of TORC2. Overall, our goal is to determine the molecular mechanisms underlying changes in VTA structural plasticity in the hopes of identifying novel targets for therapeutic intervention in addiction and depression.

Support or Funding Information

PhRMA Foundation Pre‐Doctoral Fellowship in Pharmacology/Toxicology (SK)

Integrative Pharmacological Sciences Training Program: T32 GM092715 (SK)

NIDA: R01 DA039895‐01A1 (MMR)

NIDA: R03 DA037426 (MMR)

PhRMA Foundation Research Starter Grant (MMR)
The Role of Norbuprenorphine in Buprenorphine‐Induced Neonatal Abstinence Syndrome: A Pilot Study
Authors: Brents Lisa K , Caperton Caitlin O , Patton Amy L , Owens S Michael , Moran Jeffery H , Stowe Zachary N , Fantegrossi William E
Published: November 2023

Opioid abuse during pregnancy may cause drug dependence in the fetus, and the abrupt discontinuation of opioid exposure at birth may induce neonatal abstinence syndrome (NAS) during the first week of life. NAS is characterized by inconsolable high‐pitched crying, tremor, vomiting, diarrhea, hypersensitivity, and sleep disturbances. Buprenorphine (BUP), a mu‐opioid receptor partial agonist, is administered as a substitution therapy to treat opioid use disorder during pregnancy. BUP improves neonatal outcome compared with methadone or no treatment, but BUP exposure often leads to NAS. Furthermore, NAS severity is independent of maternal BUP dose throughout pregnancy, suggesting that individual differences in maternal‐fetal BUP pharmacology may increase NAS susceptibility in some newborns. One possible explanation is that fetal exposure to norbuprenorphine (NorBUP), a metabolite of BUP that has high affinity and efficacy at the mu‐opioid receptor, differs across the BUP‐treated population and drives individual differences in the development of opioid dependence. This study tested the hypothesis that NorBUP can induce NAS. To test this hypothesis, we developed a novel model of NAS in which exposure to opioids occurs continuously across fetal development. Pregnant rats were implanted with subcutaneous minipumps containing NorBUP (0.1 or 0.3 mg/kg*day) or vehicle (5 % Emulphor) on gestation day (GD) 9. Treatment continued until delivery. Upon delivery, half of the pups were observed and scored for signs of precipitated withdrawal for 10 minutes after administration of the mu‐opioid receptor antagonist naltrexone (1 or 10 mg/kg, i.p.) or saline (10 ml/kg). Because of the CNS immaturity of the neonatal rat pup relative to the human newborn, this group models opioid dependence in preterm newborns. To model NAS at term, the other half of each litter was fostered to an untreated dam and received subcutaneous injections of NorBUP (0.05 mg/kg or 1.5 mg/kg) every 12 hours (BID) until they were tested for precipitated opioid withdrawal on postnatal day (PD) 7. Few withdrawal signs were observed in the pups tested within 24 hours of delivery, but PD7 pups exposed to the high dose of NorBUP in utero and after birth exhibited significantly more “wall climbing” and less “quiet” signs when challenged with naltrexone, indicating opioid withdrawal. The results of these preliminary studies suggest that NorBUP can induce opioid dependence in the neonatal pup. Ongoing experiments will determine fetal brain concentrations of NorBUP after maternal exposure to NorBUP (GD 9‐ GD20). Results of this experiment will be used to determine additional doses of NorBUP to be tested in the novel rat model of NAS.

Support or Funding Information

Support for this study was provided by the UAMS Fellow to Faculty award and the UAMS Tobacco Research Committee Tobacco Settlement Funds
Depression of home cage wheel running reveals greater spontaneous morphine withdrawal in rats with persistent pain compared to naïve controls
Authors: Lee Andrea T , Kandasamy Ram , Morgan Michael M
Published: November 2023

The widespread use of opioids to treat pain has led to a drastic increase in opioid dependence and overdose fatalities. The transition from the use of opioids to treat pain to abuse is driven in part by withdrawal symptoms associated with the abrupt use of opioids. The objective of the present study is to develop an animal model of withdrawal that mimics the spontaneous withdrawal that occurs in pain patients. We hypothesized that spontaneous opioid withdrawal will cause depression of home cage wheel running. This hypothesis was tested by measuring home cage wheel running before and after subcutaneous implantation of two 75 mg morphine or placebo pellets into male Sprague‐Dawley rats with and without hindpaw inflammation. Administration of the morphine pellets depressed wheel running that recovered to baseline levels by the third day after implantation in naïve rats and by the fifth day after implantation in inflamed rats. Removal of the morphine pellets depressed wheel running for 2 days in naïve animals and over 6 days in rats with an inflamed paw. These data demonstrate that home cage wheel running is a consistent and reliable method to assess spontaneous morphine withdrawal in the rat. Moreover, the magnitude of withdrawal is greatly enhanced in rats with persistent pain.

Support or Funding Information

This study was supported in part by State of Washington Initiative Measure No. 171 (to ATL) and NIH grant NS095097 (to MMM).
Interactions between Delta and Mu Opioid Agonists in Assays of Pain‐Depressed Operant Responding and Schedule‐Controlled Rate Suppression
Authors: Lanpher Janell , Kinens Abby , Richard Philomena , Rice Kenner C , Bilsky Edward J , Stevenson Glenn W
Published: November 2023

Interactions between delta and mu opioid agonists in rodents vary as a function of behavioral endpoint. Previous literature includes assessment of interactions across therapeutic (eg: antinociceptive) and side effect (eg: sedation, respiratory depression) endpoints. The present study is the first to characterize these receptor interactions using a mult‐cycle operant assay of pain‐depressed behavior as the antinociceptive measure. The selective delta agonist SNC80 (ED50 = 4.11mg/kg) and the selective mu agonist methadone (ED50 = 0.35mg/kg) both produced dose‐dependent restoration of lactic acid pain‐depressed operant responding. SNC80 (ED50 = 56.0mg/kg) and methadone (ED50 = 1.55mg/kg) also produced a dose‐dependent suppression of response rates in the operant assay in the absence of the pain‐like stimulus. Based on relative potencies in the nociceptive assay, fixed‐ratio mixtures of SNC80 and methadone (35.1:1, 11.7:1, 3.9:1) are currently being characterized in both assays. Preliminary data indicate that the 11.7:1 mixture produced additive effects in the antinociceptive assay and sub‐additive effects in the rate suppression assay, thus yielding a higher therapeutic index (TI or dose ratio) for the mixture relative to the TI of either drug alone. These results provide further evidence that delta/mu interactions depend on experimental endpoint and that assessment of these interactions utilizing an assay of pain‐depressed behavior is possible.

Support or Funding Information

AR0554975‐02A1 (NIAMS) to G.W.S.
Identification of a major QTL influencing oxycodone behavioral sensitivity and dependence in C57BL/6 substrains
Authors: Bryant Camron D , Goldberg Lisa R , Kirkpatrick Stacey L , Yazdani Neema , Wu Jiayi , Jenkins David F , Luong Alex , Luttik Kimberly P , Johnson William E , Mulligan Megan K
Published: November 2023

Opioid addiction is heritable, yet its genetic basis remains poorly understood. Mice are valuable for identifying novel genes that contribute to variation in addiction‐associated phenotypes, including acute psychomotor stimulation and conditioned reward. The closely related C57BL/6J and C57BL/6NJ substrains exhibit limited genetic diversity, yet frequently show strain differences in addiction‐associated traits, including oxycodone‐induced (OXY) locomotor activity and naloxone conditioned place aversion. Quantitative trait locus (QTL) mapping in closely related substrains drastically reduces the number of segregating genetic variants from millions to thousands, accelerating the identification of the causal genetic factors. We conducted QTL mapping for oxycodone (OXY)‐induced conditioned place preference (CPP, N=212), and naloxone (NAL)‐induced conditioned place aversion (CPA, N=209), and concomitant behaviors along with saline‐treated mice as controls (SAL, N=213). We utilized a 9 day (D) CPP/CPA protocol. Mice received drug (1.25 mg/kg OXY, 4 mg/kg NAL, or SAL, i.p.,) on D2 and D4, and SAL on D3 and D5. Mice were assessed for drug‐free CPP/CPA (D8) and drug state‐dependent CPP/CPA (D9). Mice were genotyped at 96 informative markers and QTL mapping was performed in R/qtl (scanone, 1000 permutations). We identified a major genome‐wide significant QTL for OXY‐induced locomotor activity (Chr. 1, 72.43 cM, LOD = 9.79) that co‐mapped to the same region as a QTL for anxiety‐like OXY withdrawal behavior in the elevated plus maze (chr. 1 77.33 cM; LOD = 5.33). We are currently conducting fine mapping with interval‐specific congenics and striatal transcriptome analysis via RNA‐seq to facilitate identification of genes and neurobiological mechanisms.

Support or Funding Information

R00DA029635 (NIDA; C.D.B.), R03DA038287 (C.D.B), R21DA038738 (C.D.B.), Transformative Training Program in Addiction Science (Burroughs Wellcome 9550300872; N.Y. and J.W.), and T32GM00854118
Blockade of a Novel Neuropeptide Receptor System, BigLEN‐GPR171, Reduces Adverse Effects of Prolonged Morphine Administration
Authors: Bobeck Erin N , Pena Darlene , Gomes Ivone , Fakira Amanda , Devi Lakshmi
Published: November 2023

ProSAAS derived peptides, such as BigLEN, are abundantly expressed throughout the brain. We recently identified BigLEN as the endogenous peptide for the orphan receptor GPR171. Immunohistochemical analysis shows that both BigLEN and GPR171 are highly expressed in areas of the brain involved in opioid function, such as nucleus accumbens (NAc), medial prefrontal cortex (mPFC), and basolateral amygdala (BLA). C57bl/6 mice showed an increase in GPR171 mRNA expression in NAc and BLA following conditioned place preference to morphine. Whereas there was no change in GPR171 expression under these conditions in the mPFC, but there was a reduction in BigLEN expression and enhanced cAMP signaling. Next we examined the role of GPR171 in the BLA of mice using lentiviral‐mediated local knockdown and found that this leads to a reduction in conditioned place preference to morphine. We have recently identified MS21570 as a selective antagonist of GPR171 by in silico screening of a library of small molecules using a homology model of GPR171. Pretreatment with MS21570 (5 mg/kg, i.p. in 6% DMSO) prior to 4 morphine (10 mg/kg, s.c.) pairings, reduced conditioned place preference to morphine. In addition, mice were made tolerant by twice daily morphine injections for 5 days. We found that MS21570 pretreatment reduced the development of morphine tolerance and withdrawal. Together, these data support the idea that the BigLEN‐GPR171 system plays an important role in the regulation of some of the adverse effects associated with prolonged opioid exposure.

Support or Funding Information

Supported by NIH grants, DA019521 and NS026880 (to LAD) and NIDA postdoctoral training grant DA007135 (to ENB).
Examining the capacity of nAChR antagonists to block the effects of nicotine in nicotine‐tolerant C57BL/6J mice
Authors: de Moura Fernando Barreto , McMahon Lance Richard
Published: November 2023

The clinical effectiveness of some nAChR‐based smoking cessation aids is proposed to be due to antagonism of the effects of nicotine. However, it is unclear whether nicotine antagonism varies as a function of repeated nicotine exposure. In the current stidy, nicotine, mecamylamine (nonselective nAChR antagonist), and dihydro‐β‐erythroidine (DHβE; α4β2* nAChR antagonist) were studied in male C57BL/6J mice responding under an FR20 schedule of food delivery before, during, and after discontinuation of daily nicotine treatment consisting of 1.78 mg/kg nicotine administered three times per day. Before daily nicotine treatment, nicotine decreased response rate and rectal temperature with ED50 values of 0.44 and 0.82 mg/kg, respectively. DHβE significantly antagonized the rate‐decreasing and hypothermic effects of nicotine, evidenced by 2.9‐ and 3.2‐ fold rightward shifts of the nicotine dose‐response functions, respectively. Daily nicotine treatment produced tolerance, as evidenced by 3.6‐ and 4.8‐fold rightward shifts in the nicotine dose‐response functions for rate‐decreasing and hypothermic effects, respectively. During daily nicotine treatment, mecamylamine and DHβE no longer antagonized the rate‐decreasing effects of nicotine, whereas both antagonists continued to block the hypothermic effects of nicotine. After discontinuation of daily nicotine treatment, there was a time‐related loss of tolerance to nicotine; under these conditions, mecamylamine and DHβE retained their capacity to antagonize the rate‐decreasing and hypothermic effects of nicotine. These data suggest that nicotine produces rate‐decreasing and hypothermic effects through different receptor types, and further suggest that nicotine can act at non‐nAChRs in nicotine‐tolerant animals. A potential loss of nicotine antagonist activity in cigarette smokers could underscore a potential limitation in the effectiveness of some nAChR‐based smoking cessation aids.

Support or Funding Information

Funding: USPHS DA25267