bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular characterisation of small molecule agonists and internalisation of GLP‐1R
Authors: Kanamarlapudi Venkateswarlu
Published: June 2021

The glucagon‐like peptide 1 receptor (GLP‐1R) is one of the most promising therapeutic targets for the treatment of type 2 diabetes (T2D).1 It is well documented that GLP‐1R agonists, GLP‐1 and exendin‐4, stimulate glucose‐dependent insulin secretion and increase beta cell proliferation and survival. These observations resulted in approval of both GLP‐1 agonists, Liraglutide (a stable form of GLP‐1) and Exenatide (a synthetic version of exendin‐4) for the treatment of T2D.2 The inconvenience of long‐term administration of injectable peptide therapeutics (Liraglutide and Exenatide) has necessitated discovery of orally active small molecules agonists, compounds 2 and B, of the GLP‐1R. The agonist occupied GLP‐1R signals through both the Gαs and Gαq coupled pathways to stimulate insulin secretion. The coupling of GLP‐1R to the Gαs pathway results in cAMP production whereas the receptor coupling to the Gαq pathway leads to intracellular Ca2+ accumulation and ERK phosphorylation (ERK‐p). However, our recent study revealed that compounds 2 and B induce cAMP production similar to GLP‐1, but not intracellular Ca2+ accumulation, ERK‐p.3 Agonist induced GLP‐1R internalisation is important for insulin secretion. We have also shown that inhibition of the Gαq pathway but not the Gαs pathway reduces the GLP‐1R internalisation.4 Consistent with this, compounds 2 and B, which activate only the Gαs pathway, shown to fail in inducing the GLP‐1R internalisation.3 Chemical inhibitors of the Gαq pathway significantly reduced the agonist induced GLP‐1R internalisation and suppressed ERK‐p, demonstrating phosphorylated ERK acts downstream of the Gαq pathway in hGLP‐1R internalisation.4 We have also assessed the importance of both ends (N‐ and C‐termini) of GLP‐1R for its trafficking.5,6 In my presentation, I will discuss our recent findings on small molecule agonists and internalisation of GLP‐1R.

Support or Funding Information

This work was partially supported by Biotechnologyand Biological Sciences Research Council UK (BB/C515455/2 and BB/F017596/1) and Medical Research Council UK(G0401232).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Does this gene make me look fat? ATP10A Expression in Targeted Human Populations
Authors: Hurst Sarah Elizabeth , Ward Taketa , Hall Brandon , Smolinski Kasia
Published: June 2021

Globally, the prevalence of type 2 diabetes (T2D) and diet‐induced obesity (DIO) is increasing due to higher energy intake and lower energy expenditure. However, the mechanisms that underlie these diseases remain unclear as numerous genes are implicated. ATP10A has been associated with several metabolic abnormalities in animal and in vitro models, so for this pilot study, we sought to identify the expression of ATP10A in targeted human populations. Based on the responses from the medical surveys, subjects were divided into three target groups based on body mass index (BMI), and five groups based on waist‐to‐height ratio (WtHR). From collected saliva samples, total RNA were extracted and analyzed using semi quantitative real‐time PCR (qRT‐PCR). Our results showed significant differences in the BMI groups by one‐way ANOVA [F (2, 6) = 49853, p < 0.0001] with the highest fold change in ATP10A seen in the overweight population (58.12) and the lowest in the normal weight population (32.28). When classified using WtHR, results again showed significance [F (4, 10) = 944735, p < 0.0001] with the overweight, obese, and highly obese groups having greater fold changes (10.56, 9.15, and 11.96 respectively) when compared to slender (0.19) and healthy groups (0.77). Collectively, these results suggest that ATP10A may play an important role in obesity and other metabolic disorders, and as such, we believe the results from this pilot study are promising and worth further exploration.

Support or Funding Information

Funding for this work was provided by donations to The ATP10A project.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Link between food intake and the expression of O‐linked N‐acetylglucosamine Transferase (OGT) in channel catfish
Authors: Abernathy Oaklee , Dougherty Megan , Kostner Danica , Nevarez Ericka , Schmidtberger Abigail , Spainhour Rebekah , Kobayashi Yass
Published: June 2021

The obesity epidemic in the United States has been a serious concern for many years and has driven researchers to determine genetic mechanisms that might contribute to its development. In mice, O‐linked N‐acetylglucosamine Transferase (OGT) might serve as a satiety factor responsible for controlling food intake and body weight. OGT appears to signal “fullness” to the brain, and decreased OGT expression leads to weight gain and development of an obese phenotype. In a previous study, OGT expression was examined in various tissues of channel catfish. OGT mRNA was detected in all sampled tissues, with the brain having the highest expression. Our previous study also showed that prolonged fasting appeared to decrease the expression of OGT mRNA in the brain. To determine how feeding frequency correlates with the expression of OGT mRNA in catfish, we completed a 28 day feeding study with three feeding frequencies. Fish were fed every 12 (overfed), 24 (control), or 48 (underfed) hours (n= 4 tanks per treatment, 8 fish per tank). Brain samples were collected from two juvenile catfish from each tank. Using quantitative RT‐PCR, the expression of OGT mRNA was examined in the brain samples. The expression of all OGT mRNA transcripts, as well as the expression of OGT X1 and X3 variant mRNA were not affected by feeding frequency, suggesting changes in feeding frequency might not directly influence OGT mRNA expression in the brain. We are currently examining the relationship between feeding frequency and OGT mRNA expression in the liver.

Support or Funding Information

This project was partially supported by a grant (P20GM103418) from Kansas IDeA Networks of Biomedical Research Excellence (K‐INBRE) of National Institute of Health (NIH).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Probing the Two Orientations of Pal in Vesiculating E. coli
Authors: Pannullo Nicole , Zavorin Mark , D'Arcy Brooke , Farquharson Kara , Kaur Ravinder , Surendran Naveen , Pichichero Michael , Hellman Judith , Michel Lea Vacca
Published: June 2021

Peptidoglycan associated lipoprotein (Pal) is an outer membrane protein from Gram‐negative bacteria, including Escherichia coli (E. coli). E. coli has two subpopulations of Pal: a periplasmic subpopulation bound to peptidoglycan and a surface exposed subpopulation. When the bacteria are cultured under normal conditions in rich media, only a small percentage of cells contain surface exposed Pal. Under more stressful culture conditions (ex. increased temperature, the presence of antibiotics), E. coli will release outer membrane vesicles (OMVs). Using ultracentrifugation and immunoblotting, we have shown that Pal is released from E. coli in these OMV's. We also used flow cytometry to measure the surface exposed Pal subpopulations in hyper‐vesiculating E. coli cells and their OMVs. Preliminary data suggest that the surface exposed Pal subpopulations are increased in vesiculating cells and OMVs.

Support or Funding Information

Received: Rochester Institute of Technology FEAD grant to Dr. Lea Vacca Michel Applying for: RIT Honors program $500 travel stipend to Nicole Pannullo Applying for: RIT National Technical Institute for the Deaf Student Travel Award to Nicole Pannullo

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Engineering synthetic glycan co‐receptors into the glycocalyx of muscle cells to control early stages of neuromuscular junction development
Authors: Godula Kamil
Published: June 2021

Glycans (also known as carbohydrates, saccharides or, simply, sugars) are among the most intriguing carriers of biological information in living systems. The structures of glycans not only convey the cells' physiological state, but also regulate cellular communication and responses by engaging receptors on neighboring cells and in the extracellular matrix. Despite their structural complexity, individual glycans rarely engage their protein partners with high affinity. Yet, glycans modulate biological processes with exquisite selectivity and specificity. To correctly evaluate glycan interactions and their biological consequences, one needs to look beyond individual glycan structures and consider the entirety of the cell‐surface landscape. There, glycans are presented on protein scaffolds, or are linked directly to membrane lipids, forming a complex, hierarchically organized network with specialized functions, called the glycocalyx. In this presentation, I will describe our studies toward engineering the glycocalyx of differentiated myotube cultures to present co‐receptors for the nerve‐derived synaptogenic signal, agrin, aimed at obtaining control over organization of acetylcholine receptors in the postsynaptic terminals of developing neuromuscular junctions.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The heparan sulfate degrading enzyme heparanase is up‐regulated by the EWS‐Fli1 fusion protein in Ewing sarcoma
Authors: Gaskin Shaun
Published: June 2021

Ewing sarcoma is an aggressive form of bone and soft tissue cancer that predominantly affects adolescents. It is commonly caused by the partial translocation of the Ewing sarcoma protein (EWS) gene to the gene encoding the Ets family transcription factor Fli1, resulting in the formation of the EWS‐Fli1 fusion protein. Ewing sarcoma is often highly metastatic, resulting in a poor survival rate of approximately 20%. Expression of the endoglycosidase heparanase (HPSE), a heparan sulfate‐degrading enzyme that contributes to extracellular matrix remodelling, is up‐regulated in approximately 90% of all cancers, including Ewing sarcoma, and possesses pro‐metastatic and ‐angiogenic functions. It is therefore not surprising that it is also a marker of poor prognosis for cancer patients. While increased expression of HPSE in Ewing sarcoma has been reported to correlate with increased tumour size, disease severity and a poor patient outcome, the mechanism by which HPSE expression is increased in Ewing sarcoma has not been defined. We have identified a novel link between the EWS‐Fli1 fusion protein and the expression of HPSE. Using a combination of patient‐derived samples and cell lines, we have been able to show that an increase in expression of EWS‐Fli1 correlates with increased HPSE expression and activity. We also demonstrated that increased EWS‐Fli1 expression is responsible for increased HPSE expression and enzymatic activity by binding to the HPSE gene promoter and driving its expression. These findings highlight a possible role for HPSE in the progression and prognosis of Ewing sarcoma that may contribute to the aggressive nature of the disease.

Support or Funding Information

Australian National Health and Medical Research Council

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Preclinical evaluation of a new ROS1 inhibitor to overcome resistance to EGFR therapy in glioblastoma
Authors: Aljohani Hashim M. , Bahassi El Mustapha
Published: June 2021

Glioblastoma Multiforme (GBM) is the most aggressive brain tumor in adults and remains incurable despite multimodal intensive treatment regimens. The majority of GBM tumors show a mutated or overexpressed Epidermal Growth Factor Receptor EGFR; however, tumors treated with EGFR inhibitors such as gefitinib or erlotinib inevitably recur. This recurrence highlights the need to identify signaling pathways involved in GBM resistance to EGFR therapy that may serve as targets for intervention. In our recently published work, we found that activation of the ROS1 pathway is a primary mechanism by which glioma cells become resistant to EGFR‐targeted therapy. ROS1 is a proto‐oncogene receptor tyrosine kinase activated by chromosomal rearrangement in several human cancers, including non‐small‐cell lung cancer (NSCLC), cholangiocarcinoma, gastric cancer, ovarian cancer, and glioblastoma. We therefore, designed, synthesized and screened a series of new ROS1 inhibitors. The lead compound, ROS‐Ic, is very potent (IC50 = 24nM) and highly specific to ROS1 protein. Treatment of gefitinib‐resistant GBM cells with ROS1‐lc sensitized the resistant cells to low concentrations of gefitinib resulting in apoptosis following a prolonged S phase cell cycle arrest. When compared to Crizotinib, an FDA‐approved small‐molecule tyrosine kinase inhibitor currently used to treat ALK‐ and ROS1+ NSCLC patients, ROS‐Ic showed superior cytotoxic activity in vitro towards ROS1‐positive GBM cells. Crizotinib resistance is thought to be due to diminished Crizotinib binding, due to ROS1 mutations in Crizotinib binding pocket. Interestingly, our initial analysis suggests that our lead compound ROS1‐Ic might not be affected by these mutations, highlighting the potential therapeutic value of this compound.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A High‐throughput Targeted Bisulfite Sequencing‐based Analysis for Epigenetic Age Quantification and Monitoring
Authors: Chew Yap Ching , Guo Wei , Yang Xiaojing , Jin Mingda , Booher Keith , Horvath Steve , Jia Xi Yu
Published: June 2021

Aging represents the most important risk factor for many chronic diseases including cardiovascular diseases, diabetes, and cancer, therefore understanding the mechanisms of aging is a fundamental step for designing new treatments for chronic diseases. DNA methylation is the most reliable and accurate molecular marker for aging quantification, however, genome‐wide DNA methylation profiling techniques, such as reduced representative bisulfite sequencing and Illumina Bead Array that are widely used in aging research are prohibitively expensive and have poor data quality at low‐read coverage sites. Here we report a robust targeted bisulfite sequencing approach, called SWARM™ (Simplified Whole‐panel Amplification Reaction Method), for the accurate biological age determination. SWARM™ is flexible and low cost, requires relatively low DNA input, allows the simultaneous amplification and sequencing of hundreds of loci, and has shown to increase sample throughput. Using the SWARM™ approach, we were able to analyze the methylation level of several hundreds of age‐associated loci including the published Horvath Clock sites. An age‐predictive model was built using the elastic net regression of DNA methylation levels of the loci and chronological age of blood DNA samples of over 300 healthy subjects of 18 to 89 years old. Our epigenetic age (DNAge™) predictor achieved a small median age error, and the DNAge™ of samples that were processed on both sequencing and array platforms highly correlated (r > 0.9). In brief, our versatile SWARM™ technology‐based DNAge™ platform is a very fast and accurate tool for the precise biological aging quantification and aging interventions monitoring. Dedicated DNAge™ platforms are compatible with various sample types, and can be applied for aging studies in human, and in other species including mice.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Prevalence of 22q11.2 Deletion and Genetic Characterization in Chinese Patients with Congenital Heart Disease
Authors: Hou Hai‐Tao , Chen Huan‐Xin , Wang Jun , Yuan Chao , Wang Xiu‐Liu , Yang Qin , He Guo‐Wei
Published: June 2021

Background

The 22q11.2 deletion syndrome, resulted from the rearrangements in the 22q11.2 chromosomal region, is the most common microdeletion syndrome. Most patients share a common 3Mb or 1.5Mb hemizygous deletion of 22q11.2 in the low copy repeats (LCRs). The remaining patients include smaller deletions that are nested within the 3Mb typically deleted region (TDR) and a few rare deletions that have no overlap with the TDR. The present work aimed to identify the prevalence and the detailed genetic characterization of 22q11.2 deletion or amplification in the children with any sporadic congenital heart disease (CHD), and to explore the genotype‐phenotype relationship between deletion/amplification type and clinical data.

Methods

A total of 354 CHD patients were screened by multiplex ligation‐dependent probe amplification and capillary electrophoresis methods. Universal Probe Library technology was applied to validate the screening results.

Results

We found 40 patients in 354 CHD (11.3%) carried different levels of deletions or amplifications. The phenotypes of the 40 patients included ventricular septal defect (15), atrial septal defect (12), patent ductus arteriosus (12), tetralogy of Fallot (6), right ventricular outflow tract stenosis (4), pulmonary stenosis (2), pulmonary hypertension (18), and Down's syndrome (2). Two patients with ventricular septal defect carried the 3Mb TDR from CLTCL1 to HIC2 (LCR A–D) and 1.5Mb deletions from CLTCL1 to MED15 (LCR A–B) respectively; all the others carried atypical deletions or amplifications nested within the LCRs. Based on the gene analysis, CDC45 (15), TBX1 (8), USP18 (8), RTDR1 (7), SNAP29 (6), and ZNF74 (4) exhibited a higher incidence among the 40 individuals; in addition, 6 patients presented a amplification in TOP3B gene.

Conclusions

This study revealed 3Mb TDR, smaller deletions, rare deletions, and segmental amplifications of chromosome 22q11.2 in Chinese CHD patients. The genotype‐phenotype relationship analysis indicated that those gene abnormalities may cause various types of CHD. Our data also suggest that genetic detection should be performed routinely in CHD patients and the unique deletions or amplifications may provide useful insight into individual analysis.

Support or Funding Information

Supported by grants from the National Natural Science Foundation of China (81170148 & 81641017), Zhejiang Provincial Natural Science Foundation (LY15H020008), & Tianjin Binhai Key Platform for Creative Research Program (2012‐BK110004), Binhai New Area Health Bureau (2012BWKZ008, 2016BWKY007, 2016BWKZ003).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MINIMAL EFFECT OF ALISKIREN ON MAST CELLS COUNT AND RENAL VASCULAR DAMAGE IN ACUTE RAT MODEL OF TRIOLEIN INDUCED PULMONARY FAT EMBOLISM.
Authors: Khalafi Farnaz , Onishchenko Elizabeth , Pour Mohammad , Arif Dauod , Monaghan Paula , Poisner Alan , Molteni Agostino
Published: June 2021

Introduction

In a rat model of fat embolism (FE) induced by injection of triolein (T) a severe inflammatory reaction leads to vasoconstriction and pulmonary fibrosis (1). Mast cell numbers are significantly increased in the lungs of the treated animals (2), and mast cell inhibition improves pulmonary vascular remodeling in pulmonary hypertension (3).

There is evidence that the renin angiotensin system (RAS) is involved since drugs related to this system: captopril, losartan and aliskiren, prevent the pulmonary vasculitis and fibrosis in the early phase (48 hours) (4,5). However, aliskiren (ALI), a direct renin inhibitor, had a minimal effect on renal vasculitis, and fibrotic changes (6). In this study we evaluated the effect of T and ALI on renal mast cells.

Materials and methods

22 Sprague Dawley rates (250–300g BW) received T (0.2 ml n=18) or saline n=4. The T treated rats, divided into 3 groups of 6 each, were IP injected one hour later either with 0.2 ml saline, ALI 50 mg/kg or AL I 100 mg/kg. Four controls received saline.

48 hours later rats were killed under isoflurane anesthesia and the kidneys and lungs stained with H&E, Trichrome, SMA1 for inflammation and fibrosis, and CD117 for mast cells. 10 photographs at 200× were taken for slides of each organ by two pathologists unaware of the slides' identity, mast cells were counted and pathological damage evaluated.

Results

Renal fibrosis and vasculitis were irrelevant by observation with all SM1, Trichrome or H&E stains, with the exception of the media adventitia ratio (MAR) of arteries (p= 0.007) which significantly increased after T‐saline treatment but not after ALI administration. These findings are in contrast with the observations of the lungs where the damage was severe and reduced by both ALI doses. Kidneys of ALI 100 mg/kg treated rats were similar to those ones of T+Saline or of the controls. Although a mild mast cell increase was noted in kidneys of T+Saline treated animals, no statistically significant difference in mast cells was observed.

Conclusion

Renal findings were quite different from our lung findings in either histopathological damage or mast cell accumulation. The study suggests that the RAS system influence on the kidneys differs from that of the lung at this acute stage of fat embolism.

Support or Funding Information

Supported by the Catherine T Geldmacher Foundation, St. Louis, MO

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fractones: tumor matrix in colon cancer, stomach cancer and glioblastoma
Authors: Mercier Frederic , Uguen Arnaud , Marcorelles Pascale , Fronville Alexandra , CORCOS Laurent
Published: June 2021

The basic pathological mechanisms leading to dysplasia, tumor progression and the destruction of adjacent tissues are not well known. Basement membranes and underlying connective tissue are part of the construction machinery that operates to form structures during development and throughout adult life. In cancer, basement membranes tend to restructure the tissue in conjunction with altered stem cells and their microenvironment‐specific extracellular niche. We have characterized a new type of extracellular matrix that we have named fractones. Fractones are chemically similar to basement membranes and consist at least of laminin isoforms, collagen‐IV, nidogen and heparan sulfate proteoglycan. However, fractones and basement membranes differ with respect to their location. Fractones reside in the parenchyma of tissues and organs and are visualized as particles by light microscopy whereas they often display a fractal ultrastructure (a reference to their name). In contrast, basement membranes form sheaths at the connective tissue/parenchyma interface and in between the layers of the vascular wall. We have shown that fractone‐associated heparan sulfates bind, concentrate and activate the FGF‐2, BMP‐4 and BMP‐7 growth factors to regulate stem cell proliferation in the neurogenic zone of the adult brain. Here, we hypothesized that fractones are produced by cancer stem cells and by their immediate progeny to bind and concentrate growth factors to their vicinity. This would participate in rerouting the activity of the physiological stem cell niche and benefit from neovascularization to capture essential nutrients and reach independence. Using immunohistochemistry for laminin, a marker of fractones and basement membranes, on sane and cancerous tissues, we have identified deficient basement membranes and a high expression of fractones in zones without basement membranes in colon cancer. In addition, a high ectopic expression of fractones was correlated with a significant decrease in vasculature in multiform glioblastoma. Our results show that fractones are associated with cancer‐specific morphological alterations. We anticipate that the alteration in binding of growth factors in cancer tissues could ultimately lead to increased proliferation coupled with a distorted neoangiogenesis. These results provide novel insights into hitherto unrevealed mechanisms, on which innovative future therapies may be based, for example using glycan antagonists of those expressed in tumor‐specific fractones.

Support or Funding Information

Canceropole Grand Ouest, France

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Indoleamine 2,3‐dioxygenase expression is correlated to prognosis in localized prostate cancer.
Authors: Ferreira Janaina Mendes , Pontes‐Júnior José , Matheus Luiz Henrique Gomes , Sousa Diego Mota , Lima Amanda Eunice Ramos , Almeida Robson José , Camacho Cleber Pinto , Reis Sabrina Thalita , Leite Kátia Ramos Moreira , Srougi Miguel , Dellê Humberto
Published: June 2021

Background

Indoleamine 2,3‐dioxygenase (IDO) is an enzyme that acts as an immunomodulatory molecule. It is found in several cancer types where it seems to be associated with tumor escape due to its immunosuppressive mechanisms. Some studies have reported a correlation between carcinogenesis and changes in IDO expression. However, the role of IDO expression in prostate cancer (PC) is unclear. The aim of our study was to evaluate the expression of IDO in localized PC, and to correlate its expression with the classic prognostic factor and recurrence after surgical treatment.

Methods

For this case‐control study, we retrospectively evaluated surgical specimens from 111 patients with localized PC, who underwent radical prostatectomy. Recurrence was defined as a prostate specific antigen (PSA) level exceeding 0.2 ng/ml postoperatively, and the median follow‐up was 123 months. IDO expression was evaluated by immunohistochemistry in 72 cases of which 42 (58%) had biochemical recurrence.

Results

We found that lower IDO expression was associated with higher Gleason score (p = 0.022) and PSA levels (p = 0.042). The multivariate analyses revealed that the loss of IDO expression and higher PSA levels were independently associated with biochemical recurrence. The chance of recurrence was increased by 85% in patients with lower IDO expression (OR = 0.15; p = 0.009 CI 95% [0.038–0.633]) and increased by 5.5 times in patients with higher PSA levels (OR = 5.51; p = 0.012 CI 95% [1.435–21.21]). The recurrence‐free survival curve also demonstrates that lower IDO expression (p = 0.0004) was associated with lower time to biochemical recurrence.

Conclusion

The loss of IDO expression was associated with increased chance of biochemical recurrence, higher PSA, and a Gleason score in localized PC.

Support or Funding Information

The study was supported by São Paulo Research Foundation (FAPESP)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sex Differences in Cocaine Self‐Administration Behavior are Driven by Estrous Stage Dependent Changes in Dopamine Transporter Sensitivity to Cocaine
Authors: MAUTERER MADELYN , Jones Sara R.
Published: June 2021

Traditionally, preclinical cocaine studies have focused primarily on male behavior and neurobiology, but recent work has suggested that there are distinct differences in male versus female cocaine responsivity. In humans, progression to cocaine dependence is faster in women compared to men. Women also exhibit greater craving and relapse following abstinence. Similar to humans, rodent literature suggests males and females have differential behavioral responses to cocaine, which appear to be estrous cycle dependent. For example, females exposed to cocaine during the estrous phase exhibit augmented behavioral responses, such as increased conditioned place preference and greater cocaine seeking. Furthermore, recent literature has uncovered neurobiological differences between the sexes, specifically related to estrogen, which suggest that differences within the dopamine system, specifically, may underlie the observed increase in cocaine abuse vulnerability in females.

Here, we used intravenous cocaine self‐administration to assess differences in time to acquire and motivation to seek cocaine between not only sex but also stages of the estrous cycle. We found that female rats are most likely to achieve cocaine self‐administration acquisition parameters when they are in the estrus phase of the cycle; in fact, 90% of females tested were in the estrus phase at time of acquisition. We used the threshold schedule of reinforcement, a paradigm of behavioral economics, to assess motivation to take cocaine. Females in estrus exhibit greater motivation (increased “price” paid to maintain blood cocaine levels; Pmax) which is augmented further if their first exposure to cocaine occurred during estrus as well. Our observed behavioral differences were further shown to be correlated with changes in dopamine terminal sensitivity. Using ex vivo fast scan cyclic voltammetry (FSCV) in the nucleus accumbens (NAc), we found that females in estrus show greater terminal sensitivity to cocaine which may drive the increased cocaine taking behaviors.

Support or Funding Information

R01 DA014030 T32 AA007565

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dissecting the Abuse Liability of Cathinone Derivative Psychomotor Stimulants in Female Rats
Authors: Taffe Michael A. , Harvey Eric L. , Javadi‐Paydar Mehrak , Nguyen Jacques D. , Grant Yanabel , Vandewater Sophia A. , Creehan Kevin M. , Dickerson Tobin J.
Published: June 2021

Rationale

Use of a diversity of synthetic psychoactive cathinone drugs (“bathsalts”) continues to expand worldwide despite legal control. The pyrrolidinophenones, 3,4‐methylenedioxypyrovalerone (MDPV) and α‐pyrrolidinopentiophenone (α‐PVP), cause violent and/or bizarre public behavior in some users and are potent reinforcers in rodent intravenous self‐administration (IVSA) models. Other cathinones with entactogen‐like pharmacology appear to be more efficacious reinforcers than the class‐defining 3,4‐methylenedioxymethamphetamine (MDMA).

Objectives

To determine how modification of the aromatic ring and extension of the alpha carbon alkyl chain modifies the reinforcing properties of cathinone derivatives.

Methods

Female Wistar rats were trained in IVSA of α‐PVP (0.05 mg/kg/inf; 1 h sessions), MDMA (0.5 mg/kg/inf; 6 h) or Pentylone (0.5 mg/kg/inf; 6 h). Following acquisition, the groups were subjected to dose‐substitution under FR and PR response contingencies. Experiments evaluated: α‐PVP, α‐pyrrolidinohexiophenone, pentylone, methylone, pentedrone, 4‐methylpentedrone, MDMA and an MDMA analog.

Results

Extension of the carbon chain decreased potency, but increased efficacy, of MDMA and methylone. Removal of the pyrrolidino group decreased potency of α‐PVP or MDPV. Effects of 4‐methyl vs 3,4‐methylenedioxy substitution were not consistent across core structures.

Conclusions

Recreational use of a wide variety of closely‐related cathinone derivatives drives structure‐activity inferences about abuse liability. IVSA studies in rats, however, show that predictions based on structure are often incomplete or in error. In vivo studies of abuse liability are therefore necessary to understand health risks of novel cathinone derivatives.

Support or Funding Information

These studies supported by USPHS Grant: R01 DA042211
Sex Differences in Antinociceptive Tolerance Development to Δ9‐THC and CP55,940 in Wild‐type and Desensitization‐Resistant S426A/S430A Mice
Authors: Henderson‐Redmond Angela N. , Sepulveda Diana E. , Ferguson Erin L. , Kline Aaron M. , Nealon Caitlin M. , Morgan Daniel J.
Published: June 2021

Tolerance to the antinociceptive effects of cannabinoid agonists limit their therapeutic potential. Male mice expressing a desensitization‐resistant form of the CB1 receptor (S426A/S430A) show delayed tolerance development to the hypothermic and antinociceptive effects of Δ9‐THC, a strongly desensitizing partial CB1 agonist. However, it remains unknown what effect this mutation might have on altering tolerance to the synthetic full CB1 agonist CP55,940 and whether tolerance development to Δ9‐THC and CP55,940 differ in a sex‐specific manner. The purpose of the current study was to investigate whether the S426A/S430A mutation alters antinociceptive and/or hypothermic tolerance to CP55,940 and Δ9‐THC and whether these effects are sex‐specific. Tolerance to Δ9‐THC (30 mg/kg) and CP55,940 (0.3–0.6 mg/kg) was assessed in two ways: following once‐daily injections across 7–10 consecutive days and via shifts in the cumulative dose response curves following 3–7 days of treatment with each agonist in both male and female wild‐type and S426A/S430A mice. Once‐daily drug administration resulted in slower tolerance development to the effects of CP55,940 versus Δ9‐THC in male and female wildtype mice. The S426A/S430A mutation delayed tolerance to the antinociceptive effect of CP55,940 in male mice and delayed tolerance to both the antinociceptive and hypothermic effects of CP55,940 and Δ9‐THC in female mice. In our cumulative dose responses, we found that the S426A/S430A mutation rendered both male and female mice more sensitive to the effects of Δ9‐THC compared to wild‐type mice. Overall, our results demonstrate that sex and agonist specific mechanisms may mediate cannabinoid tolerance. These studies showcase the antinociceptive therapeutic potential of cannabinoid agonists and offer potential avenues to better understand tolerance to their analgesic effects following prolonged exposure.

Support or Funding Information

NIH grants DA036385 (DJM), DA037355 (DJM), Pennsylvania Department of Health using CURE Tobacco Settlement Funds (DJM), Penn State University College of Medicine Department of Anesthesiology and Perioperative Medicine Start Up Funds

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An Internal Pool of β‐Adrenergic Receptors Activates PLC‐mediated PI4P Hydrolysis in Cardiac Myocytes
Authors: Nash Craig , Wenhui Wei , Smrcka Alan
Published: June 2021

Cardiac hypertrophy and subsequent heart failure is a leading cause of morbidity and mortality in the USA. Previously, our laboratory identified a new prohypertrophic pathway where PLCɛ, scaffolded to mAKAPβ at the nuclear envelope, hydrolyzes phosphatidylinositol 4‐phosphate (PI4P) at the Golgi, locally generating DAG and drive hypertrophic gene expression. This pathway is activated by multiple upstream signals including the Epac‐selective cAMP analogue, cpTOME. However, stimulation of β‐adrenergic receptors with Isoproterenol does not activate this pathway, despite strongly raising cAMP. We were able to uncover Iso stimulated PI4P hydrolysis only in the presence of a cAMP phosphodiesterase 3 inhibitor. Irannejad et al 2 demonstrated that β1‐adrenergic receptors are present in the Golgi membrane of Hela cells and can generate cAMP there. Also, Boivin et al 3 demonstrated that β1‐adrenergic receptors are present in the perinuclear region of adult cardiomyocytes. To determine if internal β‐adrenergic receptors can stimulate PI4P hydrolysis, we treated NRVMs with the membrane permeable β‐adrenergic agonist, dobutamine. In contrast to the membrane impermeable Iso, dobutamine induced rapid and sustained PI4P hydrolysis that was at least partially blocked by the cell permeable β‐adrenergic antagonist, metoprolol (10 μM). The cell impermeable βAR antagonist, sotalol (5 mM) could not block dobutamine effects, suggesting that a pool of internal β‐adrenergic receptors can induce PI4P hydrolysis. Over expression of the RA1 domain of PLCɛ, which competes PLCɛ away from the mAKAPβ scaffold, also inhibited dobutamine stimulation of PI4P hydrolysis. These data suggest that β‐adrenergic stimulation in internal membranes, potentially at the Golgi apparatus or nuclear envelope, can stimulate PI4P hydrolysis. These observations demonstrate a novel mechanism for PLC activation through an internal GPCR which may be responsible for cardiac hypertrophy and heart failure. Targeting internal β‐adrenergic receptors may allow for the development of selective therapies which can treat heart failure with little to no effect on other cardiac functions.

Support or Funding Information

This research was supported by National Institutes of Health Grants R01GM53536 and R01GM81772.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Synergistic Effect of Dopamine and Norepinephrine Transporter Inhibition on Cognitive Enhancement
Authors: Pantoni Madeline M. , Anagnostaras Stephan G.
Published: June 2021

Psychostimulants such as amphetamine and methylphenidate are highly effective cognitive enhancing medications, yet have a high potential for abuse and addiction. Evidence suggests that both norepinephrine transporter (NET) and dopamine transporter (DAT) inhibition is required for the procognitive effects of psychostimulants. Although DAT inhibition is also responsible for the addictive potential of psychostimulants, drugs with low activity at DAT may not produce addiction. In the present study, we examined the cognitive and reinforcing effects of various DAT inhibitors (e.g., bupropion, GBR‐12909, GBR‐12935) and NET inhibitors (e.g., atomoxetine, nisoxetine) alone and in combination, using Pavlovian fear conditioning and conditioned place preference. We found that certain dose combinations of these DAT and NET inhibitors produced enhancements in immediate and/or long‐term fear memory acquisition, but did not produce reinforcement or behavioral sensitization. We propose that a pharmaceutical combination of DAT and NET inhibition could lead to the development of a non‐stimulating, non‐addictive, and non‐controlled, yet highly effective cognitive enhancer.

Support or Funding Information

NIH DA020041 (SGA)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating Arrestin‐3 Recruitment to Nociceptin/Orphanin FQ Opioid Receptor (NOPR)
Authors: Hansen Marissa M. , Spangler Skylar M. , Bruchas Michael R.
Published: June 2021

Nociceptin/orphanin FQ opioid receptor (NOPR) has therapeutic potential for treating drug addiction as demonstrated by the FDA‐approved drug Buprenorphine, a NOPR partial agonist, and recent studies show NOPR agonism blocks reward seeking behavior in rodents across multiple drug classes. Despite this, little is known about the signaling dynamics by which NOPR exerts these effects. It has been previously determined that the endogenous ligand nociceptin induces recruitment of arrestin‐3, a scaffolding protein which mediates GPCR desensitization and internalization, through NOPR c‐terminal tail phosphorylation. This study proposes that NOPR c‐terminal tail phosphorylation encodes arrestin functionality, as the specific phosphorylation patterns that recruit arrestin‐3 and 2 remain unknown. The biased signaling of NOPR in arrestin‐3 recruitment was explored using NOPR phospho‐mutants and biased ligands (reported in Chang et al., 2015) 3‐endo‐8‐[bis(2‐methylphenyl)methyl]‐3‐phenyl‐8‐azabicyclo[3.2.1]octan‐3‐ol (SCH 221,510) and 11‐[1‐(1‐methylcyclooctyl)‐4‐piperidinyl]‐2‐(3R)‐3‐piperidinyl‐1H‐benzimidazole trihydrochloride (MCOPPB) in order to dissect the molecular mechanisms of NOPR‐arrestin recruitment. Bioluminescence resonance energy transfer (BRET) assays were conducted to quantify real‐time binding between NOPR and arrestin to construct concentration response curves as a means of identifying signaling bias. This study shows that arrestin‐3 recruitment is not dependent on phosphorylation of c‐terminal tail sites as indicated by NOPR‐arrestin interaction with the NOPR c‐terminal phospho‐null mutant, however is absent in the 10A mutant which includes the 3rd intracellular loop. Further decoding of phosphorylation sites on the third intracellular loop could reveal the mechanism by which NOPR phosphorylation initiates downstream signaling via arrestin and thus the pathways by which the receptor controls neuronal function in dopamine neurons.

Support or Funding Information

This work was funded and supported by the National Institutes of Health Blueprint Program for Enhancing Neuroscience Diversity through Undergraduate Research Education Experiences (R25) and Washington University in St. Louis.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Systems Approach to Assign Expression Based Signatures to Adrenergic Drugs
Authors: McGregor Brett Anthony , Guo Kai , Porter James E. , Hur Junguk
Published: June 2021

Adrenergic receptors and associated drugs have been among the most thoroughly studied G‐protein coupled receptors involved in a wide variety of physiological processes. There are three main groups of adrenergic receptors (alpha1, alpha2, and beta) with each group having multiple receptor subtypes which involve different targets modulating various functions. Due to the highly conserved structures of these receptors, identifying specific and non‐specific binding partners as well as activity of the receptors have been a challenge to assign within this family. In this study, we used a systems approach, involving the use of high‐throughput drug‐perturbation profiling data from the Library of Integrated Network‐Based Cellular Signatures (LINCS) database, to assign drug receptor preference as well as downstream modulation of cellular functions. LINCS, aiming to create a network‐based understanding of gene expression when cells are exposed to different perturbing agents, have extensively profiled the transcriptomic perturbations of over 20,000 small‐molecules in various cell lines and primary cells. Existing information about receptor specificity of drugs from DrugBank for 113 adrenergic drugs was used to assign binding partners to these drugs based on the LINCS drug perturbation data. Perturbed gene expression signatures for specific receptors were then examined for enriched biological functions as well as gene expression similarities. The analysis of drugs specific for activating these receptors provides an expression‐based signature demonstrating each receptors influence on cellular functions. Defining the role different receptor subtypes have on cellular function would provide an alternative way to determine receptor preference of the more promiscuous drugs based on the similarity of their functional involvement. We used this method to identify separate classes of drugs with similar effects on our model system. As an example, we had previously been using Isoprenaline as a beta‐adrenergic agonist to modulate cytokine response in SIMA9 cells, a murine microglia cell line, and needed to identify an alternative non‐catechol drug that would also influence toll like receptor signaling pathways. Our method identified a quinolone, Procaterol, as an alternative drug, which also influences toll like receptor signaling pathways and yields a similar effect in the SIMA9 cell line. Using this systems pharmacology approach may provide a way to further elucidate receptor preference of certain drugs as well as identifying drugs with similar functional influences despite other characteristics.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
New Paradigms of Mass‐Action Law Based Bio‐Dynamics and Pharmacodynamics for New Drug Evaluations
Authors: Chou Ting‐Chao
Published: June 2021

The Mass‐action law (MAL) based Median‐effect eq. (and its Combination‐index eq.) is the unified general biodynamics (BD) theory for biochemistry and biophysics (Chou TC, Pharmacol. Rev. 58: 621–681, 2006; Integr. Biol. 3: 548–559, 2011). Its eqs & algorithms provide efficient, quantitative, econo‐green, digital, automated computer simulations for bio‐medical research & development (R&D). This paper is to illustrate the fundamental principle of how the theory was derived during the 1967–1984 period, and how this theory, to date has been cited 27,594 times in >1,500 biomedical journals worldwide (Google Scholar Citations – Ting‐Chao Chou; Web of Science: www.researcherid.com/rid/B‐4111‐2009). One theoretical article alone, Chou TC & Talalay P, (Adv. Enz. Regul. 22: 27–55, 1984) has been cited 5,422 times in 1,102 journals, which is referred as “Makes History” [Elsevier News Release 3/16/2016]. The derived/proven theory reveals the following new basic paradigms: 1. “Dose” and “Effect” are interchangeable. 2. All dose‐effect curves (DEC) of MAL can be transformed into straight lines. 3. The theoretical minimum of only 2‐data points are required to simulate a DEC [and the practical minimum of 16, 10, and 10 data points for a DEC by computer simulation in vitro, animals and clinical trials, respectively]. This is the theoretical basis for simple, efficient, quantitative “econo‐green” bio‐R&D and regulations. 4. Explicit digital definition of “Synergism” of drug combination is established [in which “Synergism‐Claim” has scientific, regulatory, legal, ethical and economic consequences]. 5. Ki will never be greater than IC50. 6. The median‐dose (Dm) is the harmonic mean of kinetic constants, Kii and Kis. 7. The PD software, e.g., CompuSyn, allows 5 dynamic graphic transformations for diagnostic plots, illustrations automatically by computer simulation within 1 second after data entries. This theory/method has been cited 9,916 times during 2012 to 2017. The Scopus Analysis (11.18.2017) indicated that Chou's publications have been cited by the papers distributed in the follow disciplines: Medicine (56.2%), Biochem. (50.8%), Pharmacol. (25.3%), Chem. (15.6%), Immuno. (4.3%), Chemical Engineer. (3.5%), Mater. Sci. (2.1%), and Others (9.3%); and by number of citing papers from Institutions: Mem. Sloan‐Kettering CC (475), Harvard Med. Sch. (380), MD Anderson CC (325), NIH (305), NCI (251), Inserm (200), Chinese Aca. Sci. (155), UC‐SD (130) and Duke U. (127). The above indicate the broad utilities and high impact of the MAL based PD theory and applications.

Support or Funding Information

PD Science LLC Fund‐17

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
p23 Modulates Aryl Hydrocarbon Receptor Protein Levels in Normal Cell Lines
Authors: Chen Jinyun , Yakkundi Poonam , Chan William K.
Published: June 2021

Aryl hydrocarbon receptor (AHR) is a transcription factor activated in response to exposure of environmental toxins. p23, a co‐chaperone of AHR, was found to be capable of regulating ligand‐dependent AHR activation and functions. Previous study from our laboratory showed that low expression of the p23 protein is associated with poor AHR protein stability and function in cancer cell lines. While in a p23 null mouse model, it was reported that p23 did not affect the AHR expression. To better understand this apparent dichotomy, we explored the possibility that the AHR expression may be regulated differently in cancer cells than in normal cells. We analyzed the AHR protein levels in three normal cell lines with either wild‐type or reduced p23 protein levels (55–62% of the wild‐type p23 protein levels), namely human breast epithelial cells (MCF‐10A), human bronchial tracheal epithelial cells (HBTEC), and mouse type I regulatory T cells (Tr1). We observed that the AHR expression was significantly reduced by about 38–57% compared to the controls after 72 hours transfection of p23‐specific shRNA in all three normal cells, indicating that p23 is more important for the control of AHR expression in normal conditions than previously expected. In conclusion, our data clearly support that p23 is necessary in maintaining the AHR protein levels in normal and cancer cells. Insights on underlying mechanisms and implications will be presented and discussed.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.