bluuebunny/crossref_metadata_2025 huggingface dataset:
In utero therapy for hemophilia A using EGFP amniotic fluid stem cells
Authors: Kao Yung‐Tsung , Chen Yen‐Ting , Lan Ying‐Wei , Chen Chuan‐Mu
Published: June 2021

Hemophilia A is an X chromosome recessive genetic disease, that causes clotting factor VIII (FVIII) deficiency leading to blood coagulation dysfunction. The activity of coagulation factor in serious hemophilia patients is less than one percent. The current therapeutic methods of hemophilia A are mainly for giving either the coagulation factor supplementary injection or gene therapy. However, both methods have some shortcomings. Therefore, an alternative potential treatment might be desired. The amniotic fluid stem cells (AFSCs) are derived from amniotic fluid. The amniotic fluid is the medical waste and it don't involve in ethical issues. Moreover, AFSCs can differentiate to diverse cells of ectoderm, mesoderm, and endoderm lineages. In utero transplantation (IUT) of AFSCs is an approach used to cure inherited genetic fetal defects during the gestation period of pregnant. In human, certain disease, such as osteogenesis imperfect, was successfully treated by using this method. By transplanting AFSCs into fetus, the AFSCs can directly involve in the development of the fetus and achieve therapeutic effect. The aim of this research is to assess therapeutic effect of hemophilia A after in utero transplantation by amniotic fluid‐derived EGFP‐stem cells (EGFP‐AFSCs) into the liver of FVIII knockout mice. In this study, the B6;129S‐F8tm1Kaz/J knockout mice was used as a hemophilia A animal model. The C57BL/6 mice was used as a normal control and untreated hemophilia A mice was used as a negative control. To establish IUT animal model, the pregnant CD‐1 mice (14.5 days) underwent abdominal surgery and transplanted EGFP‐AFSCs (5 × 104 cells per fetus) into the liver of fetus. Three weeks after birth, the mice were sacrificed and the EGFP signal in organs were analyzed by RT‐PCR, histological examination and flow cytometry. Results showed that EGFP signal can be detected in the liver and intestine. Furthermore, we used several methods including activated partial thromboplastin time, FVIII activity assay, tail clip survival rate, and hemoglobin concentration to analyze whether the FVIII activity in the hemophilia A mice can be restored or not after IUT operation by EGFP‐AFSCs. By conducting this research, we expect to solve the hemophilia patient's therapeutic problems and increase better quality of life.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Correlating Increased Mechanical Forces with Tissue Lesions in Equine Navicular Disease
Authors: Uhl Elizabeth W. , Blas‐Machado Uriel , Kirejczyk Shannon GM. , Osborn Michelle L.
Published: June 2021

Navicular disease is a common form of chronic lameness in horses that currently can only be managed but not effectively treated. The navicular apparatus is a functional enthesis organ very similar to the human Achilles, and is highly adapted to dissipate forces away from the osteotendinous junction between the deep digital flexor tendon and the distal phalanx. Force analyses using 3D models constructed from CT scans of a horse's forelimb indicate that an exaggerated dorsiflexion of the foot, a posture commonly observed in horses with navicular disease, puts the deep digital flexor tendon under greater tension. With this in mind, tissue lesions in the navicular enthesis organ in horses euthanized for navicular disease were correlated with the force analysis. Areas of tension in the deep flexor tendon and underlying adipose tissue were identified by differential histological staining of stretched versus relaxed collagen using Masson's trichrome and picrosirius red. These areas correlated with tendon lesions documented by MRI and were in line with compressive lesions in the navicular bone. The dorsiflexed position of the foot was also found to increase forces on the impar and suspensory ligaments in which enthesial loss and disruption of collagen fibers and deposits of cartilage and bone was documented. These findings confirm the primary mechanical pathogenesis of navicular disease and indicate that rehabilitative therapy focused on decreasing dorsiflexion of the foot by reducing overloading of the forelimb is likely to be an effective treatment for many cases of navicular disease.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Small Molecule ER Proteostasis Regulators Rescue Activity of ATF6 Disease Mutants
Authors: Chiang Wei‐Chieh , Chan Priscilla , Paxman Ryan , Kelly Jeffery W. , Wiseman R. Luke , Lin Jonathan H.
Published: June 2021

ATF6 encodes a transcription factor that mediates the Unfolded Protein Response (UPR) to maintain protein folding homeostasis in the endoplasmic reticulum (ER). In response to ER stress, ATF6 migrates from the ER to the Golgi apparatus and undergoes regulated intramembrane proteolysis to release its cytosolic bZIP transcriptional activator domain, which upregulates chaperones and enzymes necessary for ER protein folding and homeostasis. We previously identified novel ATF6 mutations in patients with hereditary vision loss diseases by whole exome sequencing. Class I ATF6 mutants exhibit impaired ER‐to‐Golgi trafficking under ER stress conditions and therefore, impaired production of cytosolic bZIP domain through regulated intramembrane proteolysis. Class II ATF6 mutants are truncated ATF6 proteins that contain the entire cytosolic bZIP domain and they are constitutively active, even when the cells are not under ER stress. Class III ATF6 mutants have a defective or absent bZIP domain, resulting in the loss of ATF6 signaling.

To identify potential treatments to address the molecular defects found in these ATF6 mutants, we tested a recently discovered small molecule ER proteostasis regulator that selectively promotes the activation of ATF6 signaling. We found that treating the cells expressing ATF6 mutant with this molecule restores the ATF6 signaling pathway. Furthermore, treating patient fibroblasts carrying ATF6 mutations with this molecule also reduced cell death under experimentally induced protein misfolding. These findings indicate that targeting ATF6 signaling pathway with a small molecule ER proteostasis regulator may be a potential therapeutic strategy to prevent disease caused by ATF6 mutations.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Salivary Cortisol Levels and Body Composition in Mexican College Students
Authors: Suarez Paola Algara , Medina Elisa Rodriguez , Martinez Josefina Gallegos , Martinez Monica Lucia Acebo , Hernandez Jaime Reyes
Published: June 2021

Obesity is a serious public health problem worldwide, generating almos 82% of health care expenses in México. A growing young population is presenting overweight and excesive body fat mass wich in the long term evolves in metabolic syndrome. Cortisol plays a pivotal role in the generation of obestiy and inflammation, which leads to further endocrinological disorders as is insulin and leptin resistance. Therefore, it is imperative to study the association between body fat mass and cortisol levels, which can be studied in fasting saliva as a non‐invasive method.

The aim of this study was to characterize body composition and fasting salival cortisol levels in a population of mexcian college students. This protocol was accepted by the Ethics Comittee of Nursing School of UASLP.

150 female and 82 male college students aged 18–24 years were included in this study. Body composition was measured using a bioelectrical impedance scale (InBody, Korea), stadiometer and measuring rod as recomended by WHO procedures. Saliva samples were collected in a sterile plastic tube in fasting conditions, freezed and used for cortisol determination using a specific ELISA kit (Salimetrics, PA). For salivary cortisol studies, a cohort of 42 samples from women and 34 from men were selected representing all BMI categories. Psychological stress was measured in order to contol cortisol production due to anxiety.

This population showed a lower percentage of obesity and overweight than that presented for this age group. BMI: 58.2% normal, 24.1% overweight, 12% obesity and 5.6 % low weight. Nevertheless, fat body mas was elevated in 63.8% of the population, concentrated mainly in the abdominal region (71.5% showed high degrees of abdominal fat mass, 25.4 % was normal and 3 % was low). Parameters for categorization of anthropometry were based on WHO standards. Skeletal muscle mass was normal in 53.4% of the population and low in 39.2% and basal metabolic rate was low for 64.2% . Regarding cortisol, a mean value of 0.40 ± 0.19 μg/dL was found for women and 0.41 ± 0.26 μg/dL for men. Higher levels of salivary cortisol were found in the high fat mass percentaje in the male population but not among BMI categories or in the female population. Psychological stress had no significant effect on cortisol levels in either population.

We conclude that fat mass is altered earlier than BMI in this young population considered healthy. Morning cortisol levels are not significantly increased in women but are increased in men, and are neither affected by physchological stress.

Support or Funding Information

CONACYT Scholarship 793437 to ERM

UASLP Grant C17‐FAI‐06‐36.36 to PAS

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Low Intensity Ultrasound Can Affect Cell Behavior And Can Promote Drug Delivery
Authors: Lucchetti Donatella , Perelli Luigi , Colella Filomena , Scoarughi Gianluca , Barbato Gaetano , De Maria Ruggero , Sgambato Alessandro
Published: June 2021

Ultrasounds (US) represent a new frontier for medical biotechnologies for their potential applications in therapy. Several studies have shown that cellular biomechanical and biophysical changes are involved in the pathogenesis of many diseases, such as cancer. For this reason, the study of the biological effects induced by exposure of both normal and neoplastic cells to the US is important to understand the effects of these changes on cellular functions and to evaluate and justify their potential applications in oncology.

Aims of this study were to evaluate the effects of low‐intensity US on the proliferation, motility and differentiation of a human colon‐adenocarcinoma cell line (HT29). Moreover, we evaluated the effects of US on the delivery of oxaliplatin and on the uptake of exosomes.

Cell proliferation was evaluated by MTT viability test, while cell motility using the scratch test. Cell differentiation was evaluated assessing alkaline phosphatase activity and by Western blot. Exosomes were isolated by differential centrifugation and characterized by transmission electron microscopy, Dynamic Light scatter and Western Blot to evaluate the expression of exosomal markers (CD63‐CD81‐CD9). Uptake of exosomes was evaluated by flow cytometry.

All experiments were performed at four different ultrasonic frequencies (KHz), and at three acoustic intensities. Twelve hours after the treatment, the application of medium‐low intensity and frequencies US led to an increase in cell proliferation of HT29 (Fig. 1A). An inhibitory effect was observed at higher intensities. The proliferative effect was less evident at 24 hours from treatment. US exposure increased cell motility at all frequencies with a greater effect at high frequencies (Fig.1B and C). It has been described that treatment with sodium butyrate (NaBu) induces differentiation of HT29 cells, as assessed by an increased phosphatase alkaline activity, together with a reduction in the expression of the cancer stem cell marker CD133. We have shown that US treatment increases the differentiating effect of sodium butyrate by promoting alkaline phosphatase activity (Fig.2A) and CD133 reduction (Fig.2B). We also observed that USs promotes oxaliplatin‐induced cell toxicity (data not shown) and increases the uptake of exosomes likely through an ERK‐integrinβ4‐dependent mechanism (Fig. 2C and data not shown).

US treatment can affect several cells functions. US could be used to promote drug as well as exosome‐mediated drug delivery. A better knowledge of the molecular mechanisms underlying the effects of US is essential for their future use in clinical practice alone and/or in combination with chemotherapeutics.

Support or Funding Information

Promedica Bioelectronics srl

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Combined Laser Capture Microdissection and Mass Spectrometry (LCM‐MS) to Identify, Localize and Quantify Biologics in Specific Anatomic Microenvironments
Authors: O'Neil Shawn P. , Quazi Shakey A. , Dufield Dawn R. , Wei Benjamin
Published: June 2021

Biologics represent over 30% of approved drugs and have been used to treat a broad range of diseases. The toxicological effects of several biologics have been reported in preclinical studies; however, the pathogenesis for these toxicities is often poorly understood. Correlating the precise spatial biodistribution of biologic test articles with the histopathological pattern of toxicological lesions observed in exploratory toxicity studies is helpful for understanding the pathogenesis of toxicity; however, the frequent absence of test article‐specific antibodies precludes the use of immunohistochemistry, which would normally be used to localize proteins in tissues. Here, a tissue proteomics approach (LCM‐MS) that combines laser capture microdissection (LCM) and mass spectrometry (MS) was developed to identify, localize, and quantify biologics in specific anatomic microenvironments in tissue sections.

A therapeutic monoclonal antibody (mAb‐Y) that had been associated with glomerulopathy in cynomolgus monkeys was used as the substrate for developing LCM‐MS methods. Lysates prepared from glomeruli that had been collected from kidney cryosections by LCM were enriched for mAb‐Y by immunoprecipitation, digested with trypsin and injected into a ThermoFisher LTQ orbitrap mass spectrometer for identification and quantification. Two proteotypic peptides for mAb‐Y were identified and a calibration curve was generated by adding a fixed amount of “heavy‐labeled” (13C, 15N) peptide to different amounts of intact mAb‐Y and measuring the ratio of mAb‐Y light/heavy peptide concentration versus the ratio of the peak areas under the curve from heavy and light peptide spectra generated by multiple reaction monitoring MS. The calibration curve was linear, with a correlation coefficient of 0.994 (p<0.0003), and the lower limit of quantification for mAb‐Y was 2.5 ng/ml. LCM‐MS was then used to measure mAb‐Y concentrations in lysates of LCM‐captured glomeruli from six mAb‐Y‐dosed versus six vehicle‐dosed monkeys. Glomerular lysates (2.5 μg) were immunoprecipitated with biotinylated goat anti‐human Fc antibody, and the protein eluted from streptavidin dynabeads was trypsinized and injected into a Sciex QTRAP 5500 mass spectrometer. mAb‐Y was identified and quantified in glomeruli from 2 of 6 monkeys in the high‐dose group (4.0 and 5.2 ng/ml, respectively) after a six month recovery period but not in 6 vehicle controls. The LCM‐MS approach can be used to identify and quantify other protein biologics in specific anatomic microenvironments to facilitate understanding of the toxicity of biotherapeutics in development.

Support or Funding Information

Pfizer, Inc.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Methylmethacrylate Thin Sections are Superior to Thick Ground MMA Sections for Histologic Evaluation of Complex Submillimeter Metallic Medical Devices
Authors: O'Brien Maureen T. , Dorsey Theresa , Ensminger Leslie , Kuhn Lauren , Dieterle Brian , Burgenson Janet
Published: June 2021

Hard plastic resin embedding is necessary for histology of tissues containing hard and/or metallic medical devices if the device is to be retained within the section for microscopic assessment. Traditional methylmethacrylate (MMA) embedding and sectioning of tissues containing hard and/or metallic devices requires targeted sectioning of the block with a diamond blade saw followed by careful grinding of the MMA‐embedded specimen with a calibrated grinder. This method provides a tissue section for histology that is approximately 50–60 μm in thickness (MMA thick section). However, the process of creating a MMA thick section is somewhat imprecise and results in considerable sample loss due to requisite attrition during MMA block sectioning and grinding procedures. For submillimeter (<1 mm) and/or similarly small, topographically complex medical devices, this level of relative imprecision, specimen attrition, and loss of cellular detail due to inherent thickness of tissue section can result in unacceptable diagnostic slide quality or even complete loss of a valuable specimen during sectioning/grinding procedures. To circumnavigate these challenges, we utilize MMA hard plastic resin embedding of small metallic medical devices followed by acquisition of thin (3–5 μm) MMA sections using a motorized microtome. Thin MMA sections provide superior cellular detail, greater precision of sectioning, and excellent retention of high quality device‐tissue context when compared with traditional MMA thick plastic sections. Although acquisition of MMA thin sections can be limited by larger device sizes and/or composition, MMA thin sections provide superior histologic quality for permissible specimens.

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 Hypoxia Inducible Factor 1 Alpha is Required for Optimal GammaHerpes Virus Lytic Replication and Reactivation from Latency
Authors: Lopez‐Rodriguez Darlah M. , Mesri Enrique , Andreansky Samita
Published: June 2021

Lytic replication of Kaposi's Sarcoma Associated Herpes Virus (KSHV) exacerbates Kaposi's sarcoma (KS) progression, an angiogenic spindle‐cell sarcoma associated with AIDS. Current antiviral therapy targeting lytic replication can prevent KS in seropositive patients. KSHV lytic infection is not manifested in vitro, and currently there are no animal models that display pathogenic phenotypes. In this study, we employed a biological and genetically mouse pathogen similar to KSHV, the murine gammaherpes virus (gHV) 68 (MHV68), that readily infects laboratory mice providing a valuable small animal model. Moreover, MHV68 in vitro infection exhibits a default lytic infection. Several studies have shown that during infection KSHV employs several viral proteins to de‐regulate the host transcription factor hypoxia inducible factor 1 alpha (HIF1α) and KSHV‐induced activation of HIF1α is necessary for viral persistence and KS progression. However, little is known about the role of HIF1 α in gHV replication and pathogenesis. To further investigate the role of this host factor, we induced transcriptional inactivation of HIF1α in MHV68 virus‐specific cells in vivo. Conditional knock out of HIF1α was achieved by infecting transgenic mice with Cre‐recombinase (Cre) LoxP specific site within the functional domain of HIF1α gene using a recombinant MHV68 that expressing a CMV driven Cre expression.

Our data demonstrated for the first time that HIF1α inactivation during gammaherpes virus infection in vivo affects viral gene transcription resulting in impaired virus expansion and early clearance of acute infection in a mouse model. In addition, our results from in vitro MHV68 lytic infection of mouse fibroblasts lacking HIF1α showed that lytic virus production and transcription is decreased. During latency establishment in vivo, the frequency of MHV68 latent splenocytes undergoing latent to lytic replication was largely decreased as well. Our data suggest that HIF1α is required for sustained acute infection since it's deletion in virus‐infected cells resulted in early clearance of productive infection and impairment in reactivation. Moreover, HIF1α is required for mRNA upregulation of glycolytic enzymes during MHV68 lytic infection suggesting a role for HIF1a as a modulator of cell energetics and viral gene expression. We conclude that gammaherpes viruses required the function of the cellular host factor HIF1α in order to effectively replicate and establish latency within its host.

Support or Funding Information

National Cancer Institute 3R01CA136387‐08S1

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identifying Keap1‐Interacting Coxiella burnetii Proteins
Authors: Ortiz Angely , Voth Daniel E.
Published: June 2021

Coxiella burnetii is a Gram‐negative bacterium that causes an acute flu‐like illness in humans called Q fever. Human infection is initiated by inhalation of contaminated aerosols generated while working with livestock. In human cells, C. burnetii forms a lysosome‐like vacuole for replication to promote an obligate intracellular lifestyle. The pathogen also regulates multiple events during infection using a type IV secretion system and secreted effector proteins. C. burnetii activates the Nrf2‐Keap1 pathway in macrophages, potentially using an effector protein(s) to disassociate Keap1 and allow Nrf2 to move into the nucleus for transcription. p62 is known to interact with Keap1 an promote this disassociation. We predict C. burnetii uses the proteins encoded by CBU1400 and AUR58 that contain a putative Keap1‐binding domain. In this study, we derived adenylate cyclase and fluorescent constructs of each protein to determine secretion and cellular localization, respectively. Additionally, we assessed the impact of brusatol, an established Nrf2 inhibitor, on C. burnetii intracellular growth. A better understanding of the requirement of Nrf2 for infection will allow enhanced modeling of C. burnetii‐host cell interactions.

Support or Funding Information

This project was supported by the Arkansas SURP program, with grants from the NCCR (P20RR016460) and the NIGMS (P20 GM103429), and by NIAID R01AI087669 (D.E.V.)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Estrogen Related Receptor α Is Required For Efficient Arenavirus Multiplication
Authors: Cintron Kevin , Cubitt Beatrice , Iwasaki Masaharu , de la Torre Juan Carlos
Published: June 2021

Several arenaviruses cause hemorrhagic fever disease in humans and represent important public health problems in their endemic regions. In addition, evidence indicates that the worldwide‐distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an important neglected human pathogen. There are no FDA licensed arenavirus vaccines and current anti‐arenavirus therapy is limited to the use of ribavirin that is only partially effective. Therefore, there is an unmet need for novel anti‐arenaviral therapeutics. Previous studies done in our lab found that XCT790 and AM251, small molecule inhibitors of the Estrogen Related Receptor Alpha (ERRa) protein exhibited also a potent anti‐LCMV activity. We, therefore, hypothesized that ERRa is a host cell factor involved in the replication cycle of arenaviruses. To test our hypothesis, we used a recombinant LCMV that expressed GFP to perform three assays that examined the effect of the compounds in three different steps of arenavirus replication cycle. Our results revealed that XCT790 and AM251 interfered with both the activity of the LCMV RNP responsible for directing viral RNA synthesis, and the arenavirus budding process mediated by the Z protein.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SULFORAPHANE PROTECTS AGAINST ETHANOL‐INDUCED APOPTOSIS IN NEURAL CREST CELLS AND ZEBRAFISH EMBRYOS THROUGH EPIGENETICALLY MODULATING THE EXPRESSION OF SANIL1 AND RESTORING EMT
Authors: Li Yihong
Published: June 2021

Ethanol‐induced apoptosis in neural crest cells (NCCs), a multipotent progenitor cell population, are implicated in the Fetal Alcohol Spectrum Disorders (FASD). Epithelial‐mesenchymal transition (EMT) is a process by which cells lose their epithelial phenotype and acquire a mesenchymal phenotype, which is essential for NCC migration. The objective of this study is to investigate whether ethanol exposure can induce apoptosis in NCCs by inhibiting EMT and whether sulforaphane (SFN), a vegetable‐derived isothiocyanate, can prevent ethanol‐induced apoptosis by epigenetically modulating the expression of Snail1, a key transcriptional factor that can promote EMT by repressing E‐cadherin. We found that exposure to 50 or 100 mM ethanol for 24 hours resulted in a significant increase in apoptosis in NCCs. Co‐treatment with SFN significantly reduced ethanol‐induced apoptosis. Ethanol treatment also inhibited EMT in NCCs, as indicated by an increased expression of E‐cadherin1, an EMT‐suppressing marker, and a reduced expression of vimentin, an EMT‐promoting maker. Treatment with SFN significantly reversed ethanol‐induced changes in the expression of E‐cadherin1 and vimentin, and restored EMT in NCCs. We also found that ethanol exposure significantly decreased the mRNA expression of Snail1, which can be reversed by co‐treatment with SFN. To determine whether ethanol and SFN regulate the expression of Snail1 by histone modification, the expression of kdm5a, a lysine‐specific demethylase and the levels of trimethylation of histone H3 lysine 4 (H3K4me3) at the promoters of Snail1 were determined. We found that ethanol exposure increased the expression of kdm5a, which was reversed by SFN. ChIP‐qPCR analysis revealed that ethanol exposure significantly reduced the levels of H3K4me3 at the promoters of Snail1 and that SFN treatment reversed the ethanol‐induced reduction of H3K4me3 at the promoter regions of Snail1 gene. SFN treatment also significantly diminished ethanol‐induced apoptosis in zebrafish embryos. In addition, Knock‐down of kdm5a restored EMT and significantly decreased apoptosis in ethanol‐exposed NCCs and zebrafish embryos. These results demonstrate that ethanol exposure can induce apoptosis in NCCs and zebrafish embryos by inhibiting EMT and that SFN can protect against ethanol‐induced apoptosis by reversing ethanol‐induced reduction in the levels of H3K4me3 at the promoters of Snail1, restoring the expression of Snail1 and EMT in ethanol‐exposed NCCs.

Support or Funding Information

Supported by NIH grants AA021434, AA020265, and AA024337 (S‐Y.C)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Abuse‐Related Discriminative Stimulus Effects of Synthetic Cathinones
Authors: Paronis Carol A. , Kohut Stephen J. , Sherwood Alexander M. , Brown Kelly A. , Zakarian Ani S. , Prisinzano Thomas E. , Bergman Jack
Published: June 2021

The use of synthetic cathinones, or “bath salts,” has become more widespread in recent years. “Bath salts” share many chemical properties with psychostimulants like methamphetamine or with entactogens like 3,4‐methylenedioxymethamphetamine (MDMA or “Ecstasy”), presumably reflecting structural differences that lead to differing neurochemical actions. Yet, currently available synthetic cathinones have not been well‐characterized in primate species, and it is unclear which pharmacological activity mediates their abuse‐related behavioral effects. The present study was initiated to address this question, using drug discrimination procedures to evaluate several synthetic cathinones in two groups of squirrel monkeys. Each subject was trained to respond on either of two levers under a FR10 schedule of reinforcement and, during training sessions, responding on one lever was reinforced after pretreatment with vehicle whereas responding on the second lever was reinforced after pretreatment with the training drug. Under terminal conditions, one group of monkeys discriminated 0.1 mg/kg methamphetamine from saline; the other group discriminated 0.6 mg/kg MDMA from saline. Drug or saline lever assignments were counter balanced across the left and right levers within each group. Training sessions comprised multiple components, and a component consisted of a ten‐minute timeout period followed by ten presentations of the FR10 schedule, each followed by a brief 50‐second timeout. After criterion performance was met (≥ 90% correct), test days interspersed training days, with at least two training days between each test day. The effects of five different synthetic cathinones ‐ α‐pyrrolidinovalerophenone (α‐PVP), methylenedioxypyrovalerone (MDPV), mephedrone, methylone, and methcathinone) were tested in both groups. A full range of doses of each drug was administered using cumulative dosing procedures. Results show that doses of 0.1 mg/kg – 0.32 mg/kg of MDPV, α‐PVP, and methcathinone were identified as methamphetamine in subjects trained to discriminate 0.1 mg/kg methamphetamine from vehicle, whereas doses mephedrone and methylone up to those that decreased overall response rates did not generalize to MA. Preliminary results suggest an opposite profile of substitution in MDMA‐trained animals. Thus, mephedrone and methylone, but not MDPV, α‐PVP, and methcathinone did appear to generalize to MDMA. These results demonstrate that synthetic cathinones have differing substitution profiles in animals trained to discriminate methamphetamine or MDMA, which may reflect differing subjective effects and, perhaps, differing abuse liability in man.

Support or Funding Information

Supported by NIH Grants: DA002519. GM113117, DA039306, GM111385

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of novel NKCC1 inhibitors on reducing brain damage and neurological deficits after ischemic stroke in mice
Authors: HUANG HUACHEN , Bhuiyan Mohammad Iqbal Hossain , Jiang Tong , Shankar Sandhya , Schreppel Philipp , Erker Thomas , Hintersteininger Michael , Sun Dandan
Published: June 2021

Background and Purpose

Stimulation of the WNK‐SPAK/OSR1 kinases and their substrate Na+‐K+‐2Cl− cotransporter 1 (NKCC1) play critical roles in cerebral edema and neurological functional deficits after ischemic stroke. Either NKCC1 inhibitor bumetanide (BMT) or knockout of WNK3 or SPAK shows profound protective effects in a mouse model of ischemic stroke. In this study, we investigated the efficacy of two novel pharmacological inhibitors of NKCC1, a lipophilic BMT prodrug STS5 and a novel NKCC1 inhibitor STS66, on reducing ischemic stroke‐induced brain damage in mouse.

Methods

Both male and female C57BL/6j mice (2–3 months old) were used in this study. Transiant focal ischemic stroke was induced by 50‐min occlusion of the middle cerebral artery (tMCAO). At 3 h post‐tMCAO, mice were randomly assigned to receive vehicle DMSO (2 ml/kg body weight/day, i.p.), BMT (10 mg/kg/day, i.p.), STS5 (13 mg/kg, i.p.), or STS66 (12 mg/kg/day, i.p.). Infarct volume, hemisphere swelling, animal survival, and neurological deficits were analyzed.

Results

BMT, STS5 or STS66 significantly reduced infarct volume and hemisphere swelling assessed at 24 h post‐tMCAO. STS66 is less effective on reducing brain infaraction in female than in male mice. However, STS5 treatment significantly increased the mortality in mice during 1–14 days after tMCAO. Interestingly, STS66‐treated mice showed better outcome, including improving survival and sensorimotor functional recovery during 1–14 days post‐tMCAO.

Conclusion

These results demonstrate that the novel NKCC1 inhibitor STS66 is effective in reducing ischemic infarct, cerebral swelling, and improving neurological deficits after ischemic stroke in C57BL/6j mice. STS66‐mediated NKCC1 inhibition presents a novel strategy for stroke therapy.

Support or Funding Information

This research was supported in part by NIH Grant R01 NS38118 (D Sun), VA I01BX002891 (D Sun)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RGS Protein Regulation of CB1 Receptor‐Mediated Cannabinoid Behaviors
Authors: Tai Sherrica , Kandasamy Ram , Barton Brenna , Parry Logan , Traynor John R.
Published: June 2021

The cannabinoid CB1 receptor, is a G protein coupled receptor and couples to members of the Gαi/o family of heterotrimeric G proteins. As such, signaling downstream of the receptor is negatively regulated by RGS proteins, or regulator of G protein signaling proteins. RGS proteins act as GAPs or GTPase accelerating proteins and bind to activated Gα‐GTP subunit to enhancing the hydrolysis of bound Gα‐GTP to Gα‐GDP, leading to inactivation of Gα subunit and the reformation of inactive G protein heterotrimer. Therefore, inhibition of RGS proteins will prolong cannabinoid signaling and possibly the potency of ligands that bind to the CB1 receptor. However, there are over 20 members of the RGS protein family and there can be redundancy among members of the family. Consequently, we developed a mouse line that expresses Gαo protein that is insensitive to the action of all RGS GAP activity. Here, F1 cross of 129SvEv/C57BL6 mice expressing a knock‐in RGS‐insensitive Gαo (RGSi‐Gαo) protein were administered the fully efficacious CB1 agonist, CP 55,940 and examined for reduction in body temperature and antinociception, two noted behaviors of CB1 agonism. CP 55,940 induced a greater reduction in body temperature in female RGSi‐Gαo mice that in their female wild‐type littermates. Latency to respond to a noxious thermal stimulus in the hot‐plate test was also greater in female RGSi‐Gαo mice compared to their female wild‐type littermates. In contrast, no differences were observed between male RGSi‐Gαo mice and their male littermates. The reasons underlying the observed sex differences are unknown. RGS proteins present a potential target for drug discovery and development that may affect the therapeutic actions of cannabinoids ligands.

Support or Funding Information

Supported by DA035316.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rab35 and Rab39 GTP‐ases as modulators of cannabinoid type 1 receptor signaling.
Authors: Ghita Ioana , Winsauer Peter J. , Filipeanu Catalin M.
Published: June 2021

Cannabinoid type 1 receptor (CB1R) is distinct within G‐protein coupling receptors (GPCR) family, being able to trigger cellular signaling not only when present at the plasma membrane, but also within the endolysosomal system. Rab GTP‐ases are well characterized master regulators of intracellular protein trafficking, including GPCR. The classical controllers of bidirectional plasma membrane‐endosome shuttling Rab4 and Rab5 GTP‐ases were previously shown to profoundly change the subcellular distribution of CB1R, whereas Rab11 GTPase had no effect. However, the effects of recently described Rab35 and Rab39 GTPases, which are also modulating internalization/recycling process, in the regulation of GPCR trafficking are not yet characterized. In the present work, using a bioluminescent cAMP reporter, we found that overexpression of Rab35 wild‐type (Rab35wt) did not change the effects of CP55,940 at maximal concentration (10−7M), although slightly reduced its effects at lower concentrations (10−9 M). In contrast, the dominant negative mutant Rab35S25N had opposite effects. However, the effects of non‐permeable peptide receptor agonist RVD‐Hpα (10−6M) which selectively activates plasma membrane CB1R were markedly potentiated by Rab35wt and reduced by Rab35S25N. Furthermore, overexpression of Rab35wt decreased the amount of CB1R internalized in presence of CP55,940 or RVD‐Hpα as determined by ELISA. Rab35S25N did not change the number of plasma membrane receptors in basal conditions, but reduced CB1R recycling after treatment with any of the two agonists. Overexpression of Rab39A or Rab39B GTP‐ases resulted in strong reduction of the effects of RVD‐Hpα on cAMP levels, but did not change the effects of CP55,940. These results correlated with the levels of CB1R at the plasma membrane, a decrease in CB1R internalization being observed after increasing the endogenous levels of Rab39A or Rab39B. These preliminary results indicate that multiple Rab GTP‐ases modulate CB1R signaling by interfering with the receptor shuttling between plasma membrane and endosomes. In addition, our data may have clinical significance on the use of cannabinoids in specific populations, considering that mutations affecting Rab39B functions were observed in people with intellectual disability, autism spectrum disorder, and macrocephaly.

Support or Funding Information

Supported by NIH DA037255 (PJW) and Bridge Grant from Howard University (CMF).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression of Multiple Sulfotransferase (SULT) 1C4 Transcript Variants in Developing Human Liver
Authors: Dubaisi Sarah , Fang Hailin , Gaedigk Roger , Vyhlidal Carrie A. , Kocarek Thomas A. , Runge‐Morris Melissa
Published: June 2021

The cytosolic sulfotransferases (SULTs) are conjugating enzymes that catalyze the transfer of a sulfonate group from the cofactor 3′‐phosphoadenosine‐5′‐phosphosulfate to a variety of endogenous and xenobiotic substrates. The SULT1C subfamily includes three human genes, SULT1C2, SULT1C3, and SULT1C4, and relatively little is known about their expression and regulation or the functions of the enzymes. Previous studies have indicated that the human SULT1Cs are preferentially expressed during early life and are capable of metabolizing endogenous molecules such as thyroid and estrogen hormones, suggesting that these enzymes could play physiological roles during development. Several transcript variants (TVs) of SULT1C4 are indexed in GenBank, including the full‐length mRNA containing seven exons (TV1, NM_006588), a variant mRNA lacking exons 3 and 4 (TV2, NM_001321770), two non‐coding RNA variants (TV3, NR_135776 and TV4, NR_135779), and a predicted transcript variant (TVX1, XM_017003807). Using 5′‐RACE, RT‐PCR, and DNA sequencing, we detected TV1 and TV2, as well as a variant lacking only exon 3 (E3DEL) in Caco‐2 colorectal adenocarcinoma cells, HepaRG hepatic cells, and human liver specimens. The aim of the current study was to evaluate expression of the SULT1C4 TVs more thoroughly by (1) characterizing expression of individual SULT1C4 TVs in human liver specimens from prenatal, infant, and adult donors and (2) determining whether the individual mRNA variants can be translated into proteins. Using RT‐qPCR assays designed to quantify TV1, TV2, or E3DEL separately, all three variants were found to be primarily expressed in prenatal liver samples. The TV2 transcript lacking two exons was more abundant than the full‐length TV1, while E3DEL levels were minimal. In agreement with the RT‐qPCR data, RNA‐seq analysis of prenatal and pediatric liver specimens showed that TV1 and TV2 were both more abundant during early developmental stages. The RNA‐seq data also demonstrated that a non‐coding transcript variant (ENST00000494122.1) consisting of 2 exons only was expressed in the liver specimens and was predominantly expressed in prenatal liver. TV1 and TV2, but not E3DEL, were detected by western blot when plasmids expressing DDK‐tagged variants were transfected into HEK‐293T cells. These results indicate that at least four transcript variants of SULT1C4 are expressed in human liver, primarily during prenatal life, although only one of these (TV1) would encode a functional SULT enzyme. The physiological significance of this transcript diversity is unclear and will require further investigation.

Support or Funding Information

Supported by NIH grants R01 ES022606 and P30 ES020957

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of Hepatic UGT1A4 by Liver X Receptor LXRα, and not LXRβ in hUGT1 Mice
Authors: Rettenmeier Eva , Yoda Emiko , van der Schoor Lori , Chen Shujuan , Barbier Olivier , Tukey Robert
Published: June 2021

UDP‐glucuronosyltransferases (UGTs) are metabolic enzymes catalyzing the transformation of multiple xeno‐ and endobiotics into their more water‐soluble glucuronides. Among the UGT enzymes, UGT1A4 is known for the N‐glucuronidation of amine containing xenobiotics, including anticonvulsants, antidepressants and environmental mutagens. To examine the function of UGT1A4 and other genes associated with the human UGT1 locus, we have implemented humanized UGT1 mice (hUGT1). In this study we identify UGT1A4 as a positively regulated target gene of the nuclear receptor liver X‐receptor (LXR). LXR is a member of the nuclear receptor superfamily. Two isoforms of LXR exist, LXRα and LXRβ. The treatment of neonatal hUGT1 mice with the conventional LXR agonist T0901317 led to transcriptional activation of downstream LXR target genes, including UGT1A1, UGT1A4, Scd1 and Scd2. Since neonatal hUGT1 mice develop extreme hyperbilirubinemia, the induction of intestinal and liver UGT1A1 led to a dramatic reduction in total serum bilirubin. Repeating the induction patterns in hUGT1/Lxrα−/− and hUGT1/LXRβ−/− mice, expression of Scd1 and Scd2 displayed specificity for LXRα. There was significant induction of these genes in hUGT1/Lxrβ−/− mice with limited activation in hUGT1/Lxrα−/− mice. In contrast, the induction of UGT1A1 and the UGT1A1 gene by T0901317 is regulated by both activated LXRα and LXRβ. However, like Scd1 and Scd2, induction of the UGT1A4 gene was selective towards LXRα, with greatly reduced expression in hUGT1/Lxrα−/− mice. Induction of the UGT1A4 gene was comparable in hUGT1/Lxrβ+/− and hUGT1/LXRβ−/− mice. Clearly, LXRα and LXRβ can display fine selectivity for downstream target gene activation, such as the UGT1A4 gene.

Support or Funding Information

USPHS grants GM086713 and ES010337, DFG (German Research Foundation)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Positive Allosteric Modulators of the D1 Dopamine Receptor Act at Diverse Binding Sites
Authors: Luderman Kathryn D. , Conroy Jennie L. , Free R. Benjamin , Southall Noel T. , Ferrer Marc , Aubé Jeffrey , Lane J. Robert , Frankowski Kevin , Sibley David R.
Published: June 2021

The D1 dopamine receptor (D1R) and its signaling is associated with several important neurological processes and neuropsychiatric disorders. Enhancing D1R signaling in the prefrontal cortex is associated with increased cognition and therefore is an appealing treatment strategy for the cognitive decline observed in schizophrenia, Alzheimer's disease, and other disorders. Because of the clinical liability inherent with D1R agonists, positive allosteric modulators of the D1R have been proposed as an alternative, due to their potential for high selectivity and larger therapeutic windows. Currently, the location and characteristics of potential binding site(s) for allosteric modulators on the D1R is unknown. We have identified two structurally diverse D1R positive allosteric modulators, MLS1082 and MLS6585, via a high throughput screen of the NIH Molecular Libraries Program. The compounds potentiate dopamine‐stimulated G‐protein‐ (cAMP stimulation, 3–5 fold) and β‐arrestin‐mediated (6–8 fold) signaling pathways and increase the binding affinity of dopamine for the D1R (3–6 fold). Neither compound displayed agonist activity in the absence of dopamine. Taking advantage of our structurally distinct potentiators, G‐protein signaling and β‐arrestin recruitment experiments using maximally effective concentrations of MLS6585 and MLS1082 in combination were used to determine if the compounds act at similar or separate sites. In combination, MLS1082 + MLS6585 caused an additive potentiation of the potency of dopamine beyond that caused by either compound alone for both β‐arrestin recruitment (11–20 fold) and cAMP accumulation (4–6 fold). This suggests that the two compounds are acting at separate sites on the receptor. We also observed similar results using analogs of the two compounds, with analogs of MLS6585 having additive activity with MLS1082 and vice‐versa. However, analogs are not additive with their parent compound, meaning the parent compounds and their analogs act at similar sites. The combination experiments were repeated with Compound B, a known D1R positive allosteric modulator. Compound B was additive with MLS6585 but not MLS1082, further suggesting that the D1R has two separate positive allosteric modulator binding sites. Point mutation studies are currently underway to identify the location of these putative sites as well as whether these sites may contribute separately to potentiation of signaling efficacy versus dopamine binding affinity.

Support or Funding Information

NINDS IRP

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