bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative Deep Tissue Imaging of Target Engagement in Intact Live Animals
Authors: Barroso Margarida M. , Rudkouskaya Alena , Sinsuebphon Nattawut , Intes Xavier
Published: June 2021

Targeted drug delivery in cancer treatment has been a major area of development in the past decades. However, there is a great need in preclinical studies to not only assess the drug distribution but also monitor and quantify target engagement in vivo to ensure maximal drug delivery efficacy. Macroscopic Fluorescence Lifetime Imaging of Förster Resonance Energy Transfer (MFLI‐FRET) is a unique deep imaging methodology to monitor in vivo receptor‐target interactions in pre‐clinical studies. Overall, we demonstrated that MFLI‐FRET enables real time in vivo information on receptor ligand engagement in deep tissues, conversely to current commercial systems.

In this study, we capitalized on the homodimeric nature of the transferrin receptor (TfR) to quantify transferrin (Tf) internalization into cancer cells by measuring FRET between receptor‐bound Tf‐labeled donor and acceptor near‐infrared (NIR) fluorophore pairs. We found a strong correlation between FRET levels and Tf internalization into tumor cells despite the significant heterogeneity of tumors regarding their size and cellular density. In contrast, no correlation between MFLI‐FRET and TfR levels was observed, underscoring the insufficient link between receptor density and intracellular drug delivery. We present data strongly supporting that receptor expression and tumor drug delivery cannot be estimated via ex vivo immunohistochemistry but that drug delivery is highly correlated with FRET imaging. Additionally, we compared results of in vivo MFLI‐FRET imaging with intensity‐based FRET (using the IVIS pre‐clinical optical imaging system). Intensity‐based imaging failed to provide reliable and consistent results, showing significantly higher FRET quantification in negative control samples. Hence, MFLI provides the ability to measure receptor engagement in critical organs associated with targeted drug delivery efficiency, such as the liver, xenografts and bladder, without disrupting their native environment. Such unique capabilities enable the quantitative assessment of receptor engagement in relevant in vivo heterogeneous systems as well as providing crucial intra‐ and inter‐ specimen validation. Overall, we demonstrate that MFLI‐FRET enables real time in vivo information on receptor ligand engagement in deep tissues, conversely to current commercial systems. MFLI‐FRET is well positioned to play a critical role in accelerating the optimization of targeted drug delivery efficacy in pre‐clinical studies. Furthermore, this method should find broad applicability in molecular imaging both in vitro and in vivo, and could be extended to applications as diverse as image guided‐surgery or optical tomography.

Support or Funding Information

The study was funded by NIH grants R01EB019443 and R01CA207725.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel Molecular Markers in Succinic Semialdehyde Dehydrogenase Deficiency (SSADHD), an Orphan Disorder of GABA Metabolism
Authors: Walters Dana C. , Brown Madalyn N. , Schmidt Michelle A , Klein Anders B. , Wellendorph Petrine , Vogel Kara R. , Ainslie Garrett R. , Roullet Jean‐Baptiste , Gibson K. Michael
Published: June 2021

Study Objectives

SSADHD is an autosomal‐recessive disorder of GABA metabolism featuring a non‐specific neurological phenotype, which precludes clinical diagnosis. The latter is achieved through identification of elevated g‐hydroxybutyric acid (GHB) or GABA in patient physiological fluids and/or mutations in the ALDH5A1 (aldehyde dehydrogenase 5A1) gene encoding SSADH. Targeted therapeutics are lacking, as is an understanding of the natural history of the disorder. To further the latter, we continue the pursuit of novel biomarkers that might help guide natural history studies. Because BDNF (brain derived neurotrophic factor) and GABAergic signaling are closely linked (Brady, 2017) and there is evidence that miR‐29a regulates ALDH5A1 expression in vitro (Yu, 2015), we measured the expression of BDNF and miR‐29a genes in both human and murine SSADHD.

Methods

RNA was isolated from patient, obligate heterozygote (parents) and control red blood cell (RBCs) (n=9), and from brain cortex samples obtained from wild‐type and SSADH‐deficient mice (Hogema, 2001). cDNA was synthesized employing miScript II RT Kits (Qiagen) and gene expression studies performed using pre‐validated miScript Sybr Green and QuantiTect Primer Assays (Qiagen). Data analysis employed the CT method with significance (t test) set to 0.05.

Results

In patient samples, BDNF demonstrated a 4‐fold down‐regulation (p=0.0335) and miR29a a 5‐fold down‐regulation (p=0.0191). In SSADH‐deficient mouse cortex, BDNF was 6.4 (p=0.0446)‐fold down regulated and 8.5‐fold (p=0.0115) down‐regulated in heterozygous SSADH‐deficient mice. Similarly, miR29a was 2‐fold down regulated in SSADH‐deficient mice in comparison to wild‐type mice.

Discussion/Conclusion

The established role of BDNF in neuropsychiatric disorders (e.g. depression, anxiety, epilepsy; Cataneo, 2016) suggest exciting new directions in understanding SSADHD pathogenesis, as well as novel treatment paradigms in the form of SSRI (selective serotonin reuptake inhibitors) (Li, 2017) known to target CNS BDNF levels. The potential in vivo molecular feedback between ALDH5A1 and miR29a may inform inter‐individual phenotypic heterogeneity in patients in relation to residual enzyme levels. Moreover, miR‐29a regulates multiple gene products (Slusarz, 2015), and thus may be a secondary contributor to SSADHD pathophysiology. We conclude that longitudinal evaluation of BDNF and miR29a in a natural history study of SSADHD may provide new insights into disease pathogenesis.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PULMONARY CELL STAINED IN A RAT MODEL OF FAT EMBOLISM FOR RENIN AND PRORENIN ARE INCREASED AFTER ALISKIREN TREATMENT, WHICH AMELIORATES THE FAT‐INDUCED INFLAMMATORY PROCESS
Authors: Al‐Husseinawi Ethar , Colson Jordan Dane , Van Dillen Michael , Fotouhi Ariana , Asan Mohammed , White Lucille , Pour Mohammed , Arif Dauod , Monaghan Paula , Poisner Alan , Molteni Agostino
Published: June 2021

Introduction

Fat Embolism (FE) induced in rats by IV injection of Triolein (T) results in pulmonary vasculitis, inflammation, and fibrosis (1). Captopril and losartan, two drugs acting on the renin angiotensin system, prevent this damage (2). Administration of the renin inhibitor aliskiren also ameliorates the lung's histopathological changes induced by T injections with findings present as early as 48 hours after injection (3). Aliskiren also reduced the number of mast cells observed after T injection (4). This study is aimed at assessing aliskiren's effect on both the presence of vasculitis and on the number of renin/prorenin stained cells in a rat model of FE.

Materials and Methods

Sprague‐Dawley rats (250–300g) were treated with 0.2 ml IV of T (n=18) or saline (n=4). One hour later the rats were divided into three groups with injections with 0.2 ml of saline, 50 mg/kg, and 100 mg/kg of aliskiren (n=6 per group). 48 hours later all subjects were necropsied after isoflurane anesthesia, lungs removed and fixed in 10% formalin with sections submitted for H&E, Trichrome, and Abscam specific antibodies for renin/prorenin cells (R/P). Two pathologists unaware of the slide identity took 10 photographs at random at 400× and counted the number of R/P stained cells.

Results

Sections demonstrated lung arterial vasculitis, septal inflammation, and fibrosis which were evident after 48 hours. Renin‐Prorenin stained cells were present in all groups and mostly located in the arterial adventitia, the thickened septa and subpleura. R/P immunointense reactivity was observed in cells of different sizes and shapes; with some having small oval stained cytoplasm and nucleus, while others demonstrated larger cytoplasm and nucleus with less intense stain and diffuse cytoplasmic vacuoles. A significant increase was seen for the T‐saline group vs the controls (p=0.008) for the small cells, but not for the larger ones. The 50 mg dose of aliskiren did not reduce either cell numbers while the 100 mg dose however induced a marked reduction vs the T‐saline cells both for both small and large cells (p=0.007 and 0.012 respectively). The ratio of small vs large cells was approximately 75% vs 25% for all groups.

Conclusion

The increase in R/P stained cells after FE may be related to an increase in pulmonary mast cell population as seen in other pathological conditions and underlines the RAS involvement in FE.

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.
Localized Optimization of Measles Virus (MV) Hemagglutinin (H) Gene to Human Codon Usage Bias Increases Protein Expression
Authors: Uhl Elizabeth W. , Osborn Michelle L. , Michel Frank , Jelesijevic Tomislav , Hogan Robert J.
Published: June 2021

Optimization and suboptimization of MV and canine distemper virus (CDV) genes to human or canine codon usage bias (CUB) causes changes in protein expression that are independent of viral infection and vary with virus, gene and cell type. In both human (HEK 293) and canine (A72) cells optimization and suboptimization of the MV H gene to either human or canine CUB significantly increases protein expression. In contrast protein expression from the WT CDV H gene construct was high and unchanged by similar codon manipulation. The purpose of this study was to determine whether regional optimization of the MV H gene to human CUB had the same effects as global optimization. Four ½ and ½ mosaic constructs of the MV H gene were made: 1) human CUB optimized + WT, 2) WT+ human CUB optimized, 3) WT MV H + WT CDV H, 4) WT CDV H + WT MV H. Expression vectors containing the constructs were transfected into HEK 293 cells and protein expression was detected by fluorescence labeling and quantitated by flow cytometry. Protein expression was increased from both the WT/human CUB optimized constructs, while protein levels from the WT MV H/CDV H constructs were not increased over that from WT MV H construct. These results indicate that localized, as well as global, codon changes can impact viral protein expression.

Support or Funding Information

The project depicted was sponsored by the Department of Defense Threat Reduction Agency. The content of the information does not necessarily reflect the position or the policy of the federal government, and no official endorsement should be inferred.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modeling Multiple System Atrophy Prion Propagation in Astrocytes From Transgenic Mice
Authors: Krejciova Zuzana , Carlson George , Giles Kurt , Prusiner Stanley B.
Published: June 2021

Background/Aims

There is a growing body of evidence that astrocytes contain α‐synuclein deposits in Parkinson's disease (PD), multiple system atrophy (MSA), and other α‐synucleinopathies at advanced stages of disease. The native protein isoform of α‐synuclein misfolds into a prion conformation that aggregates and spreads throughout the brain, resulting in neurodegeneration in each of these conditions. We aim to use a new in vitro model of α‐synuclein prion propagation to clarify the role of astrocytes in disease progression of MSA. This system may also prove useful for elucidating the molecular mechanisms of neurotoxicity.

Methods

We isolated primary cell culture astrocytes from the brains of transgenic mice expressing either the wild‐type (wt) form of human α‐synuclein or with the A53T mutation, which is associated with familial PD. Astrocytes were exposed to brain homogenates from MSA patients, mouse‐passaged MSA, recombinant wt α‐synuclein fibrils, and cell lysate from MSA‐infected astrocyte cultures. Following exposure, cells were thoroughly washed and cultured in fresh medium. We used confocal microscopy and high content analysis to examine α‐synuclein intracellular morphology and phosphorylation at multiple time points.

Results

While exogenously added α‐synuclein fibrils persisted unphosphorylated, they efficiently induced aggregation and phosphorylation of endogenously expressed α‐synuclein in exposed astrocytes. We observed rapid, progressive, and dose‐dependent α‐synuclein aggregate formation in astrocytes expressing either wt or A53T human α‐synuclein exposed to fibrils or MSA homogenates. Moreover, confocal microscopy revealed two morphologically distinct types of α‐synuclein assemblies – filamentous and granular. Both types progressively formed glial cytoplasmic inclusions, were hyperphosphorylated, thioflavin S positive, ubiquitinated, and co‐localized with the p62 marker, as is also seen in the brains of human MSA patients.

Conclusions

Our studies investigate the formation of α‐synuclein aggregates in astrocytes and report these cells as potential candidates in which to examine various pathological forms of α‐synuclein prions. This cell culture model may allow studies on mechanisms of neurotoxicity and might provide a novel approach for drug discovery against MSA and other α‐synucleinopathies.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gene expression of host restriction factors in HIV‐1 infected monocyte‐derived macrophages
Authors: Rivera Rodriguez Dormarie E. , Godoy Muñoz Lenin J. , Hill Martin
Published: June 2021

The Human Immunodeficiency Virus 1 (HIV‐1) replication cycle has been one of the world's most significant research topics up to date. Although HIV‐1 is capable to infect CD4+ T cells, such as lymphocytes and monocytes, previous studies had demonstrated that cells can inhibit this infection using host restriction factors. Restriction factors, like SAMHD1 and SLFN11, have been shown to interfere with many stages of the retroviral life cycle. Macrophages are able to resist HIV‐1 infection much better than CD4+ T cells. A possible explanation for this is that some of the host restriction factors (SAMHD1 and SLFN11) are strongly expressed in cells of the myeloid lineage. Therefore, the overall aim of this project is to determine gene and protein expression changes of host restriction factors SAMHD1 and SLFN11 between undifferentiated and differentiated monocytes. A monocyte cell line (THP‐1) was chemically differentiated to macrophages using phorbol 12‐myristate 13‐acetate (PMA) and infected with a macrophage‐tropic HIV‐1 strain. Total proteins and RNA from undifferentiated and differentiated monocytes were isolated. Relative SAMHD1 and SLFN11 mRNA expression was quantified by RT‐qPCR and the mean change in expression was calculated using the comparative Ct method using GAPDH as a housekeeping gene. To assess differences in protein expression, western blot analysis was performed using anti‐SAMHD1 and anti‐SLFN11 antibodies. Intriguingly, mRNA expression of SAMHD1 was higher in undifferentiated monocytes when compared to monocyte‐derived macrophages (MDM). Hence, the gene expression of SLFN11 was lower in MDM than undifferentiated monocytes. Assessment of protein expression changes is currently ongoing. Our data shows that differentiation of monocytes to macrophages can alter the expression of these restriction factors. Consequently, these changes might affect the cell permissiveness to infection which may lead to involving restriction factors in anti‐retroviral therapies.

Support or Funding Information

This work was supported by NIGMS‐NIH grants: 5SC3GM088032‐08 (MH), UPR‐Ponce Research Initiative for Scientific Enhancement (R25GM096955). The author also acknowledges the support from the Molecular and Genomics Core (MAGIC) at Ponce Health Sciences University (NIMHD Grant MD007579).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein Kinase Cβ Inhibitors Attenuate Amphetamine‐Stimulated Behaviors Through Direct and Indirect Mechanisms in Different Brain Regions
Authors: Altshuler Rachel , Gnegy Margaret , Jutkiewicz Emily
Published: June 2021

Amphetamines (AMPH) are a class of stimulants that elicit their behavioral effects through an increase in extracellular dopamine levels. Protein kinase Cβ (PKCβ) is necessary for the increase in extracellular dopamine levels and inhibition of PKCβ results in a decrease in AMPH‐stimulated dopamine release and AMPH‐stimulated locomotor activity. When PKCβ inhibitors are administered directly into the nucleus accumbens (NAc) of rats, the drugs act immediately to reduce AMPH's behavioral effects. However, when PKCβ inhibitors are administered into the lateral ventricles of rats, the drug must be administered 18 hrs prior to AMPH to alter AMPH's behavioral effects. In this study, we sought to assess the actions of the PKCβ inhibitors in distinct brain regions to better understand the mechanism of action and time course of the inhibitors. Male Sprague‐Dawley rats were administered 10 pmol of ruboxistaurin, a PKCβ‐selective inhibitor, directly into the NAc or the ventral tegmental area (VTA). Following a 30‐min or 18‐hr pretreatment time, rats were injected with 1 mg/kg AMPH (s.c.) and their locomotor activity was measured. In a separate group of rats, ventral striatal and VTA tissue were collected following an 18‐hr pretreatment of ruboxistaurin in the VTA and a 30‐min pretreatment of 1 mg/kg AMPH (s.c.). Levels of PKCβ and phosphorylated phosphoser41‐growth associated protein 43 (pGAP43), a PKC substrate, were evaluated via immunoblotting. An injection of ruboxistaurin into the NAc attenuated AMPH‐stimulated locomotor activity following a 30‐min pretreatment, but not an 18‐hr pretreatment. Conversely, an 18‐hr pretreatment of ruboxistaurin, but not a 30‐min pretreatment, in the VTA attenuated AMPH‐stimulated locomotor activity. PKCβ and pGAP43 levels were decreased in the VTA and pGAP43 levels were decreased in the ventral striatum. This suggests that an 18‐hr pretreatment of ruboxistaurin may result in a downregulation of PKCβ levels and a decrease in PKC activity, which could contribute to the decrease in AMPH‐stimulated locomotor activity at this timepoint. Furthermore, these studies demonstrate that PKCβ inhibitors act through at least two mechanisms; a direct and immediate mechanism at the NAc and an indirect mechanism at the VTA through regulation of PKCβ levels in the cell bodies. Future work will examine PKCβ levels and activity following a short or long pretreatment in the NAc.

Support or Funding Information

Funded by NIH grant R01 DA11697, T32‐GM007767, Benedict and Diana Lucchesi Graduate Fellowship.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
mGluR2/3 Agonist LY379268 on G‐protein Activation and CREB Phosphorylation
Authors: Aguilar Carolina Burgos , Ferris Mark J. , Sexton Lacey L. , Childers Steve R. , Xiao Ruoyu , Howlett Allyn C.
Published: June 2021

Drug addiction is a growing issue that currently has no FDA‐approved pharmacotherapy; therefore, the concern on how to treat this rapidly increasing problem. The neuroadaptation of neurotransmitter release and signal transduction results in changes in cognitive processes that make it harder for users to stop using the addictive drug. It is believed that restoration of cognition dysfunction may be an effective treatment for addiction. Recent studies in our NIDA‐supported “Center for the Neurobiology of Addiction Treatment” indicated that the cognitive remediator LY379268, an mGluR2/3 agonist, is able to reverse self‐administration of cocaine in rats (Karkhanis, Beveridge, Blough, Jones, & Ferris, 2016, Drug and Alcohol Dependence, 166, 51–60). We hypothesized that the mechanism of LY379268 is to alter cellular signaling in parts of the brain critical for cognition. To test this hypothesis, we examined changes in cellular signal transduction, determined as mGluR2/3‐mediated Gi/o protein activation and the phosphorylation of extracellular‐regulated kinase 1 and 2 (ERK1/2) and cyclic AMP response element binding protein (CREB) in specific regions of the brain related to drug addiction and cognitive processes. Groups of Sprague‐Dawley rats were treated with acute or chronic dosing of LY379268 via subcutaneous Alzet minipump that released 1mg/kg of LY379268 for 36 hours or 14 days, respectively. The control group was subjected to the surgical procedure but did not have a pump inserted. After sacrifice, frozen brain sections were examined for LY379268‐stimulated [35S]GTPgammaS binding, and densities were quantitated in identified brain regions. Immunohistochemistry was performed in intervening sections to quantitate changes in phosphorylation levels using primary antibodies to phospho‐ or total CREB or ERK1/2, and secondary antibodies coupled to infrared dyes. The stained brain sections were imaged and densities were analyzed for those same brain regions that responded to LY379268 in G protein activation studies using LiCor Odyssey software. We observed that desensitization of the G protein‐coupled receptor resulted after the chronic treatment with LY379268. We also observed an increase in CREB phosphorylation in chronic compared with acute treatment but there were no differences between groups in ERK phosphorylation. These studies suggest that chronic treatment with an mGluR2/3 agonist results in sustained neurochemical changes that might be responsible for behavioral modifications seen in chronically‐treated animals.

Support or Funding Information

NIDA grant P50‐DA006634

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Multiple Discriminative Stimulus Effects of the α4β2*‐Selective Agonists Ispronicline and Metanicotine
Authors: Gehrlein Matthew , Winger Gail , Ansari Mohammad , Woods James
Published: June 2021

Ispronicline and metanicotine are selective ligands at central nicotinic acetylcholine α4β2* receptors (Gao et al. Pain 2010: 149, 33; Grady et al. Neuropharmacol 2010: 58, 1054). In our study, a group of rats was trained to discriminate the stimulus properties of the nicotinic acetylcholine receptor (nAChR) agonist nicotine, and a separate group of rats was trained to discriminate the stimulus properties of the muscarinic acetylcholine receptor agonist arecoline. Ispronicline and metanicotine produced full dose‐related selection of the drug‐appropriate response option in both the nicotine‐trained and the arecoline‐trained rats. The nAChR antagonist mecamylamine blocked the discriminative stimulus effects of ispronicline and metanicotine in both groups of rats, as did the α4β2*‐selective antagonist, dihydro‐beta‐erythroidine. Neither antagonist blocked the discriminative stimulus effects of arecoline in these experiments. These data raise the possibility that ispronicline, metanicotine and similar drugs may have novel effects and require a more extensive characterization of receptor activities, through both agonists and antagonists

Support or Funding Information

Research supported by NIMH Grant 107499

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 Effect of Cannabidiol on Epileptic Processes through Microglial Modulation
Authors: Victor Tanya R. , Nissen Jillian C. , Elmes Matthew W. , Deutsch Dale G. , Tsirka Stella E.
Published: June 2021

Epilepsy is a chronic disorder characterized by abnormal brain cell activity leading to recurrent, unprovoked seizures. Pharmacological therapies are commonly used for the treatment of epilepsy, although nearly 30% of patients do not respond to current medications. This highlights the need for new drug targets and treatments. Many recent studies have centered on the use of cannabidiol (CBD) to treat epilepsy. Early use of CBD, a non‐psychotropic component of Cannabis sativa, is reported to lessen the severity of seizure in pediatric cases of epilepsy, experimentally induced seizures in animals, and to suppress neuroinflammation in culture. Although CBD has been shown to increase intracellular levels of anandamide, an endogenous cannabinoid, its complete mechanism of action is still poorly understood. Previous studies have reported that in culture CBD can suppress neuroinflammation. We and others have shown that microglia, the immune cells of the central nervous system (CNS), are important mediators of seizure severity. Microglia also act as modulators of neurogenesis and synaptogenesis, contributing to the refinement of functional neuronal circuits. We postulate that CBD modulates the activation of microglial cells to exert beneficial effects in the brain. In this study, we evaluate the effects of CBD on cell death and inflammation in the hippocampus during recovery after seizures. Ongoing studies will evaluate microglial engulfment of newborn cells and the effect of CBD on signaling effectors, including Toll‐Like receptors.

Support or Funding Information

This work was supported by NIH‐IRACDA NY CAPS K12‐GM‐102778 and NIH T32GM007518.

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‐ and Delay‐Dependent Effects of Δ9‐THC on Memory in Female Rats
Authors: DeLarge Alyssa Fournett , Winsauer Peter J.
Published: June 2021

The cannabinoid receptor agonist, Δ9‐tetrahydrocannabinol (Δ9‐THC), is a psychoactive constituent of marijuana, and has been shown to produce sex‐dependent changes in learning and memory. This study examined the effects of estrogen on memory, and how these effects alter the acute disruptive effects of Δ9‐THC (0.32–3.2 mg/kg) on memory in female rats. To do this, female rats were trained to respond under a repeated acquisition and delayed‐performance procedure and then ovariectomized. During the acquisition phase of this procedure, rats acquired a 4‐response sequence on three response keys (center, left and right), and sequences changed daily (CRLC, LRCL, RLCR, etc). Sequence acquisition was followed by a delay phase (1 minute to 24 hours), and a delayed‐performance phase in which rats were required to emit the previously acquired sequence. Responding in the acquisition and delayed‐performance phases was maintained under a second‐order fixed‐ratio 3 schedule of food presentation. Δ9‐THC or vehicle (control) was administered 30 minutes prior to the delayed‐performance phase, regardless of delay duration, in order to specifically assess memory, which was quantified by a percent savings measure. Response rate and the percentage of errors were also recorded. To further examine the effects of estrogen, OVX female rats were chronically administered estradiol using a subcutaneous capsule containing a 25/75% ratio of estradiol to cholesterol. The disruptive effects of Δ9‐THC on memory were assessed prior to and during estradiol administration. Prior to estradiol administration, increasing delays ranging from 1 minute to 24 hours produced delay‐dependent decreases in percent savings. When the delay was 1 hour, acute administration of Δ9‐THC produced dose‐dependent decreases in response rate and percent savings, and increases in percent errors, that were significantly attenuated by estradiol administration. Increasing the delay from 1 to 3 hours enhanced the effects of low doses of Δ9‐THC (e.g., 0.56 mg/kg) and produced a significant decrease in percent savings. These results demonstrate the delay‐dependent effects of Δ9‐THC on memory and the involvement of estrogen in modulating Δ9‐THC's acute memory‐disrupting effects in female rats.

Support or Funding Information

–NIH‐NIDA: R01‐DA037255‐02S1

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeting of the Histone 3 Lysine 9 Methyltransferase Pathway in Kras‐Induced Cell Growth and Pancreatic Cancer
Authors: Lomberk Gwen , Urrutia Guillermo , Caraballo Mariano Colon , Salmonson Ann , Missfeldt Mckenna , Mathison Angela , Tsai Susan , Adsay Nazmi Volkan , Evans Douglas , Iovanna Juan , Urrutia Raul
Published: June 2021

Pancreatic ductular adenocarcinoma (PDAC) ranks fourth as a cause of cancer death in the USA and is almost universally fatal, with the annual number of deaths equivalent to the number of newly diagnosed cases. Valuable research in the field has revealed genetic aberrations that contribute to PDAC development and progression, with KRAS being one of the most frequent mutations in more than 90% of patient samples. However, to date, any efforts to directly target KRAS have failed in the clinic. Thus, there is indisputably an urgent need to further improve our understanding of molecular mechanisms underlying PDAC development as to identify novel therapeutic targets, including druggable important downstream targets and nodes orchestrated by oncogenic KRAS. In the current study, using a cell model that allows inducible expression of mutant KRASG12D, we find that protein levels of the dimethyl‐K9H3 histone methyl transferase (HMT), G9a, and its complex partners are increased in response to activation of the oncogenic Kras pathway. Furthermore, the activation of this oncogenic pathway results in the formation of the G9a‐GLP‐Wiz trimer complex, as determined by affinity protein purification, combined with mass spectrometry. In vivo experiments involving the cross of the Pdx1‐CRE/LSL‐KRASG12D mice with G9afl/fl animals demonstrate that a loss of the H3K9Me2 mark in the nucleus of exocrine cells is accompanied by a significantly reduced number of PanIN lesions. RNA‐Seq experiments from these animals reveal that these mice have reduced levels of typical molecular markers of PanINs and have an upregulation of p21, a cyclin‐dependent kinase inhibitor that is a major effector of p53 activity and facilitates cell cycle arrest in response to DNA damage. Increased levels of p21 protein are also observed in lysates from Pdx1‐CRE/LSL‐KRASG12D/G9a−/− animals compared to Pdx1‐CRE/LSL‐KRASG12D control mice. Pharmacological inhibition and siRNA‐mediated knockdown of G9a in cells recapitulates this effect with p21 upregulation. Furthermore, we find G9a and its H3K9Me2 mark occupy the p21 promoter, suggesting G9a is involved in the direct regulation of this gene. Congruently, pharmacological inhibition of G9a using BRD4770 displays an inhibitory effect on KRASG12D‐induced cell proliferation, as well as pancreatic tumor growth in a subcutaneous xenograft model. Combined, these data provide evidence for a key role of the meK9H3‐G9a pathway as a mediator of the oncogenic Kras response and defines a novel point of potential therapeutic intervention for PDAC.

Support or Funding Information

NIH R01CA178627; NIH R01DK58185; Advancing a Healthier Wisconsin Endowment

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Residues in the Fifth Transmembrane‐Spanning Domain of the D2‐like Dopamine Receptors that Engender Signaling Bias
Authors: Soto Marta Sanchez , Willette Blair K. A. , Gonye Elizabeth C. , Moore Annah M. , Chun Lani S. , Moritz Amy E. , Verma Ravi Kumar , Yano Hideaki , Shi Lei , Free R. Benjamin , Sibley David R.
Published: June 2021

The D2 dopamine (DA) receptor (D2R) signals through a variety of second messenger pathways making it difficult to discern which of these are linked to specific effects of D2R‐targeted drugs; however, this complexity provides a unique opportunity to develop pathway‐selective therapeutics. Structure‐activity analyses using analogs derived from our previously described D2R G protein‐biased agonist, MLS1547, coupled with molecular modeling led to a structural model for biased signaling that entails a hydrophobic binding pocket formed by residues I184, F189, and V190 at the interface between the fifth transmembrane segment (TM5) and the second extracellular loop of the D2R. In the current study, we used mutagenesis to investigate the role of these residues in regulating signaling by the D2‐like receptors (D2R, D3R, and D4R), and the β2‐adrenergic receptor (B2R). We constructed point mutations at I184, V190 and F189 of the D2R, and at the aligned residues for F189 (i.e., position 5.38) within the D3R, D4R, and B2R, and studied their effects on G protein‐mediated signaling and β‐arrestin recruitment using BRET‐based biosensors and in vitro signaling assays. The D2R point mutations I184A and V190A produced a small change in the potency of DA for stimulating β‐arrestin recruitment or G protein activation. Strikingly, the F189A mutation ablated the ability of DA and other D2R agonists to recruit β‐arrestin while G protein‐signaling efficacy was maintained. Further, the D2R F189A mutant was unable to directly interact with GRK2 and failed to internalize from the cell surface following DA stimulation. In addition, we found that mutating the residues in the D3R, D4R, and B2R at position 5.38 to Ala resulted in parallel findings (i.e., loss of agonist‐stimulated β‐arrestin recruitment, but minimal to no effects on G protein‐mediated signaling). These data demonstrate that the D2R F189A mutant, and similarly mutated D3R, D4R, and B2R, are highly biased towards G protein‐mediated signaling and suggest that the presence of a Phe or Tyr residue at this position (5.38) is important for stabilizing an activated state for recruiting β‐arrestin. Conformational changes propagated through TM5 might thus act as a molecular switch for receptor signaling via β‐arrestin recruitment. These results may have implications for the design of novel signaling‐biased compounds for the treatment of GPCR‐related disorders.

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.
Transporter Roles in the Pharmacokinetics and Tissue Distribution of Voxilaprevir, a Pan‐genotypic HCV NS3/4A Protease Inhibitor
Authors: Wang Yujin , Yang Chris , Hao Jia , MacLennan Kelly , Wong Belinda , Mwangi Judy , Murray Bernard , Liang Xiaomin , Lai Yurong , Taylor James G.
Published: June 2021

Introduction

Voxilaprevir (VOX; GS‐9857) is a pan‐genotypic hepatitis C virus nonstructural (NS) protein 3/4A protease inhibitor (HCV‐PI) with potent antiviral activity against HCV genotypes 1 to 6 and an enhanced resistance profile over previous HCV‐PIs1. VOX is a component of Vosevi™, a fixed‐dose combination of three agents (SOF/VEL/VOX) and is approved for treatment of HCV. In vitro and in vivo assessments were conducted to determine the roles of drug transporters in the pharmacokinetics (PK) and preferential tissue distribution of VOX.

Methods

Voxilaprevir metabolic stability was assessed in vitro in hepatic microsomal fraction and cryopreserved hepatocytes from the rat, dog, monkey and human. Permeability and efflux transport were characterized in Caco‐2 cells and Pgp‐ or BCRP‐overexpressing MDCKII cells. The uptake kinetics of VOX was characterized in rat and human hepatocytes. VOX PK and distribution into liver was determined following a single intravenous (IV) administration in rat, dog, and monkey. VOX elimination was evaluated in bile‐duct cannulated rat following a single IV dose. The distribution of VOX in select tissues (liver, kidney, & lung) was assessed with and without rifampicin pretreatment following IV administration in rat. PK following single ascending oral administration of VOX was also evaluated in rat.

Results

VOX was metabolically stable in hepatic microsomal fractions across all species tested, except for the monkey. In the rat, VOX was mainly eliminated via biliary excretion as unchanged parent drug (70% of dose), and to a lesser extent as its oxidated metabolites. Liver uptake of VOX was observed across the nonclinical species tested ‐ liver to plasma concentration ratio was 970, 34, and 41 in the rat, dog, and monkey, respectively. VOX was found to be a substrate of efflux transporters P‐gp and BCRP, and also a substrate of hepatic uptake transporters (OATPs). VOX displayed a non‐linear and saturable OATP‐mediated hepatic uptake kinetics in vitro. In the rat, VOX shown substantially higher liver concentration than reference tissues (kidney and lung) with and without rifampicin pretreatment. Non‐linear PK was also observed following ascending oral doses of VOX in rat.

Conclusions

Taken together, these studies demonstrate the contribution of drug transporters in the PK and preferential liver distribution of VOX; which was also corroborated in its clinical PK and efficacy studies.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Compound 21 (C21), a Selective Angiotensin Type 2 (AT2) Receptor Agonist Attenuates Bleomycin induced Alveolar Epithelial Cell Death
Authors: Nalbandyan Armen , Shenoy Anitha , Steckelings Ulrike Muscha , Katovich Michael , Shenoy Vinayak
Published: June 2021

Background

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by excessive deposition of collagen‐rich matrix, which progressively leads to respiratory failure and death. Patients with IPF have a reduced survival rate and the disease remains refractory to current therapies. Hence, identifying novel targets and developing effective drugs are of paramount importance. Accumulating evidence indicate that death of alveolar epithelial cells (AEC) is a key event for the initiation of IPF. Bleomycin, a well‐known fibrotic agent is a potent inducer of apoptotic cell death in AEC through synthesis of Angiotensin II (Ang II). While the apoptotic actions of Ang II on AEC are mediated via the angiotensin type 1 (AT1) receptor, the functional role of angiotensin type 2 (AT2) receptor in AEC has not yet been characterized. We hypothesized that stimulation of the angiotensin type 2 (AT2) receptor by a non‐peptide agonist, Compound 21 (C21) protects against bleomycin‐induced alveolar epithelial cell death.

Methods

In this study, we investigated the effects of C21, the selective AT2 receptor agonist against bleomycin‐induced death of human pulmonary alveolar epithelial cells (A549). The MTT (3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide) assay was performed to determine cell viability of A549.

Results

We first determined the concentration of bleomycin required for the induction of cell death in cultured A549 cells. A dose‐dependent induction of cell death was obtained with different concentration of bleomycin (μg/mL: % cell death; 1: 30%; 10:42% and 20:61%). Since we observed optimal cell death with 10 μg/mL concentration of bleomycin, we selected this dose for subsequent experiments. Next, we evaluated the cell‐protective effects of C21 at different drug doses (0.001, 0.005, 0.01, 0.05 and 0.1 μM). C21 treatment of bleomycin‐challenged A549 cells (10 μg/mL) produced a dose‐dependent decrease in cell death (μM: % cell death; 0.001:31%; 0.005:29%; 0.01:16%; 0.05:14% and 0.1:9%) as analyzed by the MTT assay. We also observed that treatment of control A549 cells with the above mentioned doses of C21 had little effect on cell proliferation or cell death.

Conclusion

Our results suggest that C21, the selective AT2 receptor agonist protects against bleomycin‐induced alveolar epithelial cell death. Further studies are warranted to elucidate the mechanisms underlying the cytoprotective effects of C21.

Support or Funding Information

American Heart Association Grant (SDG12080302) awarded to Vinayak Shenoy

Start‐up Grant from California Health Sciences University

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Discovery, characterization, and preclinical development of a Kir4.1 (KCNJ10) inhibitor for the treatment of hypertension
Authors: Kharade Sujay V. , Kurata Haruto , Figueroa Eric , Blobaum Anna Louise , Kramer Meghan , Lindsley Craig , Hopkins Corey R. , Denton Jerod S.
Published: June 2021

Kir4.1 (encoded by KCNJ10) inward rectifier potassium channels are emerging as key regulators of sodium reabsorption in the distal nephron of the kidney. Kir4.1 is expressed in the basolateral membrane of the thick ascending limb of Henle, distal convoluted tubule, and collecting duct of nephron. Kir4.1 can form homotetrameric channels or heterotetrameric channels with Kir5.1, although the most common channel subtype in the kidney is the heteromeric Kir4.1/5.1 form. Genetic ablation of Kir4.1 in mice or humans causes severe salt and water wasting resembling clinical phenotypes associated with diuretic use. These human validation data and recent animal studies suggest that Kir4.1 might represent a novel diuretic target for the management of hypertension through inhibition of distal nephron sodium reabsorption. The aim of this study was to develop a selective small‐molecule inhibitor for evaluating the therapeutic potential of renal Kir4.1 channels. From a high‐throughput screen of 76,575 compounds from the Vanderbilt Institute of Chemical Biology library, we identified an inhibitor, termed VU992, which inhibits homotetrameric Kir4.1 with an IC50 of ~0.9 μM and heterotetrameric Kir4.1/5.1 with an IC50 of ~ 9 μM. Lead optimization failed to identify analogs with improved potency toward Kir4.1. The selectivity screening against other Kir channels revealed that VU992 does not block Kir1.1, Kir2.x, Kir6.1/SUR1 and Kir7.1 channels. The voltage dependent unblock suggested that VU992 probably binds in the pore of the Kir4.1 channel. Computational docking analysis using a Kir4.1 homology model revealed several possible interacting residues in the pore. Experimental binding site analysis using mutagenesis and patch clamp electrophysiology suggested that the pore lining glutamic acid residue at 158 (E158) is crucial for the binding of VU992 to the Kir4.1. Mutating E158 to corresponding asparagine residue in Kir1.1 (E158N) abolished the VU992 block completely. Pharmacokinetic studies in rats showed that VU992 does not cross the blood brain barrier and therefore should not inhibit Kir4.1 channels expressed in the CNS. Finally, oral administration of VU992 dose‐dependently increased urine output and Na+ and K+ loss in volume‐loaded, freely behaving rats. This study shows proof‐of‐concept that homotetrameric Kir4.1 might represent a molecular target for a novel class of diuretic for the treatment of hypertension. Its basolateral localization might offer advantages over other diuretic targets that are located on the luminal membrane of the nephron.

Support or Funding Information

NIH 5R21 NS073097‐01S1

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Stable Pentadecapeptide BPC 157 and Vesicovaginal Fistulas in Rats
Authors: Rasic Domagoj , Sever Marko , Sever Anita Zenko , Pavlov Katarina Horvat , Baric Marko , Drmic Domagoj , Blagaic Alenka Boban , Seiwerth Sven , Sikiric Predrag
Published: June 2021

Aim

Frequently, large vesicovaginal fistulas need operation treatment. Stable gastric pentadecapeptide BPC 157, tested in clinical trials for ulcerative colitis therapy and now multiple sclerosis (Curr Pharm Des. 2017;23(27):4012–4028; Curr Pharm Des. 2014;20(7):1126–35; Curr Pharm Des. 2010;16(10):1224–34), already resolved the healing of various fistulas (Eur J Pharmacol. 2016 Jun 5;780:1–7; Life Sci. 2016 Mar 1;148:63–70; J Physiol Pharmacol. 2015 Aug;66(4):581–90; Eur J Pharmacol. 2013 Feb 15;701(1–3):203–12; J Pharmacol Sci. 2008 Sep;108(1):7–17; Dig Dis Sci. 2009 Jan;54(1):46–56). Thus, BPC 157 may be a successful therapy of vesicovaginal fistulas in rats.

Methods

Vesicovaginal fistulas were created in Wistar rats. The vesicovaginal fistula was surgically created (4mm length between posterior wall of the urinary bladder and anterior vaginal wall ). Medication. Pentadecapaptide BPC 157 (10 μg/kg/day, 10ng/kg/day) was given intraperitoneally (5 ml/kg) or in drinking water (0.16 μg/ml, 0.16 ng/ml, 12 ml/rat/day) while controls received equivolume of saline or drinking water only. Assessment (the pressure required for leakage of fluid through fistula (mLH2O), diameter of the fistula defects (mm) (bladder, vaginal side), macro/microscopical evaluation, urinary stone presentation) was 1, 2, 3, 4, and 6 weeks. Prophylactic regimen. Medication was started immediately after fistulas creation. Therapy regimen. Medication was initiated with already advanced fistula formation, after 2 weeks.

Results

Unlike poor healing in controls, BPC 157 reduced fistulas diameters (7th day) leading to the complete closure at 46th day and considerable volume needed to leakage of fluid through fistula (V: 5.3+/−0.3 mL). Finally, the fistula was successfully healed and microscopically confirmed in BPC 157 rats (day 46, 0+/−0 mm (diameter, bladder/vagina), unlike open defects in controls: 3.1+/−0.6 (mm, bladder), 3.3+/−0.8 (mm, vagina)) diameter and small volume sustained before leakage (V: 1.2+/−0.4 mL, day 14). In posttreatment BPC 157 group we noticed reducing of fistula diameter since a first posttreatment week ( con 3.8 +/−0.2 mm, BPC 157 3.1 +/−0.1 mm) leading to fistula closing during the 4th posttreatment week. In all control animals, we noticed urinary bladder stone creation which was absent in BPC 157 animals. In posttreatment animals, BPC 157 (therapy regimen) stopped further urinary stone creation.

Conclusion

Pentadecapeptide BPC 157 improved vesicovaginal fistula healing, and it could be the pharmacological solution for the complex clinical problem of vesicovaginal fistulas healing. Vesicovaginal fistula could be used as a urinary stone animal model. BPC157 reduces urinary stone creation.

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