bluuebunny/crossref_metadata_2025_split 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 InformationThe 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 ObjectivesSSADHD 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.MethodsRNA 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.ResultsIn 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/ConclusionThe 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

IntroductionFat 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 MethodsSprague‐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.ResultsSections 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.ConclusionThe 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 InformationSupported by the Catherine T Geldmacher Foundation, St. Louis, MOThis 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 InformationThe 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/AimsThere 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.MethodsWe 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.ResultsWhile 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.ConclusionsOur 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 InformationThis 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 InformationFunded 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 InformationNIDA grant P50‐DA006634This 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 antagonistsSupport or Funding InformationResearch supported by NIMH Grant 107499This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.