bluuebunny/crossref_metadata_2025_split huggingface dataset:
Codon Optimization of Gene Editing CRISPR‐SaCas9 Augments Protein Expression in Human Liver Cells to Boost in vivo Therapeutic Application
Authors: Cheng Benjamin , Groshong Tyler , Madejski Irene , Sweeney Robert , De Luke 
Published: June 2021

CRISPR‐Cas9 gene editing technology shows great promise in revolutionizing the treatment of genetic disorders; however, the current focus on treatment by ex vivo delivery is limited to blood diseases. In vivo treatment can substantially extend the clinical reach of the technology, but is limited by a challenging delivery process. The large size of the Cas9 gene (~4500bp) inhibits delivery by the small viral vectors and lipid nanoparticles used. We propose that a codon‐optimized Cas9 gene will support significantly higher protein expression than the presently used genes; this can compensate for the low delivery efficiency and has great potential to improve the viability of in vivo CRISPR‐Cas9 therapies.A commonly used parent saCas9 sequence was selected from Addgene's public plasmid database. A novel, codon‐optimized gene sequence was engineered from the parent using GenScript's OptimumGene™ algorithm. Both the optimized gene and parent gene were transfected into human HepG2 liver cells with high efficiency as visualized by GFP‐tagged saCas9. ELISA protein quantification of two sample groups demonstrated that the codon‐optimized gene expressed the saCas9 protein at 3.5× the rate of the commercial sequence. The augmented Cas9 protein expression supported by the novel saCas9 sequence can better enable in vivo CRISPR‐Cas9 treatments.Support or Funding InformationThis work was supported by The Nueva School.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Perlecan Domain V Increases Amyloid Clearance in Co‐Morbid VCID
Authors: Trout Amanda , Hartz Anika , Roberts Jill , Zhang Zouhui , Frazer Justin , Bix Gregory 
Published: June 2021

IntroductionVascular contributions to cognitive impairment and dementia (VCID) is a broad term that encompasses a spectrum of initial asymptomatic cerebrovascular changes (seen in small vessel disease and cerebral amyloid angiopathy) to the profound symptomatic damage following acute stroke(s). Besides aging, metabolic disorders such as type 2 diabetes complicate our understanding of underlying mechanisms. Cerebrovascular remodeling and angiogenesis may represent early compensatory changes to the reduced blood flow seen in VCID by increasing the proteolytic turnover of the surrounding extracellular matrix. We have demonstrated that one such extracellular matrix protein, perlecan, possesses a C terminal domain V (DV) protein that, upon cleavage from perlecan, greatly enhances brain angiogenesis.ObjectiveWe hypothesize that DV therapy can reduce pathological changes in the cerebrovasculature, an underlying component of VCID.MethodsDV's mechanism and therapeutic potential in facilitating Aβ clearance were examined in an ex vivo model of isolated mouse brain capillaries in normal and high glucose environments. We also determined the cerebrovascular changes in a comorbidity mouse model of VCID (diabetic APP/PS1 knock in (db/AD)) that is associated with hemorrhages, aneurysms, Aβ deposition, cognitive decline and increased mortality.ResultsDV (300 and 600 nM) doubled the transport of fluorescently tagged hAβ1‐42 over 24 hours from the bath (abluminal) to luminal side of the brain capillaries. One of Aβ's known transport proteins, P‐glycoprotein (P‐gp), also increased in total protein expression and in transport activity in the presence of DV. At 3–6 months in db/AD mice, we observed a decrease in BBB proteins (claudin‐5) indicating an altered cerebrovascular function correlating with DV expression increase during the asymptomatic angiogenic stage. At 9–12 months, cognitive changes were associated with altered cerebrovascular protein expression in PECAM, Collagen IV, and DV correlating with the aberrant two‐photon vascular imaging, increased saccular aneurysms, and microhemorrhages.ConclusionThese data show that DV increases cerebrovascular Aβ clearance through P‐gp. Increased DV expression correlates with early cerebrovascular changes induced by angiogenic‐remodeling in a diabetic co‐morbid mouse model of VCID. Lower DV expression is associated with aberrant vasculature that contribute to increased mortality, suggesting that DV ‘replacement therapy’ could represent a novel therapeutic for VCID.Support or Funding InformationNIH RO1 N5089515‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Opposing roles of Sigma‐1 and Sigma‐2 receptors in heavy alcohol drinking and associated mechanical allodynia in mice
Authors: Sabino Valentina , Quadir Sema G , Tanino Sean M , Rohl Christian D , Sahn James J , Moore Catherine F , Martin Stephen F , Cottone Pietro 
Published: June 2021

Alcohol Use Disorder (AUD) is a serious and complex neuropsychiatric disorder characterized by excessive alcohol intake, inability to control consumption, and the emergence of a negative emotional state in the absence of alcohol. While the role of the Sigma‐1 receptor (Sig‐1R) in animal models of AUD has been demonstrated, the role of Sigma‐2 receptor/Transmembrane Protein 97 (Sig‐2R/TMEM97), which has recently been cloned, is still unclear. Using an intermittent access, 2‐bottle choice paradigm, the effects of the Sig‐2R/TMEM97 modulator JVW‐1034 (0–30 mg/kg, i.p.) on ethanol drinking in male C57Bl/6J mice were assessed. Sucrose intake was used to control for non‐specific effects of the drug on an alternate reinforcer in a separate study, and locomotor intake was also assessed. In addition, using an automated von Frey filament test, the effects of JVW‐1034 on mechanical sensitivity in alcohol drinking mice was tested during withdrawal. The effects of the Sig‐2R modulator were compared to those of BD‐1063 (0–30 mg/kg, i.p.), a preferential Sig‐1R antagonist. Results showed that JVW‐1034 reduced alcohol intake and preference in this model, without affecting water intake. Sucrose intake was not affected by the drug, suggesting selectivity of the effect for alcohol. JVW‐1034 also blocked the mechanical allodynia induced by chronic alcohol drinking without affecting sensitivity in control mice. Lastly, JVW‐1034 did not affect locomotor activity in either group. BD‐1063 had a very similar profile. These data suggest that Sig‐1R and Sig‐2R play opposite roles in the regulation of alcohol drinking and associated allodynia and hyperalgesia, and lay the foundation for more extensive studies examining the involvement of the Sig‐2R/TMEM97 system in AUD.Support or Funding InformationR01‐AA024439This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Palmitic acid methyl ester directly induces hippocampal neuroprotection against oxygen and glucose deprivation
Authors: Neumann Jacob , Van Allen Joshua , Azizbayeva Rinata , Lin Hung Wen 
Published: June 2021

Cardiopulmonary arrest (CA) is one of the leading causes of death and disability in the USA. The prevalent quandary in this field is the multi‐factorial nature of CA (i.e., whole body ischemia), which compromises systemic blood parameters and cerebral, renal and cardiac functions. This disruption of cerebral blood flow (CBF) also results in severe and selective brain damage (i.e., neuronal cell death) in the hippocampus, especially in the cornu Ammonis 1 (CA1) region. While previous research has indicated PAME (a vasodilator) as a neuroprotective agent, the specific mechanism(s) of neuroprotection is/are currently unknown and it is currently unknown if PAME‐induced neuroprotection is from direct modulation of CBF or due to direct activation of neuroprotective pathways. Here, we tested the hypothesis that PAME induces neuroprotection independent of CBF, and that PAME alters additional signaling pathways to induce neuroprotection in the hippocampus. Our studies used an in vitro hippocampal model (hippocampal organotypic slices) to determine the requirement of CBF for PAME‐induced neuroprotection and if specific modulation of alpha‐7 nicotinic acetylcholine receptors (a potential target of PAME) can induce neuroprotection following oxygen and glucose deprivation (OGD) using propidium iodide staining. All of the protocols were approved by the West Virginia School of Osteopathic Medicine Institutional Animal Care and Use Committee. Our results suggest that the direct application of PAME or inhibition of alpha‐7 nicotinic acetylcholine receptors induced neuroprotection following OGD in the CA1 region of the hippocampus. These data suggest that PAME‐induced neuroprotection occurs independent of CBF and identifies alpha‐7 nicotinic acetylcholine receptors as a potential mechanism for PAME‐induced neuroprotection.Support or Funding InformationThis study was supported by a West Virginia School of Osteopathic Medicine intramural grant and a grant from the American Heart Association 17GRNT33660336 .This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
N‐Palmitoylethanolamide‐oxazoline Treatments Protects Against Neuroinflammatory Injury Induced by Transient Middle Cerebral Artery Occlusion in Diabetic Rats
Authors: Di Paola Rosanna , Fusco Roberta , Cordaro Marika , D'Amico Ramona , Gugliandolo Enrico , Siracusa Rosalba , Peritore Alessio , Crupi Rosalia , Impellizzeri Daniela , Cuzzocrea Salvatore 
Published: June 2021

Diabetes causes various macrovascular and microvascular alterations often culminating in major clinical complications, first of all is stroke, that, until today, lacks effective therapeutic intervention. N‐Palmitoylethanolamine‐oxazoline (PEA‐OXA) possesses anti‐inflammatory and potent neuroprotective effects. Although, recent studies have explained the neuroprotective properties of PEA‐OXA, nothing is known about its effects in treating cerebral ischemia. The aim of this study is to explore the mechanism and the effect of PEA‐OXA against cerebral ischemic/reperfusion injury using the transient middle cerebral artery occlusion (MCAo) model in diabetic rats. Neurological severity score and infarct volumes were carried out to assess the neuroprotective effects of PEA‐OXA. Moreover we observed PEA‐OXA‐mediated improvements in tissues histology shown by reduction of lesion size and improvement in apoptosis level (assessed by Caspases, Bax and Bcl‐2 modulation and TUNEL assay) further support the efficacy of PEA‐OXA therapy. We also found that PEA‐OXA treatment was able to reduce mast cell degranulation and to block infiltration of astrocytes and microglia and reduced MCAo‐induced expression of cytokines, nitrotyrosine, MDA, VEGF and TGF‐b, as well as was competent to re‐establish growth factors such as BDNF, GDNF and NGF. PEA‐OXA also inhibited the MCAo‐mediated increased expression of NAAA and NF‐κB and degradation of IκB‐α. PEA‐OXA treated injured animals also improved neurobehavioral functions as evaluated by water maze test. Based on these findings, we propose that PEA‐OXA would be useful in decreasing the risk of impairment or improving function in ischemia‐reperfusion brain injury‐related disorders.Support or Funding Informationno fundingThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Calcium‐activated K+ channel KCa3.1 Plays a Pro‐inflammatory Role in α‐Synuclein Models of Parkinson's Disease
Authors: Malovic Emir , Sarkar Souvarish , Nguyen Hai , Singh Vikrant , Jin Huajun , Anantharam Vellareddy , Kanthasamy Arthi , Wulff Heike , Kanthasamy Anumantha G. 
Published: June 2021

Chronic neuroinflammation characterized by the sustained activation of microglia and subsequent release of pro‐inflammatory mediators is concomitant with the progressive neurodegeneration observed in Parkinson's disease (PD). Microglial potassium channels have a well‐developed pharmacology and constitute attractive targets for reducing neuroinflammation. We previously implicated the voltage‐gated channel Kv1.3 in playing a proinflammatory role in cell culture and animal models of PD. Another K+ channel, the calcium activated channel KCa3.1 is also expressed in a variety of immune cells, including B cells, T cells, and microglia. However, the role of KCa3.1 in PD‐related neuroinflammatory processes has not been investigated. In this study, we examined whether KCa3.1 plays a role in regulating neuroinflammation in PD. Exposure of primary mouse microglia to aggregated α‐synuclein (α‐Synagg) led to the upregulation of KCa3.1 mRNA, as determined by qRT‐PCR. Patch‐clamp recordings revealed that KCa3.1 activity is also upregulated following α‐Synagg stimulation. We further demonstrated that α‐Synagg increases the production of pro‐inflammatory cytokines, including IL‐1β, IL‐6, IL‐12, and TNFα, while these cytokines were significantly attenuated with the KCa3.1 inhibitor, TRAM‐34 (1‐[(2‐chlorophenyl)diphenylmethyl]‐1H‐pyrazole). Using proteomics analysis, we identified potential targets that may be involved in KCa3.1 signaling in α‐Synagg‐treated samples. In animal studies, C57BL/6J mice received a unilateral stereotaxic injection of mouse α‐synuclein preformed fibrils (α‐Synpff) in the striatum. One subset of these animals received TRAM‐34 (10 mg/kg, i.p.) daily starting at 4 months and animals were sacrificed one month later. Behavioral analysis revealed α‐Synpff‐induced motor deficits at 5 months post‐injection, whereas TRAM‐34 attenuated α‐Synpff‐induced motor deficits. TRAM‐34 was also able to significantly block α‐Synpff‐induced decreases in striatal dopamine and DOPAC levels. This was further corroborated by TRAM‐34's protection against the α‐Synpff‐induced loss of tyrosine hydroxylase in both substantia nigral and striatal tissues by immunohistochemistry and immunoblotting, respectively. Collectively, our data highlight the pivotal role of KCa3.1 in PD‐associated neuroinflammatory processes and has potential therapeutic implications.Support or Funding Information(NIH grants NS100090, NS088206 and ES026892)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of matirx‐metalloproteinases mediates carnosine‐neuroprotection during ischemia
Authors: Kim Eun‐Hye , Shin Donggeun , Shin Young‐Jun , Kim Donghyun , Oh Eujin , Majid Arshad , Bae Ok‐Nam 
Published: June 2021

Despite the increasing global medical burden of ischemic stroke, there is no existing therapeutic option against ischemic stroke. It is urgent to find a new therapeutic candidates or therapeutic targets for ischemic stroke. Matrix‐metalloproteinases (MMPs) contribute to deterioration of neuronal damage by breakdown of blood‐brain barrier and edema formation during ischemic stroke, and inhibition of MMP activity is known to reduce ischemic brain injury. Our group recently showed that the endogenous dipeptide, carnosine, has a high protective activity against ischemic brain damage, suggesting that carnosine may be a new candidate for stroke treatment. In this study, we investigated whether carnosine regulates the activity of matrix‐metalloproteinases (MMPs) and brain edema by in vivo rat stroke models and in vitro enzyme activity assay. Carnosine therapy has significantly reduced neuronal infarct as well as edema in rats with ischemic stroke initiated by permanent occlusion of middle cerebra artery. Inhibitory effect of MMP activation by carnosine were observed in gelatin zymography and western blot of brain hemispheres. In the isolated enzyme assay, carnosine significantly inhibited the activity of MMP‐2 and MMP‐9 mediated by chelation of zinc, which is essential for the MMP activity. Taken together, we have found that carnosine can inhibit MMP activity, which reduces edema formation and brain damage by reducing the level of co‐factor, zinc needed. We believe that our research provides new insights into the carnosine neuroprotection mechanism of ischemic brain injury.Support or Funding InformationFunding Source: This work was supported by grants from the National Research Foundation (NRF‐2017R1C1B3002626) of Korea.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Guanabenz Modulates Microglia and Macrophages in EAE
Authors: Thompson Kaitlyn Koenig , Tsirka Stella E. 
Published: June 2021

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) characterized by peripheral immune cell infiltration, demyelination, and axonal damage. Microglial activation has also long been recognized as an early event in MS pathogenesis and is generally considered harmful as it can contribute to pro‐inflammatory responses and neuronal damage. However, microglia are also capable of releasing factors that can suppress the immune response and promote remyelination and repair. Therefore, a valid therapeutic strategy is to prevent pro‐inflammatory microglial activation and/or shift their phenotype towards neuroprotective and anti‐inflammatory. We have previously characterized the immunomodulatory tetrapeptide, tuftsin, that attenuates the experimental autoimmune encephalomyelitis (EAE) model of MS by inducing an anti‐inflammatory shift in microglia and infiltrating monocyte‐derived macrophages. As tuftsin is not FDA‐approved, we sought to investigate the repurposing of an already approved drug that may act similarly. We identified guanabenz, an FDA‐approved antihypertensive shown to possess anti‐inflammatory properties, using a virtual screen against tuftsin's receptor, neuropilin‐1 (NRP1). Although guanabenz does not bind NRP1, it does provide beneficial anti‐inflammatory effects in EAE. Using two EAE models that represent chronic and relapsing‐remitting (RR) MS disease courses we observe that guanabenz has beneficial, microglia/macrophage‐targeted immunomodulatory properties. In the chronic EAE model guanabenz improves disease scores, reduces demyelination and the amount of harmful, pro‐inflammatory microglia when administered therapeutically. In the RR‐EAE model guanabenz attenuates relapses, reduces demyelination and pro‐inflammatory microglia numbers when administered immediately after the initial symptom peak. Expression of pro‐inflammatory genes, such as TNF‐a, is reduced in stimulated primary macrophages and microglia. Overall, we report that guanabenz is capable of dampening the pro‐inflammatory responses of microglia and macrophages and improving pathological hallmarks and motor deficits in EAE. Further, combination of guanabenz with clemastine, an FDA‐approved antihistamine now in clinical trials for inducing remyelination in MS, offers additional benefits.Support or Funding InformationThis work was supported by NMSS CA1044A1 and PP1815 (SET) and PhRMA predoctoral fellowship (KKT).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The anti‐glioblastoma drug ONC201 and related analogs act as non‐competitive antagonists at the D2 dopamine receptor through unique binding site interactions
Authors: Cuoco Caroline Ann , Free R. Benjamin , Willette Blair , Lane J. Robert , Allen Joshua E. , Sibley David R. 
Published: June 2021

ONC201 is a first‐in‐class imipridone molecule that is in clinical trials for the treatment of glioblastoma. It was initially identified as a p53‐independent inducer of the immune cytokine TRAIL leading to tumor cell death. Notably, overexpression of the D2 dopamine receptor (D2R) occurs in a variety of cancers, including glioblastoma, and its antagonism imparts pro‐apoptotic and anti‐proliferative effects in tumor cells. Characterization of ONC201 and several novel analogs (ONC206 and ONC213) using functional assays (β‐arrestin recruitment and cAMP accumulation) and radioligand binding analyses revealed that all ONC compounds were full antagonists of the D2R with low micromolar affinities, similar to their potencies for anti‐proliferative effects. To investigate the mechanism of D2R antagonism by the ONC compounds, Schild‐like curve‐shift experiments were performed using the functional assays where dopamine dose‐response curves were constructed in the presence of increasing concentrations of ONC compound. The addition of ONC compounds caused both a rightward shift in the dopamine concentration‐response curve and a depression of the maximum response. Such results are consistent with a mixed/non‐competitive mechanism of antagonism. These curve shift data were fit to the operational model of allostery, which revealed that the predominant modulatory effect of the ONC compounds is on dopamine efficacy with a smaller effect on dopamine potency. To investigate how ONC201 binds to the D2R, we employed scanning mutagenesis where amino acids are systematically mutated to alanine coupled with a D2R‐mediated calcium efflux assay. In this fashion, eight residues, located in two divergent pockets were identified as being important for ONC201's functional antagonism of the D2R and postulated to represent potential ligand‐receptor interaction sites. D2Rs harboring these mutations were expressed in HEK293 cells and assayed for both binding affinity and functional efficacy of the ONC compounds. The ability of ONC201 and analogs to block dopamine‐mediated G protein responses (cAMP and Go‐activation) and to bind to the receptor were hindered by the introduction of each mutant supporting the importance of these interaction sites. Of particular note is the loss of ONC binding affinity and functional efficacy with the distant I397A and V91A mutations possibly suggesting allosteric coupling between these sites. Elucidation of the pharmacological profile and unique interaction sites of ONC201 and analogs may lead to a better understanding of its mechanism for anticancer targeting.Support or Funding InformationNINDS Intramural Research ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.