bluuebunny/crossref_metadata_2025_split huggingface dataset:
Methylmethacrylate Thin Sections are Superior to Thick Ground MMA Sections for Histologic Evaluation of Complex Submillimeter Metallic Medical Devices
Authors: O'Brien Maureen T. , Dorsey Theresa , Ensminger Leslie , Kuhn Lauren , Dieterle Brian , Burgenson Janet 
Published: June 2021

Hard plastic resin embedding is necessary for histology of tissues containing hard and/or metallic medical devices if the device is to be retained within the section for microscopic assessment. Traditional methylmethacrylate (MMA) embedding and sectioning of tissues containing hard and/or metallic devices requires targeted sectioning of the block with a diamond blade saw followed by careful grinding of the MMA‐embedded specimen with a calibrated grinder. This method provides a tissue section for histology that is approximately 50–60 μm in thickness (MMA thick section). However, the process of creating a MMA thick section is somewhat imprecise and results in considerable sample loss due to requisite attrition during MMA block sectioning and grinding procedures. For submillimeter (<1 mm) and/or similarly small, topographically complex medical devices, this level of relative imprecision, specimen attrition, and loss of cellular detail due to inherent thickness of tissue section can result in unacceptable diagnostic slide quality or even complete loss of a valuable specimen during sectioning/grinding procedures. To circumnavigate these challenges, we utilize MMA hard plastic resin embedding of small metallic medical devices followed by acquisition of thin (3–5 μm) MMA sections using a motorized microtome. Thin MMA sections provide superior cellular detail, greater precision of sectioning, and excellent retention of high quality device‐tissue context when compared with traditional MMA thick plastic sections. Although acquisition of MMA thin sections can be limited by larger device sizes and/or composition, MMA thin sections provide superior histologic quality for permissible specimens.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Hypoxia Inducible Factor 1 Alpha is Required for Optimal GammaHerpes Virus Lytic Replication and Reactivation from Latency
Authors: Lopez‐Rodriguez Darlah M. , Mesri Enrique , Andreansky Samita 
Published: June 2021

Lytic replication of Kaposi's Sarcoma Associated Herpes Virus (KSHV) exacerbates Kaposi's sarcoma (KS) progression, an angiogenic spindle‐cell sarcoma associated with AIDS. Current antiviral therapy targeting lytic replication can prevent KS in seropositive patients. KSHV lytic infection is not manifested in vitro, and currently there are no animal models that display pathogenic phenotypes. In this study, we employed a biological and genetically mouse pathogen similar to KSHV, the murine gammaherpes virus (gHV) 68 (MHV68), that readily infects laboratory mice providing a valuable small animal model. Moreover, MHV68 in vitro infection exhibits a default lytic infection. Several studies have shown that during infection KSHV employs several viral proteins to de‐regulate the host transcription factor hypoxia inducible factor 1 alpha (HIF1α) and KSHV‐induced activation of HIF1α is necessary for viral persistence and KS progression. However, little is known about the role of HIF1 α in gHV replication and pathogenesis. To further investigate the role of this host factor, we induced transcriptional inactivation of HIF1α in MHV68 virus‐specific cells in vivo. Conditional knock out of HIF1α was achieved by infecting transgenic mice with Cre‐recombinase (Cre) LoxP specific site within the functional domain of HIF1α gene using a recombinant MHV68 that expressing a CMV driven Cre expression.Our data demonstrated for the first time that HIF1α inactivation during gammaherpes virus infection in vivo affects viral gene transcription resulting in impaired virus expansion and early clearance of acute infection in a mouse model. In addition, our results from in vitro MHV68 lytic infection of mouse fibroblasts lacking HIF1α showed that lytic virus production and transcription is decreased. During latency establishment in vivo, the frequency of MHV68 latent splenocytes undergoing latent to lytic replication was largely decreased as well. Our data suggest that HIF1α is required for sustained acute infection since it's deletion in virus‐infected cells resulted in early clearance of productive infection and impairment in reactivation. Moreover, HIF1α is required for mRNA upregulation of glycolytic enzymes during MHV68 lytic infection suggesting a role for HIF1a as a modulator of cell energetics and viral gene expression. We conclude that gammaherpes viruses required the function of the cellular host factor HIF1α in order to effectively replicate and establish latency within its host.Support or Funding InformationNational Cancer Institute 3R01CA136387‐08S1This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identifying Keap1‐Interacting Coxiella burnetii Proteins
Authors: Ortiz Angely , Voth Daniel E. 
Published: June 2021

Coxiella burnetii is a Gram‐negative bacterium that causes an acute flu‐like illness in humans called Q fever. Human infection is initiated by inhalation of contaminated aerosols generated while working with livestock. In human cells, C. burnetii forms a lysosome‐like vacuole for replication to promote an obligate intracellular lifestyle. The pathogen also regulates multiple events during infection using a type IV secretion system and secreted effector proteins. C. burnetii activates the Nrf2‐Keap1 pathway in macrophages, potentially using an effector protein(s) to disassociate Keap1 and allow Nrf2 to move into the nucleus for transcription. p62 is known to interact with Keap1 an promote this disassociation. We predict C. burnetii uses the proteins encoded by CBU1400 and AUR58 that contain a putative Keap1‐binding domain. In this study, we derived adenylate cyclase and fluorescent constructs of each protein to determine secretion and cellular localization, respectively. Additionally, we assessed the impact of brusatol, an established Nrf2 inhibitor, on C. burnetii intracellular growth. A better understanding of the requirement of Nrf2 for infection will allow enhanced modeling of C. burnetii‐host cell interactions.Support or Funding InformationThis project was supported by the Arkansas SURP program, with grants from the NCCR (P20RR016460) and the NIGMS (P20 GM103429), and by NIAID R01AI087669 (D.E.V.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Estrogen Related Receptor α Is Required For Efficient Arenavirus Multiplication
Authors: Cintron Kevin , Cubitt Beatrice , Iwasaki Masaharu , de la Torre Juan Carlos 
Published: June 2021

Several arenaviruses cause hemorrhagic fever disease in humans and represent important public health problems in their endemic regions. In addition, evidence indicates that the worldwide‐distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an important neglected human pathogen. There are no FDA licensed arenavirus vaccines and current anti‐arenavirus therapy is limited to the use of ribavirin that is only partially effective. Therefore, there is an unmet need for novel anti‐arenaviral therapeutics. Previous studies done in our lab found that XCT790 and AM251, small molecule inhibitors of the Estrogen Related Receptor Alpha (ERRa) protein exhibited also a potent anti‐LCMV activity. We, therefore, hypothesized that ERRa is a host cell factor involved in the replication cycle of arenaviruses. To test our hypothesis, we used a recombinant LCMV that expressed GFP to perform three assays that examined the effect of the compounds in three different steps of arenavirus replication cycle. Our results revealed that XCT790 and AM251 interfered with both the activity of the LCMV RNP responsible for directing viral RNA synthesis, and the arenavirus budding process mediated by the Z protein.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SULFORAPHANE PROTECTS AGAINST ETHANOL‐INDUCED APOPTOSIS IN NEURAL CREST CELLS AND ZEBRAFISH EMBRYOS THROUGH EPIGENETICALLY MODULATING THE EXPRESSION OF SANIL1 AND RESTORING EMT
Authors: Li Yihong 
Published: June 2021

Ethanol‐induced apoptosis in neural crest cells (NCCs), a multipotent progenitor cell population, are implicated in the Fetal Alcohol Spectrum Disorders (FASD). Epithelial‐mesenchymal transition (EMT) is a process by which cells lose their epithelial phenotype and acquire a mesenchymal phenotype, which is essential for NCC migration. The objective of this study is to investigate whether ethanol exposure can induce apoptosis in NCCs by inhibiting EMT and whether sulforaphane (SFN), a vegetable‐derived isothiocyanate, can prevent ethanol‐induced apoptosis by epigenetically modulating the expression of Snail1, a key transcriptional factor that can promote EMT by repressing E‐cadherin. We found that exposure to 50 or 100 mM ethanol for 24 hours resulted in a significant increase in apoptosis in NCCs. Co‐treatment with SFN significantly reduced ethanol‐induced apoptosis. Ethanol treatment also inhibited EMT in NCCs, as indicated by an increased expression of E‐cadherin1, an EMT‐suppressing marker, and a reduced expression of vimentin, an EMT‐promoting maker. Treatment with SFN significantly reversed ethanol‐induced changes in the expression of E‐cadherin1 and vimentin, and restored EMT in NCCs. We also found that ethanol exposure significantly decreased the mRNA expression of Snail1, which can be reversed by co‐treatment with SFN. To determine whether ethanol and SFN regulate the expression of Snail1 by histone modification, the expression of kdm5a, a lysine‐specific demethylase and the levels of trimethylation of histone H3 lysine 4 (H3K4me3) at the promoters of Snail1 were determined. We found that ethanol exposure increased the expression of kdm5a, which was reversed by SFN. ChIP‐qPCR analysis revealed that ethanol exposure significantly reduced the levels of H3K4me3 at the promoters of Snail1 and that SFN treatment reversed the ethanol‐induced reduction of H3K4me3 at the promoter regions of Snail1 gene. SFN treatment also significantly diminished ethanol‐induced apoptosis in zebrafish embryos. In addition, Knock‐down of kdm5a restored EMT and significantly decreased apoptosis in ethanol‐exposed NCCs and zebrafish embryos. These results demonstrate that ethanol exposure can induce apoptosis in NCCs and zebrafish embryos by inhibiting EMT and that SFN can protect against ethanol‐induced apoptosis by reversing ethanol‐induced reduction in the levels of H3K4me3 at the promoters of Snail1, restoring the expression of Snail1 and EMT in ethanol‐exposed NCCs.Support or Funding InformationSupported by NIH grants AA021434, AA020265, and AA024337 (S‐Y.C)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Abuse‐Related Discriminative Stimulus Effects of Synthetic Cathinones
Authors: Paronis Carol A. , Kohut Stephen J. , Sherwood Alexander M. , Brown Kelly A. , Zakarian Ani S. , Prisinzano Thomas E. , Bergman Jack 
Published: June 2021

The use of synthetic cathinones, or “bath salts,” has become more widespread in recent years. “Bath salts” share many chemical properties with psychostimulants like methamphetamine or with entactogens like 3,4‐methylenedioxymethamphetamine (MDMA or “Ecstasy”), presumably reflecting structural differences that lead to differing neurochemical actions. Yet, currently available synthetic cathinones have not been well‐characterized in primate species, and it is unclear which pharmacological activity mediates their abuse‐related behavioral effects. The present study was initiated to address this question, using drug discrimination procedures to evaluate several synthetic cathinones in two groups of squirrel monkeys. Each subject was trained to respond on either of two levers under a FR10 schedule of reinforcement and, during training sessions, responding on one lever was reinforced after pretreatment with vehicle whereas responding on the second lever was reinforced after pretreatment with the training drug. Under terminal conditions, one group of monkeys discriminated 0.1 mg/kg methamphetamine from saline; the other group discriminated 0.6 mg/kg MDMA from saline. Drug or saline lever assignments were counter balanced across the left and right levers within each group. Training sessions comprised multiple components, and a component consisted of a ten‐minute timeout period followed by ten presentations of the FR10 schedule, each followed by a brief 50‐second timeout. After criterion performance was met (≥ 90% correct), test days interspersed training days, with at least two training days between each test day. The effects of five different synthetic cathinones ‐ α‐pyrrolidinovalerophenone (α‐PVP), methylenedioxypyrovalerone (MDPV), mephedrone, methylone, and methcathinone) were tested in both groups. A full range of doses of each drug was administered using cumulative dosing procedures. Results show that doses of 0.1 mg/kg – 0.32 mg/kg of MDPV, α‐PVP, and methcathinone were identified as methamphetamine in subjects trained to discriminate 0.1 mg/kg methamphetamine from vehicle, whereas doses mephedrone and methylone up to those that decreased overall response rates did not generalize to MA. Preliminary results suggest an opposite profile of substitution in MDMA‐trained animals. Thus, mephedrone and methylone, but not MDPV, α‐PVP, and methcathinone did appear to generalize to MDMA. These results demonstrate that synthetic cathinones have differing substitution profiles in animals trained to discriminate methamphetamine or MDMA, which may reflect differing subjective effects and, perhaps, differing abuse liability in man.Support or Funding InformationSupported by NIH Grants: DA002519. GM113117, DA039306, GM111385This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of novel NKCC1 inhibitors on reducing brain damage and neurological deficits after ischemic stroke in mice
Authors: HUANG HUACHEN , Bhuiyan Mohammad Iqbal Hossain , Jiang Tong , Shankar Sandhya , Schreppel Philipp , Erker Thomas , Hintersteininger Michael , Sun Dandan 
Published: June 2021

Background and PurposeStimulation of the WNK‐SPAK/OSR1 kinases and their substrate Na+‐K+‐2Cl− cotransporter 1 (NKCC1) play critical roles in cerebral edema and neurological functional deficits after ischemic stroke. Either NKCC1 inhibitor bumetanide (BMT) or knockout of WNK3 or SPAK shows profound protective effects in a mouse model of ischemic stroke. In this study, we investigated the efficacy of two novel pharmacological inhibitors of NKCC1, a lipophilic BMT prodrug STS5 and a novel NKCC1 inhibitor STS66, on reducing ischemic stroke‐induced brain damage in mouse.MethodsBoth male and female C57BL/6j mice (2–3 months old) were used in this study. Transiant focal ischemic stroke was induced by 50‐min occlusion of the middle cerebral artery (tMCAO). At 3 h post‐tMCAO, mice were randomly assigned to receive vehicle DMSO (2 ml/kg body weight/day, i.p.), BMT (10 mg/kg/day, i.p.), STS5 (13 mg/kg, i.p.), or STS66 (12 mg/kg/day, i.p.). Infarct volume, hemisphere swelling, animal survival, and neurological deficits were analyzed.ResultsBMT, STS5 or STS66 significantly reduced infarct volume and hemisphere swelling assessed at 24 h post‐tMCAO. STS66 is less effective on reducing brain infaraction in female than in male mice. However, STS5 treatment significantly increased the mortality in mice during 1–14 days after tMCAO. Interestingly, STS66‐treated mice showed better outcome, including improving survival and sensorimotor functional recovery during 1–14 days post‐tMCAO.ConclusionThese results demonstrate that the novel NKCC1 inhibitor STS66 is effective in reducing ischemic infarct, cerebral swelling, and improving neurological deficits after ischemic stroke in C57BL/6j mice. STS66‐mediated NKCC1 inhibition presents a novel strategy for stroke therapy.Support or Funding InformationThis research was supported in part by NIH Grant R01 NS38118 (D Sun), VA I01BX002891 (D Sun)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RGS Protein Regulation of CB1 Receptor‐Mediated Cannabinoid Behaviors
Authors: Tai Sherrica , Kandasamy Ram , Barton Brenna , Parry Logan , Traynor John R. 
Published: June 2021

The cannabinoid CB1 receptor, is a G protein coupled receptor and couples to members of the Gαi/o family of heterotrimeric G proteins. As such, signaling downstream of the receptor is negatively regulated by RGS proteins, or regulator of G protein signaling proteins. RGS proteins act as GAPs or GTPase accelerating proteins and bind to activated Gα‐GTP subunit to enhancing the hydrolysis of bound Gα‐GTP to Gα‐GDP, leading to inactivation of Gα subunit and the reformation of inactive G protein heterotrimer. Therefore, inhibition of RGS proteins will prolong cannabinoid signaling and possibly the potency of ligands that bind to the CB1 receptor. However, there are over 20 members of the RGS protein family and there can be redundancy among members of the family. Consequently, we developed a mouse line that expresses Gαo protein that is insensitive to the action of all RGS GAP activity. Here, F1 cross of 129SvEv/C57BL6 mice expressing a knock‐in RGS‐insensitive Gαo (RGSi‐Gαo) protein were administered the fully efficacious CB1 agonist, CP 55,940 and examined for reduction in body temperature and antinociception, two noted behaviors of CB1 agonism. CP 55,940 induced a greater reduction in body temperature in female RGSi‐Gαo mice that in their female wild‐type littermates. Latency to respond to a noxious thermal stimulus in the hot‐plate test was also greater in female RGSi‐Gαo mice compared to their female wild‐type littermates. In contrast, no differences were observed between male RGSi‐Gαo mice and their male littermates. The reasons underlying the observed sex differences are unknown. RGS proteins present a potential target for drug discovery and development that may affect the therapeutic actions of cannabinoids ligands.Support or Funding InformationSupported by DA035316.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rab35 and Rab39 GTP‐ases as modulators of cannabinoid type 1 receptor signaling.
Authors: Ghita Ioana , Winsauer Peter J. , Filipeanu Catalin M. 
Published: June 2021

Cannabinoid type 1 receptor (CB1R) is distinct within G‐protein coupling receptors (GPCR) family, being able to trigger cellular signaling not only when present at the plasma membrane, but also within the endolysosomal system. Rab GTP‐ases are well characterized master regulators of intracellular protein trafficking, including GPCR. The classical controllers of bidirectional plasma membrane‐endosome shuttling Rab4 and Rab5 GTP‐ases were previously shown to profoundly change the subcellular distribution of CB1R, whereas Rab11 GTPase had no effect. However, the effects of recently described Rab35 and Rab39 GTPases, which are also modulating internalization/recycling process, in the regulation of GPCR trafficking are not yet characterized. In the present work, using a bioluminescent cAMP reporter, we found that overexpression of Rab35 wild‐type (Rab35wt) did not change the effects of CP55,940 at maximal concentration (10−7M), although slightly reduced its effects at lower concentrations (10−9 M). In contrast, the dominant negative mutant Rab35S25N had opposite effects. However, the effects of non‐permeable peptide receptor agonist RVD‐Hpα (10−6M) which selectively activates plasma membrane CB1R were markedly potentiated by Rab35wt and reduced by Rab35S25N. Furthermore, overexpression of Rab35wt decreased the amount of CB1R internalized in presence of CP55,940 or RVD‐Hpα as determined by ELISA. Rab35S25N did not change the number of plasma membrane receptors in basal conditions, but reduced CB1R recycling after treatment with any of the two agonists. Overexpression of Rab39A or Rab39B GTP‐ases resulted in strong reduction of the effects of RVD‐Hpα on cAMP levels, but did not change the effects of CP55,940. These results correlated with the levels of CB1R at the plasma membrane, a decrease in CB1R internalization being observed after increasing the endogenous levels of Rab39A or Rab39B. These preliminary results indicate that multiple Rab GTP‐ases modulate CB1R signaling by interfering with the receptor shuttling between plasma membrane and endosomes. In addition, our data may have clinical significance on the use of cannabinoids in specific populations, considering that mutations affecting Rab39B functions were observed in people with intellectual disability, autism spectrum disorder, and macrocephaly.Support or Funding InformationSupported by NIH DA037255 (PJW) and Bridge Grant from Howard University (CMF).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.