bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sex‐Specific Estrogen Regulation of Hypothalamic Astrocyte Estrogen Receptor and Glycogen Metabolic Enzyme Protein Expression, and Glycogen Content in Rats
Authors: Ibrahim Mostafa M.H. , Bheemanapally Khaggeswar , Sylvester Paul W. , Briski Karen P. 
Published: April 2020

Brain astrocytes are implicated in estrogenic neuroprotection against bio‐energetic insults, which may involve their glycogen energy reserve. Forebrain estrogen receptor (ER)‐alpha (ERα) and ‐beta (ERβ) exert differential control of glycogen metabolic enzyme [glycogen synthase (GS); phosphorylase (GP)] expression in hypoglycemic male versus female rats. Studies were conducted using a rat hypothalamic astrocyte primary culture model along with selective ER agonists to investigate the premise that estradiol (E2) exerts sex‐dimorphic control over astrocyte glycogen mass and metabolism. Female astrocyte GS and GP profiles are more sensitive to E2 stimulation than the male. E2 did not regulate expression of phospho‐GS (inactive enzyme form) in either sex. Data also show that transmembrane G protein‐coupled ER‐1 (GPER) signaling is implicated in E2 control of GS profiles in each sex, and regulates GP expression in females. E2 increases total 5′‐AMK‐activated protein kinase (AMPK) protein in female astrocytes, but stimulated pAMPK (active form) expression with equivalent potency via GPER in females and ERα in males. In female astrocytes, ERα protein was up‐regulated at a lower E2 concentration and over a broader dosage range compared to males, whereas ERβ was increased after exposure to 1–10 nM versus 100 pM E2 levels in females and males, respectively. GPER profiles were stimulated by E2 in female, but not male astrocytes. E2 increased astrocyte glycogen content in female, but not male astrocytes; selective ERβ or ERα stimulation elevated glycogen levels in the female and male, respectively. Outcomes imply that dimorphic astrocyte protein and glycogen responses to E2 may reflect, in part, differential steroid induction of ER variant expression and/or regulation of post‐receptor signaling in each sex.Support or Funding InformationThis research was supported by NIH DK 109382.
MicroRNA‐21 Ablation Attenuates Acetaminophen‐Induced Hepatoxtoxicity in Male Mice
Authors: Huffman Alexandra M. , Syed Maryam , Rezq Samar , Anderson Christopher D. , Smith Stanley V. , Harmancey Romain , Yanes Cardozo Licy L. , Romero Damian G. 
Published: April 2020

IntroductionAcetaminophen (APAP) induced acute liver failure (ALF) is recognized as the most common cause of ALF in Western societies, occurring in approximately 40% of all cases. The disease is characterized by coagulopathy, hepatic encephalopathy, multi‐organ failure, and death. Micro‐RNAs are small, non‐coding RNAs capable of altering gene expression at the post‐transcriptional level. MicroRNA‐21 is dynamically expressed in the liver and several target genes of microRNA‐21 are highly expressed in the pericentral hepatocytes. We hypothesize that microRNA‐21 genetic ablation attenuates hepatotoxicity following acetaminophen intoxication.MethodsEight‐week old microRNA‐21 knockout (miR21KO) or wild‐type (WT) C57BL/6N male mice were injected with acetaminophen (APAP, 300 mg/kg BW) or saline. Animals were sacrificed 6 or 24 hours post‐injection, and serum and livers were harvested.ResultsWT animals showed significantly higher hepatic injury markers serum ALT (20,077±2,084 U/L vs 13,364±1,667 U/L, p<0.05, n=11) and AST (29,125±3,485 U/L vs 20,282±2,445 U/L, p<0.05, n=11) than their miR21KO counterparts after 6 hours of APAP treatment, while no significant difference was observed in animals treated for 24 hours. miR21KO mice presented attenuated liver necrosis compared with WT animals (59.8±5.8 vs 42.5±7.9 %, p<0.05, n=10) 24 hours post‐APAP treatment. Moreover, miR21KO mice had decreased hepatic DNA fragmentation analyzed by TUNEL assay than WT animals (58.26±4.5 vs 42.60±4.9 %, p<0.05, n=8) after 24 hours of APAP treatment. MicroRNA‐21 ablation did not modify hepatic GSSH/GSH or APAP‐Cysteine adducts at either 6 or 24 hours post treatment. MicroRNA‐21 ablation upregulated mRNA expression of autophagy (Beclin‐1, Map1LC3a, Pdcd4, p62) and decreased inflammation (PAI‐1) markers compared with WT mice 24 post APAP treatment. MicroRNA‐21 ablation exacerbated hepatic autophagy marker LC3A/B‐II protein expression at both 6 (2.8 vs 1.5‐fold) and 24 (2.2 vs 1.2‐fold 1.2) hours post APAP, correlating with increased Map1lc3a/b mRNA levels.ConclusionOur results suggest that miRNA‐21 mediates APAP‐induced hepatotoxicity. MicroRNA‐21 inhibition could be a novel therapeutic approach to abolish or mitigate APAP‐induced hepatotoxicity. The beneficial effect of microRNA‐21 is due to an upregulation of hepatic autophagy. Specifically, microRNA‐21 inhibition could be particularly useful when APAP intoxication is detected at its late stages and the only available therapy, NAC, is minimally effective.Support or Funding InformationSupported by NIH grants R21 DK‐113500 and P20 GM‐121334.
The Effects of Ciproxifan on Liver in Rats with Cerebral Ischemia
Authors: Yegin Bengi , Donmez Dilek Burukoglu , Oz Semih 
Published: April 2020

Ischemia and reperfusion occur in some tissues or organs and may develop the severity of dysfunction. It causes permanent functional or structural damage and occurs many events leading to cell death. It is possible to see the damage not only in the tissues where the blood flow is stopped but also elsewhere in the body. Ciproxifan belongs to a novel chemical series of histamine H3‐receptor antagonists. Ciproxifan has been shown to cause vigilance and consideration in animal studies. We aimed to find out whether ciproxifan has an effect on liver tissue in rats with ischemia‐reperfusion injury.Approval for the animal study was obtained from the ethical committee with file number 556, on October 27, 2016. All surgical procedures were performed under anesthesia (50 mg/kg ketamine hydrochloride +10 mg/kg xylazine). 32 Wistar Albino male rats were used in 4 groups. Control and Sham groups were given dimethyl sulfoxide for 7 days/single dose. 10 mg/kg ciproxifan were administered to the Low Dose group and 30 mg/kg ciproxifan to the High dose group for 7 days/single dose. No surgical procedure was applied to the control group. Sham, Low Dose, and High Dose groups were administered cerebral ischemia induced by common carotid artery technique for 15 minutes. Twenty‐four hours following reperfusion. Rats were sacrificed and liver samples were taken.Examination of all rat liver revealed that the liver of control rats was observed in the normal histological structure. Also; hepatocyte cells, sinusoidal structures, and vena centralis were observed normal the normal histological structure. Necrotic cells with pyknotic nuclei, eosinophilic cytoplasm, and partial sinusoidal dilatation are seen in rat liver tissues of the Sham group. In the Low Dose and High Dose, groups were observed hepatocyte cells, sinusoidal structures, and vena centralis structures together with a nearly normal liver structure.As a result, we have observed that ciproxifan has a protective effect on the liver tissue. It was observed that administered of different doses did not affect the result.Support or Funding InformationESOGU‐BAP
Intermittent hypoxic training regulates the microglia phenotype by increasing melatonin receptor expression.
Authors: Bhandari Sabita , Ryou Myoung-gwi 
Published: April 2020

Microglia is a resident immune cell in the brain. Microglia could be polarized into two phenotypes: pro‐inflammatory (M1) and anti‐inflammatory (M2). The pro‐inflammatory phenotype of microglia is known to harmful in ischemic stroke disease processes. An intervention, therefore, that could shift the phenotype of microglia toward the anti‐inflammatory phenotype could be beneficial in cerebral protection against ischemic stroke. Intermittent hypoxic training (IHT) protects the microglia against hypoxia and reoxygenation stress and increases the M2/M1 ratio. The involvement of the melatonin, an antioxidant, was proposed from the many laboratories. We studied the function of melatonin in the polarization of microglia in the IHT.MethodsEOC20, mouse microglia, have been assigned to the control, oxygen‐glucose deprivation (OGD), IHT, OGD + IHT. The cell viability of each group was assayed by using Calcein‐AM assay. The phenotype of EOC20 cells was determined with immunocytochemistry staining CD 68 (M1) and CD 206 (M2) and quantified by flow cytometry. The involvement of melatonin (30 μM) and melatonin receptor was determined by using melatonin and melatonin receptor antagonist, Luzindole (50 μM). Protein contents of melatonin receptor 1B, Akt, Erk1/2 were measured by immunoblot. Two factors (treatment and protocol) ANOVA and Tukey post hoc test was used to find statistical significance. P<0.05 was considered a significant value.ResultCell viability in the OGD group was significantly decreased comparing to the control, IHT, OGD+ IHT groups. Meaningful elevation in the melatonin 1B receptor and phosphorylation of Akt and Erk1/2 were detected, but those protein contents were blunted with Luzindole treatment. M2/M1 ratio was altered with melatonin treatment, control group 0.32 and melatonin group 2.7.ConclusionMicroglia was activated and polarized to the pro‐inflammatory phenotype after OGD and reoxygenation. However, IHT was able to mitigate cell death and shift the phenotype to the anti‐inflammatory phenotype. IHT‐induced favorable effects including phenotype of microglia were diminished by inhibiting melatonin 1B receptor with Luzindole. Results in the current study proposed that polarization of microglia influenced by the activation of the melatonin pathway.
Monosynaptic Excitatory Drive of Kölliker‐Fuse Neurons by Noradrenergic Locus Coeruleus Neurons in Mice
Authors: Varga Adrienn G. , Reid Brandon T. , Levitt Erica S. 
Published: April 2020

The opioid epidemic is still a national crisis with constantly increasing fatal overdoses. The primary cause of death from opioid overdose is respiratory failure, which is the result of muopioid‐receptor mediated inhibition in respiratory centers of the brain. Recently, we have shown that the pontine Kölliker‐Fuse nucleus (KF) is highly susceptible to inhibition by opioids and may be a compelling target to counter the respiratory side effects of opioids. The ideal strategy to reverse an opioid overdose only affects respiratory depression without causing pain or withdrawal. This approach has to rely on targeting non‐opioid respiratory pathways. Prior work has identified direct projections between the KF and the main noradrenergic arousal center of the brain, the locus coeruleus (LC). In an effort to explore non‐opioid‐mediated excitation of respiratory neurons, we studied how modulation via LC neurons may affect KF neuronal activity. We selectively expressed channelrhodopsin2 in the LC of Th‐Cre hemizygous mice. Whole‐cell patch clamp recordings combined with optogenetic stimulation of LC neurons, and immunohistochemistry confirmed channelrhodopsin2 expression in Th+ LC cells. Next, we optically stimulated LC axon terminals in coronal KF slices and recorded light‐evoked EPSCs in the presence of picrotoxin. Since light‐evoked EPSC onsets had fixed latencies with minimal jitter, followed a similar time course and resulted in no failure, our results indicate the presence of excitatory monosynaptic drive between the LC and KF. KF neurons receiving monosynaptic drive from the LC were then tested for opioid‐sensitivity. Application of a mu‐opioid agonist to KF neurons showing consistent eEPSCs resulted in a hyperpolarizing outward K+ current in voltage clamp recordings in >90% of the cases. Our results demonstrate that the majority of opioid‐sensitive KF neurons receive monosynaptic excitatory drive from noradrenergic LC neurons. This work provides exciting novel evidence for an excitatory pathway between the LC and the respiratory brainstem.Support or Funding InformationFunding: This work was supported by National Institutes of Health Grants R01 DA47976 (E.S.L.). A.G.V. was funded by the UF Breathing Research and Therapeutics Training Program (T32 HL134621) and the Center for Respiratory Research and Rehabilitation.
Examining the Role of p190RhoGAP on the Organization of the Actin Cytoskeleton in Breast Epithelial Cells
Authors: Robinson Courtney Rae , Kitt-Hopper Khameeka 
Published: April 2020

Angiogenesis (i.e. blood vessel formation) and metastasis of tumors are two of the most dangerous aspects of malignant cancer cells. Metastasis requires spatial and temporal coordination of protein complexes known to affect cell adhesiveness and migration/proliferative properties. These protein complexes include E‐cadherin, Rho GTPases (e.g., Rac1 and RhoA), and the actin cytoskeleton. Rho GTPases perform different functions in the cell and have been shown to affect cancer progression, wound healing, and inflammation through regulation of cadherin and actin. For Rho GTPases to function, additional proteins increase or decrease their activity in the cell. These include guanine‐nucleotide‐exchange factors (GEFs) and GTPase‐activating proteins (GAPs). The current study aims to examine p190RhoGAP (GTPase activation protein (GAP) for RhoA) on cell adhesion and migration dynamics of normal Human Mammary Epithelial cells (HMECs) and the metastatic adenocarcinoma breast cancer cell line, MCF‐7. Cells were examined under endogenous conditions, as well as transfected with p190RhoGAP wild‐type and a catalytically inactive p190 RhoGAP‐R1283A mutant that locks RhoA into a constitutively active state. Overexpression of wild‐type and catalytically inactive p190RhoGAP had no major effect on actin stress fiber and cortical actin at cell‐cell contacts. However, HMEC cells overexpressing p190 RhoGAP‐R1283A showed reduced cytosolic RhoA protein, suggesting that loss of RhoA activity affects RhoA protein levels. Although no significant change in wound closure was observed, transwell cell invasion assays show a decreased rate of cell invasion in the presence of the chemo‐attractant, epidermal growth factor (EGF) in MCF‐7 cells. These data indicate a role for p190RhoGAP in the regulation of RhoA during cell migration and invasion. Loss of p190RhoGAP catalytic activity could affect the production of RhoA protein, leading to a decrease in the formation of actin structures (i.e., lamellipodia and filopodia) and a decrease in cell migration and invasion. Future work aims to determine how p190RhoGAP controls spatiotemporal regulation of protein complexes involved in metastatic cancer.Support or Funding InformationSaint Mary's School of Science Research Program and the Robert W. and Beverly J. Summers Scholarship
Role of cystic fibrosis transmembrane conductance regulator in FHC colon epithelial cell dysfunction and colon cancer
Authors: Grant Garett J. , Mogan Cara A. , Liou Theodore G. , Helms My N. 
Published: April 2020

ObjectiveTo determine the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in colon cancer development, with a focus on cell migration, EGFR signaling, and inflammation using model FHC colon epithelial cells.BackgroundOur clinical observations indicate that individuals with cystic fibrosis (CF) over the age of 40 are 4–8 times more likely to get colon cancer than the general population. Moreover, the likelihood of getting colon cancer increases to over 30 times the general population’s risk in CF patients who have had an organ transplant. The mechanism(s) leading to increased colon cancer in CF patients is not fully understood, but dysfunctional Cl− and Na+ transport are highly implicated. Chronic inflammation has also been shown to play an important role in the development and progression of many cancers. We hypothesize that dysregulated ion transport in CF patients leads to chronic inflammation and increased incidence of colon cancer via altered epidermal growth factor receptor (EGFR) signaling.MethodsWe performed scratch assays on cultured human colon epithelial cells (FHC) and measured the rate of cell migration every 24 hours for 5 days in the presence of either CFTR activating or inhibiting compounds. EGFR signaling was analyzed using standard immunohistochemical labeling techniques and commercially available reagents in similarly treated FHC cells.ResultsCells treated with 500 μM IBMX and 5 μM forskolin (CFTR activators) had significantly decreased rates of cell migration (26 μm2/24 hours) compared to the control group (51 μm2/24 hours) n=3; p<0.05. Control and colon cells treated with 500 μM IBMX and 5 μM forskolin showed low levels of EGFR labelling (restricted to cell periphery), whereas cells treated with 5 mM CFTR(inh)‐172 (CFTR inhibitor) showed more robust EGFR signal throughout the cytoplasm in 3 independent observations.ConclusionOur data indicates that suppressed CFTR activity impacts colon epithelial cell migration and EGFR localization in FHC cells. Additional research on CFTR and EGFR signaling is needed to better understand and improve treatment options for both colon cancer and CF patients.Support or Funding InformationFunding: NIH R01 HL137033‐01 awarded to MNH.
Structure‐Activity Relationships for Cumyl‐Containing Synthetic Cannabinoids to Induce Hypothermic, Cataleptic and Analgesic Effects in Mice
Authors: Baumann Michael H. , Garibay Nancy , Partilla John S. , Brandt Simon D. 
Published: April 2020

Synthetic cannabinoids (SCs), also known as “spice” or “K2”, are an evolving class of new psychoactive substances that pose public health risks. SCs exert their psychoactive effects by stimulating cannabinoid‐1 receptors (CB1) in the brain, yet few studies have systemically evaluated structure‐activity relationships (SAR) for newly‐emerging compounds. Here, we investigated the pharmacology of SCs which possess a fixed cumyl ‘head group’ but a variable N‐alkyl ‘tail group’ which ranged from 3 to 7 carbons in length. Cumyl‐PINACA, which contains a 5‐carbon pentyl tail, served as a reference compound. In vitro CB1 binding was assessed by displacement of [3H]SR141716 in mouse brain membranes. In vivo effects of subcutaneously (sc) administered drugs were examined in male C57Bl/6J mice bearing surgically‐implanted telemetric temperature transponders. Mice were subjected to a ‘triad’ test battery which assessed temperature, catalepsy, and analgesia every 30 min post‐injection for 2 h. Temperature was measured via a handheld receiver. Catalepsy was measured using the bar test, with a cut off of 60 sec, whereas analgesia was measured using a hot plate set at 52°C, with a cut off of 45 sec. Cumyl‐PINACA was the most potent inhibitor of CB1 binding with an IC50=1.8 nM. Decreasing N‐alkyl chain length to a 3‐carbon propyl markedly reduced binding affinity (IC50=44.8 nM), as did increasing chain length to a 7‐carbon heptyl (IC50=124.0 nM). Consistent with the in vitro findings, cumyl‐PINACA was the most potent compound to induce cannabinoid‐like effects in the triad test battery, with ED50s ranging from 0.06–0.09 mg/kg, sc. Importantly, all cumyl compounds induced hypothermic, cataleptic, and analgesic effects with a rank order of potency that matched their IC50s for inhibition of CB1 binding. THC exhibited ED50s in the triad test ranging from 18.7–30.3 mg/kg, sc. Overall, our data reveal that certain cumyl‐containing SCs are more potent CB1 agonists than THC. Furthermore, N‐alkyl chain length is a critical determinant for the pharmacological effects of cumyl SCs, with a 5‐carbon pentyl chain conferring optimal activity. More research examining the SAR for newly‐emerging SCs is needed to inform stakeholders involved with responding to the abuse of these compounds.Support or Funding InformationThis work was supported generously through IRP, NIDA, NIH.
Effect of H9, a formula of nine medicinal herbs derived from Osuyubujaijung‐tang, on trastuzumab pharmacokinetics in mice
Authors: Choi Young-Hee , Chae Hee-Sung , Chin Young-Won 
Published: April 2020

Recently, the resistance of trastuzumab as an established monoclonal antibody therapy for women with breast cancers overexpressing human epithelial growth factor receptor 2 (HER2) has been issued and the activation of phosphoinositide 3‐kinase (PI3K) pathway was identified as a main mechanism of resistance. As the combinations of western and oriental medicines have emerged accompanying the improvement of efficacy and/or safety in chemotherapies, the cases for combinations of trastuzumab and oriental medicines has been reported. H9 is a formula of nine medicinal herbs derived from Osuyubujaijung‐tang, a traditional Korean prescription for Soeumin constitution, targeting HER2‐PI3K/Akt pathway and consequently showing anticancer effects against breast cancer and lung cancer in vitro and in vivo. The synergistic effect of trastuzumab and H9 combination was reported in vivo mice model of lung cancers. Regarding that the current drug interaction issues of therapeutic proteins including monoclonal antibody with small molecules, the effect of H9 on pharmacokinetic interaction of trastuzumab in aspect of systemic exposure and tissue distribution was investigated in this study. Prior to evaluation of pharmacokinetic interaction of trastuzumab with H9, the analytical method of trastuzumab using ELISA was validated.In the validation of ELISA analytical method, 0.1–20 mg/mL of trastuzumab were analysed showing below 13.8% of precision and 84.3–112% accuracy values, respectively. In pharmacokinetics, H9 did not change any pharmacokinetic parameters of trastuzumab and also there was no difference on tissue distribution of trastuzumab to liver, heart, lung, and spleen in trastuzumab and H9 combinations. These results provided an evidence for the development of this combination therapy in clinical levels after the further investigations of dosage regimen adjustments.Support or Funding InformationThis study was supported by National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIT) (NRF‐2016R1C1B2010849 and NRF‐2018R15A2023217).
Opioid sensitivity of projection‐specific Kölliker‐Fuse neurons
Authors: Bateman Jordan , Reid Brandon , Maletz Sebastian , Levitt Erica 
Published: April 2020

Opioid‐induced respiratory depression is the primary cause of death in individuals who overdose on opioids. Respiration is controlled by a highly interconnected neural network located in the pons and medulla. The Kölliker‐Fuse nucleus (KF), located in the dorsolateral pons, is an essential part of this network and projects to core respiratory nuclei in the ventrolateral medulla. Opioids injections into the KF disturb respiratory rate, volume, and pattern. Deletion of opioid receptors from KF neurons attenuates morphine‐induced respiratory depression. These studies suggest that the KF is a critical site in opioid‐induced respiratory depression. Mu opioid receptor agonists hyperpolarize a population of neurons in the KF due to activation of G protein‐mediated inwardly rectifying conductance. However, we do not know the projection target of the opioid‐sensitive KF neurons. Given the extensive projections from the KF to medullary respiratory rhythm generators, we hypothesize that opioid‐sensitive KF neurons project to respiratory rhythm generators. Fluorescent beads (FluoSpheres 580/605, ThermoFisher) were injected into the Bötzinger complex (BötC), pre‐Bötzinger complex (preBötC), and rostral ventral respiratory group (rVRG) of mice. Whole‐cell voltage‐clamp recordings were made from retrograde‐labeled KF neurons contained in brain slices. Mu opioid receptor‐mediated outward currents were identified in KF neurons that project to the preBötC, and rVRG, but not in KF neurons that project to the BötC. Opioid‐sensitive connections between the KF and respiratory rhythm generators may be a possible mechanism by which opioids reduce respiratory rate.Support or Funding InformationR00, DA038069