bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pharmacological Reversal of Adolescent Alcohol‐Induced Perturbation to Mesolimbic Dopamine Network Dynamics Rescues Maladaptive Decision Making in Adulthood
Authors: Schindler Abigail , Soden Marta , Weber Sophia , Zweifel Larry , Clark Jeremy 
Published: November 2023

Alcohol is the most commonly used and abused substance among adolescents, promoting the development of substance use disorders and compromised decision‐making in adulthood. Using a preclinical model we have demonstrated that adolescent alcohol use results in maladaptive adult risk‐taking behavior that positively correlates with increased mesolimbic phasic dopamine transmission in response to risky options, but the underlying mechanisms remain unknown. Here we show that increased phasic dopamine after adolescent alcohol use is attributable to a selective perturbation of midbrain circuitry. Indeed, we demonstrate that VTA dopamine neurons exhibit enhanced IPSCs, but not EPSCs, leading to decreased basal dopamine levels in adulthood that negatively correlate with risk‐taking. We conclude that a novel disinhibitory mechanism in midbrain circuitry mediates the effects of alcohol in a model where increased inhibitory tone on dopamine neurons leads to a persistent decrease in basal dopamine and promotes a potentiation in stimulus‐evoked phasic dopamine release that drives maladaptive choice behavior. Based on these findings we employ a selective GABA(A) receptor agonist to pharmacologically reverse risk‐taking in adulthood in parallel with normalization of phasic dopamine. Together these results isolate the underlying circuitry involved in maladaptive decision‐making and identify a potential therapeutic target.Support or Funding InformationR01AA021121 and T32AA07455
Neurochemical Profile of a Novel Allosteric Potentiator of the Dopamine D1 Receptor
Authors: Falcone Julie F , Mitchell Stephen N , Carter Guy , Maren Deanna L , Katner Jason , Chernet Eyassu , Wright Rebecca A , Wang Hong , Ryder John W , Morin Michelle , Gehlert Donald R , Johnson Michael P , Beck James P , Hao Junliang , Menezes Michelle M , Bruns Robert F , Svensson Kjell A 
Published: November 2023

DETQ is a potent and selective dopamine D1 receptor potentiator that is active in several behavioral models (see abstracts by Heinz et al and Svensson et al at this meeting). We wished to explore the neurochemical basis for the behavioral effects of DETQ. Due to low affinity of DETQ in rodents, the current studies were carried out in transgenic mice in which both copies of the murine D1 receptor were replaced with its human counterpart (hD1 mice) (see Svensson et al). Receptor expression in hD1 mice was characterized using autoradiography of the D1‐selective antagonist 3H‐SCH23390. The pattern of D1 receptor distribution was very similar in wild‐type and hD1 mice, but the absolute level of expression was about 50% lower in hD1 mice. Using microdialysis in freely moving hD1 mice, DETQ and the D1 agonist SKF82958 increased extracellular levels of acetylcholine and histamine in cortical and subcortical areas. At a high dose we also observed enhanced extracellular levels of norepinephrine in the prefrontal cortex. Furthermore, a low dose of DETQ elevated hippocampal acetylcholine levels in an additive fashion with the acetylcholinesterase inhibitor rivastigmine. Brain levels of the histamine metabolites tele‐methylhistamine and tele‐methylimidazole acetic acid were also elevated in both microdialysate and post‐mortem tissue samples. The increases in brain histamine metabolites produced by DETQ were not present in wild‐type mice, indicating that the effects were mediated via the D1 receptor. Overall, these neurochemical changes correlated with brain exposure of DETQ and also with the behavioral effects (see Svensson et al.)Effects on brain cyclic nucleotide levels were studied after microwave fixation of the forebran. Levels of cGMP were elevated in the striatum after both DETQ and SKF82958. Further changes in downstream signaling were explored measuring phosphorylation of the transcription factor CREB and the AMPA receptor GluR1. Both DETQ and SKF82958 increased pCREB and pGluR1 in the brain. Together, these data provide neurochemical evidence for enhanced synaptic plasticity of DETQ and SKF82958.In conclusion, our in vivo neurochemical data support the proposal that the D1 potentiator DETQ selectively enhances D1 receptor function in the brain. Studies of neurotransmitter release, second messengers, and downstream signaling support a potential utility for D1 potentiators to enhance cognitive function in CNS disorders.Support or Funding InformationAll authors are current or past employees of Eli Lilly & Co
Identification of potential PAR‐regulating proteinases in agricultural dust using an activity‐based serine proteinase probe
Authors: Hollenberg Morley D. , Polley Danny , Saifeddine Mahmoud , Gilmore Brendan , Romberger Debra J. , Heires Art J. , Nordgren Tara M , Toews Myron L 
Published: November 2023

In previous work from our laboratories (PMIDs 26092994, 24152160, 21270400) we found that environmental allergens, including cockroach‐ and barn‐derived particulates, contain proteolytic enzymes that contribute to lung inflammation by cleaving and activating proteinase‐activated receptors (PARs). Preliminary work using in‐gel zymography demonstrated the presence of multiple serine proteinase inhibitor‐sensitive enzymes in hog barn dust extract (HDE: PMID 26092994). Because these HDE enzymes were blocked by alkylation of the active serine in many of the proteinases with PMSF and related agents, we reasoned that an activity‐based serine proteinase probe (ABP: PMID16554154) would also be able to alkylate the enzymes in the HDE. Using this novel approach (PMID 21270400), we ABP‐biotinylated the enzymes in HDE with ABPs selective for tryptic, chymotryptic, and elastase enzymes, respectively. Our data show differential reactivity of these ABP probes with enzymes in the HDE, identifying several enzymes in the mass range from 10 to 36 kDa, overlapping with the mass of ABP‐labeled porcine trypsin (28 kDa). Porcine trypsin itself is a major PAR‐regulating proteinase that is present in HDE, and this enzyme may play a prominent role in triggering lung inflammation in agricultural workers. Thus, HDE trypsin and other proteinases represent novel therapeutic targets both for preventing dust‐induced disease and for monitoring as environmental risk factors in many agricultural settings.Support or Funding InformationSupported by the Canadian CIHR (MDH), The Lung Association of Alberta‐NWT (MDH), and NIOSH 2R01‐0H‐008539 (DJR)
Nephrotoxic Potential of 3,5‐Dichloroaniline Putative Metabolites in Isolated Renal Cortical Cells from Fischer 344 Rats
Authors: Rankin Gary , Racine Christopher , Ward Dakota , Tyree Conner , Pope Deborah , Anestis Dianne 
Published: November 2023

Halogenated anilines are widely used as chemical intermediates in the manufacture of dyes, agricultural agents and as industrial intermediates. Previous studies in our laboratory have found that 3,5‐dichloroaniline (3,5‐DCA) is the most potent nephrotoxicant among mono‐ and dichloroanilines, both in vivo and in vitro. The potential role of renal biotransformation in 3,5‐DCA‐induced nephrotoxicity has also been examined. When isolated renal cortical cells (IRCC) from male Fischer 344 rats were pretreated with various biotransformation enzyme inhibitors, 3,5‐DCA –induced cytotoxicity was significantly attenuated. These studies indicated that 3,5‐DCA could be biotransformed via multiple pathways in the kidney to one or more toxic metabolite(s). The current study was designed to explore the nephrotoxic potential of three putative 3,5‐DCA metabolites [2‐amino‐4,6‐dichlorophenol (2A4,6DCP), 3,5‐dichlorophenylhydroxylamine (3,5‐DCPHA), and 3,5‐dichloroacetanilide (3,5‐DCAA)] in IRCC from male Fischer 344 rats. Briefly, IRCC were isolated using a collagenase perfusion technique, and IRCC (~4.1 million cells/ml; 3 ml) were exposed to various concentrations of a putative metabolite [2A4,6DCP or 3,5‐DCAA (0.5–1.5 mM); 3,5‐DCPHA (0.25–1.0 mM)]or vehicle (DMSO) for 60 or 90 min. Cytotoxicity was determined by lactate dehydrogenase (LDH) release assays. Results showed that there were no significant changes in LDH release following exposure to 3,5‐DCAA at any time point or concentration tested. However, 2A4,6DCP was a nephrotoxicant at all concentrations tested, but only after 90 min exposure. In contrast, 3,5‐DCPHA significantly increased LDH release at concentrations of 0.5 mM or greater after 60 min and at all concentrations tested after exposure for 90 min. These results suggest that at least two putative 3,5‐DCA metabolites [3,5‐DCPHA and/or 2A4,6DCP, but not 3,5‐DCAA] may contribute to 3,5‐DCA induced nephrotoxicity in vitro.Support or Funding InformationSupported in part by NIH grant P20GM103434
Renoprotective Effects of 4‐Phenylbutyrate Against Palmitate‐Induced Renal Cell Injury
Authors: Munusamy Shankar , Alkhiyami Dania Ihssan , Berzou Souad , Gholamhussain Atefeh , Mohamed Hebat Alla Ahmed , Saleh Alaaeldin 
Published: November 2023

BackgroundIncreased dietary intake of fats rich in saturated fatty acids and dyslipidemia have been shown to significantly aggravate the risk of cardiovascular morbidity and mortality in patients with chronic kidney disease (CKD). Although multiple mechanisms including endoplasmic reticulum (ER) stress and oxidative stress are implicated in the pathophysiology of CKD, the effects of elevated free fatty acid (FFA) levels per se on renal cells is still unclear. Hence, we investigated the effects of palmitic acid (a saturated fatty acid) on rat renal proximal tubular cells in vitro, and tested the renoprotective effects of 4‐phenylbutyrate (4‐PBA), a chemical chaperone shown to inhibit ER stress in vitro and in vivo, in our model.MethodsSubconfluent rat renal proximal tubular (NRK‐52E) cells were exposed to 100 mM palmitic acid in presence and absence of 5 mM 4‐PBA for 24 hours. Cells were then assessed for alterations in cell viability (using MTT assay), oxidative stress (via Dichlorofluorescein (DCF) staining) and the expression of ER stress markers (i.e., glucose‐regulated protein, GRP78 and C/EBP homologous protein, CHOP) and apoptosis (caspase‐3 and ‐12) using western blotting. Flow cytometry for cell cycle analysis and immunoblotting for p27 (an inhibitor of cell cycle) were performed to determine the effects of palmitate and 4‐PBA on the cell cycle in renal cells.ResultsRenal cells exposed to palmitate for 24 hours showed decreased viability (76.9% to that of control), increased DCF staining (1.3‐fold increase over control), and marked upregulation of GRP78 and CHOP. Similarly, the expression of active forms of caspase‐3 (1.8‐fold increase over control) and caspase‐12 (3‐fold increase over control) were also increased in renal cells treated with palmitate. These changes were significantly attenuated in cells co‐treated with 4‐PBA. Intriguingly, exposure to palmitate did not alter the cell cycle in renal cells, whereas treatment with 4‐PBA caused downregulation of p27 (54% to that of control) and increased the transition of cells from G1 to S‐phase and G2/M phase.ConclusionsOur findings indicate that exposure to palmitate induces ER stress as well as oxidative stress in renal cells, and results in renal cell apoptosis. Co‐treatment with 4‐PBA protects renal cells against the perturbations induced by palmitate and improves renal cell viability, which underscores the renoprotective effects of 4‐PBA against fatty acid‐induced renal cell injury. Further studies are required to understand the mechanisms by which 4‐PBA prevents oxidative stress and alters cell cycle in renal cells.Support or Funding InformationSupported by student research grants # QUST‐CPH‐FALL‐12/13‐6 and QUST‐CPH‐SPR‐12/13‐3 from the Office of Academic Research, Qatar University.
CaMKK2 activates oncogenic Akt in ovarian cancer cells and is a promising therapeutic target
Authors: Gocher Angela Marie , Azabdaftari Gissou , Karacosta Loukia , Franke Thomas F , Edelman Arthur M 
Published: November 2023

BackgroundCa2+/Calmodulin‐dependent protein kinase kinase 2 (CaMKK2) is highly expressed in prostate and breast cancer and has been shown to regulate cell proliferation and survival. In ovarian cancer (OvCa) cells CaMKK2 regulates G1/S phase cell cycle progression and inhibits apoptosis (preliminary results). Akt (PKB) is an oncogenic pro‐survival protein kinase that is commonly hyperactive in OvCa and contributes to resistance to first line platinum chemotherapy. Akt is activated by the canonical PI3K/PDK1 cascade resulting in Akt phosphorylation at its primary site Thr308. Inhibitors of the canonical Akt activation pathway are in clinical trials.ResultsWe report here that RNAi‐mediated knockdown of CaMKK2 expression in OVCAR‐3 OvCa cells decreased phosphorylation of Akt at Thr308 (p‐Akt Thr308) (59.3%) to a similar extent as knockdown of PDK1 (50.4%) suggesting that in OvCa cells CaMKK2 may play a role comparable to that of PDK1 in maintaining Akt activity. Combined CaMKK2 and PDK1 knockdowns resulted in an additive (80.7%) decrease in p‐Akt Thr308 suggesting that CaMKK2 regulates Akt phosphorylation independently of the PDK1 pathway, a conclusion confirmed in another OvCa cell line SKOV‐3. Additive effects of CaMKK2 and PDK1 knockdowns on p‐Akt at the secondary activation site Ser473 were also achieved. The ability of recombinant purified Akt to phosphorylate an Akt specific peptide substrate was enhanced at least 9‐fold by purified CaMKK2, whereas kinase activity of Akt T308A/S473A was not increased by CaMKK2. In this assay direct phosphorylation of Akt Thr308 by CaMKK2 was confirmed by Western blotting. Pharmacological inhibition of CaMKK2 with STO‐609 resulted in decreased p‐Akt Thr308 and Ser473 as did the intracellular calcium chelator BAPTA‐AM and the CaM inhibitor W7 in OVCAR‐3 cells. STO‐609 treatment reduced the growth and viability of OVCAR‐3 cells. CaMKK2 knockdown in platinum‐resistant OVCAR‐3 cells significantly increased sensitization to the anti‐proliferative effects of carboplatin and of the PI3K inhibitor PX‐866, reducing the EC50 of PX‐866 more than 2‐fold.ConclusionsThese data show that CaMKK2 regulates cell growth and viability of OvCa cells and directly phosphorylates Akt Thr308 to promote its activity via a non‐PDK1‐dependent pathway. CaMKK2 silencing/inhibition potentiates the anti‐proliferative effects of compounds used to treat OvCa (carboplatin) or in development (PX‐866). CaMKK2 may be a novel therapeutic target for OvCa in combination with the PI3K inhibitors currently in clinical trials and/or with carboplatin.Support or Funding InformationUBF
In Vitro Validation of Cancer Immunotherapy Reagents Using an Impedance‐Based Platform
Authors: Cerignoli Fabio , Zhu Jenny , Guenther Garret , Radecke Chris , Xi Biao , Zhao Leyna , Abassi Yama 
Published: November 2023

Immunotherapy is emerging as one of the most promising approaches in cancer treatment, allowing specific targeting of cancer cells from immune cells, mediated by surface marker expression. In vitro characterization of the efficacy and potency of cancer immunotherapy reagents is a necessary step before moving to animal models and Phase I and II clinical studies. In this study we report the validation and optimization of protocols for analyzing the efficacy of reagents designed to stimulate the immune response against cancer cells using an impedance‐based and label‐free platform. The platform allows detection of cell adhesion and cell death trough monitoring the change in conductance of microelectrodes embedded in the bottom of 96‐wells cell culture plates. Because cell adhesion alters the conductivity of the electrodes, any change in cell morphology, cell adhesion or cell number is detected as variation of an impedance‐related parameter named Cell index (Ci). The platform avoids the use of any chemical label or sample processing, allowing continuous monitoring of cell activity over prolonged time periods. Furthermore, the untouched cells are still available for secondary assays that can better clarify the biological mechanism involved in the response.To mimic tumors that are target of immunotherapy efforts we utilized several carcinoma cell lines whit different level of expression of the EpCAM membrane protein. Such antigen is overexpressed in many tumors and is currently under evaluation in clinical trials. In PC3 prostate cancer cells we measured cell cytotoxicity of Primary Blood Mononuclear Cells (PBMCs) mediated by an EpCAM/CD3 Bispecific T cell Engager (BiTE) antibody. The use of the impedance‐based platform allowed measuring the full kinetic of the cytotoxic activity under different effector:target ratios and BiTE concentrations. Apoptosis of tumor cells was confirmed trough flow cytometer analysis. We also demonstrated the relation between amount of PBMCs cytotoxic activity and EpCAM surface expression trough testing MCF7 (high EpCAM expressor) and HeLa (low EpCAM expressor).To evaluate cancer immunotherapy protocols in lymphomas and leukemia, we developed a protocol where lymphomas cells are immobilized on electrodes‐coated plates and the T‐Lymphoblast cell line TALL‐104 is used as cytotoxic effector.Taken together our results demonstrate high sensitivity of the impedance‐based approach in measuring cytotoxic activity in sophisticated high throughput set up. Furthermore, the availability of continuous recording of the Cell Index over the temporal scale allows better characterization, comparing to alternative end point assays, of construct efficacy.
Sex Differences in Sympathetic Neurotransmission in Normotensive Rat Mesenteric Arteries
Authors: Gebremedhin Kibrom , Galligan James J , Xu Hui 
Published: November 2023

Hypertension is less common in premenopausal women than in men possibly due to high estrogen levels in females. The diameter of mesenteric arteries (MA) is an important determinant of system blood pressure and sympathetic nerve activity is a major determinant of MA diameter. Sympathetic nerve supplying MA uses norepinephrine (NE) and ATP as vasoconstrictor transmitters in male rats. We tested the hypothesis that noradrenergic/purinergic neuroeffector transmission to MA also occurs in female rats. Young (10–14 week), normotensive Sprague Dawley male and female rats were used. The estrus cycle stage in female was determined cytologically by vaginal lavage before euthanasia. MA (200 – 250 μm inner diameter) were harvested in a physiological buffer solution and mounted in a pressure (60 mmHg) myograph and inner diameter was measured continuously. Electrical field stimulation (30 stimuli at 0.2–30Hz) and drug addition to the chamber were used to constrict MA. Neurogenic constriction was greater in male compared to female arteries. Neurogenic constriction did not vary with the estrus cycle in female rats. Neurogenic constrictions were blocked by combined application of prazosin (0.1 μM, α1‐adrenergic receptor antagonist) and PPADS (10 μM, P2X‐purinergic receptor antagonist) as well as suramin (100 μM, P2X and P2Y‐purinergic receptor antagonist) in arteries from male and female rats. PPADS/Suramin inhibited ATP and α,β‐methylene ATP, but not KCl, ‐induced MA contraction. Constrictions caused by exogenous NE, ATP,α,β‐methylene ATP or elevated KCl were similar in MA from male and female rats. Immunostaining for tyrosine hydroxylase (a sympathetic nerve marker) revealed no observable differences in nerve density and distribution in MA from male and female rats. Our data indicate that there are fundamental differences in the mechanisms of neurogenic vasoconstriction in MA of male and female rats. Differences in neurotransmission and/or nerve fiber distribution in male and in female rats might contribute to and explain the sex differences in hypertension prevalence between human males and females.Support or Funding InformationSupported by NHLBI 2P01HL07687
Burn Injury Alters Cardiac β‐Adrenergic Receptor Signaling in Interleukin 6 Knockout Mice
Authors: Guillory Ashley N , El Ayadi Amina , Porter Craig , Herndon David N , Finnerty Celeste C 
Published: November 2023

IntroductionBurn injury disrupts many signaling pathways including those that are β‐adrenergic receptor (β1‐AR) dependent. Studies performed in various animal models have shown that burn injury causes cardiac dysfunction primarily attributed to altered β‐AR and pro‐inflammatory signaling. Interleukin 6 is involved in the regulation of cardiac hypertrophy as well as the development of heart failure, both of which are also regulated by β‐ARs.MethodsMale wild‐type and interleukin 6 knockout mice received a 30% total body surface area full thickness burn. Animals were sacrificed and hearts were isolated 10 days post injury. We examined the expression of β‐ARs and downstream proteins via Western blotting. Protein kinase A activity was measured in cytosolic fractions using an enzyme immunoassay kit.Resultsβ1‐AR protein expression was significantly higher in the knockout animals after burn injury (p=0.01). Protein kinase A activity and Troponin I phosphorylation were also significantly higher in the interleukin 6 knockout mice post burn (p=0.003; 0.03). Conversely, β2‐AR protein expression was significantly lower in the burned knockout animals (p=0.03). Sarcoplasmic reticulum calcium ATPase protein expression was significantly lower in both the wild‐type and interleukin 6 knockout mice after burn injury (p=0.003).ConclusionThese data indicate that interleukin 6 may play a role in the disruption of cardiac β‐AR signaling including proteins involved in calcium handling after burn injury. Burn induced cardiac dysfunction has been well documented but is not clearly understood. Understanding how these signaling pathways are altered post injury may provide insight to the appropriate method of treatment.Support or Funding InformationExternal Funding: Shriners Hospitals for Children (84291,80500, 84202,71008); National Institutes of Health (R01GM056687, R01HD049471‐07, R01AR065270, R01GM112936)
Superoxide via Sp3 but Not NF‐Kb Regulates Renal AT1 Receptor Expression and Function in Human Kidney Cells
Authors: Saleem Mohammad , Asghar Mohammad 
Published: November 2023

Oxidative stress plays a causal role in higher Angiotensin II type 1 receptor (AT1R) function and hypertension. Both redox‐sensitive transcription factors have been implicated in regulation of AT1R. Here, we tested a hypothesis that superoxide but hydrogen peroxide (H2O2) via Sp3 but not NF‐kB p‐65 transcription factor regulates renal AT1R. We tested our hypothesis in human kidney (HK2) cells by undertaking following studies. Diethyldithiocarbamate (DDC), a superoxide prodrug, but not H2O2 increased nuclear accumulation of both Sp3 and NF‐kB in HK2 cells as measured by immunoblotting. Importantly, this effect was attenuated with Tempol, a superoxide mimetic. Further, DDC increased AT1R mRNA and protein levels that were attenuated with Tempol treatment, measured respectively by RT‐qPCR and immunoblotting. However, H2O2 did not show any significant effect on AT1R mRNA and protein levels. Moreover, Sp3 plasmid increased while Sp3 siRNA decreased AT1R protein levels in HK2 cells. Interestingly, NF‐kB plasmid did not show any significant effect on AT1R protein levels. Moreover, NF‐kB shRNA and PDTC, an NF‐kB inhibitor, both increased AT1R mRNA, suggesting an inhibitory effect of NF‐kB on AT1R. In addition, DDC treatment showed an exaggerated response of Ang II on protein kinase C (PKC) activity, an indicator of AT1R function, which was attenuated with Tempol treatment. Collectively, our results suggest that superoxide but not H2O2 via Sp3 but not NF‐kB up‐regulate AT1 receptor expression and function that may contribute to hypertension.Support or Funding InformationNIH/NIA: AG039836