bluuebunny/crossref_metadata_2025 huggingface dataset:
Serine 216 Regulates ERα to Repress Inflammation in Microglia of Mouse Brains
Authors: Shindo Sawako
Published: November 2023

ERα is also phosphorylated in mouse brain microglia, but not in astrocytes and neuronal cells. ERα KI mice (Esr1S216A) which express the ERα Ser216Ala mutation were used to investigate the role of phosphorylation in the inflammatory regulations of microglia. Esr1S216A mice are fertile and their hormonal cycles appeared to be normal. Brain cells were prepared from 2 days‐old neonates of wild type and Esr1S216A mice and were cultured for 2–3 weeks prior to LPS or PBS treatments. Both fluorescence immuno‐staining and Western blot analyses revealed phosphorylated ERα present in microglia but neither astrocytes nor neuronal cells. Culture media were collected after treatments for subsequent ELISA assays to measure cytokines and inflammatory metabolites. Pro‐inflammatory cytokine IL‐6 and metabolite PGE2 were 10‐ to 5‐times higher in culture media of KI than wild type glia cells in the early stage of LPS treatment. Conversely, an anti‐inflammatory cytokine IL‐10 was higher in wild type than in KI media in the later stage of LPS treatment. In addition, IHC with an anti‐Iba1 antibody found that microglia was more activated in brains of adult KI than of wild type mice. These results suggest that phosphorylated ERα elicits anti‐inflammatory signals to repress LPS‐induced M1 activation and to stimulate M2 polarization. FACS analysis is now undergoing to confirm these findings with intracellular cytokines and examine apoptotic nature of microglia from KI mice. It appears that phosphorylated ERα acts as an anti‐inflammatory factor to protect brains from stress‐induced inflammation.
Co‐ultra PEALut enhances neuronal recovery after a moderate traumatic brain injury
Authors: Campolo Michela , Crupi Rosalia , Cordaro Marika , Bruschetta Giuseppe , Siracusa Rosalba , Esposito Emanuela , Cuzzocrea Salvatore
Published: November 2023

Traumatic brain injury (TBI) is a major health problem worldwide. Currently, there is no effective treatment to improve neural structural repair and functional recovery. Recently, has been demonstrated that hippocampal injury‐induced neurogenesis is one mechanism underlying endogenous repair following TBI. The aim of this study was to evaluate the neuro‐regenerative properties co‐ultra PEALut (constituted by the association of palmitoylethanolamide (PEA), with the flavonoid luteolin (Lut) in a mouse model of TBI until 7 days. TBI was induced in mice by the controlled cortical impact (CCI), one of the most common models of TBI; the mice were orally administered co‐ultra PEALut (1mg/Kg) 1h after trauma and daily for 72h and 7d. A conspicuously neurogenesis was seen in mice 72h and 7d after trauma. Co‐ultra PEALut treatment stimulated neuronal reconstitution process restoring the basal level of new neurons (by BrdU staining) and mature neurons (by doublecortin staining). Moreover this positive effect was associated with an important upregulation of neurotrophic factors (BDNF and NT‐3), ultimately leading to improvement in memory recall on behavioral testing (Morris water maze test).

Co‐ultra PEALut could represent a therapeutic potential of augmenting the endogenous repair response for treating TBI.
In Vitro Characterization of a Novel Allosteric Potentiator of the Dopamine D1 Receptor
Authors: Heinz Beverly A , Schaus John M , Beck James P , Hao Junliang , Wang Xushan , Krushinski Joseph , Reinhard Matthew , Hellman Sarah , Getman Brian , Suter Todd , Nelson David , Wiernicki Todd , Cohen Michael P , Yang Charles R , Svensson Kjell A , Bruns Robert F
Published: November 2023

Allosteric potentiators of the dopamine D1 receptor could be useful for treatment of Parkinson's disease, schizophrenia, depression, ADHD, and narcolepsy. We identified a novel D1 potentiator (“DETQ”)by high‐throughput screening followed by iterative chemical optimization. In a HEK293 cell line expressing the human D1 receptor, agonist‐stimulated cAMP production was measured in the presence of an EC20 concentration of dopamine using homogeneous time resolved fluorescence (HTRF) technology. DETQ potentiated the dopamine response with a mean EC50 of 5.8 nM and mean efficacy corresponding to 95% of the maximum response to dopamine. In the absence of exogenous dopamine (agonist mode), the maximum efficacy of DETQ was 11.6% with an EC50 of 30 nM; based on the initial slopes of the two curves (Ehlert R‐ratio), DETQ was thus 43‐fold less potent as an allosteric agonist than as a potentiator. When concentration‐response curves for dopamine were carried out at multiple DETQ concentrations in the cAMP assay, DETQ shifted the dopamine curve 21‐fold to the left with a KB of 26 nM. In experiments measuring binding of the D1 antagonist 3H‐SCH23390 to the human D1 receptor, 100 nM DETQ caused a 5‐fold leftward shift in the concentration‐inhibition curve for dopamine, indicating adirect effect of DETQ on the D1 receptor. The potency of DETQ in the cAMP assay was about 50‐fold lower at the mouse and rat D1 receptors than at human, rhesus, and dog D1 receptors. DETQ was inactive at concentrations up to atleast 10 uM when tested in agonist and potentiator modes at related receptors, including D2, D5, β1, β2, β3, and 5HT6. DETQ was also inactive in binding and/or functional assays at a largeset of unrelated targets.

These results suggest that DETQ could be a useful tool to probe applications of dopamine D1 potentiators for CNS disorders.
GPCRs as Possible Therapeutic Targets in Pancreatic Cancer
Authors: Chang Sarah , McCann Thalia , Zhou Shu , French Randall , Lowy Andrew M , Corriden Ross , Insel Paul A
Published: November 2023

Pancreatic ductal adenocarcinoma (PDAC, the most common type of pancreatic cancer) is the 4th leading cause of cancer deaths in the US and has a survival rate of ~5%. New therapeutic approaches are thus needed for this disease. PDAC tumors are composed of malignantductal epithelial cells, and cells of the tumor microenvironment, which includes inflammatory/immune cells, cancer‐associated fibroblasts (CAFs, stromal cells), vascular cells and dense extracellular matrix. In this study, we tested the hypothesis that PDAC cells express G‐protein‐coupled receptors (GPCRs, the largest class of targets of FDA‐approved drugs) that may be novel therapeutic targets for pancreatic cancer.

We used GPCR Taqman arrays to identify and quantify mRNA of ~350 GPCRs and assessed PDAC tumors(n=3), patient‐derived PDAC cell lines (n =2), established PDAC cell lines (Miapaca, ASPC‐1), and patient‐derived pancreatic CAF cells (n=5). We found 77 GPCRs expressed commonly by the tumor samples, 46 GPCRs expressed in common in the patient‐derived and established PDAC cell lines and 104 GPCRs shared among the CAF samples.

An orphan GPCR (termed Orphan A) was highly expressed in PDAC tumors and cells and CAFs, results confirmed by independent qPCR. OrphanA was among the most highly up‐regulated GPCRs in PDAC cells compared to their normal precursor, pancreatic ductal epithelial cells (HPDE6). Transfection of Orphan A into HPDE6 cells increased their DNA synthesis, implying that Orphan A enhances PDAC proliferation and that its inhibition may blunt tumor growth. These results indicate the utility of an unbiased Taqman array approach to identify GPCRs expressed by PDAC cells and pancreatic CAFs. We propose that GPCRs, such as Orphan A, whose expression is shared by these two cell types and more highly expressed than in normal precursor cells may be novel targets for the treatment of pancreatic cancer.

Support or Funding Information

Supported by NIH R21CA189477 and T32CA121938
Loss of brain‐expressed receptors GPR37 and/or GPR37L1 results in increased seizure susceptibility to induced seizures
Authors: Giddens Michelle M , Wong Jennifer C , Coleman Brilee M , Schroeder Jason P , Farrow Emily G , Soden Sarah E , Weinshenker David , Escayg Andrew , Hall Randy A
Published: November 2023

GPR37 and GPR37‐like‐1 (GPR37L1) are orphan G protein‐coupled receptors that are expressed almost exclusively in the brain. Recently, exome mapping in a large, consanguineous family of Middle Eastern descent identified a homozygous amino acid substitution in the GPR37L1 gene as the causal mutation for a previously undescribed recessive monogenic disorder. Affected individuals develop severe migraines and seizures in adolescence, followed by cortical atrophy, cognitive decline, and ultimately death in the late teens. We and others have shown that expression of GPR37 and/or GPR37L1 can mediate cytoprotective signaling. Therefore, we assessed the effect of loss‐of‐function mutations using GPR37 and GPR37L1 knockout mice. Here we show that a loss of GPR37, GPR37L1, or both receptors result in increased seizure susceptibility to induced seizures. These data support the potential association between GPR37L1 and the aforementioned inherited neurological disorder and also reveal a previously‐unappreciated role for GPR37 and GPR37L1 in susceptibility to induced seizures, which may have implications for future drug development efforts aimed at these receptors.
GPCRs as novel targets in pulmonary artery smooth muscle cells in pulmonary arterial hypertension
Authors: Sriram Krishna , Aroonsakool Nakon , Michkov Alexander V. , Insel Paul A.
Published: November 2023

There is an unmet need for new therapeutics to treat pulmonary arterial hypertension (PAH): currently approved therapy has limited benefit in terms of overall mortality and many such treatments not only reduce vascular resistance in the pulmonary arterial tree but also have side‐effects, including systemic hypotension. We sought to determine if certain G‐protein‐coupled receptors (GPCRs) are more highly expressed in pulmonary artery smooth muscle cells (PASMCs) from patients with PAH compared to controls and thus might be novel therapeutic target for the treatment of PAH.

METHODS

We performed GPCR expression profiling using qPCR‐based Taqman GPCR arrays that identify and quantify mRNA expression of non‐chemosensory endo‐GPCRs. Profiling was done on PASMCs isolated from PAH and non‐PAH patients. We also assessed GPCR expression in PASMCs from mice and rats and in SMCs from the human aorta and coronary artery.

RESULTS

PASMCs expressed ~120 GPCRs, including ~45 orphan (without known physiologic agonist) GPCRs. Numerous adhesion and frizzled receptors were detected: LPHN2, CD97, ELTD1 and GPR124 were among the highest expressed GPCRs in all human SMCs profiled. Expression and identities of the highest expressed GPCRs were consistent among biological replicates of non‐diseased human PASMCs. GPCR expression profiles were similar, between non‐IPAH PASMCs and SMCs from the aorta and coronary artery but PAH‐PASMCs showed changes in GPCR expression. We identified ~20 receptors with at least 2‐fold increases in expression in PASMCs from PAH patients compared to controls. These include numerous orphan receptors as well as GPCRs previously not known to play a role in PAH. In addition, a similar number of GPCRs had reduced expression in PAH‐PASMC relative to control‐PASMCs, indicating a subset of GPCRs that might be considered for gene‐based therapies of PAH. GPCR expression profiling in mouse and rat PASMCs revealed a high degree of similarity with PASMCs from human subjects, highlighting the utility of animal models and cells for further investigation of the physiological/functional role for the GPCRs we identified.

CONCLUSION

Numerous orphan and previously under‐studied GPCRs are expressed by PASMCs with a number of GPCRs having increases/decreases in PAH compared to control PASMCs. GPCR expression in PASMCs from rats and mice are similar to that of human PASMCs, highlighting the potential for use of animal models to study these GPCRs as potential therapeutic targets for PAH

Support or Funding Information

Supported by DOD AWARD: No. W81XWH‐14‐1‐0372
Bitter taste receptor agonists as potent tocolytics for preterm labor
Authors: Zheng Kaizhi , Lu Ping , Simas Tiffany A Moore , Delpapa Ellen , Lifshitz Lawrence M , Shi Fangxiong , ZhuGe Ronghua
Published: November 2023

Preterm labor (PTL), the leading cause of neonatal mortality and morbidity, occurs in ~11% of pregnancies. Its etiologic mechanisms are complex, multifactorial, and not clearly elucidated. PTL is defined by cervical change in the presence of regular uterine contractions occurring prior to 37 weeks gestation in humans. Given contractions as a central feature, tocolytics are used in its management, yet current tocolytics are not sufficiently effective. In the present study, we report that myometrial cells from mouse and human express bitter taste receptors (TAS2Rs), a class of proteins that were long thought to be solely in the taste buds of tongues, and their canonical signaling components (i.e., G‐protein gustducin and PLCβ2). Strikingly, bitter tastants (e.g., chloroquine) relax myometrium pre‐contracted by uterotonics (e.g., oxytocin and prostaglandin F2α) more completely than current commonly used tocolytics (i.e., nifedipine, indomethacin and MgSO4) and at a lower dose than MgSO4 and indomethacin. Using isolated single mouse myometrial cells, we found that bitter tastants reverse the rise in [Ca2+]i and cell shortening induced by uterotonics, and this reversal effect is inhibited by pertussis toxin treatment and α‐gustducin deletion. Moreover, bitter tastants (e.g., chloroquine) can prevent mouse PTL induced by bacterial endotoxin lipopolysaccharide more often than current commonly used tocolytics. In summary, myometrial cells express the canonical bitter taste receptor signaling system, and bitter tastants demonstrate the potential to be more effective tocolytics than those currently used for PTL management.
Compartmentalized cAMP Response to Prostaglandin EP2 Receptor Activation in Human Airway Smooth Muscle Cells
Authors: Harvey Robert D , Miyashiro Kathryn , Agarwal Shailesh R , Latt Htun , Ostrom Rennolds S
Published: November 2023

It has been previously demonstrated that prostaglandin EP2 receptors selectively couple to adenylyl cyclase type 2 in non‐lipid raft domains of human airway smooth muscle (HASM) cells, where they regulate specific cellular responses. In the present study, we used FRET‐based biosensors targeted to different subcellular locations to determine exactly where EP2 receptor production of cAMP occurs in these cells. The Epac2‐camps biosensor, which is expressed throughout the cell, was used to measure bulk cytoplasmic responses, while MyrPalm‐Epac2 and CAAX‐Epac2 were targeted to lipid raft and non‐lipid raft regions of the plasma membrane, and NLS‐Epac2 was targeted to the nucleus. All probes were able to detect cAMP responses produced by direct activation of adenylyl cyclase with forskolin or exposure to the beta‐adrenergic receptor agonist isoproterenol. On the other hand, butaprost, an EP2 receptor agonist, elicited a response that could be detected by CAAX‐Epac2, but not MyrPalm‐Epac2. The butaprost response could also be detected by Epac2‐camps, as well as NLS‐Epac2. Exposure to rolipram, a selective inhibitor of phosphodiesterase type 4 (PDE4), produced no effect on its own that could be detected by any of the probes. However, in the presence of rolipram, exposure to butaprost produced a response that could then be detected by the MyrPalm‐Epac2 probe. Rolipram also enhanced the butaprost responses detected by Epac2‐camps and CAAX‐Epac2. These results support the conclusion that EP2 receptor activation leads to cAMP production in subcellular compartments associated specifically with non‐lipid raft regions of the plasma membrane. Furthermore, the activity of PDE4 appears to play a role in maintaining the integrity of this compartmentalized response.

Support or Funding Information

Supported by NIH (GM107094)
Heat Shock Protein 90 Regulates μ‐Opioid Receptor Signaling In Vitro and in Different Regions of Mouse Brain
Authors: Lei Wei , Mullen Nate , McCarthy Sarah , Brann Courtney , Richard Philomena , Cormier James , Lindros Kayla , Edwards Katie , Bilsky Edward , Streicher John Michael
Published: November 2023

Heat shock protein 90 (Hsp90) is a ubiquitous and highly expressed regulator for various signaling pathways and proteins. Only 2 previous studies suggested that Hsp90 might be involved in the regulation of opioid receptor signaling, which is one of the primary targets for drug discovery for chronic pain. This study was designed to determine the regulatory activities of Hsp90 on μ‐opioid receptor (MOR) signaling in various types of MOR expressing cells and in mouse brain. MOR expressing CHO, HEK, U2OS, and SH‐SY5Y neuroblastoma cells were treated with an Hsp90 specific inhibitor (17‐AAG) for 24 hrs followed by treatment with the MOR full agonist DAMGO. The expression and activation of signaling proteins was investigated by Western blot. We found that the expression of MOR, βarrestin2, and Akt was reduced in some cell types by inhibiting Hsp90. In contrast, the expression of Hsp70 was increased by the treatment of 17‐AAG in all cell types, as expected. The activation of ERK MAPK was also altered by Hsp90 inhibition in a cell context dependent manner – whether increasing DAMGO stimulated activation, increasing the baseline, or blunting activation. The strongly context dependent nature of Hsp90 regulation indicated that we would need to determine the role of Hsp90 in regulating the MOR in vivo. We thus injected CD‐1 mice intracerebroventricularly with 17‐AAG for 24 hrs, followed by injection of vehicle or DAMGO into the brain. Interestingly, we found an increased ERK signaling baseline and abolished DAMGO induction in the periaqueductal grey, along with increased Hsp70 expression. Preliminary results in the brain stem suggest that ERK MAPK activation in response to DAMGO is abolished by 17‐AAG, without increasing the baseline. Furthermore, 17‐AAG treatment showed a modest decrease on the acute anti‐nociceptive tail‐flick response to DAMGO, and increased urine and feces output after naloxone precipitated withdrawal in models of acute and chronic dependence. These findings demonstrate that Hsp90 has a strong regulatory role in MOR signaling both in vitro and in vivo, and suggest that Hsp90 activators or other modulators could be potentially used as a co‐therapy for improving the therapeutic index of opioid drugs.

Support or Funding Information

Work supported by institutional startup funds from the University of Arizona, along with an internal UNE Mini‐Grant and a Pilot Grant from a UNE Centers for Biomedical Research and Excellent (COBRE) grant (Ian Meng PI, #P20GM103643).
Internalization of Protease‐Activated Receptor 4 is AP‐2 Dependent and Utilizes a Non‐canonical Sorting Motif
Authors: Smith Thomas Horace , Coronel Luisa , Nieman Marvin , Trejo JoAnn
Published: November 2023

Protease‐activated receptor‐4 (PAR4) is a GPCR that is activated by proteolytic cleavage of its N‐terminus by thrombin and other proteases. Due to this irreversible proteolytic mechanism of activation, the mechanisms that control signaling by PAR4 are important for regulating the magnitude and duration of the signaling response. While the processes that control signaling of PAR1, a related GPCR activated by thrombin, have been extensively characterized, the mechanisms that regulate PAR4 signaling remain poorly understood. Unlike PAR1, activated PAR4 is not phosphorylated and exhibits prolonged signaling. These findings raised the question of whether PAR4 signaling is regulated by intracellular trafficking, a process which remains poorly defined for the receptor. The focus of this study is to delineate the mechanisms that control PAR4 intracellular trafficking. To examine the mechanisms that control PAR4 endocytosis, we expressed FLAG‐PAR4 in HeLa cells, a cell model system used extensively to characterize clathrin‐ and dynamin‐mediated endocytosis. We showed that upon activation with the agonist peptide AYPGKF, ~25% of PAR4 internalized from the cell surface over one hour. We next employed immunofluorescence confocal microscopy to visualize the endocytic sorting of activated PAR4 and found that the majority of activated and internalized PAR4 localized to early endosomes by 30 minutes and then sorted to lysosomes between 60 and 90 minutes. We utilized siRNA knockdown of clathrin and immunofluorescence microscopy to show that PAR4 undergoes clathrin‐mediated endocytosis following activation. Consistent with clathrin‐mediated endocytosis, expressing a dominant‐negative dynamin K44A mutant also blocked activated PAR4 internalization. Interestingly, however, unlike most GPCRs, PAR4 internalization occurred independently of both β‐arrestin 1 and 2 and the C‐tail domain. Rather, we identified a highly conserved potential AP‐2 binding motif in the receptor's third intracellular loop, and thus hypothesized that PAR4 internalization was mediated by the clathrin adapter protein AP‐2. Using siRNA knockdown of AP‐2, we confirmed that the adapter protein was necessary for PAR4 endocytosis. We also observed that a PAR4 mutant lacking the putative AP‐2 binding motif exhibited a defect in agonist‐induced internalization. To confirm our findings we examined endogenous PAR4 internalization in the Dami megakaryoblastic cell line. In ongoing studies, we are investigating the effect of dysregulated PAR4 trafficking on signal regulation.

Support or Funding Information

Funding for this project was provided by NIH/NHLBI F31 HL116187 (T. Smith).
Increased Tubular Dysfunction and Injury Response Biomarkers in the Absence of Overt Clinical Nephrotoxicity in Cancer Patients Receiving Cisplatin Therapy
Authors: George Blessy , Wen Xia , Johnston Nickie , Gomez Madeleine , Ellison Lucas , Joy Melanie , Aleksunes Lauren
Published: November 2023

Novel urinary proteins have emerged as more sensitive indicators of acute kidney injury compared to serum creatinine and may provide clues regarding the mechanism and anatomic location of nephrotoxicity. We sought to characterize urinary concentrations of biomarkers that reflect an acute injury response (KIM‐1, clusterin, NGAL, TFF3), glomerular and tubular dysfunction (cystatin C, calbindin, GST‐π, albumin, β2MG) and inflammation (IL‐18, MCP‐1, osteopontin) after exposure to cisplatin. Patients (n=57) with solid tumors and scheduled for chemotherapy with cisplatin ≥ 25 mg/m2 were recruited. Urine was collected at baseline, 3, and 10 days post infusion and analyzed using a multiplex protein assay. Markers of tubular dysfunction (β2MG, cystatin C, albumin) were elevated at day 3 by 330%, 185%, and 147%, respectively. By day 10, levels of calbindin and GST‐π were increased by 827% and 160%, respectively. Markers of injury and inflammation (KIM‐1, clusterin, and MCP‐1) were also elevated at day 10. Although reported to be down‐regulated in tubular injury in rats, levels of TFF3 in cisplatin‐treated patients were increased at days 3 (170%) and 10 (203%). Interestingly, serum creatinine and urinary concentrations of pro‐inflammatory proteins IL‐18 and osteopontin were not altered. ROC curves calculated from pre‐ and post‐cisplatin infusion concentrations revealed that β2MG and TFF3 had the highest AUC for day 3 (0.79 and 0.68, p<0.002), while calbindin, TFF3, KIM‐1, and MCP‐1 AUCs (0.76, 0.69, 0.68 and 0.68, p<0.003) were highest at day 10. These data suggest that novel urinary protein biomarkers may reveal mechanisms and time‐dependent subclinical kidney injury in the absence of clinically detectable damage in patients receiving cisplatin.

Support or Funding Information

Supported by an AFPE Fellowship, DK080744, DK093903, and CA072720.
N‐acetylcysteine decreases the susceptibility to Diclofenac‐induced nephrotoxicity in diabetic patients
Authors: Yeh Ching Hua
Published: November 2023

Diabetes patients are at high risk for chronic kidney diseaseor end stage renal disease which leading to dialysis because of long‐term taking medicine. Diclofenac (DLF) is a commonly prescribed NSAID. DLF‐induced nephrotoxicity may involve production of reactive oxygen species leadingto oxidative stress and cell death in the kidney. DLF could induce nephrotoxicity by inhibiting the activity of Nrf‐2 has been discovered in our previous research. The present study clarified whether the dysfunction of Nrf‐2 mediated oxidative stress under high‐glucose oxidative stress is related to the high susceptibility to DLF induced‐nephrotoxicity in diabetes patients. In vivo, normal or STZ‐diabetic mice were intraperitoneally injected with DLF 200 mg/kg for continuous three days or 5 mg/kg for four weeks to induce acute or chronic renal damage respectively. N‐acetylcysteine (NAC; 600 mg/kg) was administrated by oral gavage to DLF‐injected mice. Tissue injury, oxidative stress and fibrotic changes were observed in the kidney of these mice. The expression changes of antioxidative genes, Nrf‐2, SOD, GPx, and BMP‐7 in normal and STZ‐diabetic mice were compared. In vitro, the oxidative stress and the gene expression changes in NRK52E cell exposed to high glucose combined with DLF stimulation were analyzed. Both acute and chronic DLF‐injection increased the BUN and creatinine levels of mice. The DLF‐induced nephrotoxicity was severe in STZ‐diabetic mice. The antioxidant‐NAC treatment could attenuate the DLF‐induced acute or chronic kidney injury as well as decrease the TGF‐β and fibronectin expression in the kidney of STZ‐induced diabetic mice. The renal expressions of BMP‐7 and Nrf‐2 as well as SOD and GPx in the DLF‐injected STZ mice were increased by NAC treatment. DLF cause high cytotoxicity when NRK52E cell was exposed to high glucose medium. The NAC treatment could decrease the significant cell death by increasing the activity of Nrf‐2 and inhibiting oxidative stress induced by high glucose combined with DLF stimulation. The present study demonstrated that NAC treatment could decrease the susceptibility of DLF‐induced kidney injury in diabetic mice by increasing the Nrf‐2‐triggered antioxidative effects in the kidney. We suggest that the low Nrf‐2 activity and extremely high oxidative stress is the main reasons of susceptibility in diabetic patients why they are more prone to the development of kidney injury than others.

Support or Funding Information

The present study was supported by grants MOST103‐2320‐B‐212‐001 from the Ministry of Science and Technology, ROC.
Evaluation of Protein Biomarkers for Renal Toxicity Induced by Exposure to Low‐dose Heavy Metals
Authors: Shin Young‐Jun , Kim Jeong‐Hyeon , Kim Hyung‐Sik , Bae Ok‐Nam
Published: November 2023

Kidney is one of the main targets for toxicity induced by xenobiotics including drugs and heavy metals. The biomarkers to detect xenobiotic‐induced nephrotoxicity are being actively developed, but the sensitivity and efficiency of these biomarkers still need to be improved. The aim of this study is to identify novel biomarkers using proteomic approaches in xenobiotic‐exposed kidney tissue. Mercuric chloride (HgCl2) was administered by oral route in the drinking water to male Sprague‐Dawley rats every two days for 14 days. Proteomic analysis with kidney samples revealed that AKR7A1 and GSTP1 were significantly elevated by mercury exposure, suggesting them as potential candidates for new nephrotoxic biomarkers. Interestingly, in normal rat kidney proximal tubular cells (NRK‐52E), the change of expression level of AKR7A1 and GSTP1 was well correlated to the extent of cytotoxicity induced by exposure to nephrotoxicants of heavy metals (Hg and Cd) and cisplatin. The sensitivity of these two biomarkers was found to be superior to that of traditional biomarkers for renal toxicity, such as Kim1, Lcn2, Spp1 and TIMP1. With this study, we identified AKR7A1 and GSTP1 as new candidates for drug or heavy metal‐induced renal toxicity. Correlation between cytotoxicity and the extent of AKR7A1 and GSTP1 change suggests that they can be useful biomarkers to evaluate or predict renal toxicity.
Pharmacokinetic Analysis of Aerosol Propellant HFA‐134a Elimination in the Human Breath
Authors: Shin Hye‐Won , Landaw Elliot M. , Barletta Barbara , Meinardi Simone , Aizik Shlomit , Leu Szu‐Yun , Blake Donald , Cooper Dan M.
Published: November 2023

The goal of the study is to model the elimination kinetics of Hydrofluoroalkane (HFA‐134a) in the exhaled breath following a typical single asthma inhaler administration. HFA‐134a is the most commonly used aerosol propellant in metered dose inhalers. We previously demonstrated that our technical capabilities allow us to measure breath HFA‐134a levels at 24 to 48 hours post asthma inhaler administration, and thus breath HFA‐134a is a promising biomarker for identifying asthma inhaler compliance (Clinical and Translational Science 2015).

Exhaled breath gases were collected in evacuated electro‐polished stainless steel canisters from healthy participants at baseline and at 5 min, 7 min, 10 min, 15 min, 30 min, 1hr, 2hr, 3hr, 4hr, 6hr, 8hr, 12hr. 24hr, 36hr, and 48hr ‐post inhaled corticosteroids administration (Flovent HFA, Glaxo Smith Kline). The corresponding ambient HFA‐134a levels were also measured. HFA‐134a levels in the breath and ambient air samples were quantified using gas chromatography with a quadrupole mass spectrometer detector. The Institutional Review Board at the University of California, Irvine approved the study, and informed consent was obtained from the participants.

HFA elimination in the breath followed a three‐exponential pharmacokinetic model. The estimated time constants (mean (SD)) for the three components were 5.5 (0.5) minutes, 62.4 (11.1) minutes, and 664 (140) minutes, with an overall mean retention time (MRT) of 71.8 (40.4) minutes. The shortest and longest half‐life components comprised 66 (17) % and 9 (7) % of the total area under the curve, respectively. Breath carbon dioxide (CO2) levels were also monitored as a reference breath gas to ensure that the gas sampled was alveolar gas. The mean (SD) CO2 level was 4.7 (0.6) % of all breath samples.

This study demonstrates that the breath HFA‐134a elimination in healthy control humans can be described by a 3rd order exponential equation. In earlier studies, we modeled bicarbonate distribution kinetics and found similar time constants (2.2 min, 11.9 min, and 83.3 min; MRT 65 min.; Barstow et al. Am. J. Physiol. 259: R163, 1990). We speculate that the first two time constants reflect compartmental kinetics with the lung and circulation while the slowest primarily reflects distribution to body tissues. The kinetics for HFA‐134a appear to be slower than for bicarbonate. The calculated HFA‐134a elimination parameters (i.e., rate constants or corresponding half‐lives) may be useful in conjunction with breath HFA‐134a levels for accessing asthma inhaler medication compliance and further to understand physiological complexity of the lungs that affect HFA‐134a elimination.

Support or Funding Information

This work was supported by NIH grants: Translational Pilot Research Award (subaward of UL1 TR000153) and Program Project Grant P01HD048721
Transcriptional Regulators Predict CYP3A4 Expression in Human Pediatric Liver
Authors: Vyhlidal Carrie A , Bi Chengpeng , Ye Shui Q , Leeder J. Steven
Published: November 2023

Members of the human CYP3A family of metabolizing enzymes exhibit extensive variability in catalytic activity. Up to 90% of the variation in CYP3A4 activity has been attributed to genetic factors; however, polymorphisms within the CYP3A4 gene do not adequately explain the observed variability. In pediatric liver, developmental changes in CYP3A4 expression are an additional source of variability. Recent studies have explored genetic and epigenetic variation and variable expression of regulatory factors as predictors of CYP3A4 activity. The objective of this study was to investigate the interactions between known regulatory factors of CYP3A4 and its expression in postnatal liver. The expression of genes encoding factors responsible for transcriptional regulation of CYP3A4 and modulators of CYP3A4 activity was determined by RNA‐Seq in 52 human postnatal liver samples (ages <1 day to 17 years of age). Individually, relative expression of total CYP3A4 mRNA in pediatric liver was significantly correlated (p<0.05 after Bonferroni correction) with the expression of PXR (r2=0.6), CAR (r2=0.59), LXRα (r2=0.46), ACSM3 (r2=0.69), AKR1D1 (r2=0.75), EHHADH (r2=0.67), FMO3 (r2=0.7), GLYAT (r2=0.64), and SLC10A1 (r2=0.68). Stepwise regression using minimum Bayesian Information Criterion was performed. The expression of CYP3A4 mRNA could be explained by a subset factors including CEBPA, FOXA3 (HNF3γ), RXRα, NFIA, AKR1D1, and FMO3 (r2=0.78, p=1.07*10−12). In conclusion, these results suggest that transcriptional regulators of CYP3A4 are important contributing factors to variability in its mRNA expression in pediatric liver.
A novel CXCR4‐ARF1‐MAPK signaling pathway controls prostate tumorigenesis
Authors: Davis Jason E , Wu William H , Surrao Kristen , Teng Yong , Wu Guangyu
Published: November 2023

Prostate cancer is one of the most common cancers in men and is the second leading cause of cancer‐related mortality in men. It is well known that enhanced activation of the MAPK pathway Raf‐MEK‐ERK1/2 directly correlates with the progression, androgen independence and poor prognosis of prostate cancer. Recent studies have also shown that the chemokine receptor CXCR4 is highly expressed in prostate cancer and contributes to prostate tumor growth and metastasis. However, the molecular mechanisms underlying the hyper‐activation of MAPK and the function of CXCR4 remain poorly elucidated. Here, we demonstrated that CXCL12‐induced CXCR4 activation markedly enhanced the activation of ADP‐ribosylation factor 1 (ARF1), a Ras‐related small G protein, in a time‐ and concentration‐dependent fashion, and siRNA‐mediated depletion of ARF1 dramatically inhibited MAPK activation by CXCR4 in prostate cancer cells. More interestingly, we found that ARF1 can mimic Ras to directly and activation‐dependently interact with Raf1, specifically its Ras‐binding domain. Mutations that disrupted ARF1 activation considerably attenuated ARF1‐Raf1 interaction and MAPK activation. These data suggest that, through its direct interaction with Raf1, ARF1 regulates the MAPK activation by CXCR4 in prostate cancer cells.

We then investigated the role of ARF1 in prostate tumorigenesis. We found that ARF1 expression and activation were markedly elevated in human prostate cancer cells and tissues. Depletion of ARF1 significantly inhibited prostate cancer cell proliferation in vitro and xenograft tumor growth in vivo, whereas over‐expression of ARF1 produced opposing effects. These data have revealed an unappreciated function of ARF1 in prostate cancer. Altogether, our studies have identified a novel CXCR4‐ARF1‐MAPK signaling pathway which plays an important role in prostate tumorigenesis and may represent a key molecular target for therapeutic innovation (R01GM076167).

Support or Funding Information

R01GM076167
MRTF‐A and YAP exert dual control in GPCR and RhoA‐mediated transcriptional regulation and cell proliferation
Authors: Yu Olivia M. , Miyamoto Shigeki , Brown Joan Heller
Published: November 2023

The ability of a subset of G‐protein coupled receptors (GPCRs) to activate RhoA endows them with unique growth regulatory properties. Two transcriptional pathways are activated through GPCRs and RhoA, one utilizing the transcriptional co‐activator MRTF‐A and SRF, the other the transcriptional co‐activator YAP and TEAD. These pathways have not been compared for their relative importance and potential interactions in RhoA target gene expression. GPCRs for thrombin and S1P on human glioblastoma cells robustly couple to RhoA and induce the matricelluar protein CCN1. Knockdown of either MRTF‐A or YAP abrogates S1P stimulated CCN1 expression, demonstrating that both co‐activators are required. MRTF‐A and YAP are also both required for transcriptional control of other S1P regulated genes in various cell types and for S1P stimulated glioblastoma cell proliferation. Interactions between MRTF‐A and YAP are suggested by their synergistic effects on SRE.L and TEAD‐luciferase expression. Moreover, MRTF‐A and YAP associate in co‐IPs from S1P stimulated cells. ChIP analysis of the CCN1 promoter demonstrates that S1P increases co‐activator binding at their canonical transcription factor sequences. Unexpectedly, SIP also enhances MRTF‐A binding at TEA sites. Our findings reveal that GPCR and RhoA‐regulated gene expression requires dual input and integration of two distinct transcriptional pathways.

Support or Funding Information

This work was supported by NIH Grant GM036927, HL028143, and PPG/HL085577 to JHB, and by HL097037 and AHA Grant 15GRNT22970009 to SM. OY was supported by T32 GM007752 (Graduate Training in Cellular and Molecular Pharmacology) and T32 DK007541 (Endocrinology and Metabolism Training Grant).
Electrophilic Nitroalkenes Inhibit Triple Negative Breast Cancer Metastasis
Authors: Woodcock Chen‐Shan , Woodcock Steven R , Cao Chunyu , Davidson Nancy E , Huang Yi , Freeman Bruce A
Published: November 2023

Triple negative breast cancer (TNBC), which lacks estrogen receptor (ER), progesterone receptor (PR) and Her2/Neu, is an aggressive and therapy‐resistant metastatic cancer. A high level of tumor necrosis factor‐α (TNFα) expression and activation in TNBC patients is associated with distant tumor metastasis. Current therapy for TNBC is limited, and there is an unmet need for novel therapeutic agents. Electrophilic fatty acid nitroalkenes are endogenous signaling mediators that modulate cell differentiation and proliferation via post‐translational modification (PTM) of functionally‐significant nucleophilic amino acids (Cys, His) of transcriptional regulatory proteins such as NFκB, PPARγ, and Keap1/Nrf2. Recent studies have revealed that an exemplary electrophilic nitroalkene, nitro‐oleic acid (NO2‐OA), inhibits TNFα‐induced TNBC cell migration. NFκB plays an important role in tumor development and progression, and its signaling actions are constitutively activated in ER‐negative breast cancer cell lines and primary tumors. Notably, NO2‐OA causes polyubiquitination and degradation of NFκB RelA protein via protein nitroalkylation in TNBC cells. Therefore, it is hypothesized that electrophilic nitroalkenes have therapeutic potential that inhibit TNBC metastasis through down‐regulation of TNFα‐NFκB signaling. We demonstrate that NO2‐OA significantly inhibits two TNFα‐induced NFκB‐regulated genes involved in tumor metastasis, ICAM‐1 and uPA, in TNBC cells. The TNBC xenograft study indicates that NO2‐OA reduces MDA‐MB‐231 tumor xenograft growth. Baseline mortality rate for vehicle treated mice was 28.6 percent due to metastasis phenotype. We show that the survival curve increased with NO2‐OA dose level, correlating with better survival of TNBC xenograft mice. Moreover, ICAM‐1 and uPA expression levels in NO2‐OA treated tumors are lower compared to vehicle treated tumors. This study serves as a prelude to the clinical study of electrophilic nitroalkenes as therapeutic agents that may display high selectivity for inhibition of TNBC progression and metastasis.
Plasticity of bone marrow‐derived dendritic cells (DC) as immunotherapy vaccines
Authors: Jungsuwadee Paiboon
Published: November 2023

Dependingon microenvironment, dendritic cells (DCs) may activate and differentiate naïveCD4+ T cells into Th1 or Th2 phenotypes when bacterial lipopolysaccharide (LPS)or allergen is present, respectively. The combination of antigens with Toll‐like receptor (TLR) ligands such as LPS enhances DCs activities compared to antigen alone. However, in such combinations, it is not clear to which direction CD4+ T cells will be polarized into. Here, we investigated there sponse of CD4+ T cell to DCs loaded with ovalbumin (OVA) or endotoxin‐depletedovalbumin (OVAED) vs. LPS‐stimulated DCs. Up on exposure to antigens, DCs became matured as indicated by up‐regulation of MHC and costimulatory molecules and increased secretion of IL‐10 and IL‐12. Interestingly, the levels of CD40 expression, and IL‐10 and IL‐12 productions appeared to be correlated with LPS levels. LPS‐stimulated DCs induced naïveCD4+ T cells to produce more IFNγ while OVAED‐stimulated DCs induced T cells to produce more IL‐4. Addition of LPS into OVAED‐stimulated DCs during T cell activation compromised the production of IL‐4. These results suggest that DC maturationcan be polarized into type 1 (LPS‐stimulated) or type 2 (Allergen‐stimulated)DCs influenced by the nature of antigens. Type 1 DCs promote Th1 while Type 2DCs promote Th2 immune responses. This plasticity of DCs provide a flexibility for applications in the development of DC‐based vaccines.
MULTI DYNAMICS MATHEMATICAL FUNCTION AND MATRIXES IN STIMULATION IMMUNE SYSTEM
Authors: Izunobi Felicia Ukamaka , Einstein George P
Published: November 2023

One of the most frequent complications of people with diabetes is foot disorders, specially foot ulcers or wounds. These wounds can easily become infected, and are known as a diabetic foot infections (DFIs). If they are not treated, the infection can progress rapidly, involving deeper tissues and threatening survival of the limb. Sometimes these infections conclude with the affected limb needing to be amputated. It is unclear whether any particular antibiotic is better than any another for curing infection or avoiding amputation.

Materials and Methods

This report focuses on Multi Dynamics Mathematical Function and Matrixes for Infections of diabetic's patients. First Dynamic Mathematical function and Matrixes presented successful G. P. Einstein Sigma Quantum Biophotonics (GPEB) Therapy Model. Second Multi Dynamic Function and Matrixes presented Sigma Quantum Natural Medicine I.F. Ukamaka Model (IFUN). In Multi Dynamics models the independent variables are time and Quantum Absorbed Energy for stimulation the immune System by Biophotonics Spectrum GPEB or Natural Medicine (IFUN).

Results

The research of investigating the marks of the cytokines profile of mononuclear layers of cells that remove infections for patients with diabetic type two. Expression Analysis mRNA (PCR): Complementary DNA test (cDNA) was carried out on synthetic matrix/dye RNA (1μg) by application of Transcriptor First Strand cDNA on Synthetic Kit (Roche). IL‐1, IL‐6, Il‐10 IL‐12 TNF‐α IFN γ.

Conclusion

Multi Dynamics Math Function and Matrixes creates hope to extend the treatments for diabetics type 2 patients with infections resistant to antibiotics

Support or Funding Information

Einstein Medical Institute USA
Sex‐based Differences in the Aortic Function of UCD‐T2DM Rats: A Novel Rat Model of Type 2 Diabetes Mellitus
Authors: Akther Farjana , Shaligram Sonali , Karimi Mujibullah D. , Graham James L. , Stanhope Kimber L. , Havel Peter J. , Rahimian Roshanak
Published: November 2023

UC Davis type 2 diabetes mellitus (UCD‐T2DM) Rat is a validated model of type 2 diabetes mellitus. UCD‐T2DM rats are characterized by polygenic, adult‐onset obesity and spontaneous β‐cell failure and, as a result, more closely models the pathophysiology of type 2 diabetes in humans than other rodent models of the disease. The objective of this study was to investigate alterations in aortic endothelial function in the UCD‐T2DM Rat model and to determine whether there is a sexual dimorphism in diabetes‐induced aortic dysfunction. Endothelium‐dependent vasodilation (EDV) to acetylcholine (ACh, 10−8 to 10−5M) was measured in intact aortic rings pre‐contracted with phenylephrine (PE, 2μM). Endothelium‐independent vasodilation to sodium nitroprusside (SNP, 10−9 to 10−5 M) was assessed in endothelium‐denuded rings pre‐contracted with PE (2μM). Furthermore, constrictor response curves to PE (10−8 to 10−5 M) were generated.

Diabetes significantly impaired relaxation responses to ACh and SNP in aortic rings from female UCD‐T2DM Rats, however, potentiated the relaxation in males. Moreover, diabetes significantly shifted PE contractile responses to the left in aortic rings of both sexes. Both maximal contraction and sensitivity to PE in aortic rings from diabetic females were significantly higher than those in control rats. In males, diabetes did not affect the maximal contraction to PE.

These data, for the first time, show that the vascular function in aortic rings of UCD‐T2DM is altered in both sexes. The decreased sensitivity of vascular smooth muscle to NO along with the enhanced contractile responsiveness to PE may in part contribute to the attenuated relaxation response to ACh in diabetic female rats. On the other hand, an elevated sensitivity of vascular smooth muscle to NO may partially explain the potentiation of ACh responses in diabetic males. The overall results indicate the presence of sex‐based differences in the aortic function of UCD‐T2DM Rats.
The Effects of Sex and Genotype on the Population Pharmacokinetics and Pharmacodynamics Modeling and Simulation of Low Dose Epinephrine and Cardiac Output
Authors: Eugene Andy Roger , Joyner Michael
Published: November 2023

OBJECTIVE

Men and women differ in cardiac output. We hypothesize a sex by genotype interaction may exist during a low‐dose adrenaline infusion affecting the β2 adrenergic receptor.

METHODS

Subjects: Ten males (mean ± SD: age=27.8±6.6, height=178.5±8.0cm, weight=83.9 ±12.4kg, BMI=26.3±2.9kg/m^2, BSA=2.0±0.2) and fourteen females (mean ± SD: age=26.3±5.9, height=164.3±7.0cm, weight=60.8±6.2kg, BMI=22.5±2.1kg/m^2, BSA=1.7±0.1) were studied. Three males were homozygous for Arginine (Arg/Arg) and 7 males were homozygous for Glycine (Gly/Gly) at position 16. In the female cohort, eight females were Arg/Arg and six females were Gly/Gly, at position 16. Procedure: A 1‐hour epinephrine infusion dosed at 5ng/kg/min was administered. BP was measured via an arterial catheter and HR via EKG. Stroke volume and Cardiac output were estimated via Modelflow and TPR was calculated. Plasma samples were collected at time: 0, 30, 45, 60, and 70 minutes and catecholamines measured using HPLC with electrochemical detection. Population Pharmacokinetic‐Pharmacodynamic Analysis: Plasma epinephrine concentrations and cardiac output responses were analyzed using the Nonlinear Mixed Effects (NLME) Stochastic Approximation Expectation‐Maximization (SAEM) algorithm in MATLAB® SimbiologyTM (version 2015a, Mathworks, Natick, MA). The Visual Predictive Check was conducted using the R statistical software (version 3.2.2, The R Foundation for Statistical Computing, Vienna, Austria).

RESULTS

Epinephrine pharmacokinetics were well‐described using a one‐compartment model with an ‘effect compartment’ and elimination from the central compartment. The population mean estimates for the volume of distribution and elimination rate were 22.81 L (CV=27.52%) and 8.77 L/hour (13.00%), respectively. Cardiac Output was modeled using the Hill Stimulation Equation and resulted in mean population estimates of E0=4.94 L/min (13.56%) for baseline, Emax=34.86 L/min (38.61%), and EC50=2486.23 ng/L (CV=5.54%). Bodyweight (mean=70kg) and Sex:Genotypes were the main covariates for the model. A visual predictive check revealed adequate central tendency for the linked PK/PD model. Dosing (300ng/kg/hour for a 70kg individual) simulations resulted in a PK/PD profile stratified for the population mean and Sex:Genotype covariates. Plasma Concentration‐Response curves produced subpopulation (Male Arg/Arg, Male Gly/Gly, Female Arg/Arg, and Female Gly/Gly) differences.

CONCLUSION

Based on the plasma concentration‐response curves, the interaction of sex and genotype influences the ability to increase cardiac output during an infusion of low‐dose epinephrine.

Support or Funding Information

NIH T32 Clinical Pharmacology Fellowship Grant GM008685‐17.