bluuebunny/crossref_metadata_2025 huggingface dataset:
60 Minutes of Tension: The Hour Long Research Talk
Authors: Marcucio Ralph
Published: November 2023

The hour‐long research talk is one of the most coveted talks to be invited to give. While the shorter talks are meant to tell single vignettes, the longer talk is a platform for you to place your work in the perspective of the larger field, and to illustrate your road map to achieve your long‐term goals. Using your time to tell a compelling story is essential to convey the mission of your laboratory, and to highlight your accomplishments and those of your trainees. There are a number of ways to prepare this talk, but they should all start with broad brush strokes about the field that you are working in, and should set the stage for the experiments and data that will follow. An overarching hypothesis can be developed in this section of the talk that is then tested in various ways throughout the talk. Breaking this talk into a series of segments that tell short related stories can be an effective means to deliver your work. Linking these segments with coherent vision is an important consideration in order to keep your audience engaged and focused on your work. An effective way to present your work is to use a literary convention called a story arc. In a story arc a protagonist is introduced and a series of events occur that generate and finally relieve tension. In each segment of the scientific talk you can introduce the specific question being asked, and then use a series of slides to slowly increase the tension as you build toward the most important pieces of data, then you relieve the tension by backing up that key information with supporting data before moving to the next linked story. Summations and transitions between the elements of the talk are essential for ensuring that the audience stays with you along the way. Finally, personal stories of discovery are often compelling because they place work in a humanistic context. Explaining how a post‐doc came to an idea or conclusion illustrates the real process of scientific discovery. There is a person (people) behind your work and telling their stories are often engaging. Sixty minutes of data is boring, telling the story behind your work is not. So, avoid a 60 minute data‐dump, and instead tell the story of your place in science.

Support or Funding Information

NIH: NIDCR: R01‐DE018234, R01‐DE019638, R01‐DE021708, NIA: R01‐AG046282
TGF‐beta induces the expression of Scleraxis in lung fibroblasts from Idiopathic Pulmonary Fibrosis
Authors: Aragon Miguel Ramirez , Oropeza Manuel Alejandro Blancas , Sanchez Fernando Hernandez , Lagunas Alfredo Cruz , Czubryt Michael P. , Milla Criselda Mendoza
Published: November 2023

Background

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and lethal lung disease. Its pathogenesis is not totally understood but is characterized by the expansion of the fibroblast population and excessive extracellular matrix accumulation with the subsequent destruction of the lung architecture. The latter processes are regulated by TGF‐beta by the activation of various transcription factors such as SMAD2/3 or bHLH family members. Scleraxis (SCX) is a transcriptional factor of the bHLH family. It has been reported active during the early stages of embryonic development, but recent evidence demonstrate that SCX participate in fibrotic processes in adults organs like heart and kidneys, promoting Collagen I synthesis, one of the most abundant matrix proteins in IPF. However the role of SCX in IPF is unknown. Therefore, taking into account recent evidence, we hypothesized that SCX can play an important role in the pathogenesis of IPF.

Aim

To determine if Scleraxis have a role in IPF.

Methods

We first examined by qPCR and Western Blot the basal expression of SCX in primary cultures of lung fibroblasts from healthy subjects and IPF patients; and in two transformed human cell lines (A549 and MLE12 derived from human and mice alveolar epithelium respectively). Then we explored the role of TGF‐beta in the activation of SCX. We stimulated epithelial cells and fibroblasts with 10 ng/ml TGF‐beta for 24 hours and examined expression changes in SCX.

Results

We found that SCX is not expressed in normal human lungs fibroblasts while in IPF lung fibroblasts it is expressed in basal conditions and increase after 48 hours from TGF‐beta stimulus. In alveolar epithelial cells SCX showed an increase in the mRNA levels after 24 hours from TGF‐beta stimulus.

Conclusions

Our findings indicate that Scleraxis is present in pulmonary fibroblasts obtained from IPF patients; therefore SCX may participate in IPF.

Support or Funding Information

Posgrado en Ciencias Biologicas, Universidad Nacional Autonoma de Mexico
BLES‐Cath‐2 Intratracheal Administration in an in vivo P. aeruginosa Pneumonia Model
Authors: Nicholson John Michael , Banachewski Brandon , McCaig Lynda , Veldhuizen Edwin , Yamashita Cory , Veldhuizen Ruud
Published: November 2023

Background

Increasing patterns of antibiotic resistance, especially in individuals with chronic lung disease, have mandated the development of novel therapies. Host‐defense peptides HDP such as cathelicidin‐2 (CATH‐2), possess excellent antimicrobial activities in vitro however in vivo efficacy has not been well established. Furthermore, in order to enhance in vivo distribution, a novel approach using CATH‐2 combined with bovine lipid extract surfactant (BLES) as a delivery vehicle was utilized. It was hypothesized that CATH‐2/ BLES would be superior to CATH‐2 alone in decreasing bacterial burden and improving physiologic parameters in an in vivo model of Pseudomonas aeruginosa (PA) infection with mechanical ventilation.

Methods

Adult male Sprague‐Dawley rats were intubated, mechanically ventilated for 30 minutes, followed by an intratracheal inoculum of 8 x 10^8 CFU/mL of PA. One hour following PA administration, rats were randomized to receive an intratracheal administration of either: i) Saline, ii) Air, iii) BLES, iv) Cath‐2, or v) BLES/Cath‐2. Mechanical ventilation continued for three hours during which peak inspiratory pressure (PIP) and arterial blood gases were recorded. At the completion of the ventilation protocol, animals were sacrificed and whole lungs were homogenized and the bacterial burden was assessed by growth analysis of lung homogenates on Cetrimide Agar plates incubated overnight at 37 C. Statistical analysis was performed using a one‐way ANOVA.

Results

Physiological measurements revealed a significant decline of arterial oxygenation (PaO2) in air and saline control groups. At the three‐hour time point, the BLES/Cath‐2 group had a significantly (p < 0.01) higher PaO2 and compared to the Cath‐2 treated animals. Interestingly, the Cath‐2 group had PIP values at 30 minutes following PA instillation that were significantly higher compared to all groups. Colony growth data demonstrated a significant difference between the Cath‐2 treated animals and the control groups; however there was no difference compared to the BLES/Cath‐2 group.

Conclusion

Our findings demonstrate that Cath‐2 has excellent anti‐microbial activity against PA in an in vivo model of pneumonia and mechanical ventilation, although significant reduction in lung function was observed. Cath‐2 alone worsens lung function by possibly causing an overwhelming inflammatory response. Furthermore, CATH‐2/ BLES significantly improved physiological parameters compared to Cath‐2 alone. This suggests that the combination of BLES/Cath‐2 allows for both improved lung function and while maintaining antimicrobial activity. Future analysis includes extension of the time points and lavage inflammatory markers.

Support or Funding Information

Physician Services Inc. and the Canadian Cystic Fibrosis foundation
A Novel Assay to Screen Porcine MHC Class II Antibodies in Potential Xenotransplant Recipients
Authors: Ladowski Joseph M , Butler James , Martens Greg , Reyes Luz , Wang Zheng‐Yu , Tune John , Tector Matt , Tector A. Joseph
Published: November 2023

Background

Well‐elucidated in clinical allotransplantation is the role of recipient's donor specific antibodies and graft rejection, specifically the antibodies targeted at Human Leukocyte Antigen (HLA) class II. To determine whether cross‐reactive or preformed antibodies exist to Swine Leukocyte Antigen (SLA) class II, an assay was developed to screen the patients on the Indiana University (IU) transplant waiting list.

Methods

69 patients on the IU transplant waiting list were selected for study based on their levels of anti‐HLA class II antibodies. Wild‐type pig red blood cells were used to absorb the sera, removing anti‐carbohydrate antibodies and leaving anti‐MHC antibodies. This sera was screened on an immortalized fibroblast cell line transfected with a version of the human class II transactivator (CIITA), producing a constitutively activate SLA class II cell line. A cell that incorporated the antibiotic resistance gene but not the CIITA was chosen as a negative control. Following incubation, the sera was evaluated for presence of IgM and IgG antibody binding by flow cytometry.

Results

Cells lacking expression of SLA class II demonstrated decreased IgM and IgG binding compared to cells with 100% SLA class II DR and DQ expression (Figure 1). Of note are several individuals whose sera preferentially binds the class II positive cell (the dots beneath the diagonal line). The difference in binding between a positive and negative cell is statistically different for IgG (p = 0.0048) but not IgM (p = 0.5102). Increased binding is found when evaluating those individuals with anti‐HLA class II antibodies (Figure 2). The statistical significance holds when comparing the change in binding on the SLA class II positive and negative cells for individuals with and without anti‐HLA class II antibodies for IgG (p = 0.0341) and IgM (p = 0.2602).

Conclusions

Expression of SLA class II results in increased IgM and IgG human antibody binding, demonstrating the presence of human anti‐pig class II antibodies. The existence of this humoral barrier suggests that antibody‐mediated binding to SLA class II may be an issue faced in clinical xenotransplantation.

Support or Funding Information

This publication was made possible in part by Grant Number UL1 TR001108 (A. Shekhar, PI) from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award.
Modeling Chronic Alcohol‐Stimulant Co‐use Shows Dampening of Alcohol Withdrawal Severity
Authors: Nanchanatt Alex J. , Popkin Shayna , Mauterer Madelyn , Rhoads Dennis E.
Published: November 2023

Co‐use of alcohol and stimulants, including caffeine and amphetamine, may be occurring at an all‐time high level of frequency among students, college age and younger. In previous studies, application of the liquid diet approach allowed us to test alcohol‐caffeine co‐administration to adolescent rats and showed that caffeine reduced alcohol withdrawal severity, but only when adolescents were administered caffeine before and then along with alcohol. The first objective of the present study was to test the hypothesis that amphetamine would have a similar dampening effect on alcohol withdrawal severity. Adolescent rats were assigned randomly to one of 4 groups: pair‐fed control, ethanol, ethanol plus amphetamine, and amphetamine alone. At the doses used, amphetamine had no significant effect on alcohol consumption. After 7 days of alcohol consumption and 6 hours of withdrawal, rats showed significant hypoactivity as measured in computer controlled activity chambers. In sharp contrast, inclusion of amphetamine with alcohol resulted in activity measures indistinguishable from pair‐fed controls. Amphetamine alone had no effect on the activity measures during the same period of administration and withdrawal. After an additional 11–14 days of alcohol consumption, a more severe withdrawal syndrome develops with high frequency of induced running and convulsions (withdrawal severity score = 3.9). Inclusion of amphetamine with ethanol under these conditions resulted in a drastic reduction in the alcohol withdrawal severity score to 1.4. This occurred without the pretreatment period required with caffeine but otherwise amphetamine resulted in a corresponding decrease in alcohol withdrawal severity. The dopamine system plays an important role in the actions of both stimulants. Thus, our second objective in the present study was to determine whether changes in dopamine receptor protein expression correlated with the observed behaviors. Membrane fractions were obtained from cortical and subcortical forebrain preparations and levels of dopamine receptor expression were evaluated by Western blotting. The most interesting results were obtained with expression of D1 receptors in subcortical preparations. D1 receptors were down‐regulated by alcohol, up‐regulated by caffeine and not significantly different from pair‐fed controls under conditions where caffeine decreased alcohol withdrawal. These findings parallel behavioral results and, while these may not be the only receptor changes occurring during administration of these drugs, they lead us to suggest that antagonistic effects of alcohol and stimulants at the level of dopamine D1 receptor expression may serve to reduce the severity of alcohol withdrawal.

Support or Funding Information

Supported by the Biology Department of Monmouth University.
The effects of ARVs on ARG‐iNOS pathway in the brain: does the interaction play a role in microglial activation?
Authors: Laudati Emilia , Lucia Lisi , Russo Cinzia Dello , Navarra Pierluigi
Published: November 2023

Neurological complications affect approximately 50% of HIV patients undergoing HAART therapy; the occurrence of these complications may result from a combination of factors, and their underlying molecular mechanisms are not fully elucidated. The central nervous system is an immunological privileged site, representing a sanctuary and a reservoir for HIV‐1. Unlike other viral infections, once in the brain HIV‐1 does not directly infect neurons, but replication is mainly observed within microglia cells and perivascular macrophages. With this in mind, we carried out a screening of different AVRs (Atazanavir, Darunavir, Efavirenz, Nevirapine, Abacavir and Tenofovir) for their potential pro‐inflammatory effects on primary microglial cells. Efavirenz, Neviparine, Darunavir and Atazanavir increase nitric oxide production in microglial cells activated with Gp120CN54 and interferon (IFN)‐γ (a scenario mimicking the late stage of HIV infection), but not iNOS gene expression. In parallel the same drugs are able to inhibit arginase I (ARG) activity. Therefore, we hypothesize that ARVs interact with ARG; the subsequent increase in L‐arginine (a substrate for both ARG and iNOS) in turn modifies iNOS activity. However, when these ARVs are tested on microglial lysates, optimizing the in vitro conditions to assess ARG activity, we observe an effect opposite to that obtained in cell cultures: ARVs significant increase ARG activity. Biochemical approaches are now carried out in our laboratory, attempting to clarify the mechanism underlying such apparently paradoxical effect. Taken together, these data suggest that ARG‐iNOS interplay can be foreseen as an additional target of different ARVs, which may contribute to their pharmacological activity as well as explain, at least in part their neurotoxic potential.
mTOR Pathway Inhibition Prevents Neuroinflammation and Neuronal Death in a Mouse Model of Spinal Cord Injury
Authors: Esposito Emanuela , Cordaro Marika , Paterniti Irene , Impellizzeri Daniela , Siracusa Rosalba , Cuzzocrea Salvatore
Published: November 2023

Mammalian target of rapamycin (mTOR) pathway signaling governs cellular responses, hypoxia and inflammation including induction of autophagy and cell survival. Spinal cord injury (SCI) is a serious and debilitating health problem that usually causes lifelong disability and leads to neurological dysfunction. While the mTOR signaling cascade has been linked to several neurological disorders, manipulation of the mTOR signaling cascade as a pre‐clinical therapeutic strategy in SCI has not been investigated. We hypothesized that mTOR pathway inhibition would diminish neuroinflammation and prevent neuronal death in a mouse model of SCI.

Spinal cord compression injury was performed in adult male mice. SCI was induced in mice by the application of an aneurysm clip at T6‐T7 level. mTOR pathway inhibition was achieved with rapamycin (a mTOR inhibitor; 1mg/kg), or temsirolimus (rapamycin analogues; 0.6mg/kg), or KU0063794 (a dual mTORC1and mTORC2 inhibitor; 8mg/kg) following spinal cord trauma (1 and 6 hours after SCI), and then for 3 subsequent days. Phospho‐activation of the mTOR effectors p70S6kinase and ribosomal S6 protein and expression of infammatory parameters in perilesional area were assayed at 24 hrs, 48 and 72 hrs. Neuronal cell death was evaluated, autophagy was measured using Beclin‐1 and LC3II expression. Iba‐1 labeled, activated microglia were quantified.

Neuronal death, and numerous Iba‐1 labeled, activated microglia were evident at 24 and 48h following SCI. Rapamycin or KU0063794 treatment significantly reduced mTOR signaling, neuronal death, and microglial activation, coincident with enhanced expression of Beclin‐1 and LC3II, markers of autophagy induction. KU0063794 was able to blunt the neuroinflammation (evaluated as iNOS, COX2, GFAP and NOS expression) better than rapamycin and temsirolimus.

mTOR pathway inhibition prevented neuronal death and diminished neuroinflammation in this model of SCI. Persistent mTOR signaling following SCI suggests a failure of autophagy induction, which may contribute to neuronal death. These results suggest that mTOR signaling may be a novel therapeutic target to reduce neuronal cell death in SCI.

Support or Funding Information

No funding
Memory enhancement induced by PDE2 knockdown in an Alzheimer's disease model of mice
Authors: Xu Ying , Chen Zhuoyou , Pan Jianchun , Zhang Han‐Ting , O'Donnell James M.
Published: November 2023

PDE2 is the most prevalent of all PDEs expressed in the hippocampus and frontal cortex, regions involved in cognition and especially vulnerable to damage at early stages of AD. Studies suggest that beta‐amyloid 1‐42 (Abeta 1‐42) has a detrimental effect on memory performance that induces the initial and major symptom of Alzheimer's disease (AD) via inhibition of both cAMP/cGMP‐dependent pathway. Microinfusion of Abeta in animal models leads to Abeta deposition, inflammation, neuroapoptosis and memory impairment. However, whether phosphodiesterase 2‐dependent cAMP and/or cGMP pathway is involved in the neuroinflammation and apoptosis that lead to the progression of AD are unclear. The present study found that intraventricular infusion of Abeta1‐42 into bilateral CA1 subregions led to impairment of learning and memory, as evidenced by an increased latency to find the platform and decreased number of platform crossing in the probe trial of the water maze task and decreased 24‐h retention in the step‐down passive avoidance test. The time period that induced the behavioral abnormalities lasted from 2 weeks to 6 months, with the maximal abnormality at 4 months after microinfusion with Abeta 1‐42. These behavioral abnormalities were blocked by microinfusion with lenti‐PDE2‐miRNA at 5′106 tu/ml. Four months after microinfusion with Abeta were also found to increase in the expression of IL‐6, IL‐10, IL‐17 and Bax. The decreased expression of BCl‐2, PKA, PKG and brain‐derived neurotrophic factor (BDNF) in the hippocampus were also significant in these Abeta1‐42‐treated mice. These effects were reversed by treatment with lenti‐PDE2‐miRNA at 5′106 tu/ml. These findings suggest that inhibition of PDE2 would reverse Abeta‐induced learning and memory deficits at least in part by its regulation of neuroinflammatory and apoptotic processes, which are involved in cAMP/cGMP‐BDNF signaling.

Support or Funding Information

This study was supported by 2014–2015 American Association of Colleges of Pharmacy New Investigator Award and Changzhou Sci&Tech Program Grant (No. CJ20130023).
Opicapone improves the effects of L‐DOPA on the MPTP‐induced Parkinson's‐like syndrome in cynomolgus monkeys
Authors: Bonifacio Maria Joao , Medakkar Sonali , Sousa Filipa , Vivian Jeffrey A , Soares‐da‐Silva Patricio
Published: November 2023

Background

Opicapone is a high binding affinity and long‐acting third generation nitrocatechol COMT inhibitor under development for use as adjunctive therapy in levodopa‐treated Parkinson's disease (PD) patients.

Objectives

The aim of this study was to evaluate the effects of opicapone, as adjunctive therapy to levodopa, in reversing a MPTP‐induced Parkinson's‐like syndrome in cynomolgus monkeys (Macaca Fascicularis).

Methods

Induction of a PD‐like syndrome was performed by daily intravenous administrations of MPTP. Characterization of the animals included scoring with the Primate Parkinsonism Motor Rating Scale (PPMRS) and assessment of locomotor activity.

Results

MPTP produced a stable Parkinson's‐like behavioural syndrome as evidenced by tremor, postural changes, rigidity, impaired movements and balance, (i.e., PPMRS scores between 10 and 15) and decreased locomotor activity (13% of their pre‐MPTP values). Opicapone treatment per se, for 14 days, did not change either the PD‐like symptoms or the decreased locomotor behaviour of the subjects. Ascending combinations of levodopa/benserazide dose‐dependently decreased PPMRS reaching statistical significance for the levodopa/benserazide doses of 18/4.5 mg/kg and that effect was further enhanced in opicapone treated subjects. Similarly, levodopa/benserazide dose‐dependently increased locomotor behaviour, reaching significance with levodopa/benserazide doses of 18/4.5 mg/kg. This increase was also potentiated in the presence of opicapone. Opicapone treated subjects as compared to those treated with vehicle, had significantly increased plasma levodopa levels (2‐fold higher AUC) with no changes in Cmax while for 3‐OMD, AUC and Cmax values were both significantly reduced 7–8 fold. Erythrocyte COMT activity was markedly reduced in opicapone treated subjects.

Conclusions

Opicapone potentiated the improvements in PD‐like symptoms produced by levodopa/benserazide combinations with a concomitant increase in plasma levodopa exposure, reduction of plasma 3‐OMD levels and erythrocyte COMT activity.
Changes in Brainstem Protein Levels During the Development and Decay of Tolerance to Morphine: Focus on PKCγ
Authors: Thompson Benjamin G , McKeel Jackie M , Taylor David A
Published: November 2023

Our hypothesis is that heterologous tolerance following chronic treatment with morphine is an adaptive process mediated by alterations in one or more cellular signaling proteins. This lab has previously characterized a time course for the onset and decay of tolerance following subcutaneous (sc) injection by assessing tolerance using paw pressure testing and the response of the longitudinal muscle/myenteric plexus (LM/MP) to DAMGO and 2‐CADO after various schedules of treatment. Western blot protein analysis in LM/MP homogenates from those animals revealed no change in the number of μ, A1, or A2a receptors, indicating that the tolerance observed in that tissue cannot be explained by a decrease in receptor abundance. Because the brainstem is involved in the analgesic effect produced by morphine, we examined the abundance of several proteins in tissue homogenates obtained from the same animals used for the previous time course study. Western blot analysis of brainstem homogenates also revealed no change in the abundance of μ, A1, and A2a receptors at all time points examined. In addition, the α2b receptor was also largely unchanged. Previous studies have shown that the α3 subunit of the Na+/K+ ATPase decreased as tolerance develops and returned to baseline as tolerance decays in the LM/MP. We therefore determined the abundance of the α1, α3, and β1 subunits of the Na+/K+ ATPase. Unlike results obtained from the LM/MP, we observed no change in any of these subunits in the brainstem. We also examined PKCγ and PKCɛ and observed that PKCγ decreases in concentration as tolerance develops and that this change reverses as tolerance decays. PKCγ is not present in the LM/MP and PKCɛ does not change significantly there. These data indicate that PKCγ may be an important mediator for the development of tolerance in the brainstem and that the cellular mechanism by which tolerance develops may be different between the LM/MP and brainstem.
Alpha‐1D‐Adrenoceptors Lacking Their Carboxyl Terminus Inhibit The MAPK/ERK Pathway in Rat‐1 Fibroblasts
Authors: Alfonzo‐Méndez Marco A. , Castillo‐Badillo Jean A. , Romero‐Ávila M. Teresa , García‐Sáinz J. Adolfo
Published: November 2023

Human alpha‐1D‐adrenoceptors are G protein‐coupled receptors that mediate the actions of adrenaline and noradrenaline participating in the maintenance of homeostasis as well as in the physiopathology of some diseases, such as hypertension and benign prostatic hyperplasia. There is a growing interest in investigating the function of their regulatory domains, such as the carboxyl tail. However, studies focused on the participation of this domain are limited. The objective of this work was to study the role of the human alpha‐1D‐adrenoceptor carboxyl tail in activation of the mitogen‐activated protein kinase pathway. To accomplish this, we assessed changes in extracellular signal‐regulated kinase (ERK) phosphorylation using biochemical and biophysical approaches.

We found that Rat‐1 fibroblast expressing amino and carboxyl termini‐truncated (ΔNΔC) alpha‐1D‐adrenoceptor mutants were hardly able to activate the MAP kinase pathway in response to noradrenaline. Furthermore, expression of this mutant also decreased ERK activation induced by activation of other endogenous G protein‐coupled receptors and receptor tyrosine kinases, i. e., this mutant receptor alters downstream step(s) in the mitogen‐activated protein kinase pathway. As part of the mechanism, we showed that alpha‐1D‐adrenoceptor‐mediated ERK activation is PKC‐dependent. Moreover, we identified the inhibition of two key elements in the pathway downstream PKC: Raf‐1 and MEK1. In addition, we found that over‐expression of the alpha‐1D‐adrenoceptor third intracellular loop cannot mimic the effect of the carboxyl tail truncation, but is enough to activate ERK pathway. Our data indicate that both the alpha‐1D‐adrenoceptor carboxyl terminus and the third intracellular loop play roles in ERK signaling.

Support or Funding Information

This research was partially supported by Grants from PAPIIT‐DGAPA‐UNAM and CONACYT.
No Longer “The Dog That Did Not Bark”: Discovery of Xenobiotic Phosphorylation Metabolism of a TNKS‐Wnt Inhibitor When Dosed To Mice or Incubated In Mouse Whole Blood
Authors: Gu Chungang (Chuck) , Doig Peter , Gangl Eric , Johannes Jeffrey W , Wen Shenghua
Published: November 2023

The phosphorylation of xenobiotics has been deemed impossible and dubbed as “the dog that did not bark” by Andrew Parkinson.1 This was rationalized for a number of reasons, such as limiting consumption of ATP in cells by xenobiotics, avoiding interference with intracellular and intranuclear signaling, and the hypothesis that the phosphorylated metabolites are unlikely to be excreted through the cell membrane if formed in hepatocytes or other cells.

A pyrimidinone nicotinamide mimetic (compound 1: (S)‐2‐(4‐(6‐(3,4‐dimethylpiperazin‐1‐yl)‐4‐methylpyridin‐3‐yl)phenyl)‐8‐(hydroxymethyl)quinazolin‐4(3H)‐one) was identified as selective tankyrase and wnt pathway inhibitor.2 When compound 1 was dosed orally in mice, an unexpected phosphorylated metabolite was formed at the benzyl alcohol. This was observed in mouse plasma with AUC at approximately 5–10% of the parent drug. The phosphorylation showed a temporal profile and a dose response that are consistent with metabolite formation. The metabolite was unambiguously characterized by accurate mass measurement and LC‐MS/MS comparison to the authentic synthetic standard in retention time and fragmentation. Numerous control experiments have been done to rule out analytical, sampling, and dosing errors.

To further confirm this xenobiotic phosphorylation, as well as our 1st step to identify the mechanism, the phosphorylated metabolite was generated after incubation of 10 μM of compound 1 at 37 °C for 2 h in mouse fresh whole blood, fortified with 5 mM ATP and 10 mM MgCl2. Heparin instead of EDTA was used as the anticoagulant to prevent chelation of the Mg2+. Interestingly, the phosphorylated metabolite was not produced in mouse fresh plasma under exactly the same incubation conditions. In addition, this phophorylated‐metabolite was not detected after incubation in a phosphate‐buffered saline solution (+ATP).
High Fat Diet Feeding Alters Expression of Hepatic Drug‐metabolizing Enzymes in Mice
Authors: Ning Miaoran , Jeong Hyunyoung
Published: November 2023

Obesity is prevalent in U.S. that more than one‐third of adults are considered to be obese. Medical conditions accompanying obesity (e.g., metabolic diseases) often require drug therapy. However, whether and how obesity alters the expression of drug‐metabolizing enzyme and thus drug pharmacokinetics is poorly defined. Previous studies have shown that high fat diet feeding and subsequent obesity in mice lead to altered expression of transcriptional regulators for cytochrome P450 (CYP) 2D6, including hepatocyte nuclear factor 4α (HNF4α, transcriptional activator of CYP2D6) and small heterodimer partner (SHP, transcriptional repressor of CYP2D6) (1, 2). The objective of this study is to examine whether diet‐induced obesity represses CYP2D6 expression by modulating HNF4α and SHP expression. To this end, male CYP2D6‐humanized transgenic (Tg‐CYP2D6) mice were fed with western diet (WD, TD.88137, 42% kJ from fat) or matching control diet (TD.08485, 13% kJ from fat) for 18 weeks. qRT‐PCR results showed that hepatic expression of CYP2D6 decreased to a small extent (1.5‐ and 1.2‐fold at mRNA and protein levels, respectively). CYP2D6 activity (tested by using a CYP2D6 probe drug debrisoquine) in S9 fractions prepared from the liver tissues did not differ between the groups. While hepatic SHP expression did not differ between the groups, HNF4α mRNA and protein levels decreased 1.5‐fold in WD group. Considering that HNF4α is a global regulator of hepatic genes including other drug‐metabolizing enzyme genes, we examined the expression of mouse endogenous CYPs (Cyp1a2, 2b10, 2c37, 2d40, 2e1, and 3a11). Cyp1a2, 2c37 and 2d40 showed significant decreases in WD group; Cyp2e1 expression did not differ between groups; and Cyp2b10 and 3a11 expression increased significantly. CYP3A activities (in S9 fractions, determined by using triazolam as a substrate) in WD‐fed mice were 2.9‐fold higher as compared with those in control diet group. Notably, the expression of transcription factors that mediate upregulation of Cyp3a11 and Cyp2b10 (pregnane X receptor and constitutive androstane receptor, respectively (3)) did not increase in WD‐fed group, suggesting that increased concentrations in ligands of these transcription factors might be responsible for Cyp3a11 and Cyp2b10 induction in obese mice. Together, these results suggest that obesity represses CYP2D6 expression potentially through decreased HNF4α expression although the magnitude of the change in CYP2D6‐mediated drug metabolism was small. Obesity had differential effects on mouse endogenous CYPs.

Support or Funding Information

This study was supported by NIH (GM112746 and HD065532)
Induction of Xenobiotic Metabolizing Enzymes and Transporters in Human Intestinal Cells by Pregnancy‐Related Hormones
Authors: Moscovitz Jamie E , Herrera‐Garcia Guadalupe , Guo Grace L , Aleksunes Lauren M
Published: November 2023

The intestine expresses many critical xenobiotic metabolizing enzymes and transporters that regulate oral absorption and fecal excretion. Important nutrients and vitamins are absorbed at this interface, and as a result expression of these enzymes and transporters are tightly regulated. While it is known that gastrointestinal motility decreases during pregnancy, additional molecular changes in intestinal cells have yet to be investigated. To determine whether pregnancy alters intestinal disposition pathways, RNA and protein were collected from human colon adenocarcinoma LS174T cells cultured in media supplemented with 20% serum from either non‐pregnant or pregnant women (weeks 31 to 39 of gestation). In intestinal cells treated with pregnant serum, the mRNA expression of the CYP3A4 enzyme, basolateral efflux transporters OSTα and MRP3, as well as the luminal efflux transporters MDR1, MRP2 and BCRP were induced 2‐ to 4‐fold as compared to cells treated with non‐pregnant serum. MRP3 protein exhibited a 2‐fold up‐regulation when treated with pregnant serum. Profiling of differentially expressed genes (induction of CYP3A4 and MDR1 along with down‐regulation of the SHP transcription factor) suggested that serum from pregnant women is activating the Pregnane X Receptor (PXR). Further mechanistic studies revealed that progesterone and its sulfated metabolite PM5S may be responsible, in part, for expression changes observed in cells treated with pregnant serum. Because MDR1, MRP2 and BCRP are important to the luminal efflux of endocrine disrupting chemicals, such as mycotoxins, perfluorinated chemicals, and pesticides, activation of intestinal PXR by hormones in pregnant serum may be an adaptive response to decrease systemic absorption and in turn, fetal exposure.

Support or Funding Information

F31HD082965, R01ES020522, T32ES007148, P30ES005022
Sex Specific Differences In Key Signaling Pathways Involved In Sympathetic Nervous System Control Within The Dorsal Medulla Of Adult Sheep With Fetal Betamethasone Exposure
Authors: Hendricks Alexa S , Diz Debra I , Shaltout Hossam A , Chappell Mark C
Published: November 2023

Glucocorticoids including betamethasone (BM) are a widely accepted and routine therapy administered to women at risk of early preterm labor to facilitate fetal lung development and reduce infant mortality rates. However, fetal steroid exposure may lead to negative long term consequences for autonomic regulation. In a sheep model of fetal programming, BM‐exposed (BMX) adult offspring exhibit elevated mean arterial pressure (MAP), decreased baroreflex sensitivity (BRS) for control of heart rate and insulin resistance accompanied by dysregulation of the brain, renal and circulating renin‐angiotensin system (RAS). In the brain solitary tract nucleus of the dorsal medulla, BMX dysregulation resulting in a shift towards the sympathetic activator angiotensin II (Ang II) actions through the AT1 receptor that oppose the beneficial actions of angiotensin‐(1–7) [Ang‐(1–7)] at the Mas receptor for BRS regulation. Therefore, we examined RAS and insulin/leptin signaling a pathways in the dorsal medulla to understand further the molecular disturbances that lead to the observed alterations in sympathetic outflow in adult male and female adult sheep (n = 16 sheep; 4 each control or BMX, male or female).

A protein array for kinases (PathScan® Intracellular Signaling Array Kit, Cell Signaling) and subsequent verification by Western blot hybridization revealed that female BMX sheep exhibit lower expression of proteins in the PI3 kinase pathway (pGSK3β: 33±0.10%, pAkt: 22±0.33%; p = 0.03) which are known to be related to regulation of metabolic function and blood pressure. In contrast, in male BMX sheep the PI3 kinase pathway components are not different from control, but there is a trend (p = 0.11) for higher expression of phosphorylated MAP kinase pathway protein ERK (37%) and p38 (68%), known to increase sympathetic activation and facilitate insulin actions. Future studies will explorethe potential mechanism for the lower PI3 Kinase related pathway in female sheep. We conclude that in utero BMX promotes an imbalance in adult brain medullary RAS and insulin signaling pathways known to influence sympathetic regulation with different patterns observed in male and female sheep.

Support or Funding Information

Support: HD47584
Modulation by NADPH Oxidase/Rho‐Kinase Oxidative Pathway of Chronic Cyclosporine‐ Diclofenac Hemodynamic Interaction in Female Rats
Authors: El‐Mas Mahmoud M , Ibrahim Karim S , El‐Yazbi Ahmed F , El‐Deeb Nevine M , El‐Gowelli Hanan M
Published: November 2023

We recently reported contrasting effects for nonsteroidal antiinflammatory drugs (NSAIDs) on the hypertensive effect of acutely administered cyclosporine (CSA) in female rats. CSA hypertension was ameliorated or exaggerated after selective cyclooxygenase‐2 (COX2) and nonselective COX1/COX2 inhibition, respectively. Here we investigated (i) whether a similar interaction could be seen when drugs were administered chronically, (ii) effect of interacting drugs on left ventricular (LV) and cardiac autonomic and histologic profiles, and (iii) role of the NADPH oxidase/Rho‐kinase pathway in the interaction. Rats were pre‐instrumented with femoral indwelling catheters and treated for 10 days with CSA (25 mg/kg/day), diclofenac (nonselective NSAIDs, 1 mg/kg/day), celecoxib (COX2 inhibitor, 10 mg/kg/day), or their combinations. CSA hypertension was maintained upon concurrent administration of either NSAID whereas the concomitant reductions in the time‐ and frequency‐domain indices of heart rate variability were accentuated in presence of diclofenac but not celecoxib. The isovolumic relaxation constant (Tau, τ), a measure of cardiac diastolic function, was similarly reduced by single or combined drug regimens whereas LV contractility (dP/dtmax) remained unaffected. Histopathologically, compared with CSA or CSA/celecoxib treatment, the CSA/diclofenac regimen caused more extensive cardiac structural damage (ischemic necrosis and edema). The co‐administration of diphenyleneiodonium (NADPH‐ox inhibitor), but not fasudil (Rho‐kinase inhibitor), reversed the CSA/diclofenac‐evoked (i) increases in MAP, (ii) decreases in LF/HF and Tau, and (iii) cardiac structural damage. Overall, female rats treated chronically with CSA plus diclofenac exhibit exacerbated hemodynamic, autonomic, histopathologic, and LV functions via Rho‐kinase‐independent upregulation of the NADPH oxidase pathway.

Support or Funding Information

Supported by the ALEX‐REP Grant Fund, Alexandria University, Egypt (Grant HLTH‐13‐01).
MSC Exosomes Deliver Cardioprotective miR‐21
Authors: Luther Kristin , McGuinness Michael , Haar Lauren , Xu Huan , Chen Jenny , Medvedovic Mario , Jones W. Keith
Published: November 2023

Introduction

Mesenchymal stem cell (MSC) transplantation has been shown to improve outcomes including infarct size in animal models of myocardial infarction (MI), but the mechanism of the beneficial effects is not well understood. Much data supports that it involves direct protection of the ischemic myocardium via paracrine transfer of factors from the MSC to cardiac myocytes. If these factors can be identified, they can provide new opportunities for therapeutic development. Exosomes are small vesicles that contain miRNA, which can be exchanged between numerous different types of cells. The objective of this study was to examine the role of miRNA in paracrine effects mediated by MSC exosomes.

Hypothesis

Our study addresses the hypothesis that exosomal miR‐21 is a mediator of protection in MSC therapy.

Methods and Results

In vitro, exosomes dose dependently decreased cell death (39% reduction at maximum) in an oxygen‐glucose deprivation model of ischemia‐reperfusion injury. To determine whether cardioprotective miRNAs are found within MSC exosomes, their RNA was isolated, and miRNA‐Seq was performed. The most highly expressed exosomal miRNA was found to be miR‐21, which is known to be upregulated by numerous cardioprotective stimuli. To determine the contribution of miR‐21 in exosome‐mediated protection, we cultured MSCs from miR‐21− mice, and compared their exosomes to those of wild‐type MSCs in vitro and in a mouse model of MI. We found that the miR‐21− exosomes were significantly less protective than wild‐type exosomes. To determine the gene regulatory mechanism behind the observed protection, we investigated the expression of miR‐21 target genes in H9c2 cells, and found significant downregulation of Programmed Cell Death 4 and Fas ligand.

Conclusions

MSC exosomes are protective against ischemia‐reperfusion injury in vitro and in vivo, and they contain miRNAs that influence expression of proteins in cell death pathways. The data support a key role for miR‐21 in these protective effects and aid in the understanding of how this mechanism could be translated into cardioprotective therapies.

Support or Funding Information

This research was supported by the National Heart, Lung, and Blood Institute grant # 5R01HL091478‐07.
Human Perivascular Adipose Tissue Contains an Adrenergic System
Authors: Ayala‐Lopez Nadia , Pereira‐Hicks Cristiane , Burnett Robert , Fink Greg D. , Watts Stephanie W.
Published: November 2023

Perivascular adipose tissue (PVAT), fat tissue that wraps most blood vessels of the body, can alter vascular tone through various mechanisms. Our team discovered a source of a releasable pool of catecholamines in PVAT from the rat but the relevance to human physiology is unknown. Increased adipose tissue mass is observed in obesity. Sympathetic activity is also increased in obesity‐associated hypertension. Thus, the link between obesity and hypertension could be the PVAT adrenergic system. PVAT may affect vascular tone negatively in disease leading to hypertensive states. We hypothesized that similar to the rat, human PVAT would contain an adrenergic system that could synthesize and store catecholamines. Tissue was collected from patients (body mass index= 43.58±3.23, age range 29–58; mean age= 41.56±3.62, 8 females and 1 male) during elective bariatric surgery and placed into physiological saline solution (PSS). Splanchnic blood vessels with associated PVAT were dissected from the tissue and snap frozen for HPLC analysis or saved for immununohistochemistry (IHC). Dopamine (DA) and norepinephrine (NE) were quantified by HPLC in human mesenteric artery PVAT. NE was the most abundant catecholamine with concentrations of 14.63±1.39 ng/g NE vs. 0.80±0.09 ng/g DA (N=9). Two key enzymes in catecholamine synthesis; tyrosine hydroxylase and dopamine β‐hydroxylase, were located to PVAT adipocytes by IHC (N=9). In order for catecholamines to be released, a storage reservoir that holds catecholamines may be present in PVAT. IHC for the vesicular monoamine transporter 2 (VMAT2) indicated the presence of the transporter in the adipocyte cytoplasm (N=9). Negative controls were performed without the inclusion of the primary antibody and these were not positive. These data indicate that human PVAT contains elements of an adrenergic system complete with capabilities of synthesis and storage of catecholamines. Sympathetic activity of an adrenergic system located within PVAT may effect vascular tone in a balance that could be altered in disease to promote contraction. Future studies aimed to understand the differences in the PVAT adrenergic system of obese hypertensives compared to obese non‐hypertensives are necessary to address the question as to why a subset and not all obese people develop hypertension.

Support or Funding Information

NIH NHLBI P01HL70687

IPSTP 5 T32 GM 92715‐4

NIH NRSA F31 HL128035‐01
Effect of Ang III on JNK mitogen activated protein (MAP) kinase in Wistar rat VSMCs
Authors: Alanazi Ahmed Z. , Clark Michelle A.
Published: November 2023

Objective

We investigated whether angiotensin (Ang) III induces JNK mitogen activated protein (MAP) kinase protein phosphorylation in isolated rat vascular smooth muscle cells (VSMCs). Moreover, we compared its effect with Ang II.

Background

The molecular mechanisms by which Ang III induces various biological effects have not been fully investigated. Most studies have shown that MAP kinases mediate Ang II apoptosis and growth promoting effects in VSMCs. In addition, Ang II induces vascular remodeling in these cells leading to increases in blood pressure. MAP kinases regulate or induce two crucial actions in VSMCs, proliferation and migration, which are associated with atherosclerosis and restenosis.

Methods

Primary cultures of VSMCs were isolated from the thoracic aorta of adult Wistar rats by the explant technique. VSMCs were treated with Ang II and Ang III ranging in concentration from 0.1 nM to 1000 nM for 10 minutes or with 100 nM Ang II and Ang III for 1 minute to 60 minutes. Western blotting technique was used to determine the effects of both peptides on JNK MAP kinase protein phosphorylation.

Results

Concentration studies showed that Ang II and Ang III caused a dose‐dependent increase in JNK MAP kinase protein phosphorylation. The effects of the peptides on this MAP kinase phosphorylation were maximal between 10 nM and 100 nM. The peptides’ effects were rapid and significant, occurring within minutes of treatment and the maximal effects on MAP kinases phosphorylation was observed by 10 minutes.

Conclusion

These findings provide insight into the molecular nature of the actions of Ang III and offer possible mechanism by which Ang III physiological actions occur in VSMCs.

Support or Funding Information

Funding for this project was provided through: Nova Southeastern University Health Professions Division Grant & Saudi Arabian Cultural Mission.
TBX20 Regulates Endothelial Angiogenesis and Migration
Authors: Meng Shu , Wong Wing Tak , Cooke John P
Published: November 2023

Background

T‐box 20 (TBX20) is a crucial transcription factor (TF) for heart development. It is expressed in myocardium and endocardium. In previous work, our lab has identified TBX20 as a key transcription factor for endothelial differentiation. In this study, we investigated the role of TBX20 in mature endothelial function.

Methods

Gain‐ and loss‐of‐function studies were performed in human umbilical vein endothelial cells (HUVEC) with overexpression (OE) and knock down (KD) of TBX20 respectively. Lentiviral shRNA targeting TBX20 gene was used to KD studies while lentiviral constructs encoding open reading frame of TBX20 were used for overexpression. Gene expression and protein level were examined by RT‐PCR and western blot. Endothelial angiogenesis was examined by tube formation assay. Angiogenesis PCR array of 84 genes was used to screen the potential angiogenesis related molecular targets. ELISA assay was used to measure secreted protein level. Migration assay and BrdU incorporation assay were used for migration and proliferation analysis.

Results

TBX20 KD in HUVECs resulted in 50% reduction in both TBX20 mRNA and protein level. KD of TBX20 significantly impaired tube formation compared with ECs transfected with control shRNA. After screening of 84 potential angiogenesis related molecular targets, hepatocyte growth factor (HGF) gene expression was found downregulated by TBX20 KD. The secreted HGF level of TBX20 KD cells was found only half of the level of CT KD cells detected by ELISA. Further, supplement of exogenous HGF reversed the impaired angiogenesis of TBX20 KD cells. TBX20 KD also impairs endothelial migration (mean migration distance 442 μm vs CT 569 μm) through downregulation of PROK2 gene. TBX20 OE upregulates PROK2 to 20 fold and significantly promote migration (mean migration distance 729.6 μm vs CT 441 μm).

Conclusion

TBX20 is a crucial transcription factor to maintain endothelial angiogenesis and migration. TBX20 regulates angiogenesis through HGF secretion. TBX20 promotes endothelial migration partially through PROK2.