bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vitro Systems Toxicology assessment of acute exposure of an aerosol from a candidate modified‐risk tobacco product on organotypic human small airways tissue culture
Authors: Martinez Yannick , Iskandar Anita , Leroy Patrice , Martin Florian , Sewer Alain , Majeed Shoaib , Trivedi Keyur , Guedj Emmanuel , Torres Laura Ortega , Mathis Carole , Frentzel Stefan , Ivanov Nikolai , Peitsch Manuel , Hoeng Julia
Published: November 2023

The primary site of morphological changes in the airways associated with chronic cigarette smoking is the small airway epithelium. The small airway refers to bronchi of less than 2 mm in diameter. It is the initial site of Chronic Obstructive Pulmonary Disease (COPD). Mechanistic investigations of the effect of inhaled substances on the small airway tissue are difficult to conduct because biopsy samples are not readily obtainable. Recently, a human small airway epithelium culture model grown at the air liquid interface, SmallAir™ (Epithelix, Switzerland) was established. The model is reconstituted from primary small airway epithelial cells isolated from the distal lungs. The morphology of the model mimics the characteristics of in vivo human small airway epithelial tissue. It offers a relevant alternative for an in‐vitro toxicity assessment of inhalation exposure. Using the newly developed human organotypic small airway model (reconstituted from a healthy, non‐smoker donor), this study assessed the biological impact of an aerosol generated from a candidate modified risk tobacco product, the tobacco heating system 2.2 (THS2.2). The biological impact of the THS2.2 aerosol was compared with that of the smoke generated from the reference cigarette 3R4F at comparable nicotine concentrations. A comprehensive set of biological endpoints were measured at various time points after exposure including cytotoxicity, culture histology, cytochrome P450 (CYP) 1A1/1B1 activity, secreted pro‐inflammatory mediators concentrations, and global mRNA/miRNA alterations. Following a 28‐min exposure to 3R4F smoke, various inflammatory markers (MMP‐9, IL‐6 and IL‐8) and genes (MGST2, GPX2, AKR1B1) known to be involved in the pathogenesis of COPD were upregulated. A significantly lower impact of THS2.2 aerosol exposure was observed not only on these COPD‐related molecular entities, but also on cytotoxicity, secreted pro‐inflammatory mediators, and global alterations of mRNA and miRNA as compared with those of 3R4F smoke at comparable nicotine concentrations. Using a network‐based Systems Toxicology approach, the gene expression profile 4 h after 3R4F smoke exposure at 0.15 mg nicotine/L elicited the highest biological impact (taken as 100% impact). By comparison, the impact of THS2.2 aerosol exposure at comparable nicotine concentration was only 14.6%. Collectively, this study shows that the biological impact of THS2.2 aerosol was significantly lower than that of 3R4F smoke at similar nicotine concentrations on the in vitro small airway cultures.

Support or Funding Information

PMI R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, CH‐2000 Neuchâtel, Switzerland (Part of Philip Morris International group of companies).
MSC‐Derived Exosomes Deliver Cardioprotective miR‐21
Authors: Haar Lauren , Luther Kristin , Phan Ahn , McGuinness Michael , Zilliox Michael , Wang Yang , Jones W. Keith
Published: November 2023

Mesenchymal stem cell (MSC) transplantation has been shown to improve outcomes after myocardial infarction (MI) in animal models and in man. Specifically, reduction of infarct size (cardioprotection), increased LV muscle, improved function, and preserved ejection fraction have variously been reported. However, the mechanism of beneficial effect is not well understood. While some data supports transdifferentiation of MSC into myocardial constituent cells, other results support direct cardioprotection against ischemia/reperfusion injury (IRI). Overall, there is a growing consensus that the MSC and their derivatives do not contribute in proportion to the extent of the reparative effects, supporting the concept of paracrine effects of MSC. If paracrine factors can be identified, they might be employed therapeutically and contribute to identification of new therapeutic targets. Several recent results, including ours, support that exosomes, and their microRNA (miR) cargo, underlie at least some of these paracrine effects. Our objective was to test the hypothesis that MSC‐derived exosomes from non‐treated MSC can mediate cardioprotection via specific miR cargo. To test this hypothesis, we identified by next generation RNA sequencing, the miRs present in exosomes isolated from murine MSC. We identified both protective and injurious miRs, and focused upon miR‐21, the most highly expressed miR in MSC derived exosomes. We also identified miRs associated with apoptotic signaling, cell proliferation, angiogenesis, and heat shock response. We report in vitro data that the exosomes and miR‐21 are protective in a dose‐dependent manner against sim I/R. We next showed in vivo that exosomes were cardioprotective, reducing infarct size (normalized for risk region) by 68%, (P≤0.01 vs. control) and that exosomes isolated from MSC having miR‐21 deleted reduced infarct size by only 28% (P=0.039 vs. control), demonstrating a major role for miR‐21. In vivo, the exosomes increased miR‐21 levels in myocardium, resulting in significant depression of four target protein levels, PDCD4, Peli1, FasL, and PTEN. These results support that suppression of cell death is by coordinated reduction of several parallel cell death pathways, and derepression of pro‐survival Akt. In conclusion, our data supports that the cardioprotective aspects of MSC are explained, at least in part, by secretion of exosomes and the action of the miR‐21 cargo. Whether other aspects of MSC action are regulated by other of the miR cargo is under study.

Support or Funding Information

NIH R01HL091478
Angiopoietin‐2 levels in the risk stratification and mortality outcome prediction of sepsis associated coagulopathy
Authors: Statz Steve , Walborn Amanda , Williams Mark , Hoppensteadt Debra , Mosier Michael , Rondina Matthew , Fareed Jawed
Published: November 2023

Background

Angiopoietin‐2 (Ang‐2) is a 70 kDa glycoprotein which plays multiple roles in angiogenesis, inflammation, and vascular development. Ang‐2 is upregulated in vascular stress, and circulates at high levels in certain pathologic conditions such as sepsis. Ang‐2 upregulation may be related to the severity of sepsis associated coagulopathy (SAC), and therefore it may have a predictive role in the clinical outcome of this syndrome.

Aim

The aim of this study is to measure Ang‐2 levels in a defined cohort of patients with sepsis and suspected disseminated intravascular coagulation (DIC), to compare the circulating levels of this biomarker to controls, and to demonstrate its predictive value for the clinical outcome and severity of SAC.

Methods

Plasma samples were collected from 102 patients with sepsis and suspected DIC at ICU admission and immediately frozen at −80°C for batch analysis. Control plasma represented 50 normal samples which were commercially obtained from George King (Orland Park, KS). Ang‐2 levels were quantified using a sandwich ELISA method (R&D Systems, Minneapolis, MN, USA). This method employs a monoclonal antibody specific for human Ang‐2. DIC scores in all 102 patients were evaluated using the ISTH scoring algorithm.

Results

Table 1 shows that in comparison to normal levels, the samples collected from all patients showed significantly higher levels of Ang‐2 for all groups (p=0.0005). Table 2 shows that the Ang‐2 levels were also significantly different between the survivors and non‐survivors for 28‐day mortality outcome by the Mann‐Whitney test (p=0.001).

Conclusion

This study demonstrates that Ang‐2 levels are significantly upregulated in SAC, and this biomarker can be used to risk stratify the severity of this disease. Ang‐2 level can also be used to differentiate non‐overt and overt DIC. Furthermore, Ang‐2 level at the time of initial diagnosis provides a predictive biomarker for mortality outcome.

Ang‐2 levels in patients with Sepsis and Suspected DIC Broken Down by ISTH DIC Score

Category
Normals
+ Sepsis−DIC
+ Sepsis + Non‐Overt DIC
+ Sepsis + Overt DIC

ISTH DIC Score
N/A
0–2
3–4
≥ 5

n
50
20
57
24

Ang‐2 Mean ± SEM (ng/mL)
1.87 ± 0.15
8.34 ± 2.50
11.53 ± 1.40
29.44 ± 6.27

Ang‐2 Std. Deviation (ng/mL)
1.07
11.19
10.54
30.73

Significant Difference in Ang‐2 Level Between Survivors and Non‐Survivors

Survivors
Non‐Survivors

n
86
15

Ang‐2 Mean ± SEM (ng/mL)
12.54 ± 1.54
30.17 ± 8.62

Ang‐2 Std. Deviation (ng/mL)
14.28
33.39
De novo Thymidylate Biosynthesis and dUTPase Interact in DNA Synthesis
Authors: Chon James , Gondokusumo Jabez C , Field Martha S , Stover Patrick J
Published: November 2023

Impaired folate mediated one‐carbon metabolism is implicated in several deleterious health outcomes including neural tube defects, neurodegeneration, and colon cancer. Folate is a cofactor for de novo thymidylate biosynthesis. The de novo thymidylate biosynthesis enzymes serine hydroxymethyl‐transferase (SHMT1), dihydrofolate reductase (DHFR), thymidylate synthase (TYMS), and methylene tetrahydrofolate dehydrogenase (MTHFD1) are SUMOylated, translocate to the nucleus, and form a complex at sites of DNA replication and repair. The nuclear de novo thymidylate biosynthesis complex is unique to mammalian cells and does not form in yeast. Impaired de novo thymidylate biosynthesis leads to increased uracil incorporation into DNA in cell culture models and in mice, subsequently leading to repair‐mediated DNA damage and cell death. In this study, we demonstrate that disruption of de novo thymidylate biosynthesis via SHMT1 CRISPR knock‐out in HeLa cells does not increase uracil levels in DNA. This suggests de novo thymidylate biosynthesis may not be a major determinant of uracil incorporation into HeLa cell nuclear DNA. We also show that dUTPase is a key regulator of uracil incorporation in HeLa cells, and that dUTPase physically interacts with SHMT1 and is a major source of dUMP substrate for de novo thymidylate biosynthesis. These data indicate that dUTPase‐catalyzed dUTP degradation and de novo thymidylate biosynthesis complex interact during DNA synthesis and repair to limit uracil accumulation in DNA.

Support or Funding Information

National Institutes of Health Grants DK58144 and HD059120
Simultaneous knockdown of toll‐like receptors 2 and 4 attenuates the severity of post‐stroke inflammation and brain damage
Authors: Nalamolu Koteswara Rao , Chelluboina Bharath , Smith Nathan J , Klopfenstein Jeffrey D , Pinson David M , Wang David Z , Veeravalli Krishna Kumar
Published: November 2023

Toll‐like receptor 2 (TLR2) and TLR4 belong to a family of highly conserved pattern recognition receptors and are well‐known upstream sensors of signaling pathways of innate immunity. Evidence suggests that the induction of TLR2 and TLR4 by endogenous molecules after ischemic stroke contributes to the brain damage. Also, TLR2 and TLR4 upregulation in stroke patients is associated with poor stroke outcome. Recent reports indicated that the genetic deletion of TLR2 or TLR4 is neuroprotective in stroke‐induced animals, but few models have sought to demonstrate this neuroprotection in a therapeutically relevant manner. Therefore, we hypothesize that the pharmacological suppression of TLR2 and TLR4 alone and in combination in stroke‐induced wild‐type animals attenuates the severity of post‐stroke inflammation and brain damage. Male Sprague‐Dawley rats were obtained and randomly assigned to various groups. Rats were subjected to a two‐hour middle cerebral artery occlusion procedure followed by reperfusion. To achieve the specific knockdown of TLR2 and/or TLR4, plasmids expressing shRNAs specific to TLR2 or TLR4 formulated as nanoparticles were administered immediately after reperfusion to various groups of rats intravenously via tail vein at a dose of 1 mg/kg body weight. Untreated and treated stroke‐induced rats along with controls were sacrificed at various post‐reperfusion time points. We performed various techniques such as TTC staining, real time PCR, immunoblot and immunofluorescence analysis in order to investigate the severity of post‐stroke inflammation and brain injury. Our results demonstrated huge upregulation of TLR2 and TLR4 in the ischemic brains of stroke‐induced rats. The expression of TLR2 and TLR4 downstream signaling molecules including the proinflammatory mediators such as IL‐1β, IL‐6, and TNFα was increased until seven days post‐reperfusion, the maximum post‐reperfusion time point tested. shRNA‐mediated TLR2 or TLR4 suppression significantly attenuated the infarct size. Additionally, the extent of ischemic brain injury is significantly inhibited with the simultaneous suppression of TLR2 and TLR4 as compared to their individual treatments. Based on our results, we conclude that the reduction of severity of TLR2‐ and TLR4‐mediated post‐stroke inflammation attenuates ischemic brain damage and can be achieved using shRNA targeting techniques.
Involvement of Tenascin‐R in Post‐Stroke Brain Damage
Authors: Nalamolu Koteswara Rao , Klopfenstein Jeffrey D , Pinson David M , Wang David Z , Veeravalli Krishna Kumar , Chelluboina Bharath
Published: November 2023

Tenascins represent an important class of extracellular matrix proteins that have a clear ability to regulate cell behavior, and display highly restricted dynamic patterns of re‐expression following a vascular injury. Tenascin‐R (TNR) is reported to be involved in regulating the action potential and the conduction of neurons during neuronal excitability. TNR serum levels were upregulated in various CNS diseases and its acute upregulation led to neuronal apoptosis, blood brain barrier disruption and neuroinflammation. Further, the functional recovery was improved in TNR knockout mice subjected to spinal cord injury. The main objective of this study is to elucidate the possible role of TNR in the pathogenesis of brain injury after ischemic stroke. Male Sprague‐Dawley rats were obtained and randomly assigned to various groups. Rats were subjected to a two‐hour middle cerebral artery occlusion procedure followed by reperfusion. Rats were sacrificed at various time points after reperfusion and the ischemic brains were analyzed for TNR expression by immunoblot analysis. To achieve the specific knockdown of TNR, plasmids expressing shRNA specific to TNR and formulated as nanoparticles were administered immediately after reperfusion to the appropriate groups of rats intravenously via tail vein at a dose of 1 mg/kg body weight. We performed TTC staining procedure on the coronal brain sections obtained from various groups to evaluate the extent of brain damage. Our results indicated a significant upregulation of TNR at all the post‐reperfusion time points tested. Further, TNR suppression by gene silencing technology revealed a significant reduction in infarct size indicating TNR's detrimental role in brain damage after ischemic stroke. Our results clearly indicate a possible role for TNR in post‐stroke pathogenesis.

Support or Funding Information

We would like to thank OSF Illinois Neurological Institute for the financial support.
The Changes of the Dopaminergic D1 Cell after Stimulation by STN‐DBS in Parkinson's Disease Rats
Authors: Duan Hubin , Hao Chunyan , Li Hao , Wang Suping , Zhang Ce , Fan Yimin , Liu Yueting , Pei Mingyang , Wang Huan
Published: November 2023

Objective

To investigate the changes of the dopaminergic D1 cell after stimulated by subthalamic nucleus deep brain stimulation (STN‐DBS) in Parkinson's Disease (PD) rats.

Methods

55 healthy male SD rats were randomly divided into normal control group (n=15)、 PD group (n=15)、 sham stimulation group (n=15) and stimulation group (n=15). The apomorphine rotation was observed as the symbol of successful PD model. Stimulation and sham stimulation groups were planted in unilateral STN‐DBS. STN‐DBS in stimulation group was powered on and was stimulated consecutive days at the regular time, while Sham stimulation group complied with the same program except power on the STN‐DBS. The latency to find the platform, swimming speed, swimming distance and crossing times were detected with Morris water maze. Immunohistochemistry was used to observe the change and count the dopaminergic D1 receptor cells in hippocampal C1 area and prefrontal cortex.

Results

After stimulated by STN‐DBS regularly for 7 days, the symptoms of PD rats were improved significantly after stimulated by STN‐DBS. Compared with PD model group and sham stimulation model group, after trained, not only the escape latency of rats in the DBS stimulated model group was decreased (P < 0.01), but also swimming distance (P < 0.05)in the plate quadrant was increased. The dopaminergic D1 receptor cell of hippocampal C1 area and prefrontal cortex in stimulation group increased by compare with that in PD model group and sham stimulation group, furthermore, which there were statistical significance.

Conclusion

STN‐DBS stimulation not only can improve the ability of PD rats' cognition, but also can protect dopaminergic receptor cells in some degree.

Support or Funding Information

This study was supported by the National Natural Science Foundation Youth Fund (Fund No. 30600637), China Postdoctral Foundation(Fund No.2014M561207), the Shanxi Province Basic Research Program Youth Science and Technology Research Fund (Fund No. 2010021034‐4), the Shanxi Scholarship Council of China (Fund No. 2011‐096 and Key Projects No.4), The science and technology research projects of health department of Shanxi Province(Fund No.2016D011119).
Chemical cross‐linking of doxorubicin to synthetic oligonucleotides by formaldehyde
Authors: Medina Juan , Nagesetti Abhignyan , McGoron Anthony J , Leng Fenfei
Published: November 2023

Anthacycline antibiotics, such as doxorubicin and daunorubicin, are among the most effective anti‐cancer drugs that treat various cancers. Previous research has shown that doxorubicin and daunorubicin were efficiently cross‐linked to double‐stranded (ds) DNA in the presence of formaldehyde in a regioselective and base specific manner. In an effort to further explore the cross‐linking of anthracycline antibiotics to DNA, doxorubicin is crosslinked to a variety of synthetic oligonucleotides including sequence specific ds and single stranded (ss) DNA oligomers. Our results demonstrate that doxorubicin is able to efficiently crosslink to the N2 amino group of guanine for the ds DNA oligomers. Intriguingly, we also discovered that doxorubicin is able to crosslink to ssDNA and ssRNA specifically to guanine residues by formaldehyde. These results suggest that ssDNA and ssRNA may serve as a new delivery system for the treatment of cancer patients by doxorubicin and daunorubicin.

Support or Funding Information

1R15GM109254‐01A1; R25GM061347
Elevated Circulating CD40‐L in Atrial Fibrillation and Its Modulation by Ablation
Authors: Otto Abigail McKenzie , Fareed Jawed , Statz Stephen , Walborn Amanda , Liles Jeffrey , Hoppensteadt Debra , Rowe Tim , Jabati Sallu , Syed Mushabbar
Published: November 2023

Introduction

Atrial fibrillation (AF) is currently the most common cardiac arrhythmia encountered in clinical practice and a major cause of morbidity and mortality among adults. Inflammation plays a crucial role in the development and progression of atrial fibrillation. CD40‐Ligand (CD40‐L) is a cellular mediator that is characteristically seen in inflammatory processes. CD40‐L can be expressed on platelets, leading to activation of endothelial cells and the release of cytokines during inflammation. The purpose of this study was to characterize and quantify the levels of the inflammatory biomarker CD40‐L to evaluate its relevance to AF both pre‐ and post‐ablation.

Methods

In a prospective study, plasma samples were collected from 55 AF patients at baseline prior to ablation, and at 1 and 3‐month follow‐up post‐ablation. Normal human plasma samples (n=50) were supplied by George King Biomedical Inc. (Overland Park, KS). ELISA kits were obtained to profile CD40‐Ligand (R&D Systems, Minnesota USA). Statistical analysis was performed using non‐parametric Mann‐Whitney t‐tests.

Results

There was a significant increase in the mean levels of CD40‐L at pre‐ablation baseline in patients with AF compared to normal (401.0±73.3 pg/mL vs. 53.6±4.74 pg/mL, p=0.0034). CD40‐L levels post‐ablation significantly decreased. At 1 month follow up, CD40‐L levels dropped (401.0±73.3 pg/mL vs. 40.6±6.86 pg/mL, p=<0.0001). At 3 months, the levels of CD40‐L remained decreased (43.3±7.98 pg/mL, p=<0.0001). There was no significant change between levels at 1 month and 3 months (p=0.8573). There was also no significant difference in post‐ablation levels of CD40‐L compared to normal human plasma levels (p=0.2095).

Conclusion

Inflammation plays an important role in the pathogenesis of atrial fibrillation. In our study, patients with atrial fibrillation have highly elevated levels of CD40‐L compared with normal healthy individuals. Contrary to previous reports, our study suggests that elevated levels of CD40‐L may play an important role in the pathogenesis of AF. It is also noted that the levels of this inflammatory mediator are modulated by atrial ablation as a medical treatment for AF.

Support or Funding Information

This project is supported by internal funds of Loyola University Medical Center.
Base unpairing at the flap junction controls the rate of FEN1‐catalyzed cleavage of DNA during replication and repair
Authors: Song Bo , Hingorani Manju M.
Published: November 2023

Flap Endonuclease 1 (FEN1) recognizes and cleaves 5′ single‐stranded DNA flaps to create nicked duplex products during DNA metabolic processes such as Okazaki fragment maturation, long‐patch base excision repair and telomere maintenance, and therefore has an essential role in genome duplication and stability (1). Defects in human FEN1 are associated with cancer and other genetic diseases (2). Hence, there is considerable interest in hFEN1 as a therapeutic target and diagnostic or prognostic biomarker (3–5), which can be aided by a detailed mechanistic understanding of the enzyme.

Previous studies have shown that hFEN1 bends its DNA substrate, and suggest that unpairing of two bases in the duplex beyond the 5′ flap helps position the scissile phosphate in the active site for cleavage (6,7). These studies have largely been performed under equilibrium conditions and in the absence of Mg2+, which is indispensible for catalysis. To study the hFEN1 mechanism under catalytically competent conditions, we utilized transient kinetic approaches and monitored individual events in the millisecond time scale, including DNA binding and bending, base unpairing and cleavage, which elucidated the order of events and rate‐determining step(s) in the reaction. Furthermore, we examined the role of several conserved active‐site residues in the reaction mechanism. A FRET assay revealed that hFEN1 binds and bends the DNA substrate rapidly at a diffusion‐limited rate (109 M−1s−1), indicating that these events occur simultaneously. Lack of Mg2+ and mutations in the active site do not affect this binding/bending rate, thus this initial event does not depend on specific contacts within the catalytic pocket. A 2‐aminopurine fluorescence‐based assay revealed unpairing of base(s) at the flap junction at a much slower rate of 20 s−1. Importantly, quench‐flow measurements revealed that base unpairing limits the cleavage rate to 20 s−1. A subsequent event, likely related to product release, limits the overall steady state turnover rate to 1 s−1. Mutations in active site residues D34, Y40, K93, R100, E160 and D181 exhibit differential effects on base unpairing and cleavage. The results show that base unpairing is a pre‐requisite for flap cleavage, and clarify the contributions of these conserved residues toward an electrostatic environment that stimulates precise cleavage of the 5′ flap. The findings also apply to other 5′‐nucleases that share similar active site residues and a two‐metal ion dependent reaction mechanism (8,9).
Curcuminoids Supress Cell Proliferation of Glioblastoma by Down‐regulation of CXCR4 Signaling Pathway
Authors: Shivachar Amruthesh C. , Nnamdiebere Joseph Ekeada , Jordan Bryan C. , Selvam Chelliah , Mathews Omana P , Ranganna Kasturi
Published: November 2023

Glioblastoma multiforme is one of the glial‐derived malignant primary brain tumors in adults with a poor prognosis. The current average survival period still remains between 14–16 months despite various treatment modalities. The robust tumor progression is mainly attributed to its pro‐angiogenic and anti‐hypoxic targets that many current chemotherapeutics may miss. Curcumin (#1) is a plant derived dietary constituent, and has been shown to play a role in preventing many cancers. Here we investigated for the first time the potencies and the molecular mechanisms of action of #1 and its three pyrazole analogs (#7, #20, and #32) that were prepared through an acid catalyzed condensation with various hydrazine derivatives. Heterocyclic pyrazole modification introduces conformational constraint that is beneficial for improving potency and in vivo chemical stability for curcumin analogs. Cultures of grade IV glioblastoma multiforme (GBM) cells (10,000 cells/cm2) in 24‐well plates were treated with various concentrations (0.01–10micrMole) of #1, #7, #20 or #32 for 24 hr. along with vehicle controls. Cell viability/toxicity was assessed using the fluorescent dyes Hoechst 33342 (1 mg/ml, 20 min at 37°C), which stains DNA of both living and dead cells and propidium iodide (PI; 5 mg/ml, 5 min, 37°C), which stains the DNA of cells with disrupted membranes. The cell death was calculated [pink cells × 100/total cells] from 3–5 non‐overlapping microscopic fields in an Olympus IX81 inverted fluorescent microscope: dead cells are double stained (pink), while viable ones remained blue and were quantified using Prism Graph Pad software. The cell proliferation was measured by the uptake of BrDU by immunofluorescent method. In the sister cultures, the effect of these analogs on the expression of chemokine receptor 4 (CXCR4) that is known to affect tumor progression and metastasis in these cells was measured by western blotting analyses. Our results show that the tested analogs dose‐dependently decreased cell viability and inhibited cell proliferation in the order #32> #20> #7> #1, suggesting that heterocyclic pyrazole modification increased the potency of the parent compound #1. Additionally, we noted that #1 and its pyrazole analogs significantly down‐regulated the expression of CXCR4 over vehicle‐treated controls. Thus our preliminary results demonstrate that curcuminoids may suppress cell proliferation of GBM by down‐regulation of CXCR4 signaling pathway.

Support or Funding Information

This work was supported, in part, by an Institutional seed grant (ACS), and by NIH‐RCMI‐Program Molecular Biology Core Facility (KR).
STK11 Loss Stabilizes MCL1 and Confers Targetable Drug Resistance in KRAS Mutant NSCLC
Authors: Kahle Michael , Akbay Esra , Christensen Camilla , Sholl Lynette , Becker Jeffrey , Soucheray Margaret , Chen Zhao , Pulido Ines , Kikuchi Eiki , Gao Yandi , Dennis Peter , Qiu Wei , Al‐Shahrour Fatima , Johnson Neil , Awad Mark , Carretero Julian , Wong Kwok‐Kin , Shimamura Takeshi
Published: November 2023

The combined KRAS/STK11 (serine‐threonine kinase 11, LKB1) mutated genotype is prevalent and predictive of poor clinical outcome in NSCLC as tumors are drug resistant. Here, we demonstrate that the bromodomain and extraterminal inhibitor (BETi) JQ1 can induce apoptosis in KRASm NSCLC by targeting the pro‐apoptotic BIM anti‐apoptotic BCL2/BCL‐XL axis, but not in KRASm/STK11m due to additional elevated MCL1. Specifically, loss of STK11 stabilizes anti‐apoptotic MCL1 protein via activation of the PI3K‐mTOR signaling axis. In NSCLC cell line models, we have demonstrated that overexpressing myristoylated‐PI3K increased MCL1 expression whereas the PI3K/mTOR inhibitor BEZ235 reduced MCL1 in a dose, time, and proteasome‐dependent, yet relatively GSK3β‐independent, manner. Furthermore, MCL1 stability also attenuates the antitumor efficacy of the direct BCL2/BCL‐XL inhibitor, ABT‐263 (Navitoclax) in KRASm/STK11m cells. However, combination of BETi or ABT‐263 with MCL1 protein reduction by siRNA or BEZ235, or direct MCL1 inhibition by the BH3 mimetic, A1210477, could sensitize resistant cells to apoptosis while no monotherapy was effective. Therefore, STK11 mutation or aberrant PI3K/mTOR pathway activations emerge as biomarkers of BET‐ or BCL2/BCL‐XL inhibitor resistance, suggesting patient selection for combined MCL1 inhibition.

Support or Funding Information

This work is supported by research Scholar grant RSG‐14‐229‐01 from the American Cancer Society and United Against Lung Cancer Legacy Program (Lung Cancer Research Foundation). This work is also supported by research fellowship from Sumitomo life social welfare services foundation (E.K), MINECO (SAF2010‐21769), ISCIII (RD12/0036/0045) and Generalitat Valenciana (ACOMP/2013/156 and ACIF/2013/239) (J.C.), and National Institutes of Health grants CA140594, CA122794, CA163896, CA166480 (K.K.W)
Exercise ameliorates endoplasmic reticulum stress‐mediated vascular dysfunction in atherosclerotic mesenteric arteries
Authors: Hong Junyoung , Kim Kwangchan , Park Eunkyung , Lee Jonghae , Park Yoonjung
Published: November 2023

Introduction

The endoplasmic reticulum (ER) stress has been known as a disruption of ER homeostasis promoting to an accumulation of misfolded and unfolded protein response (UPR) that is closely associated with cardiovascular diseases including atherosclerosis. The underlying mechanisms for atherosclerotic mesenteric arterial dysfunction, major cause of chronic mesenteric ischemia, is unclear and no study has been conducted to investigate the effect of exercise on mesenteric arterial dysfunction in atherosclerosis.

Hypothesis

We hypothesized that ER stress aggravates endothelial dysfunction in mesenteric arteries in atherosclerosis

Methods

We used wild type (WT), wild type with exercise training, running on the treadmill for 12 weeks (WT‐EX), apolipoprotein E knockout (ApoE KO) and ApoE KO with exercise training (ApoE KO‐EX) mice. To test mesenteric artery function, endothelium‐dependent (acetylcholine, ACh) vasodilation and endothelium‐independent nitroprusside (SNP) vasodilation of isolated and pressurized mesenteric arteries were measured by a concentration‐dependent manner. To determine underlying mechanism, endothelial nitric oxide synthase (eNOS) inhibitor, (NG‐nitro‐L‐arginine methyl ester (L‐NAME)), ER stress inducer, (tunicamycin), ER stress inhibitor, (Tudca), caspase‐1 inhibitor (AC‐YVARD‐cmk) and uncoupling protein‐2 (UCP‐2) inhibitor, (Genepin) were incubated and ACh‐induced dilation was measured to investigate the underlying mechanisms. To measure the expression of ER stress markers, protein kinase RNA‐like endoplasmic reticulum kinase (PERK) and C/EBPa‐homologous protein (CHOP), in the isolated superior mesentery arteries, immunofluorescence staining was performed.

Results

ACh‐induced vasodilation was attenuated in ApoE KO compared to WT but exercise training improved the ACh‐induced vasodilatation in ApoE KO‐EX while endothelium‐independent SNP‐induced vasodilation was not different among the groups. Incubation of Tudca and AC‐YVARD‐cmk improved ACh‐induced vasodilation in ApoE KO. ACh‐induced vasodilation in the presence of L‐NAME, tunicamycin and Genepin were reduced in WT, WT‐EX and ApoE KO‐EX, but not in ApoE KO. The expression of PERK and CHOP in the isolated superior mesentery arteries were increased in ApoE KO compared to WT but exercise training decreased its expression in ApoE KO‐EX compared to ApoE KO.

Conclusion

Our findings suggest that ER stress plays a significant role in vascular endothelial dysfunction in atherosclerotic mesenteric arteries and exercise restores it through ER stress and its downstream signaling pathways including NOS, UCP‐2 and caspase‐1.
Evaluation of Diuretic and Urine Electrolyte Effects of Naringenin in Hypercholesterolemic Rats
Authors: Madkhali Hassan Abdu , Ganaie Majid Ahmad , Ansari Mohammad Nazam , Khan Tajdar Husain
Published: November 2023

Hypercholesterolemia is a major risk factor in the incidence of many cardiovascular diseases and renal dysfunction. In the recent times, the natural compounds have received special attention for the treatment of the diseases due to their safety and low cost. Thus, the aim of our study was to investigate the possible diuretic and electrolyte effects of naringenin (50mg/kg and 100 mg/kg; orally) in hypercholesterolemic rats. Four groups of Wistar rats were used (6 in each group): group I, the normal control rats fed with normal pallet diet; group II, rats fed with 1% cholesterol diet for 4 weeks; group III, rats fed with 1% cholesterol diet and concomitantly treated daily with naringenin 50mg/kg orally for 4 weeks; group IV, rats fed with 1% cholesterol diet for 4 weeks and concomitantly treated daily with naringenin 100mg/kg orally for 4 weeks. Urine volume was recorded for each group after 5 hrs and 24 hrs of the last dose of treatment. Serum and urine samples of all the groups were collected and tested for serum total cholesterol and urine electrolytes. Hypercholesterolemia in rats resulted in significantly increased serum cholesterol level and decreased in urinary volume (ml) and electrolytes (sodium, potassium and chloride ions) excretion. Oral administration of naringenin significantly restore the altered parameters in hypercholesterolemic rats. The current study investigated the diuretic and urinary electrolyte effects of naringenin in hypercholesterolemic rats. Our result suggest a beneficial role for naringenin in restoring the water and electrolyte balance in hypercholesterolemia induced cardiovascular and renal dysfunction.
Adenosine receptor dependent regulation of vascular tone in L‐NAME mouse model of hypertension.
Authors: Yadav Vishal R , Teng Bunyen , Mustafa Jamal S
Published: November 2023

L‐NAME, a nitric oxide synthase inhibitor, down regulates eNOS protein expression and leads to depletion of plasma nitric oxide levels in blood. L‐NAME pre‐treatment causes systemic vasoconstriction, increased vascular resistance and high blood pressure (BP). Thus, L‐NAME model of hypertension is used to study endothelial dysfunction and related vascular effects. Adenosine is an important metabolite, which controls local blood flow through activation of four subtypes of adenosine receptors (ARs). Stimulation of A1 and A3 ARs produce contraction while A2A and A2B ARs cause relaxation in mouse aorta and mesenteric arteries (MAs). In present study, we sought to determine whether ARs play a role in altered vascular tone regulation in this model of hypertension. We used L‐NAME (1 mg/ml) in the drinking water for 4 weeks. The BP was monitored using CODA non‐invasive tail cuff system. For vascular tone studies, aortae and MAs were isolated from control and hypertensive mice. Muscle tension studies were performed using DMT for MAs and organ bath for aorta. Systolic BP recorded at the end of 28 days was (mean ± SE; mmHg) 133.9 ± 3.0 in hypertensive while 107.8 ± 2.6 in control group (n=11). Our DMT data analysis in MAs showed that acetylcholine (Ach) induced relaxation responses were (% PE) 98.5 ± 0.2 and 93.4 ± 1.4 (p<0.05, n=10) in control and L‐NAME hypertensive mice, respectively. Vascular studies in MAs revealed higher CCPA (selective A1AR agonist) induced contractions in hypertensive mice. Particularly, 100 nM CCPA (% PE) produced 24.9 ± 14.1 contraction in control whereas 89.2 ± 27.4 (p<0.05, n=7) in L‐NAME mice. Contrary, NECA (non‐selective AR agonist) induced vascular responses were comparable. Pinacidil (KATP channel opener) induced relaxation was significantly increased in hypertensive mice. Pinacidil (1 μM), produced relaxation of 51.72 ± 9.8 and 76.29 ± 5.9 (p<0.05, n=7) in control and L‐NAME mice, respectively. Phenylephrine induced contraction was reduced to (mg) 278.8 ± 32.8 in L‐NAME mice compared to 492.1 ± 33.8 in control (p<0.05, n=11). Angiotensin (ANG) II induced vascular contractions were reduced to (% KCl) 54.3 ± 9.0 in L‐NAME compared to 122.4 ± 14.0 in control mice (p<0.05, n=7). Our organ bath data in aorta showed that CCPA, NECA, pinacidil PE, KCl and Ach induced responses were comparable in both groups. In conclusion, A1AR induced contraction and KATP channel induced relaxation were both higher in MAs of L‐NAME mice. In addition, ANG II induced contraction were lower in MAs. Thus, suggesting altered responses to ARs and ANG II in L‐NAME induced mouse model of hypertension.

Support or Funding Information

Supported by HL 027339
Role of MCPIP1 in High Insulin‐Related Endothelial Cell Dysfunction
Authors: Chen Renee , Chang Yingzi
Published: November 2023

Vascular complications are one of the major adverse outcomes for diabetic individuals. Elevated inflammatory cytokines are thought to contribute to impaired vascular function. MCP‐1‐induced protein 1 (MCPIP1) is a negative regulator in inflammatory response. Previous research has shown that the proinflammatory cytokine MCP‐1 induces endothelial cell migration and proliferation that is mediated by the induction of MCPIP1. The goal of this project is to better understand the role of MCPIP1 in hyperinsulinemia‐associated endothelial dysfunction. Previous data from our lab showed that pre‐treatment of vascular smooth muscle cells suppresses MCPIP1 expression, suggesting that downregulation of MCPIP1 in diabetic patients may contribute to the development of endothelial dysfunction and subsequent vascular complications. A scratch migration assay and BrdU cell proliferation assay were conducted to determine whether or not a hyperinsulinemic state can suppress MCPIP1 mediated cell migration and proliferation. Our preliminary data revealed that morphology of Human Umbilical Vascular Endothelial Cells (HUVEC) were changed from a cobblestone morphology into an elongated fusiform morphology following transfection with adenovirus expressing GFP‐tagged MCPIP1, overexpression of MCPIP1 increased HUVEC cell migration, and pretreatment with insulin suppressed MCPIP1‐induced HUVEC cell migration. These results suggest that MCPIP1 not only affects cell morphology, but also cell function and that insulin inhibits upregulation of MCPIP1‐induced cell movement. Experiments observing the effect of MCPIP1 on HUVEC cell proliferation and the possible impact of insulin are still being conducted.
Evogliptin, a novel dipeptidyl peptidase 4 inhibitor ameliorates atherosclerosis by modulating vascular inflammatory responses
Authors: Won Jong Soon , Nguyen Phuc Anh , Cho Min Kyung
Published: November 2023

Evogliptin (SuganonTM), a potent and selective dipeptidyl peptidase 4 inhibitor, was globally approved for the treatment of type 2 diabetes mellitus. However, the effect of evogliptin on the atherosclerotic progression has not been evaluated. In the present study, we examined the effects of evogliptin on the progression of atherosclerosis and its possible mechanism of action. Evogliptin reduced high fat diet‐induced plaque area in ApoE‐knockout mouse model. Evogliptin significantly suppressed the expression of adhesion molecules including ICAM‐1 and VCAM by ox‐LDL, a proatherogenic risk factor in human endothelial cells. NF‐kB activation was dose dependently inhibited by evogliptin. PPARγ suppressed by ox‐LDL was reversed by evogliptin. The anti‐adhesive effects between endothelial cells and monocytes by evogliptin were reversed by gene knockdown of PPARγ. The present study demonstrates that evogliptin attenuates atherosclerotic progression via inhibiting of vascular inflammatory responses implying the potential therapeutic action of evogliptin on the atherosclerotic complication.
Evaluation of Antiobesity activity of Ethanolic extract of Cashew Apple against high fat diet induced obesity in rodents
Authors: Rani T Jhansy , Prasad K.V.S.R.G. , Dodoala Sujatha
Published: November 2023

Obesity has doubled since 1980, as per WHO report affecting more than 1900 million adults throughout the world. In recent studies, diets containing antioxidants and branch chain amino acids were demonstrated to have potential effects on fat utilization. Cashew apple juice comprising nutritional components including vitamin C and BCAS reported as an enhancing factor in fat oxidation during exercise and may improve endurance performance (Piyapong Prasertsri et al., 2013). Hence, the current work was planned with an objective to evaluate the anti‐obesity activity of the ethanol extract of cashew apple (ECA). To identify the active constituents, ECA was subjected for analysis using GC‐MS technique. High fat diet induced rodent model was used to induce obesity in the experimental animals. Adult male albino rats were randomized in to five groups (n=6) among which first group was kept on standard rodent chow (normal). The remaining four groups were assigned as obese control, standard & ECA treated groups which received high fat diet along with vehicle, atorvastatin (10 mg/kg, p.o) and ECA (200 & 400 mg/kg, p.o) respectively for 60 days. At the end of study, obese control group animals showed a significant increase in BMI & body wt, serum total cholesterol, triglycerides, LDL‐cholesterol and VLDL‐cholesterol compared to normal group. Administration of ECA (200mg/kg, 400mg/kg) exhibited significant decrease in serum total cholesterol, triglycerides, LDL‐cholesterol and VLDL‐cholesterol levels in ECA treated groups. In addition HDL‐cholesterol levels were also increased distinctly compared to the obese control groups. A notable elevation in the levels of serum creatinine, transaminases & alkaline phosphatase were observed with an intake of high fat diet. These effects were reversed by oral administration of ECA. A significant inhibition of serum malondialdehyde (MDA) and improvement in hepatic enzymatic (catalase, superoxide dismutase), non‐enzymatic(reduced glutathione) levels was also identified as an outcome after treatment with ECA (200mg/kg, 400mg/kg) and standarad (atorvastatin) compared to the obese control groups. Eventually, consumption of cashew apple may even reverse the fatty liver which was quite evident from histology images. GC‐MS analysis revealed the presence of 5‐Hydroxy‐2‐(hydroxymethyl)‐4H‐pyran‐4‐one; 2,3‐dihydro‐3,5‐dihydroxy‐6‐methyl‐4H‐pyran‐4‐one; Hexadecanoic acid; octadecanoic acid; 5‐(Hydroxymethyl)‐2‐furancarboxaldehyde; Furfural; 4‐H‐pyran‐4‐one,3,5, dihydro‐2‐methyl decanoic acid ethyl ester.

Based up on the available experimental evidence it can be concluded that ethanolic cashew apple extract possess anti‐obesity activity and reinforce recommending consumption of cashew apple to modulate obesity.

Support or Funding Information

1. Sri Padmavati Mahila Visvavidyalayam, Tirupati, Andhra Pradesh, India.2. DST, Government of India.
mTORC2 Facilitates the Maturation of Protein Kinase C by a Phosphorylation‐Independent Mechanism
Authors: Baffi Timothy R. , Gould Chistine M. , Wang Zhiyong , Gutkind Silvio , Newton Alexandra C.
Published: November 2023

Protein Kinase C (PKC) is primed by a series of ordered phosphorylations and conformational transitions to yield an autoinhibited enzyme that is positioned to respond to second messengers. For most PKC isozymes, this maturation to yield a signaling‐competent enzyme depends on the mammalian Target of Rapamycin Complex 2 (mTORC2); however, the molecular mechanisms by which mTORC2 regulates PKC are unknown. Here we use a variety of Förster resonance energy transfer (FRET)‐based live cell imaging assays to examine the role of mTORC2 in the the folding, phosphorylation, and activity of overexpressed PKCbII. We show that in cells lacking Sin1, a critical component of mTORC2, PKC exists in an unprimed, unphosphorylated, and open conformation, with fully‐exposed membrane‐targeting modules. In contrast, overexpression of PKCbII in cells with functional mTORC2 results in accumulation of a fully‐phosphorylated and autoinhibited enzyme. Introduction of Glu at the two phosphorylated residues on the C‐terminal tail is insufficient to bypass the requirement for mTORC2 in the folding of PKC; however, expression of Sin1 in Sin1−/− MEFs rescues the defect, allowing PKC to become phosphorylated and fold into the mature, closed conformation. Furthermore, we demonstrate that PKC is constitutively active in the absence of mTORC2. Finally, analysis of chimeric proteins of an mTORC2‐dependent (PKCbII) and mTORC2‐independent (PKCd) enzyme, coupled with peptide array experiments, identify two critical regions in the kinase domain and C‐terminal tail of PKC that each confer mTORC2‐dependence. Our data support a model in which mTORC2 serves as a molecular chaperone to permit PKC to be processed by phosphorylation, modifications that allow the enzyme to adopt an autoinhibited conformation.

Support or Funding Information

This work was supported by NIH GM43154, UCSD Graduate Training Program in Cellular and Molecular Pharmacology NIH T32 GM007752, and the PhRMA Foundation Pre‐Doctoral Fellowship in Pharmacology/Toxicology.
Exploration of the 5‐HT2A : 5‐HT2C receptor protein complex in vitro
Authors: Felsing Daniel E , Anastasio Noelle C , Gilbertson Scott R , Moeller Gerard F , Cunningham Kathryn A , Allen John A
Published: November 2023

The disruption of serotonin 5‐HT2A receptor (5‐HT2AR) and 5‐HT2CR function is implicated in psychiatric disorders that range from addiction to schizophrenia. Previous studies have demonstrated that these structurally similar class A G protein‐coupled receptors exist as homomers in living cells (Brea et al., Mol Pharm 75:1380, 2009; Herrick‐Davis et al., Biochemistry 43:13963, 2004), and we are interested in the possibility that these two receptors are in the same protein complex, which may regulate their signaling and/or trafficking. The 5‐HT2AR and 5‐HT2CR co‐localize to neurons in the medial prefrontal cortex (mPFC) of the rat (Nocjar et al, Neuroscience 25:22, 2015) and co‐immunoprecipitate from mPFC preparations (Anastasio et al, ACS Chem. Neurosci. 15:1248, 2015). To expand this work, we designed a luciferase complementation assay (LCA) to test the hypothesis that the 5‐HT2AR and 5‐HT2CR are in close proximity (~50 nm) in living cells. In the LCA, two complementary N‐terminal (NLuc) and C‐terminal (CLuc) components, which alone do not have enzymatic activity, were fused to the 5‐HT2AR and 5‐HT2CR, respectively, and transiently transfected into HEK293 cells. In the presence of the substrate D‐luciferin, we hypothesized that the association of the two receptors would bring the inactive luciferase fragments into close proximity to reconstitute the enzymatic activity of luciferase. The LCA showed robust complementation (i.e., increased luminescence) dependent on cDNA transfection ratios, with optimal signal achieved at a 3:1 ratio of 5‐HT2CR‐NLuc to 5‐HT2AR‐CLuc (n=3, p<0.001). Co‐transfection with plasmid DNA encoding unlabeled 5‐HT2AR or 5‐HT2CR, inhibited the complementation between the two tagged receptors (n=3, p<0.001, and p<0.01, respectively). In ongoing studies we are exploring the effect of receptor selective agonists and antagonists on protein complementation. Taken together, we have detected a 5‐HT2AR:5‐HT2CR complex whose formation is dependent on the relative expression of the individual receptor protomers. Because these receptors are important drug targets in psychiatric disorders, a greater understanding of this novel receptor complex may also provide insights for the development of novel pharmacotherapeutics.

Support or Funding Information

NIH NIDA P50 DA033935 and Center for Addiction Research at UTMB