bluuebunny/crossref_metadata_2025_split huggingface dataset:
Proteomic analyses of src+/+ and src−/− mouse brain microvessels reveals insights into the regulation of the blood brain barrier integrity
Authors: Minder Petra , Scott Michael , Moresco James J , Baird Andrew , Yates John R , Eliceiri Brian P , Torbett Bruce E 
Published: November 2023

The blood brain barrier (BBB) is a highly selective permeability barrier, composed of endothelial cells with tight intercellular junctions, surrounded by a basal lamina, astrocytes, and pericytes, which regulates the entry of compounds and cells between the blood and brain. The integrity of the BBB is necessary for maintaining brain function, whereas a number of severe brain diseases, such as stroke, edema, brain tumors and multiple sclerosis, have been associated with deregulation of BBB integrity. Therefore, understanding the molecular and cellular mechanisms whereby BBB integrity is maintained or deregulated, will be essential for restoration of BBB homeostasis after injury, as well as for selective therapeutic delivery.To interrogate the cellular and molecular mechanisms that maintain BBB integrity, we utilized Multidimensional Protein Identification Technology (MudPIT) proteomics to identify proteins from normal mouse (src+/+) brain microvessels and microvessels from a leakage‐resistant vascular phenotype resulting from loss of Src (src−/−). Src is a non‐receptor tyrosine kinase that phosphorylates specific tyrosine residues and has been demonstrated to be necessary for the maintenance of BBB integrity. src−/−mice support relatively normal vessel development and sprouting, but demonstrate a leakage‐resistant phenotype.We identified 2479 proteins from MudPIT proteomics from src+/+ and src−/− mouse brain microvessel membrane preparations (n=3 biological samples from each phenotype), of which 150 proteins were determined to be differentially expressed in src−/− as compared to src+/+ microvessels. The top five cellular compartments showing protein changes were membrane, cytoplasm, nucleus, mitochondrion, and cell junction. Comparing microvessels of src+/+ to src−/− mice, we found differential expression of 42 proteins involved in transport across the plasma membrane and 47 proteins involved in cell adhesion and cytoskeleton remodeling. GeneGo analysis software was used to generate Pathway Maps. The majority of pathways and the relevant proteins identified are involved in cell adhesion and cytoskeleton development and regulation. Moreover, the Src‐regulated gene, Cdc42, was downregulated in src−/− compared to src+/+ tissue. Furthermore, we identified altered regulation of proteins necessary for actin dynamics and junctional remodeling in src−/− vessels. Imaging of actin in src−/− brain microvessels revealed that actin filaments are altered as compared to src+/+ mice.Our findings reveal that specific proteins are necessary for regulating the actin cytoskeleton to maintain BBB integrity through precise control of specific transporters. No significant alterations in the BBB basement membrane appeared to be necessary for alterations in BBB permeability.
Angiogenesis by EVGATA−4 is mediated by transfer of let‐7 miRNA family
Authors: Xu Meifeng , Gong Min , Yu Bin , Wang Yi‐Gang , Ashraf Muhammad 
Published: November 2023

BackgroundIt is well known that extracellular vesicles (EVs) from stem cells are responsible for mediating diverse bio‐effects through delivering bioactive molecules. Here, we investigated whether the angiogenesis by EVs released from mesenchymal stem cells (MSC) overexpressing GATA4 (EVGATA−4) is mediated by miRs let‐7 family.Methods and ResultsMSCs were transduced GATA‐4 (MSCGATA−4) using the murine stem cell virus retroviral expression system. The conditioned medium was collected from MSCGATA−4 and its control counterpart MSCnull. EVs were isolated using exosome precipitation solution.

The data of MicroRNA‐seq of EVs shows that 358 miRs were found in EVs with 46 miRs significantly increased in EVGATA−4 compared to those in EVs derived from MSCnull (EVnull). let‐7 family not only have had a high reads in EVs, but also significantly upregulated in EVGATA−4 compared to that in EVnull.
The number of tubular structure formation was more in human umbilical vein endothelial cells (HUVECs) treated with EVGATA−4 (30μg/300μl) than in those treated with EVnull. The cumulative length of sprout per spheroid was also greater in EVGATA−4‐treated HUVECs than EVnull‐treated cells.
To further study the effect of EVs on blood vessel formation, a Matrigel containing EVs (100μg/plug) was subcutaneously injected into the abdomen of mice. Two weeks later, the Matrigel plugs were harvested. The cell number and hemoglobin were significantly increased in the plugs containing EVGATA−4 compared to the plugs containing EVnull.
The real‐time imaging capture system recorded that EVs pre‐labeled with PKH26 could be internalized by HUVECs. The expression of let‐7f in HUVECs treated with EVGATA−4 was significantly upregulated. Silence of let‐7f in EVGATA−4 by transfecting with an inhibitor of let‐7f reduced the angiogenetic effect of EVGATA−4.
Thrombospondin 1 (THBS1) is one of the potential target genes of let‐7f and an inhibitor of angiogenesis. The expression of THBS1 was significantly down‐regulated in HUVEC treated with EVGATA−4.
ConclusionThe increased pro‐angiogenic capacity of EVGATA−4 is associated with let‐7 transfer into HUVECs and down‐regulation of the expression of THBS1 in HUVECs.Support or Funding InformationThis work was supported by the National Institutes of Health grants HL105176 and HL114654 (M. Xu).
Effect of Recombinant Lubricin on Human Blood Coagulation Parameters and Platelet Aggregation
Authors: Emanuele Nicholas , Hoppensteadt Debra , Wanderling Chris , Carlsgaard Peter , Truitt Ed , Hopkinson William , Fareed Jawed 
Published: November 2023

IntroductionLubricin (proteoglycan 4; PRG4) is a proteoglycan which acts at tissue interfaces as a boundary lubricant. Lubricin is present in synovial fluid and plays a major role in synovial homeostasis. A recombinant form of human lubricin, rh‐PRG4 is being developed to treat a variety of inflammatory conditions, including osteoarthritis & dry eye (Lubris Biopharma, Boston, MA). The purpose of this study is to investigate the effect of rh‐PRG4 on blood coagulation utilizing various experimental studies.Materials and Methodsrh‐PRG4 was supplemented with citrated whole blood from normal human donors (n = 10) to obtain a concentration range of 0–200 mg/uL. Activated clotting time (ACT), thrombelastographic analysis (TEG), and whole blood prothrombin time (PT), partial thromboplastin time (aPTT), and thrombin times (TT) were measured. Similarly, citrated plasma was supplemented with rh‐PRG4 at 0–200 mg/ML to determine PT, aPTT and TT. The effect of this agent was also studied on the generation of factors Xa and IIa using a fluorokinetic method. Additionally, rh‐PRG4 was also studied for its effect on platelet aggregation induced by various agonists, such as ADP, collagen, epinephrine and thrombin at a concentration range of 0–20 ug/ml.Resultsrh‐PRG4 did not produce any effect on the whole blood clotting assays, such as the PT, aPTT and TT. Similarly, in the citrated plasma, none of the clotting assays were altered. rh‐PRG4 did not produce any inhibition of thrombin generation in plasma. Furthermore, it did not produce any inhibition or augmentation of agonist‐induced platelet aggregation. These studies suggest that rh‐PRG4 does not modify whole blood and plasma coagulation profile and produce and more relation of platelet function.ConclusionsThese studies suggest that because of no potential alteration of the coagulation system, lubricin is not expected to modulate bleeding or thrombotic processes in clinical indications.
Purification of the C‐Terminal Domain of ANAPC2 and Evidence Supporting the Interaction of Lead Compounds for Inhibition of Mitosis
Authors: Hoffman J. Mason , Al‐Eryani Laila , Saforo Douglas J. , Taylor B. Frazier , Trent John O. , Garbett Nichola C. , States J Christopher 
Published: November 2023

BackgroundMitosis‐inhibiting chemotherapeutics are a common approach to treat cancer. Successful therapy is often limited due to drug‐resistant tumors, neurotoxicity, and limited availability. There is high demand for new drugs with diverse cellular targets. The anaphase promoting complex/cyclosome (APC/C) is a multi‐subunit E3 ubiquitin ligase that controls cell cycle progression by marking key proteins for degradation, offering great potential as a target for drug development. The catalytic core consists of an ANAPC2/ANAPC11 dimer. Homology models of the ANAPC2/ANAPC11 subunits were analyzed in silico to identify compounds predicted to bind the C‐terminal domain (CTD) of ANAPC2, preventing ANAPC11 association and inhibiting APC/C function. Biological data indicates that the lead compounds identified induce mitotic arrest and cell death in multiple cancer cell lines, consistent with APC/C inhibition. Here, we aim to produce recombinant ANAPC2 CTD, and investigate compound binding via thermofluor assays, a biophysical method for monitoring protein stability in the presence of a ligand.MethodsANAPC2 CTD cDNA was optimized for expression in E. coli, and protein was purified using the NEB IMPACT® system. Thermofluor assays were performed to observe any compound‐induced changes in ANAPC2 stability.ResultsWe have successfully produced pure ANAPC2 CTD, verified by coomassie stained SDS‐PAGE and by mass spectrometry. In addition, we observed changes in the ANAPC2 CTD thermal spectra in the presence of lead compounds.SummaryThese results are consistent with observed biological effects, supporting the hypothesis that targeting the APC/C offers promise for development of novel chemotherapeutics. The melting temperature of ANAPC2 CTD, however, is too low to produce full melting curves. Therefore, in order demonstrate stabilization of ANAPC2 in the presence of lead compounds, it may be necessary to produce full‐length recombinant ANAPC2 for use in the assays.Support or Funding InformationKY Science sand Engineering Foundation grant KSEF‐3249‐RDE‐018; KY Lung Cancer Research Program grant 14‐JCS‐01; University of Louisville School of Medicine Basic Research Grant
Determination of the Membrane Topology of the PORCN O‐Acyl Transferase
Authors: Fraley J Gabriel , Galli Lisa , Burrus Laura , Sanchez Luis 
Published: November 2023

WNT signaling is critical for proper embryonic development and adult tissue homeostasis. WNT proteins require palmitoylation for optimal secretion and receptor binding. Porcupine (PORCN), a membrane bound O‐acyltransferase (MBOAT) family member is the protein that palmitoylates WNT. PORCN is localized to the Endoplasmic Reticulum (ER). Though PORCN is required for proper tissue patterning in developing embryos, its membrane topology is poorly understood. Determining the topology of PORCN may have useful applications in areas of embryonic development studies. Although numerous bioinformatics algorithms that predict membrane topology are available, no clear consensus for the topology of PORCN has emerged. Therefore, our goal is to experimentally determine the topology of PORCN.To determine topology, we developed two strategies. The first strategy utilized differential solubilization and immunofluorescent imaging of single, and dual tagged PORCN variants. Results from single Myc‐tagged N‐and C‐terminus PORCN variants in transfected COS‐7 cells orient the N‐terminus in the ER lumen, and the C‐terminus in the cytosol. To further determine topology, we developed twelve dual tagged PORCN variants with Myc epitopes within predicted loops, and a C‐terminal 3X FLAG tag. The construction of dual tagged variants provided us with qualitative information, as well as quantitative values using Mander's coefficients of colocalization. Our studies show that five of the PORCN variant loops exhibit cytosolic orientation while one loop was oriented toward the lumen. As a control, we showed that the N‐ and C‐terminal tags remained lumenal and cytosolic, respectively. However, the remaining constructs provided conflicting results. Additionally, a PORCN antibody was generated against an immunogenic region that includes residues 282–303. Immunofluorescent imaging and colocalization quantitation orient this epitope towards the cytosol.As a second strategy, we developed ten mutants which resulted in the introduction of ectopic consensus sites for the addition of N‐linked carbohydrates (NxT/NxS). These PORCN mutants were transfected into HEK‐293T cells and subjected to Western blot analysis to identify glycosylated variants. This analysis identified two glycosylated residues at A134N and D283N, which indicates localization of these two residues to the lumen of the ER. Intriguingly, these results do not agree with those from our differential solubilization studies. We are now carrying out additional controls to help us distinguish between these two results.Support or Funding InformationNIH‐MARC T34‐GM008574
Multiple Modes of Inhibition of Human Cytochrome P450 2J2 by Dronedarone, Amiodarone and their Active Metabolites
Authors: Chan Eric Chun Yong , Karkhanis Aneesh , Lam Hui Yuan , Hong Yanjun , Venkatesan Gopalakrishnan , Chai Christina Li Lin , Koh Siew Kwan , Zhou Lei , Kojodjojo Pipin 
Published: November 2023

Dronedarone, a multiple ion channel blocker is prescribed for the treatment of paroxysmal and persistent atrial fibrillation. While dronedarone does not precipitate toxicities like its predecessor amiodarone, its clinical use has been associated with idiosyncratic hepatic and cardiac adverse effects and drug‐drug interactions (DDIs). As dronedarone is a potent mechanism‐based inactivator of CYP3A4 and CYP3A5, a question arose if it exerts similar inhibitory effect on CYP2J2, a predominant cardiac CYP450 enzyme. In this study, we demonstrated that CYP2J2 is reversibly inhibited by dronedarone (Ki=0.0293 μM), amiodarone (Ki=4.782 μM) and their respective pharmacologically active metabolites namely N‐desbutyldronedarone (NDBD) (Ki=0.550 μM) and N‐desethylamiodarone (NDEA) (Ki=7.352 μM). Moreover, time‐, concentration‐ and NADPH‐dependent irreversible inactivation of CYP2J2 was investigated where inactivation kinetic parameters (KI, kinact) and partition ratio (r) of dronedarone (0.0498 μM, 0.0336 min−1, 3.3), amiodarone (0.213 μM, 0.0146 min−1, 20.7) and NDBD (0.479 μM, 0.0235 min−1, 21.7) were observed except for NDEA. The absence of the characteristic Soret's peak, lack of recovery of CYP2J2 activity upon dialysis, and biotransformation of dronedarone and NDBD to quinone‐oxime reactive metabolites further confirmed the irreversible inactivation of CYP2J2 by dronedarone and NDBD is via the covalent adduction of CYP2J2. Our novel findings illuminate the possible mechanisms of DDIs and cardiac adverse effects due to both reversible inhibition and irreversible inactivation of CYP2J2 by dronedarone, amiodarone and their active metabolites.Support or Funding InformationDean's Chair Professorship (Faculty of Science, National University of Singapore)
Discovery and development of tylophorine derived dibenzoquinolines‐33b compounds into therapeutic agents
Authors: Lee Shiow‐Ju , Li Chih‐Hung , Lee Yue‐Zhi , Yang Cheng‐Wei 
Published: November 2023

We investigated the role of the tylophorine E ring on the biological activities through synthesis of a series of derivatives, bearing modifications at the E ring and N‐substitutions. All the derivatives were submitted for a variety of tests, anti‐cell growth against a panel of cancer cell lines, suppressing nitric oxide production inLPS/IFNgamma stimulated RAW264.7 cells, and anti‐viral replication in TGEV infected ST cells detected by inhibition of TGEV N and S protein expression. The uncyclized derivatives, dibenzoquinolines, do not have the enantimerism issue at C13a position. We have synthesized a series of novel tylophorine‐derived dibenzoquinolines and evaluated for their biological activities. The role of tylophorine E ring was explored unprecedentedly for the first time. Unlike other reported tylophorine derivatives, the potent tylophorine‐derived dibenzoquinolines appear to retain similar modes of action to those of tylophorine in terms of multi‐biological activities for anti‐inflammation, anti‐cancer cell proliferation, and anti‐coronavirus. The most potent compound dibenzoquinolines‐33b (DBQ‐33b) showed improved solubility, in vivo efficacies in a murine tumor xenograft model administrated orally and a murine paw edema model, good bioavailability, and no significant neurotoxicity tested by a rota‐rod test for motor coordination. More orally active leads derived from DBQ‐33b have been designed and synthesized and development of DBQ‐33b derived lead compounds into therapeutic agents is ongoing.Support or Funding InformationThis work was supported by a grant (MOST 104‐2325‐B‐400‐006‐) from the National Research Program for Biopharmaceuticals, Ministry of Science and Technology, Taiwan ROC.
In Vitro Invastigation Effects of 4‐hydroxyacetophenone on Rat Torasic Aorta's Vasomotor Activity
Authors: GELEN Volkan , ÇELEBİ Fikret 
Published: November 2023

AimZ. clinopodioides Lam. is also known as a “Field Mint” Seven compounds which have vasodilator activity have been isolated from Z. clinopodioides Lam. and two of them are phenolics compounds and these are acetovanillone, 4‐HAP, four of them are flavonoids and these are acacetin, apigenin, chrysin, thymonin one derivat of cinnamic acid and ethyl 4‐coumarate. In this study, it was aimed to define vasodilator activiy mechanisms of fenolic compound 4‐HAP on isole rat torasic aorta.Material and MethodIn this study 40 male adult Sprague Dawley type of rat were used. Prepared rings were laid out into the 20 ml organ bath with Kreb solution. Rings were stretched by 1g and they were left to 1 hour incubation period. In the end of the incubation period, FE, KCI, nifedipine, L‐NAME, 4‐HAP, SQ22.536, ODQ, ACh, SKF96365, Propranolol, Atropin, TEA, Gibenclamide, 4‐aminopyridine and U73122 were implemented to bath with a protocol.ResultsMechanisms of this relaxed effect was assigned by using antagonists. It was observed that Vasorelaksan effect of 4‐HAP on endothelilal aorta straight muscle contractions which had been inductioned by FE under the existance of L‐NAME was considerably inhibited.ConclusionIt was stated that 4‐HAP relaxed FE and KCI contractions and owing to this activity endothelilal tissues on L‐NAME existance notably decreased because of NO networkway. It is firmly believed that as relaxed effect of 4‐HAP declines remarkably under the existance of 4‐aminopyridine and nifedipine on unendothelilal aorta rings, activity could be on K+ chanal and L‐type Ca+2 chanal.Support or Funding InformationSupported of ÖYP
Physcion Induces Vasorelaxation In Rat Aorta through Endothelium‐dependent and Endothelium‐independent Mechanisms
Authors: Zhou Zi Qi , Sun Sha Sha , Zhou Guang Hai , Kang Dae Gill , Lee Ho Sub , Yang Sun Nye , Cho Kyung Woo , Jin Song Nan , Wen Jin Fu 
Published: November 2023

BackgroundPhyscion, an active anthraquinone constituent isolated from the rhubarb, a traditional Chinese herbal medicine which is widely used in clinical treatment, has cardiovascular protective properties. However, the effects of physcion on the vascular function and its pharmacological mechanisms of action have not been studied. The aim of the present study was to evaluate the vascular effects of physcion and its action mechanisms in rats.Methods and ResultsAortic rings from Sprague‐Dawley rats were mounted in organ baths, and the effects of physcion on vascular function were studied by means of isometric tension recording. Physcion induced a concentration‐dependent relaxation of pre‐contracted aortic rings in endothelium‐intact (+E) and endothelium‐denuded (−E) vessels. The nitric oxide synthase inhibitor L‐NAME, as well as the soluble guanylate cyclase inhibitor ODQ, attenuated the relaxation in +E but not in −E vessels. Inhibition of cyclooxygenases with indomethacin had no effects on the physcion‐induced vasorelaxation in +E vessels. A modulator of the store‐operated Ca2+ entry (SOCE), 2‐aminoethyl diphenylborinate markedly attenuated the physcion‐induced vasorelaxation in +E vessels. In −E preparations, K+ channel blockers, such as tetraethylammonium, large‐conductance calcium‐activated K+ (KCa) channel blocker iberiotoxin, and KATP channel blocker glibenclamide significantly attenuated the relaxation elicited by physcion.ConclusionsThese data demonstrate that physcion induces vasorelaxation through activation of endothelium‐dependent and ‐independent signaling pathways: endothelium‐dependent NO‐cGMP signaling and endothelium‐independent KCa channel and KATP channel signaling.Support or Funding InformationAcknowledgments: This work was supported by research grants from the National Natural Science Foundation of China (Nos. 81270920, 31571175), the Natural Science Foundation of Shandong Province (No. ZR2015HM037).
Mechanisms of Dietary Fructose and Copper in the Development and Progression of NAFLD Spectrum Disorders
Authors: Morrell Austin , Burkhead Jason 
Published: November 2023

Dietary induced oxidative stress appears to be factor which affects the development and progression of non‐alcoholic fatty liver disease (NAFLD) spectrum disorders, however no clear mechanism(s) for this have been identified. Preliminary studies by the Burkhead lab and others suggest that exposure to high oxidative stresses through the diet, lowers hepatic copper import even in copper deeffiecnt conditions. This downregulation of copper import in combination with high fructose consumption could have a synergist effect increasing the levels of oxidative stress (and therefore cellular damage) experienced by hepatocytes while lowering their ability to respond to it via superoxide dismutase. Using cell as well as animal models we aim to identify the mechanisms that allow oxidative stress, particularly that caused by diet, to regulate hepatic copper import and the systematic effects of this, as they related to NAFLD. These results could help explain the rising prevalence of NAFLD/NASH as well as identifying one potential mechanism that aids the progression of NAFLD to NASH.Support or Funding InformationInstitutional Development Award (IDeA)