bluuebunny/crossref_metadata_2025_split huggingface dataset:
Determination of the P450‐ and UGT‐dependent Biotransformation of the Anti‐HIV Drugs Cabotegravir and Dolutegravir
Authors: Madeen Erin , Bumpus Namandjé 
Published: November 2023

Cabotegravir (CAB) and Dolutegravir (DTG) are FDA approved viral integrase strand transfer inhibitors in oral formulation as antiretroviral therapeutics, while CAB is additionally in clinical trials as pre‐exposure prophylaxis (PrEP) for the prevention of HIV transmission. The molecular structure of CAB and DTG are remarkably similar, only differing in the terminal ring, a pentane ring on CAB and a hexane ring on DTG. The impact of structure on the metabolism of CAB and DTG were investigated. In order to investigate CAB and DTG metabolite formation human liver microsomes (HLM), c‐DNA expressed cytochrome P450 isozymes (P450s: 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, and 3A5), or c‐DNA expressed UDP‐glucuronosyltransferase (UGT) isozymes (UGTs 1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B15, 2B17) were employed. Separation, detection and identification were performed using UPLC‐MS/MS. HLM incubation resulted in the formation of two mono‐oxygenated metabolites of each CAB and DTG. The P450s that produced the greatest abundance of the CAB mono‐oxygenated metabolite M1 were P450s 2D6, 3A4, and 3A5, while DTG mono‐oxygenated metabolite M1 was produced by P450s 2B6, 2D6, 3A4, and 3A5. The P450 that produced the greatest abundance of mono‐oxygenated CAB metabolite M2 was CYP3A4 while DTG M2 was produced by P450s 1A2 and 3A4. Both CAB and DTG were directly glucuronidated in HLM incubations. The primary UGTs responsible for CAB direct conjugation were found to be UGT1A1, 1A3, and 1A9. In addition, a mono‐oxy‐CAB‐glucuronide conjugate was detected and formed by UGT2B7, while no glucuronide conjugate species of mono‐oxy‐DTG were detected. These data suggest that, despite the similarity of the molecular structure of these compounds, there are differing routes of P450 and UGT mediated metabolism.Support or Funding InformationR01 GM103853
Development of novel substrate cocktail to applicate in vivo drug‐drug interaction study
Authors: Kim Sunjoo , Jo Jung Je , Na Ann‐Yae , Lee Sangkyu 
Published: November 2023

Cocktail approach is very useful method for investigating drug‐drug interaction that can reduce a number of sacrificed animals and rapidly identify the interacted cytochrome P450 (CYP) isoforms. In this study, we aimed to assess whether drug interactions have influence on metabolism of 5 CYP enzymes using five probe drugs; caffeine for CYP1A, bupropion for CYP2B, omeprazole for CYP2C, dextromethorphan for CYP2D and midazolam for CYP3A, respectively. The cocktail, consist of caffeine (2 mg/kg), bupropion (20 mg/kg), omeprazole (2 mg/kg), dextromethorphan (10 mg/kg) and midazolam (2 mg/kg) is administered orally to ICR mice. Heparinized blood samples are collected and the plasma concentrations of the probe cocktail drugs and their metabolites are simultaneously quantified by LCMS/MS. In conclusion, the concentration of cocktail substrates and method of LC/MS/MS had been optimized in mice. Now we can use this study to investigate CYP interaction when herbs or other drugs are co‐administered with cocktails.Support or Funding InformationBK21 Plus KNU Multi‐Omics based Creative Drug Research Team
A multidimensional FPLC approach for the purification of recombinant non‐coding RNAs
Authors: Duan Zhijian , Ho Pui Yan , Jilek Joeseph , Batra Neelu , White Ralph DeVere , Wun Theodore , Lara Primo , Yu Aiming 
Published: November 2023

Translational and clinical research on RNA therapeutics for the treatment of cancer diseases requires the use of high‐purity RNA agents. Here we present a new, single‐run, multidimensional fast performance liquid chromatography (MD‐FPLC) approach for the purification of non‐coding RNAs that are bioengineered in bacteria using a novel tRNA/pre‐miRNA carrier we have invented recently. Total RNAs are separated on a combination of columns including size exclusion, weak anion exchange and strong anion exchange in a single run using a multidimensional NGC Chromatography System. The MD‐FPLC workflow is fast and highly reproducible. This MD‐FPLC platform is reliable, allowing a rapid and consistent production of multi‐milligrams of biological RNAs in a single run and offering an overall yield of 10–15% (target RNA/total RNAs). The purified ncRNAs are highly homogenous (> 97%, determined by a HPLC assay) and have minimal endotoxin activities (< 20 EU/mg RNA). In addition, the resultant biologic RNA products (e.g., let‐7c, miR‐34a and miR‐124a) are active in the regulation of target gene expression (e.g., BCL‐2/BCL‐xL, CDK6 and STAT3, respectively) and suppression of human cancer cell proliferation. Therefore, this MD‐FPLC approach offers a robust platform for the production of high‐purity biologic RNAs for research and development.Support or Funding InformationThis study was supported by grants R01GM133888, U01CA175315, and P30CA093373 from NIH.
Mechanisms of Jzu4Me, a novel inhibitor of vascular endothelial growth factor induced angiogenesis and tumor angiogenesis
Authors: Huang Shiu‐Wen , Huang Tur‐Fu 
Published: November 2023

BackgroundAngiogenesis is the formation of new blood vessels. This process involves the migration, growth, and differentiation of endothelial cells. It plays a critical role in the growth and spread of cancer, due to the new vessels supply of oxygen and nutrients for tumors to grow, allowing the cancer cells to invade nearby tissue, leading to tumor metastases. Therefore, novel effective therapeutic agents of anti‐angiogenesis are greatly needed. Further studies established that vascular endothelial growth factor (VEGF)‐A is an attractive anti‐angiogenesis target for therapeutic interventions in cancer.Material and methodsIn an effort to develop inhibitors to block VEGF signaling and angiogenesis, benzimidazole derivatives were designed and synthesized, and their angiogenesis inhibitory effects both in vitro and in vivo were evaluated.ResultsAmong these benzimidazole derivatives, Jzu4Me significantly inhibited VEGF‐A‐induced endothelial cells proliferation, migration, invasion and tubular formation, and it also suppressed ex vivo microvessels formation of rat aorta ring induced by VEGF‐A. Moreover, in Matrigel plug implantation mice, Jzu4Me dose‐dependently suppressed the VEGF‐A or tumor‐induced neovascularization in vivo. We also found that Jzu4Me may suppress VEGF‐A signaling in modulating vascular endothelial cell remodeling.ConclusionsOur findings reveal that Jzu4Me inhibits VEGF‐induced angiogenesis via downregulation of VEGFR2‐mediated signaling, suggesting that Jzu4Me has high potential to be developed into a novel therapeutic agent for treating angiogenesis.
A Dock‐Derived PEDF Mimic Targeting Laminin Receptor Recapitulates PEDF Signaling
Authors: Umbaugh Charles Samuel , Diaz‐Quinones Adriana , Figueiredo Marxa 
Published: November 2023

Pigment epithelium derived factor (PEDF) is regarded as an exceptionally potent inhibitor of angiogenesis. Our lab and others have used PEDF as a novel prostate cancer therapy, as PEDF inhibits tumor establishment, vascularization, and alters malignant signaling. PEDF has been identified as a binding partner for the cancer associated protein Laminin Receptor (LAMR1). LAMR1 overexpression is common in a variety of cancers including breast, colon, and prostate. Recently, we attempted to mimic the protein‐protein interaction between PEDF and LAMR1 using a structure based drug design approach. From our 14,000 compound screen, we identified a hit compound with anti‐proliferative capacity against several prostate cancer cell lines.We tested this hit compound in a medium‐throughput wound healing assay using the program tscratch and explored mechanisms of action at the molecular level. Cell pellets from treated and untreated prostate cancer cells were submitted for proteomic analysis using an Orbitrap Fusion and a label free quantitative approach coupled with MaxQuant and Perseus analysis. Significantly changed proteins were analyzed at the pathway level using Ingenuity Pathway Analysis.High doses of our hit compound were able to markedly reduce wound‐healing in a scratch based assay. This compound also elicits proteome wide changes to metabolic, proliferation, and genome maintenance systems. Most notably, a 10 uM dose of hit compound was able to active P53 associated pathways while inactivating MYC associated pathways. We will orthogonally validate our results using western blotting and confirm biochemical binding of our lead compound to LAMR1 in a scalable binding assay.Support or Funding InformationCTSI Core GrantPurdue Cancer Research CenterDepartment of Basic Medical Sciences
Haptoglobin prevents atherosclerosis by inflammation regulation: A prevention role of haptoglobin Hp for atherosclerosis
Authors: Cheng Tsai‐Mu , Chu Hsueh‐Liang , Pan Ju‐Pin 
Published: November 2023

Coronary artery disease is the leading cause death all over the world. Dysfunction of the endothelial cells in blood vessels is one of the major events in the development of atherosclerosis. Inflammation involve in the initiative of atherosclerotic lesion process. In the progression, monocytes recruitment contributes to the early stage of atherosclerosis. Oxidation stress is another remarkable proinflammatory factor will induce endothelial cell inflammation and stimulate monocytes recruitment and differentiated to resident macrophages in vascular wall.In this study, we estimate that haptoglobin have strong anti‐oxidant activity and decrease the differentiated adhesive monocytes amount in vitro. In addition, we find haptoglobin inhibit monocyte adhesion molecular vascular cell adhesion molecule 1 (VCAM‐1) and Intercellular Adhesion Molecule 1 (ICAM‐1) expression in PMA‐induced inflammation endothelial cells. According, haptoglobin has the abilities to prevent LDL oxidation to mm‐LDL and ox‐LDL and decrease inflammation driven monocyte recruitment and differentiate into resident macrophages in vascular wall intimal space. Furthermore, we find haptoglobin enhance human umbilical vein endothelial cells (HUVECs) proliferation, migration, and angiogenesis in vitro. The abilities can accelerate wound healing process in the oxidative and inflammation initiated atherosclerotic lesion and improve cardiomyocyte function in ischemic damaged lesion.Currently, anti‐oxidant, anti‐inflammation, and angiogenesis enhance agents are the major studies trend for coronary artery diseases therapeutic application. In this study, we find haptoglobin has the activities to prevent atherosclerotic process and to improve functional activities in damaged atherosclerotic lesion. In conclusion, haptoglobin has strong potential as a protein drug for atherosclerosis prevention and therapeutic application.Support or Funding InformationThis work was supported by the grants MOST 105‐2314‐B‐038‐016 and MOST 105‐2119‐M‐038‐001 from Ministry of Science and Technology, Taiwan.
The Effect of MCPIP1 Deficiency on Age‐related Lipid Profile Changes
Authors: Yang Chalen , Deaton Evan , Chang Yingzi 
Published: November 2023

BackgroundChronic inflammation is associated with metabolic disorders, such as impaired lipid homeostasis, which can lead to atherosclerotic plaque formation and ultimately myocardial infarction or stroke. Monocyte chemotactic protein‐induced protein‐1 (MCPIP1) is a negative regulator in inflammatory processes. Previous studies have shown that MCPIP1 gene deficient mice display phenotypic changes, such as splenomegaly, lymphadenopathy, growth retardation, decrease adipose deposit, and increased pro‐inflammatory cytokines. Our current project is to investigate the age‐related lipid profile changes in MCPIP1 deficient mice.MethodsPlasma and liver tissues are collected from wild‐type (WT) and MCPIP1 knockout (KO) C57/BL6 mice at ages 2, 4, and 6 weeks. Plasma HDL, LDL‐VLDL, and liver total cholesterol are measured by using HDL and LDL/VLDL quantitation kit from Sigma‐Aldrich. Plasma and liver total triglycerides are quantified by using Cayman Chemical Triglyceride Assay Kit.ResultsWe found that MCPIP1 deficiency significantly: 1) lowered plasma HDL levels in mice at the age of 6 weeks; 2) caused age‐related reduction of HDL‐cholesterol; 3) increased plasma LDL‐VLDL in mice at ages of 2 and 4 weeks; and 4) increased total plasma triglyceride in the mice at the age of 6 weeks. No differences of total triglyceride and cholesterol were observed between WT and KO livers.ConclusionThe data collected thus far suggest that MCPIP1 is an important regulator in maintaining plasma lipid homeostasis. MCPIP1 deficiency may contribute to the age‐related changes of lipid profile changes.Support or Funding InformationATSU Masters of Biomedical Sciences Program
Distinct roles of extracellular vesicles from macrophages and endothelial cells in regulation of endothelial inflammation
Authors: Lin Xingguang , Zuo Ping , Khanal Grishma , Zhang Youzhi , Li Xiang , Zhang Yang 
Published: November 2023

Background and aimsExtracellular vesicles (EVs) play an important role in maintaining endothelial cell (EC) homeostasis. Here, we examined the effects of exosomes and microvesicles, two main subtypes of EVs, derived from either ECs or macrophages on endothelial inflammation.MethodsExosomes and microvesicles were isolated from the cell culture medium from mouse microvascular ECs and J774 macrophages by sequential ultracentrifugation. Exosomes were detected by Western blotting analysis of marker protein CD63 and AIP‐1. Microvesicles were stained with FITC‐annexin V and Hoechst 33342 and analyzed by flow cytometry. Endothelial inflammation was examined by analyzing mRNA and protein expression of VCAM‐1 and monocyte adhesion using real time RT‐PCR and fluorescence microscopy.Result and conclusionBoth macrophages and ECs spontaneously released CD63 and AIP‐1 positive exosomes under resting condition, whereas the expressions of these exosome markers were not affected by TNFα stimulation. In contrast, TNFα markedly increased the release of annexin V+ microvesicles from either ECs or macrophages. Interestingly, among these microvesicles, we identified a novel subpopulation of microvesicles that are positively stained with both annexin V and Hoechst 33342. This result suggests that TNFα increased the release of a unique subset of microvesicles containing DNA fragments. Functionally, EVs (microvesicles and exosomes) derived from TNFα‐treated macrophages, but not from TNFα‐treated ECs, increased VCAM‐1 expression and monocyte adhesion in naïve ECs. Together, these results suggest that EVs may exert distinct effects (pro‐inflammatory v.s. anti‐inflammatory) on endothelial homeostasis depending on their subtypes and/or cellular sources.Support or Funding InformationSupported by NIH grants HL122769 and HL122937
Arachidonic acid metabolites induce TRPV1‐mediated inflammation
Authors: Hamers Alexander Jozua Pedro , Ahluwalia Amrita 
Published: November 2023

IntroductionTransient receptor potential cation channel subfamily V member 1 (TRPV1) is a non‐specific cation channel localised to sensory C‐fibres. Opening of the TRPV1 channel leads to cell membrane depolarization, resulting in the release of neuropeptides including calcitonin gene‐related peptide and substance P (SP). These neuropeptides play an important role in vasodilatation, vascular leakage and cell migration during acute inflammatory responses [1, 2]. Endogenous activators of TRPV1 are thought to include the arachidonic acid (AA) metabolites 12‐hydroperoxyeicosatetraenoyl acid (12‐HpETE) and 20‐hydroxyeicosatetraenoic acid (20‐HETE) [3]. However, whether these lipids underlie TRPV1 activation in inflammatory responses is unknown.AimTo investigate the role of TRPV1 in the acute inflammatory response and to determine whether arachidonic acid metabolites might play a role in its endogenous activation.MethodsLipopolysaccharide (LPS, 100 μg Ecoli 055:B5), zymosan activated serum (ZAS) (50%), SP (1 nmol) or AA (30, 100 and 300 nmol)‐induced skin oedema formation were determined in TRPV1 knockout (KO) and wild type (WT) littermate mice using a modified Miles assay method. The roles of 12‐HpETE and 20‐HETE were investigated by treating animals with the 12‐HpETE synthesis inhibitor cinnamyl‐3, 4‐dihydroxy‐a‐cyanocinnamate (CDC) (8 mg/kg, i.p.) or the 20‐HETE synthesis inhibitor 17‐octadecynoic acid (17‐ODYA, 35 mg/kg,i.p.). The effects of pharmacological blockade of TRPV1 and the SP receptor neurokinin 1 (NK1) were tested using AMG‐9810 (30 mg/kg, i.p.) and SR‐140333 (0.1 μmol/kg i.v) respectively.ResultsThe oedema response to LPS (P<0.01) and ZAS (P<0.05) were reduced by ~ 53 and 43% respectively in TRPV1 KO animals compared to WT mice (LPS: 24.6 ± 3.8 μl, ZAS: 48.4 ± 9.0 μl n=13). In WT mice treatment with AMG‐9810 (n=13) reduced the LPS (P<0.01), ZAS (P<0.01) and AA (P<0.001) response, by ~ 43, 22 and 36% respectively compared with vehicle treated mice (LPS: 19.4 ± 2.2, ZAS: 42.2 ± 3.2, AA: 47.6 ± 5.9 μl n=13). 17‐ODYA (n=15) significantly reduced LPS (P<0.05), ZAS (P<0.01) and AA (P<0.05,) induced oedema by ~ 47, 24 and 29% compared to vehicle treated mice (LPS: 19.6 ± 2.7, ZAS: 48.1 ± 4.7, arachidonic acid: 47.0 ± 6.6 μl n=13). NK1 receptor blockade reduced AA‐induced (300 nmol) oedema from 43.4 ± 3.9 μl to 29.0 ± 1.8 μl (n=13, p<0.001).ConclusionOur findings suggest that TRPV1 plays an important role in vascular permeability during an acute inflammatory response induced by diverse stimuli. In addition our findings suggest that the endogenous activator of TRPV1 in these scenarios is 20‐HETE. Furthermore, the NK1 receptor plays a key role in mediating the AA oedema response induced by endogenous TRPV1 activation, indicating that 20‐HETE is the endogenous activator and SP is the downstream mediator of TRPV1.Support or Funding InformationA.H. is supported by The Trustees of the Professor Derek Willoughby PhD Fund for Inflammatory Research.
Nicotine induced endothelial dysfunction through cathepsin B‐dependent activation of endothelial Nlrp3 Inflammasome
Authors: Chen Yang , Li Xiang , Li Pinlan , Zhang Yang 
Published: November 2023

Recent studies indicate that endothelial dysfunction occurs in association with several cardiovascular risk factors and endothelial Nlrp3 inflammasomes play a crucial role in the pathogenesis of cardiovascular diseases. But the precise innate immunity mechanisms that endothelium cell how to been trigger or promote the inflammatory response during the development of early stage of smoking‐associated vascular complications remains poorly understood. The present study explored the role of endothelial Nlrp3 inflammasome as a major injurious factor in mediating gap junction disruption, an important event of endothelial barrier dysfunction leading to endothelial hyperpermeability during initiate stage of smoking addiction. In vivo study, confocal microscopy analysis demonstrated that nicotine stimulation had significantly increased endothelium gap junction disruption and Nlrp3 inflammasome induced HMGB1 release compared with animals fed normal diet (ND). And the Nlrp3−/− mice significantly attenuated the endothelium gap junction disruption and HMGB1 release. In vitro, these Nlrp3 inflammasome molecules could be aggregated to form an inflammasome complex on stimulation of nicotine, as shown by fluorescence confocal microscopy. Correspondingly, nicotine significantly increased caspase‐1 activity and IL‐1β and HMGB1 release in microvascular endothelial cells, indicating an activation of Nlrp3 inflammasomes. Further pharmacological inhibition and genetic intervention experiments showed that the nicotine‐induced endothelium cell permeability was abolished in MVECs when cells were treated with either a HMGB1 inhibitor glycyrrhizin (GLY), caspase‐1 inhibitor or Nlrp3 gene silencing. In order to further study the mechanism, we used fluorescence staining experiments. It's showed that nicotine‐induced endothelium cell cathepsin B activation was abolished in MVECs when pretreatment the lysosome stabilizer (Dexamethasone). The present study revealed that nicotine‐induced endothelium Nlrp3 inflammasome formation and activation is mediated through cathepsin B pathway and the injurious effect of nicotine on the endothelium is associated with the HMGB1 via Nlrp3 inflammasome activation. Endothelial Nlrp3 inflammasome system may serve as an important pathological mechanism in the development of early stage of smoking‐associated vascular complications.Support or Funding InformationThis study was supported by grants from the National Institutes of Health (HL057244, HL122769 and HL122937) and the National Natural Science Foundation of China (NO.81603587).
Inhibitory Effects of Growth Differentiation Factor 11 on Phenotypic Transition Associated with Autophagy Deficiency in Mouse Coronary Arterial Myocytes
Authors: Yuan Xinxu , Bhat Owais , Lee RaMi , Lohner Hannah , Zhang Yang , Li Pin‐Lan 
Published: November 2023

Growth‐differentiation factor 11 (GDF11) as an anti‐aging factor may reverse age‐related cardiac hypertrophy in mice and cause youthful regeneration of cardiomyocytes. The role of GDF11 in the growth and phenotypic transition of vascular smooth muscle cells remains poorly understood. The present study first examined the effects of GDF11 to induce differentiation of cultured mouse coronary arterial myocytes (CAMs). By real time RT‐PCR and Western blot analysis, GDF11 was found to increase CAMs differentiation, as shown by increased expression of various muscle cell differentiation markers such as α‐SMA, myogenin, MyoD, and MyHC, but decreased expression of dedifferentiation markers such as vimentin and proliferating cell nuclear antigen (PCNA). Upregulation of GDF11 gene by trichostatin A (TSA) or specific CRISPR‐cas9 activating plasmids also stimulated the differentiation of CAMs with a remarkable decrease in the ratio of vimentin vs. SM22α. In CAMs treated with 7‐ketocholesterol (7‐keto), a phonotypic transition into dedifferentiation status was observed with significant increases in the ratio of vimentin vs SM22α, which was companied by autophagosome (AP) accumulation with large increase in LC3‐II. This 7‐keto‐induced CAMs dedifferentiation and AP accumulation were almost completely blocked by GDF11 or TSA treatment. Similarly, in CAMs pretreated with lysosome function inhibitor, bafilomycin or chloroquine, both GDF11 and TSA markedly attenuated the phenotypic transition and AP accumulation. Moreover, in CAMs from mice lacking CD38 gene that produces lysosome trafficking regulator, NAADP, GDF11 was also shown to inhibit phenotype transition to dedifferentiation status with or without 7‐keto pretreatment. It is concluded that both exogenous and endogenous GDF11 promote CAMs differentiation and prevent a phenotypic transition of these cells by reduction of lysosome accumulation induced by different pathological stimuli.Support or Funding InformationSupported by NIH grants HL057244, HL075316 and HL122937