bluuebunny/crossref_metadata_2025_split huggingface dataset:
Glycine Formation Drives Bile Acid Toxicity in Sandwich‐Cultured Human Hepatocyes
Authors: Deferm Neel , Chatterjee Sagnik , Richert Lysiane , de Witte Peter , Bouillon Thomas , Annaert Pieter 
Published: June 2021

Disturbances in bile acid (BA) disposition and consequent intracellular accumulation of bile acids (BAs) are hypothesized to be associated with toxicity observed in cholestatic disease. The aim of the current study was to assess the link between toxicity and disposition of both chenodeoxycholic acid (CDCA) and its glycine conjugate glycochenodeoxycholic acid (GCDCA) in sandwich‐cultured human hepatocytes (SCHH). Biochemical integrity of the hepatocytes was assessed on multiple days of culture (day‐2 and day‐6) by determining the urea formation capacity of the cultured cells. CDCA exerted culture‐time dependent toxicity, as reflected by a more prominent reduction in relative urea formation in day‐2 SCHH, compared to day‐6 SCHH. To further explore the observed culture time‐dependent toxicity, in vitro disposition data were obtained by treating day‐2 and day‐6 SCHH with either 2.5, 10 or 25 μM CDCA. Media containing CDCA and GCDCA were collected, after which cells were lysed and intracellular accumulation was determined. Additionally, amounts excreted into the bile canaliculi were calculated. Concentration‐time profiles were fitted to a mechanistic ten‐compartment model using NONMEM v7.3.0. The model includes compartments representing the media, cells, bile and cells+bile with compound distributed among them through linear processes. Culture time (day‐2 and day‐6) was implemented as a covariate in the dataset and its effect on kinetic parameter estimations was assessed by the difference in successive objective function values. Covariate analysis showed a significant effect (p < 0.01) of culture time on both sinusoidal efflux clearance of GCDCA (day‐2: 0.6 (± 1%) μL/min/106 cells, day‐6: 1.7 (± 2%) μL/min/106 cells) and metabolic clearance (day‐2: 17.7 (± 3%) μL/min/106 cells, day‐6: 7.2 (± 11%) μL/min/106 cells), indicating that the lower toxicity of CDCA in day‐6 SCHH can be explained by decreased intracellular levels of GCDCA due to reduced formation and increased sinusoidal efflux of GCDCA. Interestingly, culture time‐dependent changes in mRNA and protein levels of the conjugating enzyme bile acid‐coenzyme A:amino acid N‐acetyltransferase (BAAT) did not correlate with the observed culture time‐dependent decrease in GCDCA formation. In conclusion, our results indicate that CDCA exerts culture‐time dependent toxicity in SCHH which seems to be related to changes in both sinusoidal efflux clearance of GCDCA and metabolic clearance of CDCA.Support or Funding InformationFinancial support for this research was provided by the Research Foundation ‐ Flanders (FWO)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Role of Primary Cilia in Breast Cancer Recurrence
Authors: Davis Jason E , Walens Andrea , Mabe Nathaniel W , Fox Douglas , Hernandez Dulcemaria , Alvarez James V , Goetz Sarah C 
Published: June 2021

Recurrent tumors are the predominant cause of breast cancer‐related deaths, therefore there is an urgent need for more effective therapeutic strategies. However, a critical barrier to their development is a lack of understanding of the molecular mechanisms involved in breast tumor recurrence. Recently, primary cilia, microtubule‐based organelles that project from the surface of vertebrate cells, have emerged into the spotlight for their link to numerous human diseases, including cancer. However, the relationship between primary cilia and breast cancer recurrence remains unknown. Here, using a genetically engineered mouse model of breast cancer, which closely recapitulates the clinical phenomenon of breast cancer recurrence, we have uncovered that primary cilia are more highly enriched in recurrent mammary tumors than in both primary mammary tumors and asymptomatic/contralateral mouse mammary tissues. Congruently, we found that the percentage of ciliated cells was substantially elevated in recurrent breast cancer cells compared to primary breast cancer cells. Moreover, RNA‐sequence analysis also revealed that GLI2 and GLI3, which are major downstream effectors of the morphogenic Sonic Hedgehog signaling pathway and are also well‐known to localize to primary cilia, were upregulated in recurrent breast cancer cells compared to primary breast cancer cells. Collectively, these data suggest that primary cilia may have a role in breast cancer recurrence. Our study also indicates that, at least within the recurrent stages of the disease, the primary cilium may serve as a specialized oncogenic signaling organelle.Support or Funding InformationTo Sarah C. Goetz: R00 HD076444 (from the National Institute of Child Health and Human Development).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interferon regulatory factor 3 and NF kappa b are required for expression of immune susceptibility genes in human lung and breast carcinoma cells treated with poly I:C
Authors: Kester Jessica M. , Bortz Eric 
Published: June 2021

Immunostimulatory Toll‐like receptor (TLR) and RIG‐I‐like receptor (RLR) agonists, including the viral RNA mimic poly I:C, have been proposed as adjuvant the efficacy of anti‐tumor immunotherapy. In combination with interferon‐gamma, poly I:C may function by direct activation of anti‐tumor M1 macrophages and natural killer (NK) cells. Poly I:C may also drive potent stimulation of dendritic cells, and in turn CD8+ and/or CD4+ T cell responses to enhance recognition of tumor‐associated neoantigens, making it an attractive adjuvant for use in combination with immune checkpoint inhibitors or therapeutic vaccines. However, the potential direct effects of poly I:C on heterogeneic tumor cells in the context of immunotherapeutic strategies are poorly understood. We hypothesized that poly I:C may also directly induce NF‐kappa B activation, and expression of interferon‐stimulated genes (ISG), in tumor cells, thus impacting tumor susceptibility to immune responses. We found that poly I:C and authentic viral RNA induced robust expression of ISG MXA in cultured human lung adenocarcinoma (A549) and metastatic breast carcinoma (ZR‐75‐1) cell lines. In contrast, the type III interferon cytokine IL28A/B was induced in both A549 and ZR‐75‐1 by viral RNA, but only in ZR‐75‐1 by poly I:C, suggesting that tumor cells may differentially retain intact innate immune transcriptional programs. In these tumor cells, poly I:C also induced NKG2D ligands ULBP 1/2/3, MHC I pathway genes TAP, MECL1‐beta, and LMP2, and the immune checkpoint protein PDL2, indicating the poly I:C may directly modulate the susceptibility of tumor cells to NK and CD8+ T cell responses. To understand transcriptional activation driven by poly I:C, we examined the transcription factors IRF3 and NF‐kB. In A549 cells treated with poly I:C, IRF3 translocated to the nucleus, and IRF3 was required for secretion of bioactive cytokines using an interferon‐sensitive VSV‐GFP reporter bioassay. Similarly, poly I:C induced the NF‐kB target gene IL8 in both A549 and ZR‐75‐1, which could be abrogated by NF‐kB inhibitor bay 11‐7082. Taken together, it may be promising to explore the efficacy of poly I:C and other viral RNA mimics to directly adjuvant tumor cell susceptibility to cytotoxic T lymphocytes, adding another dimension to immunotherapy against human tumors.Support or Funding InformationNIGMS Alaska INBRE; Portland State University‐University of Alaska Anchorage BUILD EXITO Program; UAA OURS & CASThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hormetic Response of H1299 Proliferation to Extracts of Hydnora Johannis Becc (Kausen Kasa) is Mediated Via Estrogen Receptor/EGFR and PKC
Authors: Morgem Mounira Omar , Oyagbemi Ademola Adetokunbo , Omobowale Temidayo Olutayo , Fagbohun Olusegun , Adedapo Adeolu Alex , Yakubu Momoh Audu 
Published: June 2021

Hydnora Johannis Becc (HJ) is a herbal plant used in treating menopausal symptoms and other diseases in different countries, but the mechanism of actions are not known. The effect of HJ (0.1–2 mg/ml) extract on the proliferation of H1299 cell line was investigated. Cells were plated in 96 well plates and the effects of HJ were evaluated using MTT assay. Treatment of H1299 with HJ for 24 hours induced a dose‐dependent increase in cell proliferation increasing growth by 165%, 188%, 203% and 263% at 0.1, 0.25, 0.5 and 2 mg/ml, respectively compared to the control. Paradoxically at 48 hrs, the proliferative effects of the extract were turned into antiproliferative reducing cell growth by −74%, −65%, −37%, −17%, and 108% for 0.1, 0.25, 0.5, 1 and 2 mg/ml, respectively compared to the control. This effects showed a time‐dependent hermetic responses of H1299 to the HJ treatment. To investigate the mechanism(s) by which HJ regulate H1299 growth, cells were treated with HJ in the presence or absence of inhibitors of estrogen receptor (ER; Tamoxifen 10 and 20μg/mL), epidermal growth factor receptor (EGFR; Tyrphostin 5 μM) or Protein Kinase C (PKC; Calphostin 5 μM) and the effects on cell proliferation determined. Inhibition of ER, EGFR, and PKC significantly attenuated the proliferative effects of HJ treatment at 24 hrs. The antiproliferative effects observed at 48 hrs following HJ treatment were reverted to proliferation in the presence of inhibitors. These results suggest that HJ displays paradoxical phenomena which may be mediated by activation/inhibition of both proliferative and apoptotic signaling pathways in H1299 and mediated through interaction with ER, EGFR, and PKC signaling pathways. Further studies are warranted to investigate other signaling pathways involved in the Hydnora Johannis Becc actions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modeling Potenial BACE1 Inhibitors
Authors: Guevara Johnny , Zahran Mai , Department Alberto 
Published: June 2021

A wealth of evidence suggests that aggregated amyloid beta (AB) peptide deposits in the brain play a central role in the pathogenesis of Alzheimer's disease (AD). The rate determining step in AB production is catalyzed by the beta‐site amyloid precursor protein cleaving enzyme 1 (BACE1); hence, BACE1 is a therapeutic target for AD. In our previous study, six potential BACE1 inhibitors (compounds 1–6) were synthesized and biologically evaluated by in vitro BACE1 inhibition assays. The aim of this work was to implement an in silico approach to study the underlying molecular interactions between compounds 1–6 and BACE1 on an atomic level, to ultimately understand and validate experimental results. We performed molecular docking studies using AutoDock Vina to predict favorable binding modes and calculated non‐covalent intermolecular interactions using Arpeggio to investigate important BACE1 inhibitory drug characteristics, such as the ability to interact with the catalytic Asp dyad (Asp32 and Asp228) in the active site of BACE1. Docking results showed compounds 3, 5 and 1 exhibiting the most favorable binding affinities: −8.2, −7.9 and −7.8 kcal/mol, respectively; while retaining binding orientations relative to the Asp dyad sustained by hydrogen bonds, as well as several stabilizing hydrophobic and aromatic interactions. Compounds 2, 4 and 6 retained binding affinities −7.7, 7.2, and −6.9 kcal/mol, respectively, but did not interact with the Asp dyad, forming various stabilizing interactions deemed non‐inhibiting. Known BACE1 inhibitors (OM99‐2 and LY2811376) and resveratrol were also studied for comparison purposes. Overall, our computational results correlate well with our experimental results, they indicate that several of our compounds can serve as potential BACE1 inhibitors and aid in future AD drug design.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functional Role of Microbiota‐derived Metabolites in the GPCR‐mediated Regulation of Intestinal Wound Healing and Barrier Function
Authors: Alam Ashfaqul , Boyer Darra , Matthews Jason , Saeedi Bejan , Wu Huixia , Neish Andrew S. 
Published: June 2021

The mammalian intestine houses a complex and abundant ensemble of commensal bacteria, which are in constant cross‐talk with the intestinal epithelial cells, and thereby, ensure gut homeostasis, development, permeability, and repair. Wound regeneration is a complex but coordinated cellular events to repair injured gut mucosa, which involves both cellular proliferation and migration. Gut barrier function is central to the health and is critical in a wide variety of diseases including inflammatory bowel diseases (IBD). Our recent metagenomics study discovered a consortium of novel gut mucosa‐associated proregenerative symbiotic bacteria, which preferentially enrich in and colonize the injured mucosa to augment epithelial repair. The resident microbiota of the gut is endowed with over a hundred times more genes than those in our genome. However, the microbiota's more than nine million genes, and their metabolic functions remained mostly unrecognized. We performed an unbiased screening analysis by mass spectrometry‐based metabolomic profiling of intact and wounded mucosa in germ‐free and conventional mice. Unlike the germ‐free animals, the colon of the conventional mice demonstrated increased amounts of acidic microbial metabolites, which established an extracellular pH gradient with more acidic pH at the apex of the colonic crypt. We found that the murine and human intestinal epithelial cells express the proton‐sensing G‐protein coupled‐receptor GPCR68. Intriguingly, the murine colonic contents of the conventional mouse or a specific metabolite, 2‐hydroxy‐4‐methylpentanoic acid (HMP) decreases the extracellular pH to modulate claudin‐4 dependent paracellular permeability and to regulate the proliferation of intestinal epithelial cells in a GPCR68‐dependent manner. Importantly, our findings highlighted that the HMP‐stimulated intestinal epithelial wound closure requires GPR68. Collectively, our data underscore the significance of intestinal metabolites in modulating the extracellular proton concentration in the gut mucosal microenvironment. Furthermore, our findings demonstrate that the GPCR68 functions as the sensor of microbially modulated proton gradient that is potentially involved in the maintenance of gut mucosal homeostasis, wound healing, and restoration of the intestinal barrier function.Support or Funding InformationK01DK114391 (National Institute of Diabetes and Digestive and Kidney Diseases), R01DK089763 (National Institute of Diabetes and Digestive and Kidney Diseases), R01AI064462 (National Institute of Allergy and Infectious Diseases)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Research‐Driven Laboratory and Literature Course for Senior Biochemistry/Molecular Biology Undergraduates
Authors: Fuleihan Christina , Parsley Luke , Riley Kasandra 
Published: June 2021

Biochemistry/molecular biology (BMB) course‐based undergraduate research experiences (CUREs) are part of a growing movement to involve a greater number of undergraduates in authentic research experiences. Positive outcomes of CUREs are manifold, including improvement in scientific aptitude, confidence, and critical thinking skills that make students more likely to become career scientists. Here we describe the structure and outcomes of a one‐semester, senior BMB capstone CURE that pairs two 75‐min weekly discussion/problem‐solving sessions with semester‐long, weekly laboratory research. Within the context of two complex systems (bacterial and influenza infections), students were taught how to approach BMB literature. In the first three weeks of the course, students read and discussed primary literature directly relevant to their laboratory project. Additional literature selected for its demonstration of BMB techniques was presented by students throughout the remainder of the term in an alternating seminar/problem solving format. In the laboratory, all of the students studied homologs of the RNA‐binding protein Hfq, which is small (~11 kDa), thermostable, and present in about half of sequenced bacterial species. All students expressed and purified a previously uncharacterized, wild type Hfq homolog, enabling them to improve their technique with some instructor guidance. Simultaneously, students used databases and primary literature to design independent projects to test their own hypotheses about Hfq from various bacterial species. All pairs of students cloned novel proteins using synthetic biology and successfully purified Hfq at least once. This scaffolded approach to experimentation contextualized fundamental BMB technical skills, boosting understanding of sequence databases, cloning, SDS‐PAGE, protein expression/purification, three‐dimensional protein structure, and activity assays. Final laboratory papers were written using a manuscript submission template from a professional journal and a class‐wide, blinded peer review, furthering the emphasis on an authentic research experience. Students gained an appreciation for the medical impact of basic science and a multidimensional understanding of our discipline. This type of integrated course can be easily customized to emphasize other themes (e.g. cancer) and enhance undergraduate contributions to other original research projects within the time frame of a single semester.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determination of the binding affinity of RNA aptamer to B‐cell activating factor receptor protein from non‐Hodgkin lymphoma
Authors: Blade Thanh Phan , Roa Stephanie , Avrit Tara 
Published: June 2021

Non‐Hodgkin lymphoma originates from B‐cells, a type of lymphocytes. Other research labs have shown a high expression of B‐cell activating factor protein (BAFF) binding its protein receptor BAFF‐R on the surface membrane of the malignant B cells. An RNA aptamer was designed not only to bind to BAFF‐R but also to deliver a siRNA into malignant B‐cells to induce cellular apoptosis. The ability of an RNA aptamer to deliver a siRNA in to the cells depends greatly on its high binding affinity to BAFF‐R by yielding a low dissociation constant or KD. We chose an electrophoresis mobility shift assay method to study protein‐RNA interaction using radioactive labeled RNA. After allowing various concentrations of BAFF‐R to incubate with a fixed concentration of RNA to form RNA‐protein complexes, we loaded the samples into wells of a 5% native polyacrylamide gel to separate unbound RNAs and RNA‐protein complexes. Then we exposed the gel to a film which registered the signals from radioactive phosphate RNA. We used Typhoon FLA 7000, a phosphorimager, to convert the film into an image which we analyzed to determine BAFF‐R concentration, which had fifty percent RNA bound to protein. The KD was calculated to be 495nM. In short, the affinity of RNA to BAFF‐R can be determined using EMSA, but we can lower the KD by optimizing the binding‐reaction conditions such as temperature or buffer condition.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biochemical and Functional Characterization of a Novel Mutation in the NADPH Cytochrome P450 Oxidoreductase
Authors: Pandey Amit V , Benito‐Sanz Sara , Parween Shaheena , López‐Siguero Juan‐Pedro , Camats Núria , Fernández‐Cancio Mónica , Udhane Sameer S , Kagawa Norio , Flück Christa E , Audí Laura 
Published: June 2021

BackgroundCytochrome P450 oxidoreductase (POR) deficiency (PORD) is a form of congenital adrenal hyperplasia (CAH) and results in loss of steroid and drug metabolizing cytochrome P450 activities. Mutations in POR cause mild to severe forms of CAH with/without bone malformation. We report a novel POR Arg550Trp mutation identified in a 46, XX patient with signs of CYP19A1 deficiency. Child and mother had signs of virilization. Mother had elevated T on the fifth day that returned to normal in the fourth month. The daughter was born with fused labioscrotal folds. Ultrasound revealed the presence of uterus and ovaries. Sequencing of the CYP19A1 gene did not reveal any defects, and further analysis revealed compound heterozygous mutations c.70_71delTC/p.Leu25PhefsTer93 and c.1648C>T/p.Arg550Trp in POR. At eight years the adrenal function of the patient is normal except for slightly elevated 17‐OH‐prog.MethodsDNA was analyzed with a targeted Disorders of Sexual Development NGS panel (DSDSeq.V1, 111 genes) on a NextSeq (Illumina) platform. We analyzed the ability of POR wild‐type (WT) and Arg550Trp to reduce ferricyanide, MTT, cytochrome c and activity towards the drug and steroid metabolizing cytochromes P450. The POR WT and Arg550Trp variants were expressed and produced as recombinant proteins in bacteria and combined with recombinant P450 proteins and small molecule substrates for enzyme assays.ResultsWe found severe effects of Arg550Trp mutation on activities with different substrates. Arg550Trp showed 41% of the WT activity in cytochrome c and only 7.7% activity towards the reduction of MTT. A 2.75 fold increase in Michaelis constant (Km) was observed in ferricyanide reduction assays compared to WT POR. Severe effect on NADPH binding by R550W mutation was observed using both cytochrome c and MTT as substrates with variable NADPH concentration. Assays of CYP19A1 activity showed loss of >90% activities using androstenedione as substrate.ConclusionThe mutation Arg550Trp is located in the NADPH binding region of POR. Computational analysis predicted instability in the NADPH binding region of POR by R550W mutation, Computationally predicted adverse effect on aromatase activity as well as a reduction in binding of NADPH was confirmed by experiments using recombinant proteins. These results suggest a pathological effect of POR R550W and a diagnosis of PORD in the patient with p.Arg550Trp/p.Leu25PhefsTer93 in POR.Support or Funding InformationSupported by grants to Amit V Pandey from the Swiss National Science Foundation, and Novartis Foundation for Medical Biological Research and to Mónica Fernández‐Cancio and Sara Benito from Instituto de Salud Carlos III, Madrid, Spain.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.