bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cell culture confluency estimation, transfection efficiency and total cell counting applications on the InCellis® Smart Cell Imaging System
Authors: Antanaviciute Laima , Dubacq Sophie , Varet Olivier 
Published: June 2021

Cell biology studies of cell proliferation, invasive behaviour of cells, and cell line expression require a rapid and robust cell line confluency estimation, transfection efficiency investigation, and accurate total cell counting. Accuracy and efficiency are extremely important in cell culture quality check applications. Incorrect observations might lead not only to inaccurate results but also to incorrect conclusions and recommendations. In this study, breast cancer cell line (MCF‐7) was used to accurately estimate cell line confluency and total cell counting in an automated manner using embedded applications on the InCellis® Smart Cell Imaging System (Bertin Technologies). A series of images of the cell line were taken in phase contrast mode using 10× and 20× objectives on day‐1, day‐2 and day‐3 at the same time in order to check the accuracy of cell confluency. The cell line confluency ranged from 12% – 76% on day‐1 through day‐3 with the 10× objective, and ranged from 14% to 77% with the 20× objective respectively. Furthermore, the results obtained from the cell counting application embedded on the InCellis® were compared to the manual approach performed with Malassez cell counting method that was treated as a control across the 3‐day experiment. The results demonstrated that both methods are correlated and showed an increase of the total cell number as expected (40K on day‐1 ‐ 270K on day‐3). To validate the automated transfection efficiency application, Hela cell culture lines and GFP label were used. The 3‐step process allowed easy and spontaneous observation of the percentage of transfected cells and the overall quality inspection of the cell line. The confluency and cell counting applications supply robust results with a stain‐free method in comparison to the standard method. The transfection application provided a rapid and user‐friendly method for estimation of transfected cells before use in further analysis. The automated applications not only provide consistent results but also significantly reduce the hands‐on time for all cell based assays.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential Expression of Cannabinoid Receptors, CB1R and CB2R, in Prostate Tumor Samples with Perineural Invasion
Authors: Alicea Reyes Tatiana M , Flores‐Otero Jacqueline 
Published: June 2021

Perineural invasion (PNI) affects over 20% of patients with prostate cancer who often recur with metastasis primarily to the bones. Despite its association with the patient's poor prognosis and dissemination of various cancers, knowledge of the regulatory mechanisms that drive invasion of prostate cancer cells through and around nerves is limited. Cannabinoid receptors (CBRs) are highly enriched in prostate cancer cells and exogenous application of CBR ligands attenuates the cell's survival, migration and invasion ability. Our objective is to define whether activation of the endocannabinoid system (ECS) associates with PNI and to identify the endogenous regulatory mechanisms through which the ECS promotes prostate cancer cell invasion through nerves. We used immunohistochemistry technique to determine the expression levels of the CB1R and the CB2R in formalin‐fixed paraffin embedded (FFPE) prostate tumor samples with PNI. Preliminary data from our laboratory show that CB1R is highly enriched in FFPE prostate tumor samples with PNI when compared to CB2R. Given the increase of the CB1R in PNI FFPE samples, we used total internal fluorescence reflection microscopy (TIRFm) to elucidate in vivo the trafficking kinetics of CB1Rs in highly invasive versus less invasive prostate cancer cells, PC3 and 22RV1, respectively. We found that PC3 cells display constitutive CB1R trafficking. However, this was not the case for the less invasive prostate cancer cells, 22RV1. Our results suggest that the elevated CB1R levels detected in the FFPE PNI positive samples is not coincidental, but rather an indication of an association of the ECS and PNI. Ongoing studies aim to determine how nerve‐PC tumor interactions encourage PNI by using an in vitro model in which we can modulate the ECS.Support or Funding InformationGrants: RCMI‐NIMHD 8G12MD007600 to JFO.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Streptococcus Pneumoniae Infection in Respiratory Syncytial Virus Infected Neonatal Lambs
Authors: Alnajjar Sarhad SA , Sitthicharoenchai Panchan , Gallup Jack , Ackermann Mark , Verhoeven David 
Published: June 2021

Respiratory syncytial virus (RSV) is the main cause of viral bronchiolitis resulting in hospitalization and a frequent cause of secondary respiratory bacterial infection especially by Streptococcus pneumoniae (Sp) in infants. While murine studies have demonstrated enhanced morbidity during a viral/bacterial co‐infection, human meta‐studies have been mixed. Moreover, less is known about pathogenesis of Sp serotype 22 and especially the co‐pathologies between RSV and Sp dual infections.Here, we sought to examine mechanisms contributing to co‐pathogen‐induced morbidity using a large neonatal lamb animal model naturally permissive to infection by both pathogens.Colostrum deprived lambs (aged 3–5 days) were randomly divided into four groups. Two of the groups were nebulized with RSV M37 (1.27 × 107 IFFU/mL), and the other two group nebulized with cell–conditioned mock media. At day 3 post‐infection, one RSV group (RSV/Sp) and one mock‐nebulized group (Sp only) were infected with (2×106 cfu of Sp) intratracheally. At day 6 post‐infection all lambs were humanely euthanized and bacterial/viral pathogeneses were assessed by culture, focus forming unit, qPCR, IHC, and histopathology.Lambs dually infected with RSV and Sp had higher RSV titers by qPCR but lower Sp than the other comparable groups. Additionally, lung lesions were more intense in the RSV/Sp group as characterized by increased interalveolar wall thickness with neutrophils and lymphocyte infiltration.Despite lower Sp in lungs, lambs co‐infected with RSV exhibited greater morbidity and tissue histopathology. Thus, enhanced disease severity may be due more to elevated immunopathogenesis than elevated bacterial pathogenesisSupport or Funding Informationfunded by Merck & Company Inc.55211 Verhoeven, 11/01/16–10/31/18This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Ang III on Nuclear Factor Kappa Beta in Wistar Rat VSMCs
Authors: Samman Waad , Clark Michelle 
Published: June 2021

ObjectiveWe investigated whether angiotensin (Ang) III induces nuclear factor kappa beta (NF‐kb) phosphorylation in isolated rat vascular smooth muscle cells (VSMCs).BackgroundThe molecular mechanisms by which Ang III induces various biological effects have not been fully investigated. Most studies have shown that NF‐kb mediates Ang II inflammatory responses effects in VSMCs. Inflammation of vascular smooth muscle cells (VSMCs) is a critical action associated with cardiovascular diseases including hypertension. The role of Ang III to induce NF‐kb in VSMCs is unknown and is a focus of these studies.MethodsPrimary cultures of VSMCs were isolated from the thoracic aorta of adult Wistar rats by the explant technique. VSMCs were treated with Ang III ranging in concentration from 0.1 nM to 1000 nM for 10 minutes or with 100 nM Ang III for 1 minute to 30 minutes. The western blotting technique was used to determine the effects of the peptide on NF‐kb protein phosphorylation.ResultsConcentration studies showed that Ang III caused a dose‐dependent increase in NF‐kb protein phosphorylation. The effects of the peptide on NF‐kb phosphorylation were maximal between 10 nM and 100 nM. The peptide's effects were rapid and significant; occurring within minutes of treatment and the maximal effects on NF‐kb phosphorylation was observed by 10 minutes.ConclusionThese findings provide insight into the molecular nature of the actions of Ang III and offer possible mechanism by which Ang III physiological actions occur in VSMCs.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibitors of flavivirus by targeting the NS2B‐NS3 protease interface
Authors: Li Hongmin , Brecher Matthew , Li Zhong , Ndacayisaba Libère , Zhang Jing , Parmar Sunjit S , Banavali Nilesh , Kramer Laura 
Published: June 2021

Flaviviruses require the activity of their viral protease in order to process the single polyprotein encoded by their genome into individual proteins and assemble an infectious virion. This protease consists of a large catalytic subunit (NS3) and a smaller accessory subunit (NS2B) important for function. Using the previously solved flavivirus protease crystal structures, we identified three putative sites of interaction between NS2B and NS3 which we then queried with a virtual screen of NCI library compounds, followed by subsequent in vitro biochemical screening of the resultant candidates. Of more than one hundred initial candidate compounds that passed the virtual screen, thirty‐six demonstrated significant inhibition in vitro. Three most potent compounds, 227186, 73101, and 65828 were further investigated and found to significantly reduce titers of Dengue virus 2 on A549 cells.Support or Funding InformationAI133219, AI134568, AI131669This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In Vitro Model of Naphthalene Lung Epithelial Cell Toxicity Enabled by Human Hepatocyte Metabolism
Authors: Kelty Jacklyn Skye , Ding Xinxin , Van Winkle Laura 
Published: June 2021

RationaleNaphthalene causes toxicity in the lung, but this is thought to require metabolic activation by cytochrome P450 monooxygenases (P450s) in either the liver or the lung. We are developing an in vitro approach to determine whether human hepatic bioactivation of naphthalene can contribute to lung toxicity. We hypothesize that hepatic enzymes are capable of escalating lung epithelial cell damage caused by naphthalene.MethodsAirway epithelial cells from either female primate trachea (PEC) or the human bronchiolar epithelial cell line, HBE1, were grown and differentiated at the air‐liquid interface in Transwell inserts. 6‐hour exposures to naphthalene occurred with different batches of pooled human liver microsomes or human hepatocytes from female, non‐smoker, non‐fatty liver donors in the basal well. The dose of 80μM of naphthalene represents about 4 times the highest blood concentrations reported in human occupational exposures. Liver microsomes were boiled to inactivate P450s for negative control experiments. Diethyl maleate pretreatment of airway cells and/or hepatocytes before exposure to naphthalene was used to reduce naphthalene detoxification by depleting glutathione. Lung epithelial cell toxicity was assessed by measuring permeability and density of airway cells attached to the Transwell membrane.ResultsHBE1 cells exposed to 80μM naphthalene in cultures containing human liver microsomes resulted in a 30% decrease in cell density and an ~15% reduction in viability for one batch of microsomes, and no reduction in these endpoints for another batch of microsomes. HBE1 cells exposed to 80μM naphthalene with human hepatocytes present resulted in >50% decrease in cell density when glutathione was depleted in the hepatocytes with diethyl maleate. However, no significant cell death was detected in PEC cells under these conditions.ConclusionsThe presence of human liver microsomes enabled naphthalene toxicity in HBE1 cells in a pool‐dependent manner, a finding possibly related to the differing P450 activity profiles between the two pools analyzed. The presence of intact human hepatocytes from a similar liver donor population also enabled naphthalene toxicity in HBE1 cells. PEC cells were less susceptible to cell death, relative HBE1 cells, after exposure to naphthalene with human liver microsomes present. These data indicate that the airway toxicity of naphthalene is determined by the metabolic activation and detoxification capabilities of both the lung and liver cells in this system.Support or Funding InformationSupported by: R01 ES020867, P30 ES023513, NIOSH 2U54OH007550, and T32 HL007013.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Preliminary Characterization of Desulfinase DszB for use in high‐throughput validation of DszC mutant proteins
Authors: Sanchez Misael , Truong Sharon 
Published: June 2021

High rates of fossil fuel consumption around the world have given rise to increasing levels of pollutants in the atmosphere. Sulfur Dioxide, a chief example of this problem, causes acid rain, which can result in land deterioration and respiratory illnesses around the globe. Hydrodesulfurization is a method developed to remove a large portion of sulfur from natural gas and refined fuels; however, it remains costly and highly inefficient on polycyclic aromatic compounds. Biodesulfurization is another method that employs a naturally occurring pathway in certain bacteria to eliminate sulfur from said fuels. R. erythropolis has a four‐step biodesulfurization pathway using four enzymes that convert dibenzothiophene (DBT) into 2‐ hydroxybiphenyl and sulfite, without compromising the value of the fuel. Our goal is to rationally design the biodesulfurization pathway to accommodate a larger subset of sulfur‐containing polycyclic aromatic compounds as substrates, beginning with dibenzothiophene monooxygenase (DszC), the first enzyme in the pathway. To facilitate high‐throughput validation of our rationally designed DszC mutants, we will generate a strain of E. coli that harbors the three downstream biodesulfurization genes (DszA, DszB and DszD). To develop our high‐throughput E. coli strain, we must first show successful recombinant expression of DszABC. DszB is a desulfinase enzyme that catalyzes the last step of this biodesulfurization process, converting 2′‐hydroxybiphenyl‐2‐sulfinic acid into 2‐hydroxybiphenyl and sulfite. Herein, we show the optimal conditions for DszB overexpression and solubility. DszB showed highest solubility at pH 7.5 when induced at 12C. We will purify the enzyme using metal affinity chromatography and then replicate steady state kinetic experiments to demonstrate presence of active enzyme.Support or Funding InformationResearch Initiative for Scientific Enhancement (RISE)National Institute of Health (NIH score 5SC2AI109500)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Stigmasterol solid‐lipid nanoparticle development for lung cancer therapy
Authors: Torres Zally , Delgado Yamixa , Griebenow Kai 
Published: June 2021

Development of new therapies for lung cancer using nano‐formulations has become strategy to minimize the lack of drug inactivation after administration into patients. An alternative to increasing the effectiveness of the drugs is by development of drug delivery systems using solid‐lipid nanoparticles (SLN). Stigmasterol (Stg), an unsaturated phytosterol found in plant oils, has shown potent anti‐carcinogenic properties. Herein, we developed a Stigmasterol‐SLNs (~200 nm and polydispersity index value of 0.2) nano‐formulation by use of a microemulsion method. This size is compatible with tumor accumulation through the irregular vasculature observed in cancer tissues. Our system demonstrates strong cytotoxicity in lung cancer A549 cells with an IC50 of 11 μM after 24 h. The mechanism of action of the Stg‐SNL has been confirmed to involve activation of caspase‐3/7 by extracellular assay. In addition, the established involvement of oxidative stress by determining reduced glutathione levels. The mechanism is necrosis independent. Physical stability of the Stg‐SLN formulation in the liquid phase was investigated by incubation at 37ºC by for three days and was maintained. More in vitro studies will be completed to confirm the selectivity of Stg‐SLN. In conclusion, we have designed a system for phytosterol delivery from nanoparticles with low poly‐dispersity which potentially could accumulate in cancerous tissues.Support or Funding InformationNIH grant (RISE): 5R25GM061151‐16This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Predicting the Effects of GSTPπ Polymorphism on Resistance to Ezatiostat
Authors: Risbud Kajal S. , Muwonge Alecia N. , Frey Kathleen M. 
Published: June 2021

Glutathione S‐transferases (GSTs) are a family of phase II metabolic enzymes. These enzymes catalyze the conjugation of glutathione (GSH) to xenobiotic substrates for detoxification. There is evidence that supports the role of GSTπ in cancer development. Ezatiostat (EZT) has received orphan drug status for Myelodysplastic Syndrome (MDS). A single nucleotide polymorphism (SNP) Ile105Val (I105V) near the Glutathione binding site of GSTπ affects its structure, and this may cause chemotherapeutic resistance. We hypothesize that this mutation expressed in some individuals may cause resistance to EZT. This study incorporates computational analysis of the binding mode for EZT with wild‐type and I105V. We used residue scanning and molecular dynamics (MD) to compare the interactions for EZT. Results from MD show differences in the EZT binding mode in wildtype and I105V GST. GST with the I105V mutation makes less interactions with EZT. Future work involves inhibition assays and x‐ray crystallography to validate the computational results.Support or Funding InformationDean's Internal Research Support GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Neutrophil‐induced genomic instability impedes resolution of inflammation and wound healing
Authors: Sumagin Ronen , Bui Triet M , Wiesolek Hannah L , Butin‐Israeli Veronika 
Published: June 2021

Neutrophil (PMN) infiltration of the intestinal mucosa is a hallmark of tissue injury associated with inflammatory bowel diseases (IBD). The pathological effects of PMNs are largely attributed to the release of soluble mediators and reactive oxygen species (ROS). We identified a new, ROS‐independent mechanism, whereby activated, tissue‐infiltrating PMNs release microparticles armed with pro‐inflammatory microRNAs (miR‐23a and miR‐155). Using IBD clinical samples, in vitro and in vivo injury models, we show that PMN‐derived miR‐23a/155 promote accumulation of double‐strand breaks (DSBs) by inducing Lamin B1‐dependent replication fork collapse, and inhibition of homologous recombination (HR) by targeting HR‐regulator RAD51. DSB accumulation in injured epithelium led to impaired colonic healing and genomic instability. Targeted inhibition of miR‐23a/155 in cultured intestinal epithelial cells and in acutely injured mucosa decreased the detrimental effects of PMNs and enhanced tissue healing responses, suggesting that this approach can be used in therapies aimed at resolution of inflammation, wound healing and potentially prevent neoplasia.Support or Funding InformationThis work was supported by grants from the National Institutes of Health (NIH) DK101675, Digestive Health Foundation, Chicago and by the Robert H. Lurie Comprehensive Cancer Center(Eisenberg Scholar grant).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glycerolipid intermediates as signaling mediators in physiology and disease
Authors: Finck Brian N 
Published: June 2021

Lipids have a number of essential biological roles that, despite a wealth of knowledge, somehow remain understudied. Glycerolipids are a major form of stored energy, play important structural functions as hydrophobic components of cell membranes, and have dynamic effects on signaling mediators within the cell and to distant tissues. Generally, it was believed that the pathways that synthesized lipids for these functions were distinct, compartmentalized, and did not widely intersect. For example, the main pathways for triglyceride storage as an energy source were not thought to play major roles in regulating the synthesis of lipids for signaling cascades even though many intermediates play roles as activators of intracellular kinases. However, it is now better appreciated that some of these storage intermediates also have signaling functions. In most cells, a pathway utilizing glycerol‐3‐phosphate as its initial substrate is the major route of triglyceride synthesis. Glycerol‐3‐phosphate undergoes two sequential acylations and then is dephosphorylated by lipin proteins to form diacylglycerol. Modulating the activity of lipin 1, a phosphatidic acid phosphohydrolase, has profound effects on skeletal muscle, adipose tissue, cardiac, and hepatic signaling by regulating the cellular abundance of phosphatidic acid. Evidence will be presented that lipin 1‐mediated signaling effects regulate metabolic flux, mitochondrial homeostasis, autophagy, cardiac and skeletal myocyte function, and impact cell death processes. These studies demonstrate that lipin 1 mediated effects on the concentrations of these intermediates are not important for storing energy, but also impact critical signaling cascades that control cellular homeostasis.Support or Funding InformationSupported by NIH grants R01 HL119225 and R01 DK78187This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanical Stretching Simulates Cardiac Physiology and Pathology through Mechanosensor Piezo1
Authors: Wong Tzyy‐Yue 
Published: June 2021

Dynamics of a living body enables organs to experience mechanical stimulation at cellular level. Human cardiomyocytes cell line provides a source for simulating the heart dynamics; however, lack of understanding on mechanical stimulation effect on them limits potential applications. Here, we investigated the effect of mechanical stimulation on the cardiac function‐associated protein expressions in human cardiomyocytes. Human cardiomyocyte cell line AC16 was subjected to different stresses: 5% mild and 25% aggressive, at 1Hz for 24h. The stretched cardiomyocytes showed down‐regulated Piezo1, P‐AKTS473, and P‐GSK3bS9 compared to no stretch. In addition, the stretched cardiomyocytes showed increased LRP6, and P‐JNKT183/T185. When Piezo inhibitor was added to the cells during stretching, the LRP6, and P‐JNKT183/T185 were further increased under 25%, but not 5%, suggesting that higher mechanical stress further activated the Wnt‐related signaling pathway when Piezo1 was inhibited. Supporting this idea, we found expression of eNOS decreased, and release of calcium ions significantly reduced under 25% compared to 5%. These studies demonstrate that cyclic mechanical stimulation affects cardiac function‐associated protein expressions, and Piezo1 plays a role in the protein regulation.Support or Funding InformationThis research is supported by VGHKS107‐076, VGHKS107‐168, VGHKS107‐175, MOST104‐2320‐B‐0751B‐003‐MY3, and MOST106‐2320‐B‐075B‐001.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.