bluuebunny/crossref_metadata_2025 huggingface dataset:
Rhinovirus infection alters cellular architecture of highly differentiated airway epithelial cells
Authors: Michi Aubrey , Proud David
Published: June 2021

RATIONALE

Human rhinoviruses (HRV) are responsible for over 50% of viral respiratory tract infections, and pose a significant health challenge for sufferers of chronic inflammatory airway diseases. Much of our current knowledge of the HRV lifecycle is based on other picornavirus models using cell lines of non‐airway origin (e.g. HeLa cells). However, the human airway epithelial cell (HAE) is the natural host cell that is subject to HRV infection and replication. Using highly‐differentiated HAE, we seek to elucidate the mechanisms that HRV uses to remodel the cellular architecture of HAE during replication, and to temporally relate these alterations to transient intercellular junctional changes.

METHODS

Highly differentiated air‐liquid interface (ALI) cultures of HAE, isolated from normal human lungs, were treated with three different HRV serotypes: HRV‐16, HRV‐1A, and HRV‐C15 for 24, 48, 72, 96, 120, 144 hours, and the junctional protein, ZO‐1, was examined. Studies of the architecture of the cytoskeleton were conducted using HAEs grown in monolayer submersion culture. Other stimuli include replication‐deficient HRV, poly(I:C) (a mimic of the double‐stranded RNA produced during HRV replication), cytochalasin D (actin disruptor), and paclitaxel (microtubule stabilizer). Cells were fixed for immunofluorescence using a resonance scanning confocal microscope. HRV replication kinetics were determined by serially washing ALI cultures every 24 hours to determine viral release by RT‐qPCR. Barrier function studies in Ussing chambers used FITC‐dextran (permeability) and measured real‐time transepithelial electrical resistance (TEER).

RESULTS

HRV‐1A and HRV‐C15, but not HRV‐16 induce dramatic remodeling of cellular architecture in highly differentiated HAE. These changes included the co‐localization of nuclei with apically expressed ZO‐1. This response depends upon the replication cycle of HRV, as replication‐deficient HRV does not induce these changes. Additionally, the cells associated with remodeled architecture are positive for HRV infection, as assessed by expression of replication intermediates. Preliminary data suggests that cytoskeletal inhibitors diminish the ability of HRV replication to occur, indicating a critical role for the actin cytoskeleton in HRV replication.

CONCLUSION

We conclude that some, but not all, HRV serotypes induce altered cellular architecture in HAE that is associated with disruption of the actin cytoskeleton. Future studies will seek to temporally relate altered cytoskeletal architecture with HRV replication sites.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
P311 Modulates Adipocyte Development
Authors: Nunez Sha'Kayla , Kotha Peddanna , Young Corey , Adebayo Olayinka , Badri Kameswara Rao
Published: June 2021

Adipogenesis is a complex process of adipocytes formed from progenitor cells that involves changes in gene expression, cell morphology and hormone sensitivity. The differentiation of adipocytes is regulated by a well‐orchestrated network of transcriptional factors and associated cell signaling proteins responsible for adipocyte development and maturation. An in‐depth understanding of adipogenesis is essential to fight against metabolic disorders including obesity. Our lab investigates the P311 protein that plays a key role in adipocyte development. P311 has been shown to play an essential role in blood pressure regulation and vascular contractility (Badri et al., J Clin Inv 2013). In this study, we evaluate the adipogenic function of P311 using NIH3T3‐L1 cell culture model and genetically modified animal models. We show that P311 expression, initiated with the induction of adipogenesis in 3T3‐L1 cells, increased during the adipogenesis process. Additionally, siRNA mediated P311 knockdown inhibited adipogenic differentiation in 3T3‐L1 cells. The increase in P311 expression during adipogenesis correlated with the increase in the expression of key adipogenic transcriptional factors PPARγ2 and C/EBPα. Further, we observed nuclear localization of P311 in immunofluorescence studies indicating potential transcriptional regulatory/co‐regulatory function/s of P311. To verify the transcriptional regulatory/co‐regulatory functions of P311, we conducted chromatin immunoprecipitation (ChIP) assays using adipogenesis induced 3T3‐L1 cells transfected with pCMV‐P311 or an empty vector, pCMV. P311 overexpressing adipocytes demonstrated the recruitment of P311 to the PPARγ2 promoter regions (~800 fold) compared to empty vector transfected adipocytes. However, P311 immunocomplexes failed to amplify C/EBPα promoter. Further, to verify the involvement of P311 in adipose tissue development, we conducted animal studies and did not find any differences in the white fat weights (gonadal and subcutaneous fat) between wild type mice (WT) and P311 knockout mice (KO) (18–21 weeks). However, we observed an increase in the numbers of LipidTox stained adipocytes as detected in fluorescenceactivated cell sorting (FACS) analysis in white fat of WT mice than P311 KO mice. Our current studies demonstrate the critical role of P311 in adipocyte development through the transcriptional regulation of PPARγ2.

Support or Funding Information

This study is supported by grant funds from NIMHD/NIH (U54MD008621 to Hampton University and S21MD000101).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chronic Pain Induced Inflammation in the Hypothalamus of Rats
Authors: DeForest Elizabeth , Semke Lori , Duric Vanja , Clayton Sarah C
Published: June 2021

Chronic pain has been shown in humans to be associated with cardiovascular disease and hypertension but the mechanism of that association is unclear. Our overall hypothesis of this study is that chronic pain has a sensitizing effect on the central nervous system (CNS) that increases sympathetic nerve activity and predisposes to hypertension. Because inflammation can influence neuronal activation, in this study, we hypothesized that chronic pain generates a pro‐inflammatory phenotype in the cardiovascular control centers in the brain (e.g. the paraventricular nucleus of the hypothalamus). A chronic inflammatory pain model was generated by CFA injection into the left hind paw. Animals were then sacrificed 24 hours after the injection (acute phase) or 14 days following the injection (Chronic phase). RNA was isolated from the samples and expression of the pro‐ and anti‐inflammatory markers were assessed by RT‐qPCR. The expression of the pro‐inflammatory marker interleukin ‐ 1β (IL‐1β) was elevated in the hypothalamus 24 hours after CFA injection (2.3‐fold vs sham) and remained elevated at 14 days (2.5‐fold vs. sham) (P= .002). The expression of an anti‐inflammatory marker, transforming growth factor‐β 1 (TGF‐β 1), was decreased in the hypothalamus 24 hours after CFA injection (.8‐fold vs. sham) and remained decrease at 14 days (.63‐fold vs. sham) (P=.05) This data suggests that chronic pain induces a rapid and sustained pro‐inflammatory phenotype in the hypothalamus. This could lead to inappropriate activation of the sympathetic nervous system that could contribute to the development of hypertension.

Support or Funding Information

SC IOER

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Functional Organization and Integration of Purkinje Cells in the Cerebellum
Authors: Heck Daniel Glenn , Person Abigail , Fukushima Sawako
Published: June 2021

The overall object of this project is to create a 3D model of the cerebellum in order to illustrate the anatomical organization of Purkinje neurons, and to provide a better understanding of how they are integrated into other locations of the brain. Because the communication between the cerebrum and cerebellum is a closed system of polysynaptic circuits, understanding the underlying anatomy and physiology is a significant challenge. Classic staining techniques do not have the resolution nor the sophistication to capture the complete organization of the cerebellar cortex, let alone the entire cerebrocerebellar circuit. To overcome this challenge, a more precise staining technique has been developed to understand the complex functional organization of the cerebellum. Using a genetically modified variant of the rabies virus, neural connectivity can be imaged with extreme detail. The rabies is modified so that the glycoprotein required for the trans‐synaptic jump is deleted and replaced with a GFP protein, thus creating the ability to accurately label the connections to a single neuron. As a result of this staining technique, the Purkinje neurons can be traced using the cutting edge tracing software Neurolucida. The traced Purkinje cell somas and their axons can then be rendered in 3D and used for analysis and quantification. Having the ability to visualize the origin of the axons of specific cell types that communicate with the deep cerebellar nuclei in high 3D resolution may begin to provide an understanding of how the input of Purkinje cells is integrated with other excitatory inputs from elsewhere in the brain. Moreover, a 3D model of the cerebellum could also provide a more complete explanation of how the cerebellum quickly and accurately coordinates the inputs and outputs into movement, and is able to predict body movements in space.

Support or Funding Information

https://www.sciencedirect.com/science/article/pii/0306452284901933

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The anti‐apoptotic activity of protein S worsens liver fibrosis by activating the Akt pathway
Authors: Totoki Toshiaki , D'Alessandro‐Gabazza Corina N. , Toda Masaaki , Takeshita Atsuro , Yasuma Taro , Nishihama Kota , Horiki Noriyuki , Takei Yoshiyuki , Gabazza Esteban C
Published: June 2021

Protein S is a vitamin K‐dependent glycoprotein produced mainly in the liver with anticoagulant, anti‐inflammatory, immune‐modulatory and anti‐apoptotic properties. The anti‐apoptotic activity of protein S can ameliorate or worsen a disease state depending on the organ involved. For example, protein S attenuates pulmonary fibrosis by inhibiting apoptosis of lung alveolar epithelial cells or improve diabetes mellitus by inhibiting apoptosis of pancreatic islet b‐cells. On the contrary, exogenous protein S exerts detrimental effects during acute alcoholic hepatitis by its ability to activate and prolong the survival of liver natural killer T cells. In the present study, we investigated whether human protein S can affect chronic liver injury and fibrosis. Liver injury/fibrosis was induced by carbon tetrachloride injection in mice overexpressing human protein S and in wild type mice and the degree of liver injury and fibrotic changes were evaluated. Human protein S transgenic mice receiving carbon tetrachloride showed significantly higher circulating levels of liver transaminases, increased liver expression of inflammatory cytokines and chemokines, significantly much extended liver fatty degeneration and fibrosis and increased areas with DNA breakage after chronic injury compared to wild‐type mice. Liver injury was also exacerbated in mice after one bolus injection of carbon tetrachloride. Wild‐type mice infused with exogenous human protein S exhibited exacerbated liver injury, had significantly less apoptosis associated with increased number of hepatic stellate cells compared to untreated mice. An in vitro study using primary human hepatic stellate cells demonstrated that protein S inhibits apoptosis and increases the degree of Akt pathway phosphorylation in these liver cells. Western analysis showed significantly less cleavage of caspase‐3 in liver tissue from protein S transgenic mice compared to wild type mice. In conclusion, the results of this investigation showed that the anti‐apoptotic activity of protein S may play a role in chronic liver injury and subsequent liver fibrosis.

Support or Funding Information

I have no financial relationships to disclose.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lack of a‐CGRP Prevents High Fat Diet Induced Biliary Proliferation and Hepatic Steatosis
Authors: O'Brien April , Kyritsi Tina , Chen Lixian , Glaser Shannon , Alpini Gianfranco
Published: June 2021

Nonalcoholic fatty liver disease (NAFLD) has become a prevalent problem in westernized societies where incidents of obesity lead to a reduction in blood flow and a deposition of fat into the liver increasing incidents of maladies ranging from minor nonclinical hepatic steatosis (NASH) to severe cirrhosis and cholangiocarcinoma (CCA). We have previously shown that cholangiocytes participate in a sub epithelial fibrotic process that is the hallmark of hepatobiliary injury. Employing a well‐established mouse model of mechanical liver injury through bile duct ligation (BDL), we have shown that cholangiocytes are regulated, in part, through sensory innervation, and manipulation of the innervation pathway through a‐calcitonin gene‐related peptide (a‐CGRP) signaling results in a reduction in cholangiocyte proliferation as well as liver fibrosis. Due to the fibrotic nature of fatty liver disease and our previous research demonstrating the interplay between cholangiocytes, neuropeptides, and sensory receptors, we AIM to evaluate the role a‐CGRP plays in the biliary progression from hepatic steatosis to the profibrotic phenotype found in the damaged biliary epithelium.

METHODS

Studies were performed in C57 (WT) and CGRP KO mice (4 wk of age). Mice were treated with Control and High Fat Diet (0.2% cholesterol, 45% fat calories, with 30% of the fat in the form of partially hydrogenated vegetable oil) with 45% fructose, 55% glucose (42g/L) drinking water for 20 wks. Fat deposition was visualized via Oil Red O staining. Ductular reaction (IBDM), CGRP accessory protein and collagen deposition was evaluated by IHC for CK‐19, RAMP1 and Sirius Red, respectively, in total liver sections. a‐CGRP was evaluated via protein expression in mouse total liver and mRNA in human tissue by IHC and qPCR. mRNA data for CLR, CALCRL, RAMP1, and CGRP was obtained via qPCR in pure cholangiocytes in WT CD and WT HFD.

RESULTS

There was a significant decrease in the fat deposition in the a‐CGRP KO HFD compared to the WT HFD. There was a significant decrease in IBDM and collagen deposition in CGRP KO HFD mice compared to WT HFD. Both mouse HFD and human steatotic livers showed significant increases in a‐CGRP when compared to their WT counterpart. Additionally, IHC demonstrated a significant increase in RAMP1 in the WT HFD when compared to CD. Cholangiocytes had significant increase in the expression of mRNA in the CGRP receptor (CLR), the receptor component (CGRPRC), its accessory, RAMP1, and the protein CGRP in HFD when compared to the WT CD.

CONCLUSION

a‐CGRP plays a substantial role in proliferation and hepatic fibrosis. Modulation of the a‐CGRP axis may represent a novel therapeutic approach for the management of NAFLD.

Support or Funding Information

NIH R01 DK76898

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Spatial Organization of a cAMP/Ca2+‐Regulated Signaling Complex: A Solution Structural Small‐Angle X‐ray and Neutron Scattering Study of an AKAP79‐Scaffolded Complex Containing Type IIβ PKA and Calcineurin
Authors: Hall James , Copps Jeffery , Zhang Ping , Heck Albert , Blumenthal Donald , Taylor Susan
Published: June 2021

cAMP‐dependent protein kinase (PKA) regulates many cell processes including development, differentiation, and memory. In its resting state, PKA is a tetrameric holoenzyme consisting of a regulatory (R) subunit dimer and two catalytic (C) subunits. Four isoforms of the R‐subunit exist (RIα, RIβ, RIIα, and RIIβ). Although each isoform varies in sequence and quaternary structure, all share a common domain organization. The strength, duration, and specificity of PKA activity within the cell are mostly dependent upon isoform specific subcellular localization. The D/D domain interface of the R‐subunits serves as a docking site for A Kinase Anchoring Proteins (AKAPs) that target PKA to specific cellular locations. In cardiac myocytes and neurons, PKA is localized to Cav1.2 L‐type calcium channels by AKAP79 and up‐regulates calcium influx by phosphorylating the channel's cytosolic C‐terminal tail. AKAP79 also binds the Ca2+/calmodulin‐dependent protein phosphatase type 2B (PP2B, or calcineurin) at a site very close to that of PKA binding. Given the close proximity of the calcineurin and PKA binding sites on AKAP79, calcineurin has been suggested to play roles in regulation of calcium channels, along with regulation of PKA activity itself. Using a variety of biochemical and biophysical techniques, we have isolated and characterized a stable complex of the C‐terminal tail of AKAP79, RIIβ holoenzyme, and calcuneurin (“ARC” for short). We show a stoichiometry of 1:1:1 (AKAP79: RIIβ2C2:CN) using native mass spectroscopy, and SAXS/SANS techniques confirm that this is a compact complex, suggesting a close interaction between PKA and calcineurin when bound to AKAP79. Altogether, we suggest beyond its function of localizing PKA and CN in close spatial proximity to the Cav1.2 L‐type calcium channel, the ARC signalosome fosters extended protein‐protein interactions and regulations that suggest tight spatiotemporal control.

Support or Funding Information

James Hall is a San Diego IRACDA Fellow supported by National Institutes of Health (NIH)/NIGMS K12 GM06852 Award. Support for this work was provided through NIH GM034921.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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 Information

Grants: 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 pathogenesis

Support or Funding Information

funded by Merck & Company Inc.55211 Verhoeven, 11/01/16–10/31/18

This 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

Objective

We investigated whether angiotensin (Ang) III induces nuclear factor kappa beta (NF‐kb) phosphorylation in isolated rat vascular smooth muscle cells (VSMCs).

Background

The 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.

Methods

Primary 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.

Results

Concentration 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.

Conclusion

These 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 Information

AI133219, AI134568, AI131669

This 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

Rationale

Naphthalene 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.

Methods

Airway 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.

Results

HBE1 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.

Conclusions

The 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 Information

Supported 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 Information

Research 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 Information

NIH grant (RISE): 5R25GM061151‐16

This 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 Information

Dean's Internal Research Support Grant

This 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 Information

This 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 Information

Supported by NIH grants R01 HL119225 and R01 DK78187

This 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 Information

This 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.
Evolution of differential 4‐1BB signaling in Human and Murine immune system
Authors: Bitra Aruna , Zajonc Dirk M
Published: June 2021

4‐1BB (CD137) is an inducible tumor necrosis factor receptor superfamily (TNFRSF) member and is currently being targeted in cancer immunotherapy and autoimmunity. 4‐1BB interacts with its cognate ligand 4‐1BBL to promote T lymphocyte activation and proliferation, but the dissimilarities in mouse and human molecules might result in differential engagement of this pathway. To elucidate the molecular mechanism underlying this distinct behavior, we have determined the crystal structures of human and mouse 4‐1BB/4‐1BBL complexes. Collectively, these structures provide insights into the structural differences that drive the species‐specific receptor‐ligand interactions. The most significant feature is that the TNF homology domain of human (h)4‐1BBL form non‐covalent homo‐trimeric arrangements, whereas the mouse (m)4‐1BBL formed a novel disulfide linked homo‐dimer. In the human 4‐1BB/4‐1BBL complex, each receptor binds at the crevice formed between two protomers of h4‐1BBL resulting in the formation of a “hetero‐hexameric” functional signaling complex (1), whereas each m4‐1BB receptor binds exclusively to a single m4‐1BBL protomer leading to a “tetrameric” signaling unit (2). As multimerization and clustering is a prerequisite for TNFR intracellular signaling, appearance of differential functional signaling units in human and mouse supports a unique mechanism of 4‐1BB signaling in both of these species. Of note, we have discovered that h4‐1BB forms a disulfide‐linked dimer and this covalent receptor dimerization amplifies h4‐1BB signaling by cross‐linking individual ligand‐receptor complexes to form a 2D network. Since the m4‐1BBL dimer can only recruit two monomeric 4‐1BB receptors, cross‐linking of two hetero‐tetrameric signaling units via Galectin‐9 binding to the N‐linked glycans on m‐41BB is required for receptor oligomerization and strong signaling (3).

While all characterized TNFR/TNF members exhibit interspecies cross reactivity between human and mouse molecules, 4‐1BB and 4‐1BBL lacks this distinguishing feature. Our structural studies together with comparison of human and mouse 4‐1BB/4‐1BBL complexes highlighted several differences between the ligand and receptor binding interfaces and provide an explanation for the absence of inter species cross‐reactivity between them (2). Furthermore, our biochemical and mutational analysis discloses more variations between mouse and human molecules that could affect signaling and thus suggest the generation of better animal models to study 4‐1BB biology in humans.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring the Stability and Degradation of the Lipin‐1 Protein
Authors: Giebink Faye E , Harris Thurl E
Published: June 2021

Lipin 1 plays a central role in the regulation of lipid metabolism, which is crucial to maintaining systemic energy homeostasis in the body. Lipin 1 functions as a phosphatidic acid phosphatase to convert phosphatidic acid (PA) to diacylglycerol (DAG) as part of the Kennedy pathway. Phosphorylation of lipin 1 at Ser483 and Ser487 creates a phosphodegron signal that is recognized by βTRCP and promotes lipin 1 ubiquitination and degradation by the proteasome. The polybasic domain (PBD) of lipin 1 is a stretch of nine contiguous Arg and Lys residues within the central domain that are essential for interaction with PA. Mutation of these residues to Ala greatly increases protein expression of lipin 1. We show that this increase in expression is due to an increase in protein half‐life and we suggest this is due to alterations in lipin 1 ubiquitination and proteasomal degradation. During proteomic analysis of lipin 1 we identified at least one Lys within the PBD as acetylated. Acetylated Lys cannot be ubiquitinated. We have previously demonstrated an interaction between lipin 1 and HDAC1 and 3 and lipin 1, and so we treated cells with Trichostatin A (TSA) to prevent the removal of acetyl groups and cause the accumulation of hyperacetylated lipin 1. We find that pre‐treatment of cells with TSA increases lipin 1 protein half‐life. We hypothesize that accumulation of acetylated Lys within the PBD may prevent lipin 1 ubiquitination and degradation. Alternatively, lipin 1 acetylation may affect stability through Lys residues outside of the PBD or through other mechanisms, such as altering phosphorylation. Understanding what regulates lipin 1 stability and its role as a magnesium‐dependent phosphatide phosphatase (PAP) enzyme in triacylglycerol synthesis is important to comprehend mechanisms involved in metabolic disorders that lead to obesity.

Support or Funding Information

NIH DK101946 (TEH), UVA Harrison Undergraduate Research Award (FEG)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.