bluuebunny/crossref_metadata_2025 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 Information

Financial 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 Information

To 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 Information

NIGMS Alaska INBRE; Portland State University‐University of Alaska Anchorage BUILD EXITO Program; UAA OURS & CAS

This 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 Information

K01DK114391 (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.
Insight into flavonoid structure for mammalian α‐amylase and α‐glucosidase inhibition to control starch digestion rate
Authors: Lim Jongbin , Zhang Xiaowei , Ferruzzi Mario , Hamaker Bruce R.
Published: June 2021

In this study we investigated the structural specificity of flavonoids for starch digestive enzyme inhibition to modulate glucose release from glycemic carbohydrate digestion. Using three different flavonoids [eriodictyol, luteolin, and quercetin], chosen based on the degree of hydroxylation and planarity of the C‐ring, structural specificities were found for selectively inhibiting α‐amylase and α‐glucosidases, resulting in different inhibition constants (Ki) and mechanisms. The double bond between C2 and C3 on the C‐ring of flavonoids was particularly important for α‐amylase inhibition, which leads to a π‐stacking interaction between flavonoids and α‐amylase, while the hydroxyl group at C3 of the C‐ring played a key role in inhibiting α‐glucosidases. These structural specificities of flavonoids toward starch degrading enzymes are likely the result of different protein structures of α‐amylase and α‐glucosidases, as they belong to different families, GH13 and GH31. Our findings provide insights into structure‐function aspects of flavonoids in controlling starch digestion rate.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
O‐GlcNAc signaling orchestrates metabolic adaptation to prolonged fasting
Authors: Li Mindian , Liu Jiayu , Sehgal Raghav , Wu Jing , Zhang Pei , Han Weiping , Jha Abhishek , Yang Xiaoyong
Published: June 2021

Prolonged fasting reprograms metabolic pathways to foster stress resistance. O‐linked b‐D‐N‐acetylglucosamine (O‐GlcNAc) glycosylation is essential in metabolic regulation and stress resistance. It is unknown whether and how O‐GlcNAc signaling catalyzed by O‐GlcNAc transferase (OGT) reprograms metabolic pathways during prolonged fasting. Here we report that OGT coordinates purine, lipid, amino acid and carbohydrate metabolism during prolonged fasting. Unbiased metabolomic profiling of mouse livers after 48‐hour fasting reveals that hepatic ablation of OGT impedes fasting‐associated changes in purine metabolism, phospholipid degradation and fatty acid oxidation. Loss of OGT also impedes amino acid catabolism through reduced flux through the urea cycle and reduced aspartate‐argininosuccinate shunt. Notably, the citric acid cycle is also inhibited in part through inhibition of succinate dehydrogenase activity, which is consistent with decreased oxygen consumption and energy production. These catabolic defects are correlated with reduced gluconeogenesis. Together, these results define OGT as a master regulator of metabolic adaptation during prolonged fasting in liver.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Diacylglycerol kinase δ interacts with sphingomyelin synthase‐related protein via their sterile α motif domains
Authors: Murakami Chiaki , Sakane Fumio
Published: June 2021

Introduction

Diacyglycerol kinase δ isozyme (DGKδ) plays critical roles in lipid signaling by phosphorylating diacylglycerol (DG) into phosphatidic acid (PA). DGKδ regulates a wide variety of physiological and pathological events, such as type II diabetes and obsessive compulsive disorder. Because DGK is one of the components of phosphatidylinositol (PI) turnover, it is thought that DGKδ also utilizes mainly 18:0/20:4‐DG (X:Y; the total number of carbon atoms: the total number of double bonds) derived from PI turnover. Interestingly, we recently demonstrated that DGKδ preferably metabolized palmitic acid (16:0)‐containing DG molecular species, but not arachidonic acid (20:4)‐containing DG species, in response to high glucose stimulation. However, it is still unclear what kind of DG‐generating enzyme provides palmitic acid‐containing DG species.

Sphingomyelin synthase (SMS) 1, SMS2 and SMS‐related protein (SMSr) are DG‐generating enzymes utilizing phosphatidylcholine/phosphatidylethanolamine and ceramide. SMS1 and SMSr contain a sterile α motif domain (SAM), which is a protein‐protein interaction module, at their N‐termini. DGKδ also possesses SAM at its C‐terminus, and DGKδ‐SAM is highly homologous to SMSr‐SAM. Therefore, we hypothesised that DGKδ interacts with SMSr through their SAMs.

In the present study, we investigated the interaction between DGKδ‐SAM and SMSr‐SAM.

Results

We first examined whether DGKδ‐SAM interacts with SMSr‐SAM by immunoprecipitation analysis. We found that SMSr‐SAM, but not SMS1‐SAM, was co‐immunoprecipitated with DGKδ‐SAM. Full‐length DGKδ (DGKδ‐FL) was also co‐immunoprecipitated with SMSr‐FL more strongly than with SMS1‐FL and with SMS2‐FL. To examine whether SMSr‐SAM contribute to the interaction, we performed immunoprecipitation analysis using SMSr‐FL and a SAM‐deletion mutant (SMSr‐DSAM). DGKδ‐FL was strongly co‐immunoprecipitated with SMSr, whereas SMSr‐DSAM only weakly interacted with DGKδ‐FL. Immunostaining analysis demonstrated that DGKδ‐FL co‐localized partly with SMSr‐FL in COS‐7 cells overexpressing these proteins. These results strongly suggest that DGKδ interacts with SMSr through their SAMs.

Conclusion

In summary, the present study for the first time showed that DGKδ interacted with SMSr mainly through their SAMs. It is possible that SMSr is one of the candidates of up stream DG‐providing enzymes of DGKδ, which composes a new pathway independent of PI turnover.

Support or Funding Information

This work was supported by JSPS KAKENHI (Grant Number JP18J20003) and supported in part by Venture Business Laboratory in Chiba University (Nanohana Competition 2018 Award).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of the Perilipin 5 C‐Terminus
Authors: Clinton Tim D , Wei Derek T , Tansey John T
Published: June 2021

Many countries around the globe have been plagued with a recent epidemic in lipid‐associated diseases. These diseases include obesity, diabetes, and non‐alcoholic fatty liver disease among many others. The United States itself has fallen heavily victim to these ailments, placing first in worldwide rankings for obese persons and within the top fiftieth in rankings for the percentage of persons afflicted with diabetes. These diseases pose clear threats to modern society and having a greater understanding of the mechanistic background of them may lead to better treatments options for those afflicted. A common thread through all these devastating illnesses is their connection to lipids and more specifically lipid storage. Cellular lipids are stored in lipid storage droplets, and these droplets are coated by the perilipins, five regulatory proteins that have an integral role in regulating the breakdown of fats. One such of these family members is perilipin 3, a protein that's structure has been partially determined. In Perilipin 5 this structure has not been determined and is the current focus of this study. Previously a crystal structure of the C‐terminal region of perilipin 3 was determined for residues 206 to 431. These regions were aligned with perilipin 5 using BLAST and their analogous location within the Perilipin 5 DNA sequence was determined. This region of perilipin 5 is being expressed as a 6‐his fusion protein for subsequent characterization and structure determination. Using the predicative programs RaptorX and Phyre2 theoretical protein structures were compiled. These were labeled for regions of interest including a putative hydrophobic cleft found in perilipin 3 that was previously found to recruit that protein to lipid droplets. Collectively these findings indicate that it is highly likely that perilipin 5 has a similar structure to perilipin 3 in the carboxy terminus.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of a Shortened Form of the Perilipin 5 Protein
Authors: Dalton Rebekah C , Tansey John T
Published: June 2021

Many modern health issues arise from aberrant lipid metabolism, among these are type II diabetes and non‐alcoholic fatty liver disease. One commonality these diseases share is neutral lipid metabolism and storage. Regulation of neutral lipid metabolism is of vital importance in the body, and among the proteins responsible for such regulation are the perilipins. Perilipins are a family of conserved proteins that are found on the surface of lipid storage droplets and play a central role in the regulation of cellular neutral lipid metabolism. Perilipin 5, specifically, is expressed in tissues with a high capacity for fatty acid oxidation such as cardiac muscle, oxidative muscle, and fasting liver. Using western blotting and reverse transcriptase PCR, we have identified a splice variant of perilipin 5 which has been termed perilipin 5b. This shortened form of the protein retains the amino terminal 35 kDa of the protein but lacks the 4‐helix bundle found in the C‐terminus. Based on the reports in the literature, this protein would lack the carboxy terminal domains reported to be necessary for interactions with lipases and other proteins but would retain serine 155 that is requisite for PKA phosphorylation and translocation to the nucleus. We have constructed a carboxy terminal truncation of perilipin 5 and are expressing it in CHO cells to further define the function of this protein. When compared with CHO cellular expression of perilipin 5, 5b appears to have a higher number of smaller lipid droplets per cell. Collectively these data indicate that perilipin 5b plays a role in lipid storage, but its specific function of this shortened form is still unknown.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cost‐effective synthesis of polyethylene glycol diacrylate‐acrylic acid (PEGDA‐AA), a tissue engineering hydrogel ideal for C2C12 mouse muscle cells
Authors: Lee Hunter M. , Fischer Kristin M.
Published: June 2021

Current research in tissue engineering focuses on growing and functionalizing cells in vitro to assemble viable constructs that restore, maintain, or improve damaged tissues or whole organs. Specific to the field of tissue engineering, hydrogels have proven successful in the development of multiple cell types with the goal of one day serving as grafts that will replace large areas of lost or damaged tissue. This assembly depends on the presence of a scaffold, or three‐dimensional structure that guides the growth of new tissue, and hydrogel polymers in particular have been known to be an excellent building block for scaffold formation. This research focused on the synthesis and characterization of the hydrogel polyethylene glycol diacrylate‐acrylic acid (PEGDA‐AA), a hydrophilic material that provides a viable environment for cell maturation because of its favorable characteristics for the development and maturation of mouse C2C12 muscle cells. Using modified procedures adapted from Browe et al. (2016) to create a cost‐effective way of synthesizing this precursor, PEGDA was successfully synthesized and characterized through 13C and 1H‐NMR spectroscopy. Current research focuses on analyzing the effects of the hydrogel on the growth and development of C2C12 cells with the long‐term goal of establishing an efficient model for human tissue engineering.

Support or Funding Information

Hampden‐Sydney College Office of Undergraduate Research

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating Pericyte Dynamics during Angiogenesis in the Mouse Mesometrium Culture Model
Authors: Suarez‐Martinez Ariana Diandra , Wang Jack , Murfee Walter Lee
Published: June 2021

Angiogenesis requires multiple cells and systems working in concert to develop new blood vessels from existing ones. Hence, understanding the exact dynamics between cells and systems proves to be beneficial when developing innovative therapies. In the microcirculation, endothelial cells that line the blood vessels are supported by perivascular cells that are elongated cells called pericytes. Cell membrane proteins that modulate cellular responses of different growth factors play an important role during microvascular remodeling. For pericytes, the neural glial antigen‐2 (NG2) proteoglycan is a cell membrane protein that is exclusively expressed by mural cells in mouse microvasculature. Pericytes' multifaceted roles in angiogenesis and where they originate still requires further research. Our lab has recently introduced the mouse mesometrium culture model that allows for the investigation of cell dynamics within intact microvascular networks. The objective of this study was to demonstrate the usefulness of the mouse mesometrium culture model for investigating NG2 inhibition and cell‐lineage tracing. For the NG2 inhibition studies, female C57BL/6 (WT) and NG2 KO were euthanized to aseptically harvest mesometrium tissues and culture them in MEM + 1% PS and 20% FBS at standard incubating conditions. WT and NG2 KO tissues were cultured for 3 days before labeling with PECAM and NG2 antibodies to quantify angiogenesis. PECAM and NG2 labeling confirmed the lack of NG2‐positive pericytes on capillaries from NG2 KO tissues. The angiogenic response after culture from NG2 KO tissues was significantly decreased when compared to WT tissues (NG2 KO: 0.98 ± 0.29; WT: 2.11 ± 0.32 number of sprouts per total vessel length, p = 0.019). For cell‐lineage tracing studies, female NG2dsRed mice were given a daily IP injection of 0.1 mL of sunflower oil for 5 consecutive days to increase microvascular density. Mesometrium tissues were harvested 21 days post last injection and cultured in MEM + 1% PS and 20% FBS at standard incubating conditions for 5 days. The tissues were imaged for time‐lapse at least every 12 hours to track individual cell interactions. Time‐lapse imaging taken every 2 hours for 12 hours on days 1–3 of NG2dsRed tissues revealed that NG2‐positive pericytes migrated along pre‐existing vessels to new vessel segments. Moreover, a subpopulation of NG2‐positive interstitial cells were observed to migrate throughout the tissue. These results validate that inhibiting NG2 expression has a direct effect on angiogenesis in the mouse mesometrium culture model. Furthermore, it highlights the usage of the mouse mesometrium culture model to track individual cell‐to‐cell dynamics of pericytes in intact microvasculature during angiogenesis.

Support or Funding Information

NIH R01AG049821

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chronic GPER1 Activation Prevents H9c2 Cardiac Myoblast Death after Oxidative Stress by Protecting Mitochondrial Structure and Function Via a Non‐Canonical MST1/YAP Pathway
Authors: Madungwe Ngonidzashe , Feng Yansheng , Bopassa Jean Chrisostome
Published: June 2021

Introduction

Estrogen (17β‐estradiol; E2) is known to be cardioprotective against the damaging effects of ischemia/reperfusion (I/R) injury. We found that acute pre‐ischemic E2 treatment reduces cell death in cardiac tissue via direct effects on mitochondria by the G protein‐coupled estrogen receptor 1 (GPER1). We recently showed that acute application of E2 at the onset of reperfusion results in similar cardioprotection against I/R injury via GPER1, a non‐genomic receptor that mediates rapid E2 actions. Here, we investigated the impact and mechanism underlying chronic E2 protective actions in H9c2 cardiac myoblasts subjected to treatment with the cytotoxic agent, H2O2.

Methods

Using the H9c2 cardiac myoblasts, we subjected cells to H2O2 oxidative stress with no serum, followed by reoxygenation with E2 for 2 and 4 days. Cells were then collected and analyzed for cellular, mitochondrial and genetic function. We measured cell death, gene upregulation, mitochondrial structure, morphology and function using flow cytometry, cell assays, qRT‐PCR, along with fluorescence and electron microscopy. We also collected cell lysates and analyzed them by Western blot to assess the mechanism involved.

Results

We found that chronic treatment of cells with E2 after oxidative stress results in decreased cell death but remarkably does not affect cell cycle activity. Surviving cells showed improved mitochondrial dynamics as evidenced by better cristae morphology and increased resistance to opening of the mitochondrial permeability transition pore, but with little change to mitochondrial fusion/fission. Additionally, we found that this chronic E2 action predominantly occurs through mitochondria to reduce cytochrome c release and decrease phosphorylation of mammalian sterile‐20‐like kinase (MST1) and increase yes‐associated protein (YAP) translocation to the nucleus for upregulation of TGF‐β and PGC‐1α. All these E2‐observed effects were prevented by G15, a GPER1 antagonist, suggesting a GPER1‐dependent mechanism.

Conclusion

We have found that chronic E2 treatment to H9c2 cardiac myoblasts after stress leads to rescue from cell death by reducing apoptotic signaling and protecting mitochondrial morphology and function. We also found that this process occurs via GPER1 acting directly on mitochondria in a non‐canonical MST1/YAP mechanism that leads to genetic upregulation of cell growth.

Support or Funding Information

NIH RO1 Grant/Award number: HL138093;American Heart Association, Grant/Award Numbers:18PRE34030307, 17SDG33100000

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Contribution of Membrane Raft Redox Signaling to Visfatin‐Induced Inflammasome Activation and Podocyte Injury
Authors: Singh Gurinder Bir , Patibandla Sai , Zhang Yang , Li Pin‐Lan , Koka Saisudha , Boini Krishna M
Published: June 2021

Recently we have shown that adipokine visfatin‐induced NLRP3 inflammasome activation contributes to podocyte injury. However, the molecular mechanisms of how visfatin‐induces the Nlrp3 inflammasome activation and podocyte damage still unknown. The present study tested whether ceramide and membrane raft (MR) redox signalling pathway plays a central role in visfatin‐induced inflammasomes formation and activation in podocytes. Using confocal microscopy, visfatin was found to increase MRs clustering in the membrane of podocytes in a dose dependent manner. Upon visfatin stimulation an aggregation of ASMase product, ceramide and NADPH oxidase subunits, gp91(phox) and p47(phox) was observed in the MR clusters, forming a MR redox signaling platform. The formation of this signalling platform was blocked by prior treatment with MR disruptor MCD, NOX inhibitor DPI, ASMase inhibitor amitriptyline. In addition, electron spin resonance (ESR) spectrometry analysis showed that visfatin treatment significantly increased the superoxide (O2·−) production compared to control cells. However, prior treatment with ASMase inhibitor amitriptyline significantly attenuated the visfatin‐induced superoxide production. Furthermore, immunofluorescence analysis demonstrated that visfatin treatment significantly decreased the podocin and nephrin expression (podocyte damage), and prior treatments with DPI, WEHD (inflammasome inhibitor), MCD and amitriptyline attenuated this visfatin‐induced podocin reduction. In conclusion, our results demonstrate that visfatin‐induced the ceramide production via ASMase and thereby stimulates membrane raft clustering in the membrane of podocytes to form redox signalling platforms by aggregation and activation of NADPH oxidase subunits, enhancing O2·− production and leading to Nlrp3 inflammasome formation and activation in podocytes and ultimate glomerular injury (supported by NIH grant, DK104031).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Carcinogenic arecoline shows a paradoxical effect in modulating nitric oxide production and the subsequent protein S‐nitrosylation in endothelial cells
Authors: Cheng Bi‐Ling , Fu Yaw‐Syan , Su Yuan‐Shun , Huang Bin
Published: June 2021

Arecoline exhibits a carcinogenic tone and is remarkable for the refreshing effect due to the increase of vasodilation and blood flow. Therefore, if arecoline can promote the production of nitric oxide (NO) and the subsequent protein S‐nitrosylation in endothelial cells (ECs) is interesting. Treating arecoline increased the phosphorylation of eNOSSer1177. NO molecular was observed by a fluorescent probe FA‐OMe followed with the measurement of flowcytometry. Arecoline treatment up‐/down‐regulated 914/647 proteins identified by showing by iTRAQ 114/115 labeling and LC‐MS/MS. IPA analysis indicated that these proteins were implicated in eNOS signaling. By using biotin switch, 224/159 S‐nitrosoproteins were up‐/down‐regulated by arecoline. The increased S‐nitrosylation level of chaperonin and the decreased S‐nitrosylation level of calnexin were verified by western blot. Our results suggest a paradoxical effect of arecoline in producing NO and therefore maintains endothelial homeostasis, even though the toxicological tone in carcinogenesis remains concerned.

Support or Funding Information

TB0357 from Ministry of Education, Taiwan

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Upregulation of cellular protective mechanisms against oxidative damage via pharmacological intervention
Authors: Allani Shailaja Kesaraju , Chen Xi , Ramirez‐Alcántara Verónica , Canzoneri Joshua , Prentice Howard , Huang Xupei , Piazza Gary , Weissbach Herbert
Published: June 2021

Oxidative damage is widely accepted as a major stress factor responsible for many age‐related diseases, as well as other degenerative conditions associated with normal aging. We initially became interested in activating or upregulating cellular protective mechanisms against oxidative damage after identifying methionine sulfoxide reductase A (MSRA), the first member of the MSR family of enzymes. The MSR system, MSRA and MSRB, reduces methionine sulfoxide residues in proteins, both the S and R epimers, back to methionine, repairing oxidative damage to proteins. MSRA can also reduce small molecules that contain a methyl sulfoxide S epimer, including the prodrug sulindac, that is converted to sulindac sulfide, the active NSAID (nonsteroidal anti‐inflammatory drug). We reported that sulindac can also protect both retinal pigmented epithelial (RPE) cells against chemical oxidative damage, and the heart against ischemia/reperfusion damage (I/R), by initiating a protective response, similar to ischemic preconditioning. This mechanism of sulindac protection was shown to be independent of its NSAID activity. In contrast, sulindac under similar conditions, sensitizes cancer cells to oxidative stress, resulting in enhanced killing of the cancer cells when they are exposed to additional stress. Because of sulindac's unique properties the National Institute of Aging recently selected sulindac to test for life span extension in mice in their Interventions Testing Program. In order to find a more active sulindac derivative that could protect cells against oxidative damage and lacked NSAID activity, we screened a series of sulindac derivatives containing a central indenyl core present in the sulindac scaffold. We have identified a compound, MCI‐100, that is not an NSAID, that is >20 fold more active than sulindac in protecting RPE cells from oxidative damage and also more active than sulindac in protecting the heart against I/R damage in a Langendorff ex‐vivo model. Additional experiments suggest that the protective mechanism of both sulndac and MCI‐100 may be related to their ability to inhibit PDE10, resulting in an increase in intracellular cGMP levels, that could initiate a pharmacological preconditioning response through PKG activation. However, unlike sulindac, MCI‐100 does not sensitize cancer cells to oxidative stress.

Our eventual goal is to develop drug combinations that up‐regulate several different mechanisms that cells use to protect against oxidative damage, as a therapeutic approach to slow the progression of age related diseases, and treat other diseases that involve oxidative damage.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of LAT‐1 inhibitors for the treatment of pancreatic cancers
Authors: Ikhlef Houssine , Phanstiel Otto
Published: June 2021

Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer and represents one of the world's most deadly cancers with a five year survival of only 8%. As such there is a desperate need to develop effective new medicines for pancreatic cancers. The large neutral aminoacid transporter 1 (LAT‐1) is highly expressed in PDAC tumors and provides a conduit for these tumors to acquire essential amino acids like leucine and methionine. Beyond their incorporation into proteins, leucine is an important regulator of mTOR signaling and methionine is essential for one carbon metabolism and polyamine biosynthesis. Targeting LAT‐1 inhibits the uptake of these essential building blocks and inhibits cell growth.

There has been significant interest in developing LAT‐1 inhibitors for oncology applications and virtually all of the known LAT‐1 inhibitors are predicated upon a phenylalanine scaffold. Here, we showed that piperazine‐based materials effectively inhibited leucine and methionine import into L3.6pl human pancreatic cancer cells. We also demonstrated that our lead LAT‐1 inhibitor resulted in a dose‐dependent decrease in intracellular methionine, leucine, and depleted polyamine pools in L3.6pl cells in vitro. Moreover, these treated cells were not rescued by exogenous polyamines indicating that polyamine import could not revive the treated cells. Our different molecular design provides new opportunities for enhanced targeting and both the synthesis and bioevaluation of these new LAT‐1 inhibitor designs will be discussed.

Support or Funding Information

UCF College of Medicine (#25014202)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using simulation to probe membrane defects with a pH‐sensitive peptide
Authors: Burns Violetta , Mertz Blake
Published: June 2021

The pH Low Insertion Peptide (pHLIP) binds, folds and inserts spontaneously into the membrane as a transmembrane helix under acidic conditions. This functionality makes pHLIP a promising tool for drug delivery and diagnostic imaging of disorders associated with acidosis, such as arthritis, cancer, and ischemia. The acidic sensitivity of pHLIP arises from the sequential protonation of its acidic residues which, after binding to the membrane, induces simultaneous folding and insertion in order to seclude its hydrophobic segment from aqueous solvent. However, the peptide‐bilayer interactions of pHLIP in the fully inserted state are poorly understood. Our aim is to characterize the effect of deprotonation of the acidic residues of pHLIP on the stability of the peptide and the bilayer in its inserted state. Our hypothesis is that increasing the degree of deprotonation of pHLIP disrupts favorable interactions between pHLIP and the bilayer. In order to test our hypothesis, we carried out molecular dynamics (MD) simulations of α‐helical pHLIP inserted into a 1‐palmitoyl‐2‐oleoyl‐sn‐3‐phosphocholine (POPC) bilayer. Our variable was the protonation state of the acidic residues in pHLIP. Our results show that in the fully protonated state, pHLIP remains stable. Interestingly, in the fully deprotonated state, the negatively‐charged residues remain embedded in the interior of the bilayer, stabilized by a defect that allows for interactions with waters from bulk solvent. In addition, deprotonation of the C‐terminal acidic residues leads to greater destabilization of pHLIP in the bilayer. Together, these results reveal several of the interactions between pHLIP and the aqueous and lipid phases that are critical in maintaining the fully inserted state of the peptide.

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