bluuebunny/crossref_metadata_2025 huggingface dataset:
Falcon Biomanufacturing: Teaching the core concepts of biochemistry and molecular biology through the model of a biotechnology company
Authors: Koci Matt , Ali Rizwana , Boller Bruce
Published: June 2021

To meet the challenges of the 21st century, students need a higher level of competency in science, technology, engineering, and mathematics than at any point in our history. This goes beyond terminology and abstract theory. They need to understand how different concepts relate, and most importantly how they form the basis for the technology economy. To address this need, we have developed a novel teaching platform built around the idea of a “mini‐biotech company”. The students in the biotechnology class in Bertie Early College High School, in Windsor, North Carolina learn Essential and Common Core Standards of biology and chemistry by going through the steps of making recombinant protein to be used in university research and related to the industries in their community. The year‐long program starts with a one‐week summer camp, biotech boot camp, held at NC State University. During this week, the students are introduced to concepts and techniques related to initial process development, process scale‐up, production, and validation. In addition, the students are taken to different area biotech companies where they meet working scientists and learn about their career paths. Then over the course of the next school year, the students work in their high school laboratory to develop their own process for optimal protein expression based on their equipment and available time during a school day. At the end of the school year, each student presents a poster at a biotechnology symposium held at their school, and the students that put forth the most work and leadership are selected to attend a national level science conference. Over the course of this program, students gain a deeper understanding of fundamental biochemical concepts while gaining practical, relevant work experience leading to an understanding of biotechnology beyond that of most undergraduate students.

Support or Funding Information

Burroughs Wellcome Fund SSEP

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 silico research as an active learning platform in a molecular biology course
Authors: Beaulieu Elaine
Published: June 2021

Active learning approaches can deepen the students' understanding of major biological and biochemical concepts by integrating course content with practical applications. In order to facilitate the merging of theory and application, a blended course format was developed for a 4th year class on the Structure and Function of the Human Genome. Briefly, a third of the lectures were replaced by online tutorials, called Webquests. The Webquests were constructed such that the students could independently follow both written instructions and instructions given by a series of video tutorials embedded in the Webquests. Each Webquest required the students to do specific tasks researchers in molecular biology would normally do, using specialized but freely available online software such as UCSC and Ensembl genome browsers, IGV genome viewer, Galaxy/cistrome bioinformatics data analysis platform, NCBI GEO platform and more. These tasks consisted of, for example, planning cloning experiments for protein expression or a luciferase assay or using the various software or platforms to view, manipulate and analyze high throughput sequencing data and discuss the functional organization of different gene loci when looking at several functional makers (CpG islands, histone mark, nucleosome mapping, DNase hypersensitivity, FAIRE‐seq and transcription factor binding sites). Students had to write down and explain their problem solving strategies, results and discussion in a laboratory book, as they would in laboratory research. These were also discussed in class, as a research group would discuss group member's results. Course evaluations revealed this practical aspect of the course was appreciated by students and most thought the Webquests were interesting but very demanding and generally put them in the uncomfortable position of not knowing whether their considerable amount of work was good or bad. Therefore, a teaching strategy using immediate and substantial feedback closer to one‐on‐one mentoring seems to be important for this type of course content and activity in order to nurture and guide students even in basic skills such as scientific writing and maximize their learning experience. Using this blended learning strategy allowed the students to put in practice the theoretical knowledge acquired in class in a novel and engaging manner, better reflecting the process of scientific inquiry in a real world environment.

Support or Funding Information

The University of Ottawa Teaching and Learning Support Services (TLSS)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of the Role of abl‐1 in the Apoptosis Pathways in C. elegans
Authors: Vos Margaret R. , Bennett Randy L.
Published: June 2021

Apoptosis and engulfment of apoptotic cells is necessary for normal tissue development and remodeling. Although apoptosis is a natural phenomenon, it can be induced or halted by a variety of stressors. Induction of apoptosis is a possible way to treat cancerous cells in which desirable apoptosis has been halted. In our study, apoptosis was triggered in the model organism, Caenorhabditis elegans, using carcinogens to induce DNA damage in germline cells and then the different rates of apoptosis in various mutants were measured. This assay was performed on single‐mutant strains, to establish base levels of apoptosis for each strain, and then these single‐mutants were crossed with an abl‐1 mutant strain in order to analyze the impact of these loss‐of‐function mutation interactions. We showed that a loss of function in abl‐1, when combined with another loss‐of‐function mutation directly involved in the targeted apoptosis pathway, aided in anti‐apoptotic promotion. CRISPR technology was used to increase the expression of abl‐1 in single mutants and wildtypes to see if a statistically significant impact on apoptosis occurred, and to serve as a comparison to the loss‐of‐function research. We found that increasing abl‐1 expression in wildtype worms acted in an anti‐apoptotic way, but in mutants where the traditional apoptosis pathway was impaired (e.g. those with loss‐of‐function mutations such as ced‐3), abl‐1 had a pro‐apoptotic function and could partially revive the pathway and promote apoptosis. This research is currently expanding to include a protocol focusing on analysis of somatic cell death, investigating the impact abl‐1 may have on EGL‐1. Our results demonstrate that C. elegans can serve as an in vivo model to study the mechanisms of mammalian DNA damage‐induced apoptosis. The concentration and time‐dependent responses in several mutant types and the manipulation of gene expression may provide information needed to develop more effective apoptosis‐based drug therapies.

Support or Funding Information

Funding was provided by the Biology Department at Juniata College. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). Assistance in developing CRISPR vector provided by Jason Chan, Ph.D., Juniata College.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Follicular Fluid Promotes Anoikis Resistance in TP53 Mutated Fallopian Tube Epithelial Cells in Ovarian Cancer
Authors: Flanigan W. R. , Fleszar A. , Kreeger P.
Published: June 2021

Ovarian cancer is the most lethal gynecological disease and the high rate of mortality can be largely attributed to an inability to diagnose it in its early stages, stemming from a general lack of understanding of the disease's origins. In the formation of the disease, fallopian tube epithelial (FTE) cells—ovarian cancer progenitor cells—accrue a TP53 mutation and metastasize to the ovary or omentum. To survive in suspension in the peritoneal cavity, FTE cells must evade anoikis, a form of apoptosis that occurs when anchorage‐dependent cells become detached from the extracellular matrix (ECM). During ovulation, the oocyte‐releasing follicle discharges significant quantities of follicular fluid (FF), bathing the fimbria in reactive oxygen species, hormones, and growth factors. These microenvironmental factors are believed to have a synergistic effect with genetic mutations to initiate ovarian carcinogenesis. We have found that FF significantly (p<0.0001) increased resistance to anoikis in TP53‐mutated FTE cells, facilitating metastasis. This ubiquitous trend was observed in all FF patient samples (n=7). These assays employed flow cytometry with live/dead staining to quantify cell survival. To elucidate the mechanism governing this increase in survival of FTE cells, we fractioned FF by molecular weight, denatured proteins in FF, and added exogenous factors in anoikis assays. We discovered that hyaluronic acid (HA)—a glycosaminoglycan found in ECM—significantly (p=0.0017) increased survival of TP53‐mutated FTE, indicating its role as a constituent in the anoikis signaling cascade. FF protein denaturation results pointed to a mechanism that is likely multifaceted involving both protein and non‐protein components. By understanding the interplay of genetic mutations and microenvironmental factors, we hope to reveal the complete mechanism behind anoikis resistance to develop earlier detection mechanisms and preventative methods for ovarian cancer in its earliest stage.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SIKE affects processes involving cytoskeletal rearrangement
Authors: McCool Ryan , Quintero Christian A. , Bell J. Ellis , Bell Jessica K.
Published: June 2021

Suppressor of IKK Epsilon (SIKE) is a protein in the virus‐ and TRL3‐triggered cascading signal pathway. SIKE has been shown to directly interact with alpha‐actinin, a protein involved in cytoskeletal rearrangement, in coimmunoprecipitation and in vitro precipitation experiments. However, the exact cellular processes SIKE affects are still unknown. To bridge this gap, we examined two processes that require cytoskeletal rearrangement, migration and phagocytosis, to determine if SIKE functions in these processes. Our model system is a parental cell line, HAP1, and a CRISPR/Cas9 knockout of SIKE, SIKE‐CR. Scratch assays were performed on parental HAP1 cells and SIKE knockout cells. To induce an anti‐viral response, dsRNA, a viral mimic, was applied to cells prior to the start of scratch assays in a second set of experiments. Phagocytosis assays, using latex beads coated with FITC labeled IgG, were performed and analyzed with a novel ImageJ procedure to determine the effects of SIKE on phagocytosis. The scratch assays indicate that loss of SIKE decreases cellular motility by ~30% whereas SIKE decreases cellular ability to perform phagocytosis. These studies suggest that SIKE links the innate immune system's antiviral response to cytoskeletal rearrangements necessary to host defenses.

Support or Funding Information

Studies were funded by USD SURE and NIH‐NIAID R21 to JKB.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Erlotinib inhibits epithelial mesenchymal transition of cigarette smoke‐exposed human retinal pigment epithelial cells through regulation of the FAK‐Syk/Src pathway
Authors: PARK GA‐BIN , KIM DAEJIN
Published: June 2021

Cigarette smoke (CS)‐induced oxidative stress modifies the characteristics of retina pigmental epithelial (RPE) cells to express growth factors, including epidermal growth factor (EGF). Stimulation of lung cancer cells with CS activates aberrant downstream signaling pathways of EGF receptor (EGFR). However, the molecular mechanism of epithelial‐mesenchymal transition (EMT) processes in RPE cells stimulated by cigarette smoke extract (CSE) remains largely unknown. In this study, we focused on the underlying signaling pathways to identify the key molecules that are involved in EMT of CSE‐exposed RPE cells. Cultured human ARPE‐19 cells were cultured in the presence of 5% CSE for 4 weeks. Expression and activation of mesenchymal markers and EMT‐related signaling pathways (FAK, Src family, and Smad2/3) were determined by immunoblot analysis. Secretion of VEGF, IL‐6, IL‐8, and TGF‐β1 were investigated by ELISA assay. CSE‐induced migration was measured by wound healing assay. Exposure to CSE of RPE cells induced the secretion of VEGF, IL‐6, IL‐8, and TGF‐β1 as well as the expression of vimentin and α‐SMA. CSE‐mediated Syk/Src activation resulted in the phosphorylation of focal adhesion kinase (FAK), leading to migration and invasion of RPE cells. Pharmacological inhibition of Syk/Src or gene silencing of FAK with siRNA prevented the CSE‐mediated VEGF and TGF‐β1 production and blocked the phosphorylation of Smad2/3 in CSE‐stimulated RPE cells. Erlotinib, an EGF receptor tyrosine kinase inhibitor, profoundly attenuated the level of mesenchymal phenotypes in EGF and CSE‐treated RPE cells. Our results provide new concepts about EGF‐like role of CSE against RPE cells and suggest a possible new therapeutic target and measure for proliferative retinopathy (PVR).

Support or Funding Information

The present study was supported by the Basic Science Research Program of the Ministry of Education (grant no. NRF‐2015R1D1A1A01056672) and Ministry of Science, ICT & Future Planning (grant no. NRF‐2015R1C1A2A01053732) through the National Research Foundation (NRF) of the Republic of Korea.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Converting the tumor suppressor function of WWOX to tumor promoting by Serine 14 phosphorylation
Authors: Ho Pei‐Chuan , Kuo Hsiang‐Lin , Chang Nan‐Shan
Published: June 2021

Accumulation of tumor suppressor WWOX with Ser14 phosphorylation has been shown in growing solid tumors and brain lesions with Alzheimer's disease. Functional significance of this regard is unknown. A portion of cytosolic WWOX is recruited to the cell membrane/cytoskeleton by binding hyaluronidase Hyal‐2. Here, we identified a cell surface‐exposed N‐terminal epitope WWOX7‐21 (amino acid #7 to 21), which is in front of the first WW domain. Treatment of breast 4T1 cancer stem cell spheres with the WWOX7‐21 antibody resulted in dramatic inhibition of ceritinib‐mediated cell death (>85% inhibition). Similar results were observed using other therapeutic chemicals. In contrast, when 4T1 stem cell spheres were treated with micromolar levels of synthetic WWOX7‐21 and WWOX7‐11 peptides, 4T1 cells in the spheres became highly susceptible to death by ceritinib. Notably, when pS14‐WWOX7‐11 peptide was used to treat the 4T1 spheres, cells significantly acquired resistance to ceritinib. WWOX7‐21 and WWOX7‐11 peptides caused spleen Z cell expansion and activation by expressing Hyal‐2, Zfra, and Noxa to block 4T1 growth and metastasis in mice. Accordingly, stimulating membrane Hyal‐2 with agonist Hyal‐2 antibody, sonicated hyaluronan and Zfra peptide primed Z cells for activation and blocking cancer growth in vivo. Purified activated Z cells aggressively attacked and caused death of 4T1 stem cells in spheres in vitro, as determined by time‐lapse microscopy. In stark contrast, pSer14‐WWOX7‐21 peptide significantly induced clonal expansion of spleen CD8alpha+ T and CD19+ B cells in vivo and enhanced cancer growth. Together, phosphorylation at Ser14 turns WWOX from tumor suppressor into tumor promoter.

Support or Funding Information

MOST, Taiwan

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MED28 modulates transcription factor FOXM1‐associated migration and invasion in human non‐small cell lung cancer (NSCLC) cells
Authors: Hsieh Nien‐Tsu , Huang Chun‐Yin , Li Chien‐Cheng , Wang I‐Ching , Lee Ming‐Fen
Published: June 2021

Lung cancer has become a leading cause of cancer deaths worldwide, where non‐small cell lung cancer (NSCLC) accounts for the majority of the diagnosed cases. Increased expression of transcription factor Forkhead Box M1 (FOXM1) is involved in the unfavorable development of several types of cancer, including lung tumor aggressiveness. MED28 exhibits multiple cellular roles, including a Mediator subunit for transcription, an interactor with merlin, Src kinases, Grb2 and cytoskeleton‐associated proteins, which is involved in many cellular signaling pathways. Our laboratory has previously found that MED28 appears to modulate the development of epithelial‐mesenchymal transition and invasion in human NSCLC cells. The objective of the current study is to investigate the potential role of MED28 in FOXM1‐associated tumorigenesis in NSCLC. MED28 expression positively corresponded to both migration and Matrigel invasion events. Inducible expression of FOXM1 not only enhanced NSCLC cell migration and Matrigel invasion but also significantly inhibited siMED28‐mediated suppression of cell invasiveness. Suppression of MED28 reduced the expression of FOXM1 as well as MMP2, a metastasis marker and downstream target of FOXM1. MED28 also regulated the luciferase activity of the MMP2‐reporter gene. Taken together, our data indicate an important connection between MED28 and FOXM1 in the progression of malignancy in NSCLC.

Support or Funding Information

This work was supported by the grants NSC102‐2320‐B‐309‐001‐MY3 and MOST106‐2320‐B‐309‐001‐MY3 to M‐F Lee.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Serotonin Promotes Enterohemorrhagic Escherichia Coli Pathogenesis Through Altered AI‐2 Production by Gut Microbiota
Authors: Menon Rani , Jani Sneha , Riordan Rebekah , Jayaraman Arul
Published: June 2021

The overall goal of this study is to investigate the cross‐talk between a host metabolite (serotonin), the commensal bacterial community, and a microbial signaling molecule (Autoinducer‐2; AI‐2), in the etiology of Enterohemorrhagic Escherichia coli (EHEC) infections. A broad range of microbial (e.g., AI‐2, indole) and host metabolites (e.g., neuroendocrine hormones such as serotonin and norepinephrine) are present in the GI tract microenvironment and often function together to modulate the virulence of enteric pathogens. Serotonin is of specific interest as it is abundant in the GI tract and has pleiotropic roles on intestinal physiology. While it is well established that serotonin levels increase during inflammation, its impact on the commensal microbiota composition and function, and thereby, on enteric pathogen infections is poorly understood. Here we show that serotonin exposure results in a statistically significant increase (p<0.05) in the production of AI‐2 produced by non‐pathogenic E. coli and EHEC. Serotonin exposure also increased the expression of virulence genes (eaeA, escC, and escV) in EHEC in vitro and this increase depends on the luxS gene that is required for AI‐2 production. Based on these results, we hypothesize that serotonin's effects on EHEC virulence are mediated through AI‐2. Administering serotonin in mice resulted in pronounced dysbiosis of the microbiota composition. Interestingly, approximately 50% of the bacterial communities that showed a significant fold change (p<0.05) at the genus level had representative members with the luxS gene. Significant changes were also observed in the levels of six tryptophan‐derived microbiota metabolites including a reduction in the levels of indole. Together, our results support a model in which an increase in serotonin causes microbiota dysbiosis, increases AI‐2 levels, downregulates the beneficial anti‐inflammatory metabolites, thereby creating a niche favorable for EHEC colonization. Understanding the specific mechanisms underlying the effect of serotonin on the microbial community can lead to the development of new therapeutic approaches for combating enteric infections.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterizing nucleic acid association with bacterial membrane vesicles and their transfer to host cells
Authors: Rodriguez Blanca Victoria , Kuehn Meta J.
Published: June 2021

Numerous studies have shown that extracellular nucleic acids can be secreted by bacteria via membrane vesicles (MVs) which can serve as protected vehicles for nucleic acid transfer into host cells. MVs are known to carry biologically active macromolecules that play a significant role in bacteria‐host cell communication. A potential role for prokaryotic MV‐associated RNA in pathogen‐host interactions was proposed recently but intensive investigation into this hypothesis has not been conducted. It has been revealed that vesicles contain a distinct profile of RNA molecules relative to the parent cell RNA profile of multiple bacterial organisms, thereby suggesting a selective export or protective mechanism for RNAs associated with vesicles. Previous studies have highlighted the immunomodulatory characteristics of RNA produced by the human pathogen Staphylococcus aureus during infection of host immune cells but it is currently unknown whether immunomodulatory RNAs are secreted in association with MVs. As such, we are interested in gaining a thorough understanding of the identity, selection, localization, and host cell interaction of MV‐associated RNAs derived from S. aureus.

Using electrophoretic analysis and microscopy imaging approaches, I have found that RNAs are associated with purified MVs produced by S. aureus. By size distribution analysis, I discovered that the MV‐associated RNAs appeared to be <50 nucleotides in length, consistent with previously published reports for MVs produced by other bacteria. When examining the functional roles of MV associated RNA, I discovered that S. aureus MVs induced the production of a cytokine that is typically induced in response to signaling cascades initiated by nucleic acid receptors in cultured macrophage cells. Thus, my results suggest a role for MV‐associated nucleic acids in stimulating a host immune response that could be distal to the site of infection. Based on these findings, I hypothesize that MVs are enriched with nuclease‐protected nucleic acid fragments containing immunostimulatory sequences. Using RNA‐seq and qRT‐PCR of MV RNA cargo, those sequences can be identified. Nuclease protection assays suggest a portion of the nucleic acids associated with the MVs are protected by a location inside the vesicle membrane or by association with vesicle cargo proteins. By screening for the presence and importance of RNA‐binding proteins in the MVs, I can identify mechanisms of RNA selection and protection. This research will uncover mechanisms of RNA association with bacterial MVs and mechanisms of host immune cell modulation by the MV‐associated RNAs.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Legionella Effector RavD Binds Host Phosphoinositide‐3‐phosphate and Contributes to Lysosomal Avoidance
Authors: Neunuebel Ramona
Published: June 2021

Subversion of phosphoinositide lipids is a key strategy that bacterial pathogens employ to survive within hosts. Due to the importance of phosphoinositides in phagocytosis, bacterial pathogens that infect innate immune cells manipulate these lipids to alter phagosome maturation with the purpose of converting the nascent phagosome into a replication‐permissive niche. The intracellular bacterial pathogen, Legionella pneumophila, the causative agent of a severe pneumonia known as Legionnaires' disease, infects alveolar macrophages where it proliferates within the safety of a vacuole. Upon phagocytosis, L. pneumophila hijacks the nascent phagosome and remodels the cytosolic façade of the phagosomal membrane by stimulating fusion with vesicles derived from the endoplasmic reticulum. This process relies on the function of bacterial effector proteins translocated directly into the host cell via a specialized secretion system. A number of L. pneumophila effector proteins have been shown to bind phosphoinositides either for the purpose of manipulating their metabolism or as a means of targeting specific host membrane compartments. Our preliminary data indicate that many more phosphoinositide‐binding effectors remain to be discovered. Here, we show that the L. pneumophila effector, RavD, is a novel phosphoinositide‐3‐phosphate (PI3P) binding‐protein. We establish that RavD is found on the Legionella‐containing vacuole (LCV) at early stages of infection and harbors its PI3P binding domain within the C terminal region of the protein. In the absence of the ravD gene, the LCV accumulates lysosomal markers at 2 hours post‐infection, consistent with the notion that RavD function is necessary to arrest phagosome maturation.

Support or Funding Information

This work was supported by the University of Delaware Research Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Metabolic Reprogramming of Macrophages Exposed to Pseudomonas aeruginosa Biofilm
Authors: Ammons Mary Cloud Bosworth , Fuchs Amanda , Copie Valerie
Published: June 2021

Currently, the annual economic cost of chronic wounds exceeds $1 billion in the United States and the incidence of non‐healing wounds is expected to dramatically increase in the next several years due to a Type 2 diabetes epidemic. Several common characteristics typical of chronic wounds include the development of persistent antibiotic‐resistant colonization by microbial biofilms, excessive inflammation, and failure of human cells to respond to reparative stimuli. Pseudomonas aeruginosa is one of the predominant opportunistic pathogens that colonizes greater than 50% of all chronic wounds in the U.S. In an effort to better understand the molecular mechanisms by which P. aeruginosa biofilms interfere with human macrophage responses to pathogens, we have undertaken comprehensive metabolomics studies of activated macrophages. These studies have been designed to probe the metabolic reprogramming of these immune cells as result of exposure to secreted molecules produced by P. aeruginosa biofilms, using an in vitro host‐pathogen co‐culture model. Herein, we present our most recent NMR‐based metabolomics results. This metabolic phenotyping is correlated with fluorescence‐activated cell sorting (FACS) analysis to determine the relationship between metabolic regulation and macrophage immunomodulation (i.e macrophage polarization into pro‐ or anti‐inflammatory M1 or M2 subpopulations, respectively) in response to exposure of macrophages to co‐culture with P. aeruginosa biofilms.

Support or Funding Information

A.L.F. was supported by funds provided through a Kopriva Fellowship. A.L.F. and M.C.B.A. are suppported by funds provided by the National Institute of Health (NIH) grant number 1KO1GM103821. M.C.B.A. was also supported by the NIH grant number 1KO1GM103821. NMR spectra were recorded at MSU on a Bruker AVANCE III 600 MHz NMR spectrometer. Funding for this instrument and corresponding upgrade was provided by the NIH SIG program (grant numbers 1S10RR13878 and 1S10RR026659). M.C.B.A. is currently supported by a NIH Grant No. P20GM109007 (National Institute of General Medical Sciences).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
25‐Hydroxycholesterol inhibits viral infection in a liver X receptor dependent manner
Authors: Wei Zhuo
Published: June 2021

Cholesterol 25‐hydroxylase (CH25H) catalyzes the production of 25‐hydroxycholesterol (25HC), which is an endogenous liver X receptor (LXR) agonist. The recent studies have demonstrated that CH25H is an interferon (IFN)‐induced gene and IFN‐activated 25HC functions as a molecule potently inhibiting viral infection. Previously, besides IFN‐γ, we also identified several LXR‐responsive elements (LXREs) in the CH25H promoter and determined LXR activation induced CH25H expression. Thus, we hypothesized that the LXR‐activated CH25H expression can play an important role in antiviral infection. In this study, we determined that 25HC induced its own production by activating CH25H expression. We further determined that 25HC and synthetic LXR ligands (T0901317/GW3965) inhibited the infection of HSV‐1 as well as MLV‐(VSV)‐GFP in hepatocytes and the infection of HSV‐1 in macrophages in an LXR‐dependent manner. Therefore, genetic deletion of LXRα, LXRβ or CH25H by CRISPR/Cas9 system in HepG2 cells increased cell susceptibility to HSV‐1 infection, and attenuated 25HC or T0901317‐inhibited viral infection. Lacking of IFN‐γ expression also attenuated, but not blocked, LXR‐induced CH25H expression and LXR‐inhibited HSV‐1 infection. 25HC can be further metabolized to 25‐hydroxycholesterol‐3‐sulfate (25HC3S) by sterol sulfotransferase‐2B1b (SULT2B1b). We observed that SULT2B1b moderately reduced the antiviral actions of 25HC based on the determination that 25HC3S was a weaker inhibitor of HSV‐1 infection than 25HC. In summary, our study demonstrates 25HC/T0901317 inhibits viral infection in an LXR‐dependent manner.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reverse Correlation between Body Mass Index and Post‐Transplant Hypertension by a New Diagnostic Criteria
Authors: Tantisattamo Ekamol , Vutthikraivit Possawat , Ratanasrimetha Praveen
Published: June 2021

Introduction

Obesity is associated with hypertension (HTN). Recent new diagnostic criteria for HTN with systolic blood pressure (SBP) and diastolic blood pressure of >130 and >80 mmHg, respectively associated with body mass index (BMI) in kidney transplant recipients is unknown.

Methods

Systolic hypertension (SHTN) and diastolic hypertension (DHTN) after 1‐month post‐transplantation is determined by old and new diagnostic criteria. A time‐to‐events of developing post‐transplant SHTN and DHTN associated with pre‐transplant BMI are compared between HTN defined by old and new criteria by using Cox proportional hazard regression analysis.

Results

Seventy kidney transplant recipients are reviewed and followed for 96 months. Mean age was 52.7±1.4 years (mean±SEM) and 59% are males. Half of the study population is White, and 34% and 16% are Black and others, respectively. Mean pre‐transplant BMI is 27.64±0.67 kg/m2. At 1‐month post‐transplant, a mean pre‐transplant SBP and DBP are 130.91±2.14 and 77.71±1.51 mmHg, respectively. With an old diagnostic criterion for HTN, 70% have SHTN but up to 90% have SHTN diagnosed by the new criteria. At 96‐month post‐transplant, obese patients has 15% lower risk for SHTN diagnosed with the old criterion (Hazard ratio (HR) 0.850; 95% CI 0.473 – 1.529; p‐value 0.588); whereas, these patients have 2.3% greater risk for SHTN by new criteria (HR 1.023; 95% CI 0.601 – 1.741; p‐value 0.934) [figure 1]. For DHTN, obese patients have 23.7% decreased risk for DHTN diagnosed with the old criterion (HR 0.763; 95% CI 0.394 – 1.478; p‐value 0.422), and also have 14.9% lower risk for DHTN by new criteria (HR 0.851; 95% CI 0.496 – 1.461; p‐value 0.559) [figure 2].

Conclusion

The incidence of SHTN and DHTN are increased with the new diagnostic criterion for HTN. The new criteria appear to have the reverse effect on the association between BMI and SHTN, but not DHTN. Further studies related to SHTN are required to elucidate the outcomes of SHTN after kidney transplantation.

Support or Funding Information

None

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 Expression Differences of Cyclin Dependent Kinase Inhibitors In Aged and Young Pancreatic Beta Cells
Authors: Aitken Talon J. , Grover Samuel G. , Booren Parker L. , Tessem Jeffery Sivert
Published: June 2021

The number of people affected by diabetes is continually rising, affecting over 400 million people worldwide. The insulin secreting pancreatic beta cells are essential to control proper glucose absorption and storage in insulin sensitive peripheral tissue. Both Type 1 and Type 2 diabetes are characterized by decreased functional beta cell mass and, consequently, decreased insulin production. One potential intervention is the use of beta cell transplantation from cadaveric donors. A major impediment to greater application of this treatment is the scarcity of transplant ready beta cells. Increasing the amount of functional beta cells will lead to increased insulin production and better management or a possible treatment strategy for the disease. Various genes have been defined that can induce beta cell replication. A major caveat of these findings, however, is that these factors induce replication in young beta cells and not aged beta cells. Given that the majority of beta cells that will be used for transplant will come from aged donors, it is imperative to understand why aged beta cells are refractory to these proliferative mechanisms. We aimed to determine why aged beta cells do not replicate as well as young beta cells. We hypothesized that one reason for why aged beta cells are refractory to genes that induce proliferation is greater expression of cell cycle inhibitors. The cell cycle is tightly regulated by cyclin‐dependent kinases. Cells also produce proteins known as cyclin‐dependent kinase inhibitors(CDKI's), which bind to cyclin dependent kinases, effectively shutting down cell proliferation. Here we demonstrate the expression of the INK4 and Cip/Kip family of CDKI's by mRNA, protein and histological expression in 5 week old and 5 month old primary rat beta cells. These results begin to define the functional changes that result in the inability of aged beta cells to replicate.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synergistic Anti‐adipogenic Effect of 3,3′‐diindolylmethane and Capsaicin in 3T3‐L1 Adipocytes
Authors: Lee Jihye , Lee Seong‐Ho
Published: June 2021

3,3′‐diindolylmethane is a metabolite of indole‐3‐carbinole abundant in cruciferous vegetables and capsaicin is an active component of chili pepper that belongs to Capsicum. Recently we reported that 3,3′‐diindolylmethane repressed fat accumulation via AMPK‐dependent manner in differentiated adipocytes and C. elegans. Based on our previous findings that 3,3′‐diindolylmethane and capsaicin possess synergistic anti‐cancer activity, the current study was conducted to explore if 3,3′‐diindolylmethane in combination with capsaicin synergistically suppresses adipogenesis using differentiated 3T3‐L1 adipocytes. Co‐treatment of 3,3′‐diindolylmethane and capsaicin significantly reduced triglycerides accumulation and expression of adipogenic marker proteins (PPARγ and C/EBPα). Combinational treatment of 3,3′‐diindolylmethane and capsaicin also modulated expression of several apoptosis‐ and cell cycle‐regulating proteins. Multiple mechanisms appear to be involved in synergistic anti‐adipogenic effect by combinational use of 3,3′‐diindolylmethane and capsaicin.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
TISSUE‐SPECIFIC GENETIC TARGETING OF INOS REVEALS AN UNEXPECTED ROLE OF ADIPOCYTE INOS IN THE DEVELOPMENT OF INSULIN RESISTANCE, DYSLIPIDEMIA AND ALTERED ENERGY METABOLISM IN DIET‐INDUCED OBESE MICE
Authors: Vilela Vanessa Rodrigues , Lachance Gabriel , Nachbar Renato Tadeu , Centano‐Baez Carolina , Bellmann Kerstin , Marette André
Published: June 2021

Nitric oxide (NO) is synthesized from L‐arginine by NO synthase (NOS) enzymes. The expression of the inducible member of the NOS family, iNOS, is markedly enhanced in adipose, skeletal muscle and liver tissues in response to bacterial endotoxins (LPS), inflammatory cytokines, and diet‐induced obesity. We have previously showed that mice lacking iNOS are protected from insulin resistance induced by high‐fat feeding; acute exposure to LPS or lipid infusion, providing genetic evidence that iNOS is a key inflammatory mediator in these settings. It is believed that in immune cells within adipose tissue of obese animals mainly contribute to iNOS expression and the immunometabolic perturbances of obesity. To clarify the specific role of iNOS induction in adipocytes, muscle and liver in the development of insulin resistance and altered energy metabolism of obese animals, mice containing flox sites flanking Exon 2 of nos2 were created. Myocyte‐specific (MuKO), hepatocyte‐specific (HeKO) and adipocyte‐specific (AdKO) iNOS knockout mice were generated on a C57BL/6J background by crossing mice homozygous for floxed iNOS (nos2f/f) with Ckmm‐cre, Alb‐Cre and Adipoq‐Cre, respectively. KO mice and their respective wild‐type controls were fed regular chow or a high‐fat/high sucrose (HFHS) diet for 8 weeks and then extensively phenotyped for metabolic outcomes. We found that AdKO mice were partially protected from HFHS‐diet insulin resistance as compared to WT littermates on the same diet, and that this beneficial effect was associated with increased energy expenditure as measured by indirect calorimetry. These effects were observed despite no differences in body weight gain and adiposity. Adipocyte‐specific iNOS KO was also associated with a reduction in circulating triglyceride levels upon HFHS feeding. However, disrupting iNOS selectively in skeletal muscle and liver did not improve metabolic phenotypes after 2 months of HFHS feeding. Our results reveal an unexpected role of iNOS specifically expressed in adipocytes in the development of systemic insulin resistance, dyslipidemia and altered energy metabolism in diet‐induced obesity, suggesting an important role of adipocyte NO release in the metabolic complications of obesity. The role of muscle and liver iNOS appears limited after 2 months of HFHS diet but studies are underway to document their potential impact after longer‐term feeding experiments.

Support or Funding Information

Canadian Institutes of Health Research

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
IDH2 Deficiency Aggravates Fructose‐Induced NAFLD by Activating Inflammatory Signaling in Female Mice
Authors: Beane Kaleigh E. , Montalbano Allison M. , Pan Jeong Hoon , Kong Byungwhi C. , Kim Jae Kyeom
Published: June 2021

Fructose is a strong risk factor of non‐alcoholic fatty liver disease (NAFLD), resulting from disruption of redox systems by excessive reactive oxygen species production in the liver cells. Importantly, recent epidemiological studies indicated that women are more prone to develop metabolic syndrome in response to fructose‐sweetened beverages. Here, we examined whether disruption of redox system exacerbates fructose‐induced NAFLD conditions in female mice. The redox system was disrupted by blocking NADPH supply through mitochondrial NADP+‐dependent isocitrate dehydrogenase knock‐out (IDH2 KO) in C57BL/6 female mice. After, wild type (WT) and IDH2 KO mice were treated with either water or 34% fructose water over six weeks. NAFLD phenotypes and key proteins and mRNAs involved in inflammatory pathway (e.g., NF‐κB p65 and IL‐1β) were assessed. As results, hepatic lipid accumulation was significantly increased in IDH2 KO mice fed fructose compared to its WT counterpart. Neutrophil infiltration was observed only in IDH2 KO mice fed fructose. Further, phosphorylation of NF‐κB p65 and expression of IL‐1β was remarkably upregulated in IDH2 KO mice fed fructose, and expression of IκBα was decreased by fructose treatment in both WT and IDH2 KO groups. For the first time, we report that IDH2 KO significantly aggravates fructose‐induced NAFLD phenotypes (fatty liver and neutrophil infiltration) and related inflammatory responses (i.e., NF‐κB p65 pathway). Collectively, this study provides evidence that IDH2 deficiency may have implications on fructose‐induced NAFLD through suppression of fatty acid β‐oxidation and/or activation of the NF‐κB pathway. Given our significant phenotypes in response to fructose intervention, the present study will provide a good justification for further characterization thus warrants additional investigations to clarify mechanistic roles of mice IDH2 in metabolic diseases.

Support or Funding Information

This work was supported by the University of Arkansas, VPRED Start‐up fund (KJK, and PJH). Support has been provided in part by the Arkansas Biosciences Institute, a partnership of scientists from Arkansas Children's Hospital, Arkansas State University, the University of Arkansas‐Division of Agriculture, the University of Arkansas, Fayetteville, and the University of Arkansas for Medical Sciences. The Arkansas Biosciences Institute is the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000 (JKK, JHP, KEB, and BCK).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enhancement of biological activity through conversion of rutin to quercetin using acid treatment
Authors: Yang Jinwoo , Lee Junsoo
Published: June 2021

Flavonoids attract much attention due to their various physiological properties and these compounds naturally exist as their glycoside forms. Rutin, one of the most abundant flavonoids in nature, is composed of the aglycone quercetin and the disaccharide rutinose. Although, there is a structural similarity in rutin and quercetin, there are some obvious differences in physical, chemical, and biological characters. It is reported that quercetin has a higher biological activity than other glycosidic quercetin derivatives. Thus, de‐glycosylation of rutin would be an important process for enhanced biological activities. Therefore, in this study, to convert rutin to quercetin, the effects of acid concentrations and solvents were investigated and biological activities were evaluated. As a results, when 80 % ethanol was used, the hydrolysis of rutin was more rapid than water as solvent for reaction. Hydrolysis of rutin was also accelerated as the acid concentration increased from 0.1 to 1.0 M of HCl. Anti‐oxidant, anti‐inflammatory, and anti‐adipogenic activities were increased with the increased conversion rate of rutin to quercetin. These results suggest that the acid treatment can be used as a process to improve the physiological activity of glycoside flavonoids.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enhanced Mitochondrial DNA Repair Confers Protection Against Obesity and Metabolic Syndrome by Altering White Adipose Tissue Energetics
Authors: Komakula Sai Santosh Babu , Tumova Jana , Ye Hong , Vartanian Vladimir , Lloyd R Stephen , Akal Tasleen , Sampath Harini
Published: June 2021

Oxidative DNA damage is characterized by oxidative modifications of nuclear and mitochondrial DNA. Unrepaired DNA lesions can cause mutations. Mitochondrial DNA is more prone to oxidative damage when compared to nuclear DNA. 8‐oxo‐Guanine (8‐oxoG) is the most abundant oxidative DNA lesion, which is primarily repaired by the base excision repair (BER) pathway. BER is initiated by DNA glycosyslases such as 8‐oxo‐Guanine DNA glycosylase (OGG1) that recognizes and cleaves 8‐oxoG from nuclear and mitochondrial DNA. Previously, we reported that mice lacking OGG1 both in the nucleus and mitochondria are more prone to obesity and metabolic dysfunction compared to wild type controls. However, the contribution of mitochondrial vs. nuclear OGG1 to this metabolic phenotype have not been reported. We report here that transgenic mice over expressing human OGG1 only in the mitochondria (Ogg1Tg mice) are protected from high fat diet induced obesity, insulin resistance, fatty liver, and other features of metabolic syndrome, relative to diet‐matched wild type counterparts. The observed phenotype is a consequence of altered energy metabolism in white adipose tissue. Our results indicate that white adipose tissue of Ogg1Tg mice have increased respiration due to mitochondrial uncoupling and increased expression of Pgc‐1α, Sirtuin‐1, and uncoupling protein‐3. Plasma adiponectin levels are also elevated in Ogg1Tg mice. Electron microscopy results indicate that, Ogg1Tg mice on high fat diet have elongated and more electron dense mitochondria. These results confirm a novel and critical role for the mitochondrial DNA repair and structure on whole body energy homeostasis. These findings also indicate an important role for white adipose tissue mitochondrial metabolism on whole body energy balance.

Support or Funding Information

The Sampath lab is supported by the NJ Institute for Food Nutrition and Health, NIH grant R00DK100640 (Sampath PI) and a Euro Bio4Med grant (Sampath Co‐PI)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fibroblast Growth Factor 21 Promotes Glucose Uptake in Adult Skeletal Muscle Fibers from Mice
Authors: Rosales‐Soto Giovanni , Díaz‐Vegas Alexis , Llanos Paola , Jaimovich Enrique , Contreras‐Ferrat Ariel
Published: June 2021

Introduction

Fibroblast growth factor 21 (FGF21) is a pleiotropic peptide hormone that has effects in several organs including skeletal muscle, liver and adipose tissue. FGF21 improves the glucose tolerance test in obese‐insulin resistance mice in vivo and increases the glucose uptake in both primary myotubes and C2C12 myoblasts in vitro. However, the effect of FGF21 on glucose uptake and the cellular mechanism involved in this process in adult skeletal muscle fibers is poorly understood. The aim of this study is to assess the effect of FGF21 on GLUT4‐mediated glucose uptake in isolated skeletal muscle fibers.

Material and Methods

Male mice were used at 6–8 weeks of age. The effect of FGF21 on GLUT4‐mediated glucose uptake was evaluated in single living fibers from flexor digitorum brevis muscle. To determine the glucose uptake, we used 2‐[N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino]‐2‐deoxy‐D‐glucose (2‐NBDG) (300 μM) a phosphorylable, non‐metabolizable fluorescent glucose analog that has been used to monitor glucose uptake in single living cells.

Results

FGF21 induces a dose‐response effect, increasing the glucose uptake in isolated muscle fibers. This effect is prevented by the use of either Cytochalasin B (5 μM) or Indinavir (100 μM), both antagonists of GLUT4 activity. The use of PI3K inhibitors such as Wortmannin (100 nM) and LY294002 (50 μM) prevents the FGF21‐dependent glucose uptake. Furthermore, FGF21 does not promote Akt phosphorylation in isolated FDB fibers. In fibers electroporated with the construct encoding GLUT4myc‐eGFP chimera and stimulated with FGF21 (100 ng/mL) for 20 min, a strong sarcolemmal GLUT4 presence was detected.

Conclusions

These results suggest that FGF21 promotes GLUT4‐dependent glucose uptake by a mechanism independent of the serine/threonine kinase Akt phosphorylation. This mechanism could be an important therapeutic target to treatment of insulin resistance and type 2 diabetes.

Support or Funding Information

Financed by FONDECYT 11130267 (AC‐F), 1151293 (EJ) & 11150243 (PL).

CONICYT‐PFCHA/Doctorado Nacional/2017‐21171449

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