bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a Naturally‐occurring Heparin‐binding Peptide Preferentially Targeting the Nucleolus
Authors: Loo Shining , Kam Antony , Tam James P
Published: June 2021

Organelle‐specific, cell‐penetrating peptides are useful for targeting intracellular proteins. Recent results showed that certain cationic cysteine‐rich peptides (CRPs) can target intracellular proteins, and hold promises for intracellular drug delivery [1]. Previously, we identified a family of CRPs, designated as bleogens, from Pereskia bleo, a medicinal paint found in South‐east Asia [2]. Our results showed that the prototypic bleogen pB1 is a heparin‐binding, anti‐microbial peptide with the potentials of intracellular targeting. Here we report that bleogen pB1 is an organelle‐specific cell‐penetrating peptide that targets the nucleolus. The linear precursor of bleogen pB1 was prepared using a stepwise solid‐phase synthesis and oxidative folded to its native state. We confirmed the purity and integrity of the synthetic bleogen pB1 using reversed‐phase high‐performance liquid chromatography, heparin‐affinity chromatography, and nuclear magnetic resonance spectroscopy. TAMRA‐pB1 was prepared using on‐resin TAMRA labeling at the N‐terminus of bleogen pB1. We demonstrated that TAMRA‐pB1 internalized into living cells via endocytosis using flow cytometry and live‐cell confocal microscopy. The cellular uptake of TAMRA‐pB1 was found to be dependent on glycosaminoglycan expressions. Upon cell entry, TAMRA‐pB1 preferentially targets the nucleus and eventually accumulates in the nucleolus. Taken together, this study highlights the discovery of bleogen pB1 as the first naturally‐occurring cell‐penetrating nucleolus‐targeting CRP. The ability of bleogen pB1 for specific delivery and accumulation of drugs in the nucleolus can be useful to address the problem of efflux‐mediated drug resistance encountered during cancer chemotherapy.

Support or Funding Information

This research was supported in part by Nanyang Technological University Internal Funding ‐ Synzymes and Natural Products (SYNC) and the AcRF Tier 3 funding (MOE2016‐T3‐1‐003).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antimicrobial VBT:TMQ:DMHDQ (1‐(4‐vinylbenzyl) thymine, vinylbenzyl trimethyl QUAT, and vinylbenzyl dimethylhexadecyl QUAT) terpolymer permanence and performance on hospital scrubs
Authors: Baulsir Elaine , Laemmle Ruby , Jaber Aliya , Yandian Megan , Gurney Richard
Published: June 2021

Due to the prevalence of nosocomial infections, the need for enhanced precaution between health care provider and patient has increased. In attempt to eliminate scrubs as a transmission vector, this project focused on the methodology behind the integrity of known antibacterial terpolymers on scrub fabric. Terpolymer function of immobilization, water solubility, and antibacterial activity was tested through manipulation of the ratios of 1‐(4‐vinylbenzyl) thymine (VBT), vinylbenzyl trimethyl QUAT (TMQ), and vinylbenzyl dimethylhexadecyl QUAT (DMHDQ), respectively. To begin, solubility testing of varying ratios of VBT: TMQ: DMHDQ was conducted to assess polymeric ability to dissolve in solution for ease in application. This ease in application allows for versatility in use beyond nursing scrubs in less porous surfaces such as light switches and phone screens. In addition to variation in monomer ratios, soaking and pre‐treatment mordants were examined. Electrostatic attraction of an anionic dye, trypan blue, to the polymer on treated scrubs was used for quantification of terpolymer permanence. In a period of time that the toxicology of commercially available unbound quaternary ammonium compounds (QAC) is highly scrutinized, this method addresses the growing concerns associated with excess use of cleaning solutions in attempt to prevent infection. The permanence testing of this terpolymer will determine if this method may be used in application to prevent the spread and increased resistance of bacterium in healthcare settings.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optimization of Protein Extraction and Identification from the European Green Crab
Authors: Wrobel Neal , Dachel Gabriel , Shipman Richard , Myracle Angela D. , Grant Jennifer E.
Published: June 2021

Carcinus maenas, or European green crab, is an invasive species off the coast of Maine frequently trapped alongside more desirable crab species, but is too small to provide economic benefits to fishermen. Currently, Marine organisms are widely recognized as intriguing potential sources of peptide antibiotics. Indeed, shells from the green crab may provide an untapped source of protein, potentially providing useful leads for pharmaceutical applications. However, crab shells prose a unique purification problem, in that the hard shells that are rich in hydroxyapatite must first be dissolved. Furthermore, calcium and phosphate must be separated from soluble proteins lest purification strategies result in precipitation of protein‐laden calcium deposits. Here we discuss the optimization of protein extraction protocols, as evidenced by use of the Bradford protein assay and SDS‐PAGE gel analysis. The ultimate goal of this work is to identify novel proteins in these shells by mass spectrometry.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identifying ErbB Proteins as Potential Receptors for Nephronectin during Mouse Corneal Development
Authors: Ponce Aiyana , Ma Justin , Lwigale Peter
Published: June 2021

Corneal defects are a major cause of blindness, but treatment options remain limited. Advancements require improved understanding of molecular processes involved in corneal development. Corneal development is an intricate process that involves migration, proliferation, and differentiation of embryonic neural stem cells known as neural crest cells. However, very little is known about the molecular mechanisms involved in these processes. Our recent studies identified Nephronectin, an extracellular matrix protein, in the developing cornea. The current study is to determine whether Nephronectin signals through ErbB proteins (ErbB1, ErbB2, ErbB3) during corneal development. To test the hypothesis that neural crest cells co‐localize and interact with nephronectin through ErbB proteins during corneal development, we first isolated cDNA from corneal tissues, then identified the expression of the ErbB genes by reverse transcription polymerase chain reaction (RT‐PCR), and finally determined their localization by in situ hybridization. Our initial analysis by RT‐PCR indicates that ErbB1 is highly expressed during corneal development. Next, we synthesized RNA probes using a cloning vector to analyze their spatial expression on paraffin‐embedded embryonic corneal sections. Our preliminary analyses showed that ErbB1 is expressed in the presumptive corneal stroma and epithelium, suggesting a potential role of Nephronectin/ErbB1 signaling during cell migration, proliferation, or differentiation into corneal cells. Additionally, ErbB2 and ERbB3 are expressed in the epithelium, indicating a role in proliferation. Identifying signaling pathways that govern corneal development can lead to better strategies to repair corneal defects.

Support or Funding Information

Research reported in this abstract was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under linked Award Numbers RL5GM118969, TL4GM118971, and UL1GM118970. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Prediction of Damage‐Associated Non‐Synonymous Single Nucleotide Polymorphisms in the Human LAMB2 Gene: A Computational Study
Authors: Herath Venura , Denipitiyage Shanika
Published: June 2021

Laminins are a major component of basement membranes, and they consist of a unique combination of three non‐identical sub‐chains, giving rise to at least 15 laminin isoforms. The laminin subunit β2 (LAMB2) gene encodes laminin β2, a sub‐chain of several laminins. LAMB2 has been identified as a risk locus for Pierson syndrome, nephrotic syndrome type 5 as well as chronic kidney disease of unknown etiology (CKDu). We conducted this study to predict damage‐associated non‐synonymous single nucleotide polymorphisms (nsSNPs) in LAMB2. nsSNPs were analyzed using 22 in silico prediction tools based on evolutionary, structural and consensus approaches. All tools showed a significant correlation with each other (p < 0.05) and a significant association between their categorical predictions (p < 0.05). Out of the 853 nsSNPs located in LAMB2, 221 nsSNPs within the functional domains were identified as damaging by ≥10 individual tools and by at least two consensus tools. These nsSNPs were distributed among EGF‐like (136), Laminin IV type B (25) and laminin N‐terminal domains (60). The effects of the damage‐associated nsSNPs located in these domains were found to be more deleterious than those found in other regions (Kruskal‐Wallis test H: 99.570, p = 0.001, df = 3). Overlaps between the identified damage‐associated nsSNPs and post‐translational modification sites were also identified. Future work will include modeling of the predicted damage‐associated nsSNPs, identification of the potential consequences to LAMB2 structure, and its disease association. The predicted damage‐associated nsSNPs will provide insight into the possible causes of LAMB2‐associated 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.
Plasma Protein Regulation during COPD‐Related Muscle Wasting Using Mass Spectrometry‐Based Proteomics
Authors: Utegg Rachael , Minogue Catherine
Published: June 2021

Chronic Obstructive Pulmonary Disease (COPD) affects 15.7 million Americans. Skeletal muscle dysfunction is a frequent comorbidity of COPD, which associates with worse prognosis including higher mortality and hospitalization rates, independently of the level of pulmonary disease. Moreover, recovery of skeletal muscle integrity in the context of rehabilitation exercises is associated with improved outcomes. To better understand the mechanisms underlying muscle wasting in COPD patients, a mouse model conditionally over expressing interleukin 13 (IL‐13+) in pulmonary Club cells has been developed. This animal demonstrates a pattern on skeletal muscle wasting that is highly reminiscent of the human counterpart. A comprehensive mass spectrometry‐based proteomics study demonstrated differences in skeletal muscle protein expression in wild type versus IL‐13+ mice. In this study, we used plasma samples to interrogate the proteomic profile of the wild type versus the IL‐13+ mice. Our hypothesis is that the proteomic signature found in skeletal muscle will, to some extent, overlap the pattern present in the plasma from the same animals indicating a systemic readout of skeletal muscle dysfunction. If our hypothesis is confirmed, we plan further validation studies using human plasma samples, which could facilitate early detection and potential interventions to ameliorate this devastating condition and have a significant impact on patients' outcomes.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Stimulation of Gαq Promotes Stress Granule Formation
Authors: Scarlata Suzanne , Qifti Androniqi , Jackson Lela , Singla Ashima , Garwain Osama
Published: June 2021

During adverse environmental conditions, mammalian cells regulate protein production by sequestering the translation machinery in membraneless organelles (i.e. stress granules) whose formation is carefully regulated. In this study, we show a direct connection between G protein signaling and stress granule formation through phospholipase Cβ (PLCβ). In cells, PLCβ localizes to both the cytoplasm and plasma membrane. We find that a major population of cytosolic PLCβ binds to stress granule proteins; specifically, eIF5A and Ago2, whose RNA‐induced silencing activity is halted under stress. PLCβ1 is activated by Gαq in response to hormones and neurotransmitters and we find that activation of Gαq shifts the cytosolic population of PLCβ1 to the plasma membrane, releasing stress granule proteins. This release is accompanied by the formation of intracellular particles containing stress granule markers, an increase in the size and number of particles, and a shift of cytosolic RNAs to larger sizes consistent with cessation of transcription. Arrest of protein synthesis is seen when the cytosolic level of PLCβ1 is lowered by siRNA or by osmotic stress, but not cold, heat or oxidative stress causes similar behavior. Our results fit a simple thermodynamic model in which eIF5a and its associated proteins partition into particles after release from PLCβ1 due to Gαq stimulation. Taken together, our studies show a link between Gαq‐coupled signals and transcription through stress granule formation.

Support or Funding Information

NIH GM116187

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cytokine‐stimulated iNOS Expression Requires Long Chain Fatty Acyl CoA Synthetase (ACSL)
Authors: Ali Yousuf , Weis Margaret Teresa
Published: June 2021

Rationale

ACSL catalyzes the ligation of fatty acid to coenzyme A, the first and rate limiting step for fatty acid utilization. Triacsin C is a fungal metabolite which inhibits ACSL1, ACSL3, and ACSL4. Earlier, we showed that Triacsin C reduces the infarct volume and inhibits iNOS expression in the MCAO model of stroke. As iNOS is up‐regulated in stroke, we wished to determine whether the Triacsin C effect was linked to iNOS expression.

Methods

bEnd.3 cells were stimulated with cytokine mix, (TNFα, 60 ng/mL; Ilβ,2 ng/mL; IFNγ, 100 units/mL), in the presence or absence of 5μM Triacsin C for 24 hours. RNA was extracted following RNeasy Plus Mini Kit(Qiagaen) instruction. The concentration and purity of RNA was determined by Spectrophometer. 20 μl first strand cDNA was synthesized from 1 μg total RNA for each sample using Superscript III First‐Strand(Invitrogen). One μl cDNA was amplified using SYBR Green PCR Master Mix (applied Biosystems) in CFX96 Touch Real‐Time PCR Detection System (BioRad). β‐actin (cat 330001 PPM02945B) and iNOS (cat 330001PPM02928B) primers were purchased from Qiagen. Expression level in fold change was determined by the comparative threshold cycle method (2−ΔΔCt) with β‐actin as the control gene.

Results

Cytomix stimulated iNOS expression in the presence of Triacsin C was 52.8 ± 8.33% of control. (p=0.0005; n=5 independent experiments).

Conclusions

Our data confirm that iNOS expression is modulated by ACSL activity, and support our hypothesis that the Triacsin C effect on stroke infarct volume is related to suppression of iNOS expression.

Support or Funding Information

Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, Amarillo, TX

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Skeletal Muscle Metabolic Enzyme Activity at Varying Temperatures in the Thirteen‐lined Ground Squirrel (Ictidomys tridecemlineatus)
Authors: Conley Emily , Symanietz Sarah , Loeza‐Rodriguez Monserrat , Schlater Amber E
Published: June 2021

Hibernation is a prolonged season where some mammals experience a dramatic reduction in food intake while undergoing a series of torpor bouts for weeks or months at a time. During individual torpor bouts, metabolic rate decreases and hibernators will thermoconform to their environment, dropping their core body temperature (TB) close to ambient temperature (TA). The causality of the reduction of metabolic rate has yet to be fully understood. Thirteen lined ground squirrels, Ictidomys tridecemlineatus, endure a hibernation season of about 8 months. Although, the thirteen‐lined ground squirrels undergo hibernation for a long period of time, they still manage to preserve and maintain their organs without any greater damage and are able to achieve physiological sustainability. Given the extreme range of TB experienced throughout the hibernation period, which is unlike most other mammalian endotherms (that typically reside TB at a constant 37°C), in addition to the unique metabolic challenges of thermoregulation within and between torpor bouts, we explored function of metabolic enzymes and subsequent substrate utilization across a range of physiologic temperatures in skeletal muscle tissue. We performed functional enzyme assays of lactate dehydrogenase (LDH, marker of anaerobic metabolism) and citrate synthase (CS, marker of aerobic metabolism) in the gastrocnemius muscle from control, non‐hibernating squirrels. Preliminary LDH data suggest that anaerobic activity is higher at 37°C as compared to thermoconforming temperature ranges of 30°C and 25°C. Alternatively, preliminary CS data suggest that aerobic activity does not change across a range of physiologic temperatures. Collectively, these aerobic and anaerobic comparisons across three different physiologic temperatures suggest that squirrels may not be suppressing aerobic metabolism when thermoconforming to lower temperatures, despite overall metabolic rate suppression during torpor bouts. Future studies may look to characterizing enzyme activity at an even lower range of physiologic temperatures, and may look to include data from hibernating squirrels in addition to non‐hibernating squirrels. These kinds of preliminary baseline data can ultimately be used to better understand the physiologic basis governing reduction of metabolic rate and subsequent substrate utilization during prolonged torpor bouts.

Support or Funding Information

McNair Scholars Program & The College of Saint Scholastica.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In Vitro and In Vivo Anti‐Hyperglycemic and Antioxidant Effects of Phenolic Compounds isolated from Oryza sativa L.(Keun‐nun‐Jami) Variety
Authors: Kim Tae‐Yang , Kang Hanna , Lee Jung‐Yun , Yu Jin‐a , Hong Sung‐Gil , Kwon Young‐In
Published: June 2021

Type 2 diabetes, one of the chronic diseases, is a problem in both developed and developing countries. In particular, excessive ingestion of grain, such as rice, can result in hyperglycemia and type 2 diabetes. Therefore, a Oryza sativa L. variety, Keun‐nun‐Jami (KNJM) extracts were investigated for their anti‐hyperglycemic effect using in vitro and animal models. In vitro study inhibitory activities of both ethyl alcohol extracts of bran and germ against rat intestinal sucrase and maltase were evaluated. Oxygen radical absorbance capacity (ORAC) was also investigated. Ethyl alcohol extract of KNJM bran (EKB) had the highest sucrase inhibitory activity followed by ethyl alcohol extract of KNJM germ (EKG). Taxifolin, a flavonol, was isolated from the extract of KNJM along with three unknown compounds using HPLC and semi‐prep HPLC systems. Their structures were elucidated by extensive spectroscopic analysis, including 1D and 2D NMR, and FTICR‐MS. Furthermore, post‐prandial blood anti‐hyperglycemic effect of the extacts and taxifolin were measured using SD (Sprague Dawley) rat model. Postprandial blood glucose levels were tested for 3 hr after sucrose and starch administration with or without 500 mg of EKB, EKG, and taxifolin, respectively. Similar to result in vitro study, EKB which has higher content of taxifolin than that of EKG had stronger anti‐hyperglycemic effect than that of EKG. These results indicate that taxifolin from KNJM serves to reduce post‐prandial blood glucose rise and slower absorption of glucose in the small intestine, via carbohydrate hydrolyzing enzymes inhibition. These findings suggest that EKB has better potential for this bioactivity and this observation possibly correlates with the taxifolin contents observed in both extracts. Selected variety of colored rice, KNJM may provide the potential to contribute as a dietary strategy for controlling postprandial hyperglycemia.

Support or Funding Information

This work was supported by a grant from the Next‐Generation BioGreen 21 Program (No.PJ01314003), Rural Development Administration, Republic of Korea

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The relationship between food exposure frequency and expression of AMP‐activated protein kinase (AMPK) mRNA in the brain, liver, and muscle of channel catfish
Authors: Kostner Danica LeAnn , Dougherty Megan , Schmidtberger Abigail , Kobayashi Yass
Published: June 2021

Obesity poses serious challenges to our healthcare system with serious consequences of associated diseases. Our laboratory has been examining how the expression of genes involved in food intake and tissue nutrient metabolism is associated with the development of obese‐like phenotypes in channel catfish. The objective of the study was to examine changes in expression of AMPK mRNA in response to changes in feeding frequency. Fish were fed every 12 (overfed), 24 (control), or 48 (underfed) hours (n= 4 tanks per treatment, 8 fish per tank) for 28 days. Brain, liver, and muscle tissue were collected at the end of the study. The expression of the AMPK mRNA was examined using quantitative RT‐PCR. The muscle expression of all AMPK subunit mRNA was unaffected by feeding frequency. In contrast, brain expression of alpha 1 and beta 1 subunit mRNA had a linear tendency for increase in response to increased feeding frequency. Hepatic expression of alpha 2 and beta 1 mRNA was lower in the overfeed group compared to those assigned as control or underfeed. Hepatic expression of alpha 1 mRNA decreased linearly in response to increased feeding frequency. Our results demonstrated that the hepatic and the brain expression of AMPK mRNA was influenced by increase in exposure to food in a transcript‐specific manner. Given that the AMPK serves as a regulator of tissue nutrient sensing and metabolism in mammals, it is possible that AMPK may share a similar function in the brain and liver but not in the muscle of channel catfish.

Support or Funding Information

Kansas IDeA Network of Biomedical Research Excellence Network (NIH/NIGMS grant number P20GM103418)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bisphenol A, but not Bisphenol S, exposure increases lipid deposition by acting on the PI3K pathway in vivo
Authors: Gibert Yann , YOGANANTHARAJAH Prusothman , McGee Sean , Meikle Peter , Mellett Natalie , Basheer Faiza , Hall Liam
Published: June 2021

Bisphenol A (BPA) is an endocrine disrupter present in many daily products including food and beverage packaging. BPA exposure has been linked to many disorders in humans including cancer, neurological disorders, diabetes and obesity and has therefore been declared toxic and a partial ban is in effect in several countries. Recently the plastic industry has come with a bisphenol analog as a substitute for BPA in BPA free products: BPS. However to date, little is known regarding the toxicity of BPS in vivo. In this study, we used the zebrafish as a vertebrate model to understand the mechanism of action of BPA and BPS in lipid usage during embryogenesis. Zebrafish embryos exposed to the extreme low dose 1 nM BPA (over 6,000‐fold lower than the accepted human daily exposure), have a 65% increase in lipid deposition. Interestingly, BPS exposure, at the same concentration, has no effect on lipid deposition. BPA is classified as an estrogenic compound however, pharmacological blockage of the nuclear estrogen receptors does not suppress the induced lipid deposition. To identify which pathway might be involved in this increased lipid deposition, we performed a full embryo lipidomic analysis. This revealed that BPA exposed embryos had a dramatic increase of diacylglycerol (DAG) and Phosphatidylinositol (PI) lipids. Strikingly, lipidomic analysis of BPS exposed embryos showed a decrease of DAGs and PIs. This is the first study to demonstrate a phenotypical difference between BPA and BPS at similar doses. Double exposure of BPA and LY294002, a strong inhibitor of phosphoinositide 3‐kinases, abolishes the BPA induced increase in lipid deposition and lipid usage. At the molecular level, BPA exposure increases the expression of key enzymes of the PI3K pathway such as: cdipt, plcb3, vps34p and p110. Our study allows us to propose a model in which BPA, but not BPS, at very low concentration can activate the PI3K pathway leading to an accumulation of lipid during embryogenesis in an estrogen receptors independent manner. Our results confirm that, at least in terms a of lipid usages, BPS is safer than BPA exposure and support the ban on BPA from consumer merchandise.

Support or Funding Information

This project was supported by an Australian Diabetes Research Trust (DART) research grant to YG

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phosphorylation Dependent Interactions of Perilipin 5 with Adipocyte Triacylglycerol Lipase
Authors: Osborn Rachel E , Tansey John T
Published: June 2021

Diseases such as non‐alcoholic fatty liver disease, obesity and type II diabetes are on the rise in the US, and the dysfunction of cellular lipid metabolism is a major contributor to them. Perilipins are a class of five proteins found on the exterior of lipid droplets which play a role in proper lipid metabolism. Perilipin 5 is expressed in tissues that are oxidative such as the liver, heart and skeletal muscle. We used CHO cells expressing perilipin 5 protein and immunofluorescence microscopy to examine lipase interactions with perilipin 5 under different phosphorylation states. We hypothesized that the interactions of lipases such as adipocyte triacylglyerol lipase (ATGL) and perilipin 5 were phosphorylation dependent. Using immunofluorescence microscopy, we confirmed that perilipin 5 changes its subcellular localization and moves from lipid droplets and cytosol to the nucleus when stimulated with forskolin and IBMX to activate the PKA signaling pathway. Identification of lipase‐perilipin 5 interactions are ongoing. Collectively these data show that perilipin 5 interactions and localization in the cell are phosphorylation dependent.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lipid Droplet Protein Binding in Response to Altered Phospholipid Composition
Authors: Renier Timothy , Paetz Olivia , Vuong Sunny , Listenberger Laura
Published: June 2021

Alcoholic Fatty Liver Disease (AFLD) is the alcohol‐induced accumulation of excess lipids in hepatocyte lipid droplets. Lipid droplet surfaces are phospholipid monolayers predominantly composed of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Protein binding to lipid droplets affects droplet size and activity. Previous experiments with a rat model of AFLD show lipid droplet surfaces have a decreased ratio of PC:PE. We aimed to determine if the altered phospholipid composition of AFLD lipid droplets leads to differential binding of several proteins of interest, selected for their membrane binding motifs and roles in lipid droplet homeostasis. By culturing AML12 cells in choline‐deficient media with oleate, an unsaturated fatty acid, we simulated the altered phospholipid composition of AFLD lipid droplets. Next, we isolated lipid droplet fractions and examined proteins of interest by SDS‐PAGE and western blotting. We found equivalent levels of rab18 and lanosterol synthase associating with lipid droplet fractions from cells grown with and without choline. Alternatively, there was an increase in the level of αCCT1 and perilipin 2 in lipid droplet fractions from cells grown without choline. By evaluating how altered phospholipid monolayer composition affects the binding of lipid droplet proteins, we seek to further elucidate the biochemical pathways that contribute to AFLD.

Support or Funding Information

This work was supported by St. Olaf College Collaborative Undergraduate Research and Inquiry, Howard Hughes Medical Institute, the St. Olaf TRIO McNair Scholars Program, and LSAMP Alliance.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of Phosphorylation Sites in Perilipin 5
Authors: Sumser Natalie M , Tansey John T
Published: June 2021

In modern times, there has been a significant increase in metabolic disease. Obesity has been a growing concern in the U.S, leading to a rise in various health problems, such as type II diabetes and cardiovascular disease. One major factor in these diseases is lipid metabolism. The understanding of this process would lead to a better understanding of these diseases. Perilipins, a family of five proteins associated with lipid droplets, play an important role in lipid metabolism. Perilipin 5 is primarily seen in oxidative tissue, acting to regulate fatty acid storage and release, as well as fatty acid oxidation. It is known that phosphorylation partially regulates the activity of Perilipin 5, particularly due to PKA activation, and we hypothesized that there are multiple phosphorylation sites on the protein. Treatment of cells with forskolin and IBMX was used to increase cyclic AMP to activate PKA. This was followed by immunopreciptation and trypsin digestion of perilipin 5. Phosphopeptides are enriched through affinity chromatography with a Fe‐NTA column prior to conducting mass spectrometry on the samples to differentiate between phosphorylation sites. We hypothesize that both a combination of where perilipin 5 is phosphorylated and when the sites are phosphorylated cooperate to regulate the function of the protein.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interaction of Perilipin 5 with Mitochondria
Authors: Todd Claire I , Tansey John T
Published: June 2021

Problems with lipid metabolism can lead to array of diseases such as fatty liver disease, obesity, and diabetes. Nearly one‐third of the world's population is obese or overweight, and struggle with these diseases. Cellular lipids are found in lipid storage droplets. Perilipins are a family of proteins that associate with the surface of lipid storage droplets and regulate their contents. We used CHO cells expressing perilipin 5 protein and assayed them using immunofluorescent microscopy to determine if mitochondria interact with perilipin 5 under different phosphorylation states. We hypothesized that the interaction of mitochondria and perilipin 5 was phosphorylation dependent. We used antibodies that detect either FLAG tagged and native perilipin 5 and Mitotracker red to stain cells and forskolin and IBMX to activate the PKA signaling pathway. Following one hour of stimulation, perilipin 5 appeared in the lipid droplets and cytosol of the cell but interactions with mitochondria were not apparent. In this model 3 hours of stimulation is required for perilipin 5 to translocate to the nucleus. Collectively these ongoing studies show that the cellular partitioning of perilipin 5 is in part phosphorylation dependent.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The chinchilla as a novel animal model of gestational diabetes
Authors: Duvald Christina Søndergaard , Overgaard Maria Dahl , Ovesen Per Glud , Jeppesen Per Bendix , Pedersen Michael
Published: June 2021

Background

Gestational diabetes occurs in 3–4% of all pregnancies, thereby being one of the leading pregnancy diseases in developed countries. The disease poses important health implications for both mother and child. Several animal models exist in this field, but with significant limitations. Consequently we want to introduce the pregnant chinchilla as a novel animal model of gestational diabetes. The chinchilla is excelled for various reasons; it gives birth to one or two cubs, is pregnant for 115 day and has a histological similar placenta barrier as compared with the human one. Since the chinchilla has never been used in diabetic studies, we aimed to show that it is possible to make it diabetic, before making a gestational model.

Streptozotocin is by far the most applied method to induce diabetes in animal models, mimicking the ethology of diabetes type 1. However, since gestational diabetes is due to insulin resistance, we wanted to use high fructose feed as a method to induce diabetes.

Methods

Twelve chinchillas were included in the study, six of which received high fructose diet to induce diabetes and six who received control diets. Blood glucose was monitored using Abbott Libre Freestyle glucometer. After 19 weeks the animals were euthanized and organs and blood withdrawn for analysis.

Preliminary results

Non‐fasting plasma glucose levels in chinchillas fed with high fructose diet was 19.3 ± 6.9 mmol/l, compared to controls 11.1 ± 2.2 mmol/l, p=0.056. Furthermore, DXA scans showed significant higher body fat percentage among fructose animals, p=0.043 compared to controls. HBA1C was, however, not affected in any of the groups.

Conclusion

We have proven it possible to induce high blood glucose levels in the chinchilla by using high fructose diet. Because of the low sample size the blood glucose levels were, however, not statistically significant between groups. In spite of this, we consider the chinchilla a promising option for a future model of gestational diabetes.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antioxidant and Haematological Activities of Ethanolic Extract of Ricinus communis
Authors: Ihekuna Ogechi , Imaga Ngozi Awa , Adewusi Banke
Published: June 2021

Ricinus communis commonly known as castor oil is used as a traditional natural remedy or folkloric herb for the control and treatment of a wide range of diseases around the globe. Various studies have revealed the presence of diverse phytochemicals such as alkaloids, flavonoids, terpenes, saponins, phenolic compounds such as kaempferol, gallic acid, ricin, rutin, lupeol, and ricinoleic acid. The present study investigates the in vivo and in vitro analyses of the effect of ethanolic extract of ricinus communis leaf and seed on animal models. The antioxidant potential of Ethanolic leaf and seed extract of Ricinus communis was investigated. The result shows that the ethanolic extract of the seed and leaf exhibited IC50 values of 77.17± 0.36 and 69.33 ± 0.26 μg/mL, 62.86±0.54 and 62.39±0.18 μg/mL respectively in DPPH and nitric oxide radical inhibition assays these values were less than those obtained for ascorbic acid used as standard. In the in vivo experiments the extract treatment caused a significant increase in the level of catalase in the liver and the kidneys. A significant increase in the level of SOD in the liver was observed. The acute toxicity study was carried out in both male and female wistar rats; the results reveal that even at the highest dose 2000mg/kg body weight R.communis seed and leaf extract showed no significant toxic effects showing that LD50 is above 2000 and belongs to class 5. The haematological and biochemical parameters of the treatment groups were compared with the control group, result shows that there were no significant differences (mean ± SEM n=6 *p < 0.05) in the biochemical and haematological parameters of treatment groups compared to the control group. These findings suggest that the ethanolic extract of Ricinus communis seed and leaf exhibited a strong antioxidant activity which may be responsible for some of its reported pharmacological activities and can be used as antioxidant supplement.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comprehensive snapshots of an unusual reaction cycle for an atypical protein tyrosine phosphatases (PTP)
Authors: Wang Huanchen , Jessen Henning J. , Shears Stephen B
Published: June 2021

Inositol pyrophosphates (PP‐InsPs) are “energetic” intracellular signals that are ubiquitous in animals, plants, and fungi. We are characterizing new proteins that regulate PP‐InsP metabolism and/or function as their ‘receptors'. Here, we describe our structural characterization of At1g05000, an Arabidopsis member of the cysteine‐based protein tyrosine phosphatase family that exhibits higher reactivity towards PP‐InsPs than phosphoproteins. We provide 20 high resolution crystal structures which describe how this atypical PTP has been repurposed to become the most active of known PP‐IPs phosphatases. These structures include complexes with different ligands including inositol pyrophosphates, polyphosphates and nucleotides; together they reveal pre‐reactant, reactant, intermediate and product states. As a consequence, we describe an atypical mechanism: an exceptionally electropositive ‘P’‐loop that immobilizes the β‐phosphate for hydrolysis; the opposite end of the substrate is anchored by Lys and Arg residues in a loop that is pushed into the binding pocket due to intramolecular crowding; a canonical catalytic acid is sacrificed to avoid electrostatic clashing with the substrate; a conserved water is identified to function as the proton donor; two catalytic important residues, His156 and Arg188, form π‐cation interactions that are important for positioning substrate and the catalytic water; hydrolyzed phosphate ion is rotated during catalysis. Overall, our data support a substrate‐driven reaction mechanism. Furthermore, this work opens up new directions for studying regulatory interactions of PP‐InsPs with human protein tyrosine phosphatases.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Codon Optimization of Gene Editing CRISPR‐SaCas9 Augments Protein Expression in Human Liver Cells to Boost in vivo Therapeutic Application
Authors: Cheng Benjamin , Groshong Tyler , Madejski Irene , Sweeney Robert , De Luke
Published: June 2021

CRISPR‐Cas9 gene editing technology shows great promise in revolutionizing the treatment of genetic disorders; however, the current focus on treatment by ex vivo delivery is limited to blood diseases. In vivo treatment can substantially extend the clinical reach of the technology, but is limited by a challenging delivery process. The large size of the Cas9 gene (~4500bp) inhibits delivery by the small viral vectors and lipid nanoparticles used. We propose that a codon‐optimized Cas9 gene will support significantly higher protein expression than the presently used genes; this can compensate for the low delivery efficiency and has great potential to improve the viability of in vivo CRISPR‐Cas9 therapies.

A commonly used parent saCas9 sequence was selected from Addgene's public plasmid database. A novel, codon‐optimized gene sequence was engineered from the parent using GenScript's OptimumGene™ algorithm. Both the optimized gene and parent gene were transfected into human HepG2 liver cells with high efficiency as visualized by GFP‐tagged saCas9. ELISA protein quantification of two sample groups demonstrated that the codon‐optimized gene expressed the saCas9 protein at 3.5× the rate of the commercial sequence. The augmented Cas9 protein expression supported by the novel saCas9 sequence can better enable in vivo CRISPR‐Cas9 treatments.

Support or Funding Information

This work was supported by The Nueva School.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Perlecan Domain V Increases Amyloid Clearance in Co‐Morbid VCID
Authors: Trout Amanda , Hartz Anika , Roberts Jill , Zhang Zouhui , Frazer Justin , Bix Gregory
Published: June 2021

Introduction

Vascular contributions to cognitive impairment and dementia (VCID) is a broad term that encompasses a spectrum of initial asymptomatic cerebrovascular changes (seen in small vessel disease and cerebral amyloid angiopathy) to the profound symptomatic damage following acute stroke(s). Besides aging, metabolic disorders such as type 2 diabetes complicate our understanding of underlying mechanisms. Cerebrovascular remodeling and angiogenesis may represent early compensatory changes to the reduced blood flow seen in VCID by increasing the proteolytic turnover of the surrounding extracellular matrix. We have demonstrated that one such extracellular matrix protein, perlecan, possesses a C terminal domain V (DV) protein that, upon cleavage from perlecan, greatly enhances brain angiogenesis.

Objective

We hypothesize that DV therapy can reduce pathological changes in the cerebrovasculature, an underlying component of VCID.

Methods

DV's mechanism and therapeutic potential in facilitating Aβ clearance were examined in an ex vivo model of isolated mouse brain capillaries in normal and high glucose environments. We also determined the cerebrovascular changes in a comorbidity mouse model of VCID (diabetic APP/PS1 knock in (db/AD)) that is associated with hemorrhages, aneurysms, Aβ deposition, cognitive decline and increased mortality.

Results

DV (300 and 600 nM) doubled the transport of fluorescently tagged hAβ1‐42 over 24 hours from the bath (abluminal) to luminal side of the brain capillaries. One of Aβ's known transport proteins, P‐glycoprotein (P‐gp), also increased in total protein expression and in transport activity in the presence of DV. At 3–6 months in db/AD mice, we observed a decrease in BBB proteins (claudin‐5) indicating an altered cerebrovascular function correlating with DV expression increase during the asymptomatic angiogenic stage. At 9–12 months, cognitive changes were associated with altered cerebrovascular protein expression in PECAM, Collagen IV, and DV correlating with the aberrant two‐photon vascular imaging, increased saccular aneurysms, and microhemorrhages.

Conclusion

These data show that DV increases cerebrovascular Aβ clearance through P‐gp. Increased DV expression correlates with early cerebrovascular changes induced by angiogenic‐remodeling in a diabetic co‐morbid mouse model of VCID. Lower DV expression is associated with aberrant vasculature that contribute to increased mortality, suggesting that DV ‘replacement therapy’ could represent a novel therapeutic for VCID.

Support or Funding Information

NIH RO1 N5089515‐01

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Opposing roles of Sigma‐1 and Sigma‐2 receptors in heavy alcohol drinking and associated mechanical allodynia in mice
Authors: Sabino Valentina , Quadir Sema G , Tanino Sean M , Rohl Christian D , Sahn James J , Moore Catherine F , Martin Stephen F , Cottone Pietro
Published: June 2021

Alcohol Use Disorder (AUD) is a serious and complex neuropsychiatric disorder characterized by excessive alcohol intake, inability to control consumption, and the emergence of a negative emotional state in the absence of alcohol. While the role of the Sigma‐1 receptor (Sig‐1R) in animal models of AUD has been demonstrated, the role of Sigma‐2 receptor/Transmembrane Protein 97 (Sig‐2R/TMEM97), which has recently been cloned, is still unclear. Using an intermittent access, 2‐bottle choice paradigm, the effects of the Sig‐2R/TMEM97 modulator JVW‐1034 (0–30 mg/kg, i.p.) on ethanol drinking in male C57Bl/6J mice were assessed. Sucrose intake was used to control for non‐specific effects of the drug on an alternate reinforcer in a separate study, and locomotor intake was also assessed. In addition, using an automated von Frey filament test, the effects of JVW‐1034 on mechanical sensitivity in alcohol drinking mice was tested during withdrawal. The effects of the Sig‐2R modulator were compared to those of BD‐1063 (0–30 mg/kg, i.p.), a preferential Sig‐1R antagonist. Results showed that JVW‐1034 reduced alcohol intake and preference in this model, without affecting water intake. Sucrose intake was not affected by the drug, suggesting selectivity of the effect for alcohol. JVW‐1034 also blocked the mechanical allodynia induced by chronic alcohol drinking without affecting sensitivity in control mice. Lastly, JVW‐1034 did not affect locomotor activity in either group. BD‐1063 had a very similar profile. These data suggest that Sig‐1R and Sig‐2R play opposite roles in the regulation of alcohol drinking and associated allodynia and hyperalgesia, and lay the foundation for more extensive studies examining the involvement of the Sig‐2R/TMEM97 system in AUD.

Support or Funding Information

R01‐AA024439

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Palmitic acid methyl ester directly induces hippocampal neuroprotection against oxygen and glucose deprivation
Authors: Neumann Jacob , Van Allen Joshua , Azizbayeva Rinata , Lin Hung Wen
Published: June 2021

Cardiopulmonary arrest (CA) is one of the leading causes of death and disability in the USA. The prevalent quandary in this field is the multi‐factorial nature of CA (i.e., whole body ischemia), which compromises systemic blood parameters and cerebral, renal and cardiac functions. This disruption of cerebral blood flow (CBF) also results in severe and selective brain damage (i.e., neuronal cell death) in the hippocampus, especially in the cornu Ammonis 1 (CA1) region. While previous research has indicated PAME (a vasodilator) as a neuroprotective agent, the specific mechanism(s) of neuroprotection is/are currently unknown and it is currently unknown if PAME‐induced neuroprotection is from direct modulation of CBF or due to direct activation of neuroprotective pathways. Here, we tested the hypothesis that PAME induces neuroprotection independent of CBF, and that PAME alters additional signaling pathways to induce neuroprotection in the hippocampus. Our studies used an in vitro hippocampal model (hippocampal organotypic slices) to determine the requirement of CBF for PAME‐induced neuroprotection and if specific modulation of alpha‐7 nicotinic acetylcholine receptors (a potential target of PAME) can induce neuroprotection following oxygen and glucose deprivation (OGD) using propidium iodide staining. All of the protocols were approved by the West Virginia School of Osteopathic Medicine Institutional Animal Care and Use Committee. Our results suggest that the direct application of PAME or inhibition of alpha‐7 nicotinic acetylcholine receptors induced neuroprotection following OGD in the CA1 region of the hippocampus. These data suggest that PAME‐induced neuroprotection occurs independent of CBF and identifies alpha‐7 nicotinic acetylcholine receptors as a potential mechanism for PAME‐induced neuroprotection.

Support or Funding Information

This study was supported by a West Virginia School of Osteopathic Medicine intramural grant and a grant from the American Heart Association 17GRNT33660336 .

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